extfieldsofstudy: Chemistry Medicine

s2fieldsofstudy: Biology Chemistry

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Nucleotide-binding sites on Escherichia coli F1-ATPase. Specificity of noncatalytic sites and inhibition at catalytic sites by MgADP.

Nucleotide-depleted EcF1 binds a maximum of two GTP, ATP, or ADP at noncatalytic sites, whereas all three sites can only be filled by a combination of nucleoside di- and triphosphates. MgPPi prevents binding of GTP and significantly slows ATP binding, suggesting that non-catalytic sites also bind PPi. No binding of GDP at non-catalytic sites could be detected. The slow rate of GTP dissociation from noncatalytic sites (t1/2 = 175 min) is increased 2-8-fold by EDTA, MgPPi, MgADP, or EDTA/ATP, but 23-fold by conditions for ATP hydrolysis. ATP hydrolysis by EcF1, depleted of both its inhibitory epsilon-subunit and endogenous nucleotides, shows a burst of activity. However, it shows a lag if preincubated with MgADP but not when preincubated with Mg2+ alone. For epsilon-depleted EcF1 containing endogenous inhibitory ADP, preincubation with an ATP-regenerating system results in a burst of activity, whereas the control shows a lag. This same enzyme form shows significant inhibition with increasing concentrations of Mg2+ during ATP hydrolysis but lesser levels of inhibition when other NTP substrates are used. With the five-subunit enzyme, increasing amounts of azide cause an increase in the level of inhibition with a corresponding increase in amount of bound nucleotide resistant to rapid chase. Azide-trappable nucleotide is bound at catalytic sites as shown by covalent incorporation of 2-azido-ADP. The results suggest that ligand specificity may not be a reliable means of distinguishing between catalytic and noncatalytic sites and that MgADP inhibition should be taken into account in the kinetic analysis of EcF1 mutants.

F,-ATPase, the catalytic component of proton-translocating ATP-synthase complexes from eubacteria, mitochondria, and chloroplasts, has a subunit structure of cr,p,y& and a total of six nucleotide-binding sites. Three of the sites exchange bound nucleotide rapidly during catalytic turnover and are considered to be catalytic sites. The remaining three sites do not exchange rapidly and are referred to as noncatalytic sites (for reviews see Futai et al., 1989;Senior, 1990;Fillingame, 1990;Penefsky and Cross, 1991;Allison et al., 1992;Issartel et al., 1992;Boyer, 1993).
For mitochondrial and chloroplast F,, it has been shown that MgADP can bind at a catalytic site to form an inhibitory complex. This is evident as a lag in ATP hydrolysis when these enzymes are preincubated with ADP in the presence of M e (Vasilyeva et al., 1982a;Feldman et al., 1985). Preincubation with Mg2+ alone has no effect if endogenous nucleotides are previously removed (Drobinskaya et al., 1985). Azide prevents the subsequent reacti-* This work was supported by Research Grant GM 23152 from the National Institutes of Health, United States Public Health Service. The costs of publication of this article were defrayed in part by the payment tisernent" in accordance with 18 U.S.C. Section 1734 solely to indicate of page charges. This article must therefore be hereby marked "aduerthis fact.
vation of the enzyme, whereas anions such as sulfite activate the enzyme by facilitating release of the inhibitory MgADP (Vasilyeva et al., 1982b). In the dark, 2-azido-ADP gives inhibition with identical properties to that of MgADP. Photolysis of the inhibitory complex results in the covalent labeling of a catalytic-site peptide on CF,' (Zhou et al., 1988) and MF, (Milgrom and Boyer, 1990;Chernyak and Cross, 1992).
The F,-ATPase from Escherichia coli has been shown to bind Mg2' tightly (Senior et al., 1980) and to be inhibited when magnesium is added in excess ofATP (Kanazawa et al., 1980). It has been argued that this inhibition is not due to inhibitory MgADP bound at a catalytic site since, unlike the mitochondrial and chloroplast enzymes, no dependence on ADP could be demonstrated (Senior et al., 1992). Although potential support for the presence of catalytic site inhibitory MgADP comes from the observation that azide inhibits EcF,, this has been attributed to a direct effect of azide on subunit cooperativity (Noumi et al., 1987). Additional suggestive evidence is that sulfite activates the E. coli enzyme (Dunn et al., 1987). Factors that could mask an ADP-dependent lag include the presence of an inhibitory subunit, E, which can dissociate slowly upon dilution of EcF, into an ATP hydrolysis assay (Laget and Smith, 1979) and the presence of endogenous catalytic site ADP on the isolated enzyme.
Over the past 15 years, many studies of the nucleotide-binding sites on F,-ATPases have relied on ligand specificity to distinguish between catalytic and noncatalytic sites (Harris, 1978;Harris et al., 1978;Perlin et al., 1984). Catalytic sites are known to be nonspecific toward purine nucleotide substrates. They bind and hydrolyze GTP and ITP nearly as well as ATP. In contrast, noncatalytic sites have been considered to be highly specific for adenine nucleotides. However, recent results have shown that CF, (Guerrero et al., 1990;Milgrom et al., 1991) and MF, (Milgrom and Cross, 1993) can bind GTP at noncatalytic sites.
In this study we have reexamined the specificity of nucleotide sites and the question of inhibitory catalytic site-bound MgADP. The results indicate that both GTP and PP, can bind to noncatalytic sites on EcF, and that the enzyme is inhibited by binding MgADP at a catalytic site. [P,y3'P12-Azido-ATP was synthesized as described by Melese and Boyer (1985). Enzyme for Noncatalytic Site Specificity Studies-EcF, was isolated from E. coli strainAN1460 (Downie et al., 1980) by the method of Senior et al. (1979aSenior et al. ( , 1979b as modified by Wise et al. (1981). Purified EcF, was stored at -70 "C at a concentration of about 20 mg/ml in buffer containing 50 mM Tris-HC1, pH 7.4, 2 mM EDTA, 1 mMATP, 1 mM dithiothreitol, 40 mM E-aminocaproic acid, and 10% glycerol. Just before use, EcF, was diluted to a concentration of about 2 mg/ml with buffer containing 20 mM MOPS/Tris, pH 8.0, 150 mM sucrose, and 0.2 mM EDTA and passed through two sequential 1-ml Sephadex centrifuge columns (Penefsky, 1977) equilibrated with the same buffer. This form of the enzyme (referred to as native or EcF,[2,11) contained 2.8 mol of endogenous ANP/ mol, of which 2.0 mol/mol was not chaseable by GTP in agreement with Beharry and Bragg (1992). The nonchaseable nucleotides consisted of 0.9 mol of ADP and 1.1 mol of ATP. Nucleotide-depleted enzyme (EcF,[O,OI) was prepared by published procedures (Garrett and Penefsky, 1975;Senior et al., 1992). Two ammonium-sulfate precipitation steps preceded gel filtration. The enzyme was eluted from a 1.0 x 100-cm column of Sephadex G-50 (medium) at a flow rate of 0.6-1.2 mVh with 100 mM Tris-H,SO,, pH 8.0,4 mM EDTA, 100 mM Na,SO,, and 50% glycerol. Selected fractions containing EcF, were combined and concentrated using a Centricon-10 microconcentrator (Amicon). The enzyme was diluted 20-fold and reconcentrated twice in order to transfer it to storage buffer containing 100 mM Tris-H,SO,, pH 8.0, 4 mM EDTA, and 50% glycerol. EcF,[O,Ol was stored a t a concentration of about 20 mg/ml at -20 "C. The nucleotide content of EcF,[O,Ol was less than 0.2 mol/mol of enzyme, as determined by subjecting a neutralized perchloric acid extract to ion-exchange HPLC (Milgrom and Cross, 1993).

EXPERIMENTAL PROCEDURES
Enzyme for MgADP Inhibition Studies-EcF, was isolated from strain JP17 containing the wild-type plasmid pJWl and prepared to the solubilization stage as described by Senior et al. (1979a, 197913). Chromatographic purification employed a Productiv DE cartridge, and the enzyme was judged to be greater than 95% pure by SDS-polyacrylamide gel electrophoresis analysis. Enzyme was stored at 20-30 mg/ml at -70 "C. Epsilon-depleted enzyme, ~d-EcF,[2,11, was prepared as described (Dunn, 1986). Unlike the 5-subunit enzyme, we found that ed-EcF, could be depleted of endogenous nucleotides simply by ammonium sulfate precipitation. This involved adding 2 volumes of saturated ammonium sulfate to Ed-EcF, in TE buffer (50 mM Tris-C1, pH 8.0, 0.5 mM EDTA), incubating at 0 "C for 2.5 h, pelleting 10 min in a Beckman microfuge, and redissolving in TE buffer followed by passage through a 1-ml centrifuge column equilibrated in the same buffer. A luciferase assay of neutralized, perchloric acid extracts showed ~d-EcF,[2,11 to contain 2.5 mol ANP/mol of enzyme, whereas ~d-EcF,[O,01 contained less than 0.1 mol ANP/mol.

Methods
Protein concentration was determined by a modified Lowry method according to Peterson (1977). The concentrations of free and complexed metals and nucleotides were calculated using the program Bound and Determined (Brooks and Storey, 1992).
Noncatalytic Site Specificity St~dies-[~H]Nucleotide binding was measured by a centrifuge column assay. Columns containing 1 or 2 ml of swollen Sephadex were equilibrated with MTSEM buffer (20 mM MOPS/Tris, pH 8.0, 150 mM sucrose, 0.2 mM EDTA, and 2 mM Mg(CH,COO),). When a n NTP-regenerating system was present during incubation with L3H]GTP or [3H]ATP, it consisted of 50 mM KC1, 10 mM phosphoenolpyruvate, and 1 mg/ml pyruvate kinase. When the EcF, concentration was less than 3 PM, 1.1 mg/ml bovine serum albumin was included in all buffers to enhance recovery of the enzyme through centrifuge columns (Cross and Nalin, 1982). Under these conditions, the recovery from 100-pl samples was 90% when applied to 1-ml columns and 85% when applied to 2-ml columns. When column effluents were to be counted, they were collected directly in liquid scintillation vials. Data were corrected for radioactivity eluting in the absence of EcF, (usually less than 5%). The standard deviation for replicate measurements is *lo% using these procedures.
The composition of [3H]nucleotides bound to EcF, was determined by anion-exchange HPLC using a Partisil PX3 10/25 SAX column (4.6 x 250 mm). Bound nucleotides were extracted by perchloric acid precipitation of protein in the presence of carrier ADP, ATP, GDP, and GTP. Extracts were neutralized with KHCO,, centrifuged, and diluted prior to loading on the HPLC column. Nucleotides were eluted using a phosphate gradient as described (Milgrom and Cross, 1993). Aliquots of the fractions were counted by liquid scintillation using 5-10 volumes of Bio-Safe I1 (Research Products International).
MgADP Inhibition Studies-Enzyme stocks were diluted to 1-2 mg/ml with either TE buffer or TM buffer (50 mM Tris-C1, pH 8.0,0.5 mM MgCl,) and passed through two sequential 1-ml centrifuge columns equilibrated in the same buffer. When the enzyme concentration was less than 0.5 mg/ml, buffers were supplemented with 1 mg/ml bovine serum albumin (TEB and TMB buffer (TE or TM buffers containing 1.0 mg/ml bovine serum albumin)).
Except where noted, hydrolysis assays were performed by dilution of the preincubated enzyme into 50 mM Tris-C1, pH 8.0, 50 mM KCl, 1 mg/ml bovine serum albumin, 0.5 mg/ml pyruvate kinase, 2 mM phosphoenolpyruvate (reaction buffer). plus MgC1, and [y3'P1ATP to give the indicated concentrations of MgATP and free Mg2'. The amount of inorganic 32P released was determined after removal of residual [Y-~~PIATP by adsorption to charcoal. Aliquots were subjected to Cerenkov counting with correction for 32P1 present in controls lacking EcF,.
Binding of Ipy-32P12-azido-ATP was initiated by addition of 100 pg of EcF, in TM buffer to reaction buffer containing 0.05 mg/ml pyruvate kinase and 5 mM phosphoenolpyruvate plus MgC1, and the nucleotide affinity probe to give final concentrations of 260 nM EcF,, 460 nM Mg(2azido)ATP, and 1 mM free Mg". Binding of the probe was terminated after 20 min by exposure to a 30-s cold chase containing 50 p MgATP, 1 mM free Mg2+, and either 1 mM azide or 7.5 mM sulfite followed by passage of 0.5-ml aliquots through 5-ml centrifuge columns equilibrated in TM buffer plus 0.5 mM azide. Column efiluents were irradiated for 10 min with a Mineralight lamp set at short wavelength. Noncovalently bound probe was removed by acid precipitation in the presence of ATP, followed by two brief washes with 80% acetone (-20 "C). The pellets were dissolved in 75 p1 of 8 M urea made fresh in 100 mM EPPS, pH 8.0, diluted 8-fold, and digested by 4 pg of trypsin for 4 h followed by a second equal addition of trypsin overnight. Samples were diluted 2-fold with 0.2% trifluoroacetic acid, filtered, and applied to a Vydac C, reversed-phase column. Peptides were eluted with the indicated gradient of acetonitrile in 0.1% trifluoroacetic acid at 1 mumin with 2-ml fractions collected and subjected to Cerenkov counting.

Specificity of Noncatalytic Nucleotide-binding
Sites-Incubation of nucleotide-depleted enzyme (EcF,[O,OI) with 2 mM MgL3H1GTP for 2 min results in the binding of about 3 mol of [3HIGNP/mol of enzyme (Table I, Experiment 1). If the enzyme is then incubated for 1 min without further addition, all of the [3H]GNP remains bound during passage through a second centrifuge column (Experiment 2). However, if millimolar concentrations of unlabeled GTP (Experiment 3 ) or ATP (Experiment 4 ) are present during the second incubation, 1 mol of GNP/mol of enzyme is released. L3H]GNP displaced from catalytic sites during this cold-chase step consists of nearly equal amounts of GDP and GTP. In contrast, the 2 moVmol remaining bound at noncatalytic sites is mostly GTP (-90%).
When the experiments described above were repeated using native enzyme (EcF,[2,11), about 2 mol of L3H1GNP boundmol of enzyme during incubation with 2 mM [3HlGTP (Table I, Experiments 5 and 6 ) . Again, close to 1 mol of labeled nucleotide is displaced from catalytic sites during subsequent incubation with unlabeled GTP (Experiment 7 ) or ATP (Experiment 8). However, native enzyme retained only 1 mol of labeled nucleotide at noncatalytic sites. Again this was mostly GTP. In summary, the results presented in Table I show that nucleotidedepleted enzyme can bind 2 GTP at noncatalytic sites, whereas the native enzyme binds 1 GTP at a noncatalytic site.
A time course for binding GNP to EcF,[O,Ol during incubation with 100 VM L3H]GTP in the presence of a GTP-regenerating system is presented in Fig. 1. A total of 2.7 mol of L3H1GNP boundmol of enzyme (circles), with 1.7 moVmol bound at noncatalytic sites (triangles), and 1.0 mol/mol bound a t catalytic sites (squares). Binding reached maximal levels within 3-4 min. Bound nucleotides were extracted from aliquots removed at 10 min, and their composition analyzed by anion-exchange  1.1 mg/ml bovine serum albumin without (circles) or following (tri-(squares) was calculated as the difference between total and nonchaseable (noncatalytic) binding. An additional incubation (inverted triangles) contained enzyme that was preincubated for 5 min with 100 p~ unlabeled ATP in the presence of an ATP-regenerating system and passed through a centrifuge column prior to incubation with r3H1GTP.
HPLC. Again, nearly all of the r3HIGNP bound a t noncatalytic sites was GTP (95%), whereas catalytic sites contained a mixture of GDP and GTP.
In contrast to the results obtained with GTP, we were unable to detect significant GDP binding at noncatalytic sites. Incubation of EcF, [O,O] with 100 p [3HIGDP for up to 90 min gave a total of 1.6 moVmol bound with only 0.2 moVmol bound at nonchaseable sites. This experiment demonstrates the efficacy o f the conditions used to chase catalytic site-bound nucleotide.
In order to compare these results to the binding of adenine nucleotides, EcF, [O,O] was incubated with 100 VM F3H1ATP in the presence of an ATP-regenerating system (Fig. 2) or with 100 p ['HIADP (Fig. 3). Binding stoichiometries were very similar in all cases, although the rate of binding adenine nucleotides to noncatalytic sites (Figs. 2 and 3, triangles) appears to be faster than the rate of binding GTP (Fig. 1, triangles). Bound nucleotides were extracted from aliquots, taken after 3 min of incubation of EcF,[O,OI with L3HIATP and a regenerating system (Fig. 21, and their composition was analyzed by HPLC. ATP accounted for 96% of the nucleotide bound at noncatalytic sites. To determine whether prior binding of ATP at noncatalytic sites on EcF, [O,O] blocks subsequent binding of GTP, we preincubated the enzyme with 100 p~ ATP and an ATP-regenerating Nucleotide binding was measured as described for Fig. 1, except that 3.7 m~ MgATP was used in the cold chase step. An additional incubation (inverted triangles) contained enzyme that was preincubated for 5 min with 100 PM ATP in the presence of an ATP-regenincubation with l3H1ADP.
erating system and passed through a centrifuge column prior to system. After unbound nucleotides were removed, the enzyme was incubated with 100 p L3H1GTP in the presence of a GTPregenerating system. The results show that prior exposure to ATP prevents ['HIGTP binding at noncatalytic sites (Fig. 1, inverted triangles versus triangles), while having no effect on EcF,[O,Ol was incubated at 60 n M for 10 min in MTSEM buffer containing 100 VM C3H]GTP and a GTP-regenerating system. T3H]GNP was displaced from catalytic sites by incubating with 3.3 m M MgATP for 1 min and unbound nucleotides were removed on a centrifuge column as described ("Experimental Procedures"). The effluent was incubated without further addition (Experiment 1) or with the additions shown. Half-times were calculated from the measured rate constants. binding a t catalytic sites (data not shown). This suggests that GTP binds at the same two noncatalytic sites that bind ATP or that cooperative interactions prevent the binding of more than two nucleoside triphosphates at the three noncatalytic sites.

Experiment
Preincubation of EcF, [O,O] with unlabeled ATP was also very effective in slowing L3H1ADP binding at noncatalytic sites (Fig.  3, inverted triangles versus triangles), again having no effect on catalytic site binding (data not shown). It was of interest, however, that in contrast to L3H1GTP (Fig. l, inverted triangles), [3H]ADP appears either to slowly exchange for noncatalytic site-bound ATP or to bind at the third noncatalytic site (Fig. 3,  inverted triangles).
In order to distinguish between these possibilities, EcF,LO,Ol was incubated briefly with 100 p~ ATP and an ATP-regenerating system to bind 1.4 mol ATP at noncatalytic sites. Unbound ligand was removed, and the enzyme was further incubated with 100 1.1~ ADP for 1 h. This resulted in the binding of 1.5 mol of ADP at nonexchangeable sites, with loss of only 0.1 mol of the ATP to give a total of 2.8 mol ofANP bound at noncatalytic sitedmol EcF,. It was also shown in Table I (Experiments 7 and 8) that native enzyme, which has one noncatalytic site filled with ADP and one with ATP ("Experimental Procedures"), can still bind one GTP at the third noncatalytic site. Hence, the results show that although only 2 mol/mol of GTP (Fig. l), ATP (Fig. 2), or ADP (Fig. 3) can bind at noncatalytic sites during incubation with 100 p~ nucleotide for up to 1 h, all three sites can be filled by a combination of nucleoside di-and triphosphates.
GTP bound at noncatalytic sites on EcF, is quite stable. In MTSEM buffer, the half-time for dissociation is 175 min ( Table  11). The addition of EDTA in excess of Mg2' causes a 2-fold increase in the rate of release. A 5-8-fold increase is obtained by addition of MgPP,, MgADP, or EDTNATP. The greatest increase (23-fold), however, is obtained with the addition of 100 p~ ATP and an ATP-regenerating system. This decreases the half-time for dissociation of L3H]GTP to 8 min (Table 11). This is very close to the value for L3H1GTP dissociation from noncatalytic sites on the mitochondrial enzyme (tu2 = 10 min) obtained under similar conditions (Milgrom and Cross, 1993).
We have recently found that MgPP, effectively competes with nucleotides for binding at noncatalytic sites on MF, (Milgrom and Cross, 1993), and this also appears to be the case for the E.
coli enzyme. At a concentration of 1 mM, MgPP, nearly completely prevents GTP binding at noncatalytic sites on EcF,[O,Ol (Fig. 4, triangles versus Fig. 1, triangles) and native enzyme (data not shown), and significantly slows ATP binding at noncatalytic sites on EcF,[O,Ol (Fig. 2, inverted triangles  during hydrolysis of 100 p~ L3H]GTP by more than 2-fold (data not shown). As with MF, (Milgrom and Cross, 19931, MgPPi has little effect on the binding of either GNP (Fig. 4, squares) or ANP (data not shown) a t catalytic sites.

Inhibition by MgADP Bound at Catalytic
Sites-Studies of MgADP inhibition of MF, and CF, have shown that a steadystate level of the inhibitory complex exists during ATP hydrolysis. Increased free Mg2' shifts the distribution of enzyme forms toward the inhibited state. A similar effect of Mg2' is shown for EcF, in Fig. 5 (circles). The fact that considerably less inhibition is observed over the same concentration range of Mg2' during GTP and ITP hydrolysis (Fig. 5 , squares and triangles) indicates that MgNDP rather than Mg2' alone is responsible for the inhibition.
When ed-EcF1[2,1] is assayed for ATP hydrolysis activity, a lag in product release is seen (Fig. 6, closed squares). In contrast, enzyme preincubated with pyruvate kinase and phosphoenolpyruvate to remove endogenous exchangeable ADP exhibits a burst in product release (open squares) at the same Mg2' concentration.
To further confirm the ability of MgADP t o inhibit EcF,, the epsilon-depleted enzyme was stripped of endogenous nucleotides by ammonium sulfate precipitation ("Experimental Procedures"). A burst in activity of ~d-EcF,[O,01 was seen after preincubation with either EDTA (Fig. 7, open

squares) or Mg2' (open circles), whereas MgADP resulted in a lag (closed circles).
Preincubation with MgATP under conditions that allowed complete conversion to ADP also produced a lag (data not shown).
Azide has been shown to trap the MgADP complex formed at catalytic sites on MF, and CF, (Vasilyeva et al., 1982b;Murataliev et al., 1991). Preliminary experiments with native EcF, indicated that retention of labeled ANP during a cold chase could be enhanced by azide. With increase in azide concentration during cold chase, an increase in azide-trappable ANP showed a linear correlation with the loss of hydrolytic activity (Fig. 8). Extrapolation to complete inactivation corresponded to 0.45 mol of azide-trappable ANP/mol of enzyme.
In order to identify the nucleotide site occupied by the azidetrappable ADP, 2-azido-adenine nucleotide was used. In the dark, this photoafflnity analog is trapped by azide during a cold chase in a similar manner to ADP. The trapped azido-adenine nucleotide was covalently incorporated into the protein by photolysis, and reversed-phase HPLC elution profiles of digested samples were determined (Fig. 9). Photoincorporation following a cold chase in the presence of azide resulted in significant labeling of a catalytic site peptide (Cat) eluting at 23% acetonitrile (top panel). In contrast, a cold chase in the presence of sulfite resulted in near complete disappearance of the catalytic site peptide (bottom panel). In both cases, the amount of labeled noncatalytic peptide ( N C ) eluting at 18% was small and nearly constant. The positions of catalytic and noncatalytic site peptides in the elution profile were assigned based on controls where EcF, was selectively labeled at either catalytic (pY331) or noncatalytic (pY354) sites as described by Wise et al. (1987). Also, using anion-exchange HPLC which separates peptides labeled by the di-and triphosphate forms of the probe, it was found that the azide-trappable catalytic site peptide is labeled exclusively by 2-azido-ADP (data not shown), DISCUSSION Past studies of the nucleotide-binding sites of F,-ATPases have relied on three different criteria for distinguishing between catalytic and noncatalytic sites. The first was based on affinity. Noncatalytic sites were thought to be very tight and catalytic sites to be loose (Harris, 1978). The three tightly bound endogenous nucleotides retained during purification of MF, were assumed to be bound at the three noncatalytic sites. Aliquots of 100 pl were applied to 2-ml centrifuge columns equilibrated with TMB buffer containing 0.5 mM azide. Binding was determined by Cerenkov counting of the column effluents. Azide-trappable ADP was calculated by subtracting the amount bound in the absence of azide (noncatalytic site binding) from the amount bound in the presence of azide. Activity assays were performed with the same reaction buffer using unlabeled ATP and supplemented with 0.25 mg/ml lactate dehydrogenase and 300 p~ NADH. The activity of the 50 p~ MgATP chase reaction was followed spectrophotometrically at 340 nm and was linear for 5 min.
However, this was later shown not to be the case. One of the endogenous nucleotides exchanges rapidly during catalytic turnover (Kironde and Cross, 19861, and the first catalytic site to bind ADP (K,(ADP) = 1 nM, Cunningham and Cross, 1988) is tighter than the loosest noncatalytic site (K,(ADP) = 50 nM, Kironde and Cross, 1987). Another reason for not considering catalytic sites to be loose comes from the finding that the first catalytic site on MF, to bind ATP does so with a remarkably high affinity (K,(ATP) = 1 0 " ' M, Grubmeyer et al., 1982). Hence, in recent years few laboratories have relied on affinity as a means for distinguishing between catalytic and noncatalytic nucleotide sites. A second widely used criterion is based on the nucleotide specificities of the sites. Catalytic sites clearly tolerate variations in the structure of the base moiety as GTP and ITP are hydrolyzed at rapid rates. In contrast, noncatalytic sites have been thought to be highly specific for adenine nucleotide (Harris, 1978;Harris et al., 1978;Perlin et al., 1984). However, it was recently shown that CF, binds GTP a t noncatalytic sites (Guerrero et al., 1990;Milgrom et al., 19911, and we find the same is true for MF, (Milgrom and Cross, 1993) and EcF, (Table I and Fig. 1; see also Weber et al., 1994). In addition, it appears that pyrophosphate can bind at noncatalytic sites on EcF, (Figs. 2 and 4) and MF, (Milgrom and Cross, 1993).
There are several reasons why this lack of specificity was not previously detected. The first is that GTP binding is not as tight as ATP binding. Under conditions of catalytic turnover, GTP bound at noncatalytic sites on EcF, dissociates with a half-time of 8 min (Table 11), whereas little dissociation of ATP could be detected (Pagan and Senior, 1990). In testing for GNP binding at noncatalytic sites, Perlin et al. (1984) used a 30-min incubation with unlabeled ATP to chase [3HlGNP from catalytic sites. In light of the data presented in Table 11, it can be predicted that GTP bound at noncatalytic sites would also have dissociated under these conditions. A lower affinity for GTP than for ATP may also explain why binding to the isolated a-subunit, which appears to retain part of the noncatalytic binding domain (Dunn and Futai, 19801, was not detected (Senda et al., 1983;Perlin et al., 1984).
A second reason is that the affinity of noncatalytic sites for GDP is much lower than for GTP, just as ADP binds less tightly than ATP. This may preclude detection of GDP binding by a centrifuge column assay ("Results," and Kironde and Cross, 1986) or when the enzyme is incubated with GTP over a period of hours under conditions where the GTP is rapidly converted to GDP (Perlin et al., 1984).
A final explanation for the failure t o readily detect guanine nucleotide binding at noncatalytic sites relates to the very slow rate of dissociation of adenine nucleotides from these sites. With EcF, having ANP prebound at noncatalytic sites, Senior and co-workers (Perlin et al., 1984;Senior et al., 1992) observed no exchange for medium GTP. Even when GTP is maintained by a regenerating system, no exchange is seen over a 20-min period if enzyme is pretreated with ATP ( Fig. 1, inverted triangles).
A third criterion for distinguishing between catalytic and noncatalytic sites is based on the fact that catalytic sites on fully active MF, exchange bound ligand rapidly during turnover, whereas noncatalytic sites do not (Cross and Nalin, 1982). The validity of this criterion is supported by the finding that 2-azido-ATP labels a single, specific p-tyrosyl residue when loaded at exchangeable sites and a single, but different, p-tyrosyl residue when loaded at nonexchangeable sites on MF, (Cross et al., 1987), CF, (Xue et al., 19871, and EcF, (Wise, et al., 1987).
However, this criterion also has limitations. The first is due to the fact that noncatalytic sites are not nonexchangeable. The noncatalytic site on MF, having the lowest affinity for ADP releases bound ligand with a half-time of 3 min during catalytic turnover (Kironde and Cross, 1987). However, as originally defined (Cross and Nalin, 19821, exchangeable (catalytic) sites exchange with medium ATP very rapidly during a brief episode of catalytic turnover. Typically ATP is added in a 1OOO:l molar ratio to F,, and cleavage is complete within a few seconds. Under these conditions, noncatalytic sites do not exchange. However, it should be noted that pH values below 7 (Harris et al., 1978) and nonaqueous solvents such as glycerol (Garrett and Penefsky, 1975) can accelerate release of nucleotide from noncatalytic sites.
A second limitation results from the fact that inhibitory MgADP bound at a single catalytic site on CF, (Feldman and Zhou et al., 1988) or MF, (Drobinskaya et aE., 1985;Milgrom and Boyer, 1990;Chernyak and Cross, 1992) can dissociate slowly, under certain conditions, upon addition of MgATP. Hence, nucleotide bound at catalytic sites does not always exchange rapidly. However, with MF, this can be avoided simply by use of buffers containing 1 mM Pi (Grubmeyer et al., 1982).
In contrast, the formation of an inhibitory catalytic site MgADP complex has not been thought to play a significant role in the kinetics of EcF,, making this enzyme unique. Instead it has been argued that Mg2' inhibits directly and that azide inhibition of the enzyme is related to changes in subunit cooperativity.
The ability to demonstrate MgADP inhibition of EcF, has been complicated by the presence of the inhibitory €-subunit. When EcF, is diluted into an ATP hydrolysis assay to a concentration below the Kd (2-10 nM) for the €-subunit (Sternweis and Smith, 1980;Wood et al., 1987), its slow dissociation results in a steady increase in activity (Laget and Smith, 1979). This lag would mask a delay caused by the slow release of inhibitory MgADP such as that observed with MF, (Minkov et al., 1979;Drobinskaya et al., 1985) and CF, (Feldman and Boyer, 1985).
The successful demonstration of MgADP inhibition of EcF, obtained in the present study was aided by the use of €-depleted enzyme, With four-subunit EcF,, elimination of endogenous ADP by preincubation with an ATP regenerating system (Fig.  6) or by removal of all bound nucleotides (Fig. 7) resulted in a burst of ATP hydrolysis activity which could be prevented by MgADP but not by Mg2' alone. In addition, only ~d-EcF,[O,01 gave equal rates for hydrolysis and the loading of a catalytic site with labeled nucleotide (results not shown). All other enzyme forms loaded one catalytic site at a rate too slow t o be consistent with a population of fully active enzyme. This reinforces the proposal that native EcF,[2,11 is a heterogeneous mixture of active and inactive enzyme as suggested by kinetic studies from Allison's laboratory (Muneyuki et al., 19911, as shown by the lag in Fig. 6 (closed squares) and as quantitated by the extrapolation to 0.5 mol of azide-trappable ANP/mol of enzyme at zero activity (Fig. 8). Presumably the other half of the enzyme is already inhibited by unlabeled endogenous ADP.
A second criterion for demonstrating MgADP inhibition of MF, and CF, has been its nucleotide specificity. The ability of different nucleotides to differentially inhibit Ed-EcF, with in- creasing free M e (Fig. 5) is a strong indication that M e is not solely responsible for the observed inhibition. In a similar manner, submitochondrial particles are inhibited by preincubation with ADP but not by GDP or IDP (Vasilyeva et al., 1980). A third criterion to show the presence of inhibitory MgADP at catalytic sites has been the photolabeling of a catalytic-site peptide by Mg[a-azido]ADP (Zhou et al., 1988;Milgrom and Boyer, 1990;Chernyak and Cross, 1992). Using azide to trap 2-azido-ADP on EcF,, photolysis of the inhibitory complex resulted in the modification of a catalytic site peptide (Fig. 9).
These observations can be explained by the scheme presented in Fig. 10. Under normal conditions, a heterogeneous mix of enzyme forms includes both €-inhibited (E.*F,) and MgADP-inhibited enzyme (*F,.ADP). Removal of E (step 5) and inhibitory MgADP (step 7) will generate a population of fully active enzyme (steps 14). Sulfite (Vasilyeva et al., 198213) or the filling of the noncatalytic sites with ATP (Jault and Allison, 1993;Milgrom and Cross, 1993) facilitates removal of catalyticsite bound MgADP (step 7). Azide has the opposite effect by stabilizing the inhibitory complex (step 8). We exploited this property t o trap inhibitory MgADP (Fig. 8) and Mg(2azido)ADP ( Fig. 9) under cold chase conditions. Structural and mechanistic features of EcF, have been extensively studied by site-directed mutagenesis. Knowledge of the fact that EcF, is inhibited by catalytic site-bound MgADP is thus of major importance in addition to its sharing this property with MF, and CF,. The relative susceptibility of each mutant enzyme to inhibition by MgADP should be taken into account before binding or kinetic analyses are interpreted. One mutant EcF, analyzed in our laboratory, PESllD, shows significantly lower activity than wild-type enzyme under normal assay conditions, but this has been shown to be due to a heightened sensitivity to MgADP inhibition rather than to a catalytic defect.2 When assaying ATP hydrolysis, we currently minimize the fraction of enzyme in the e*F, form by diluting below the Kd for E and by adding lauryldimethylamine oxide which helps reverse E inhibition (Lotscher et al., 1984). To minimize the level of *F,.ADP, we prefill noncatalytic sites with ATP, add sulfite or selenite to the assay mixture, and maintain low concentrations of free M e .
In summary, with the proper precautions, measuring differences in the exchangeability of bound ligands can provide a reliable means of distinguishing catalytic from noncatalytic sites on F,-ATPases, whereas differences in substrate specificities or affinities may not. Also, in analyzing EcF, mutants, it is important to distinguish between alterations in sensitivity to inhibition by MgADP and changes in catalytic efficiency.
T. M. Duncan and R. L. Cross, unpublished data.


extfieldsofstudy: Medicine Chemistry

s2fieldsofstudy: Biology Medicine Chemistry

text:
Involvement of A pertussis Toxin Sensitive G-Protein in the Inhibition of Inwardly Rectifying K+ Currents by Platelet-Activating Factor in Guinea-Pig Atrial Cardiomyocytes

Platelet-activating factor (PAF) inhibits single inwardly rectifying K+ channels in guinea-pig ventricular cells. There is currently little information as to the mechanism by which these channels are modulated. The effect of PAF on quasi steady-state inwardly rectifying K+ currents (presumably of the IK1 type) of auricular, atrial and ventricular cardiomyocytes from guinea-pig were studied. Applying the patch-clamp technique in the whole-cell configuration, PAF (10 nM) reduced the K+ currents in all three cell types. The inhibitory effect of PAF occurred within seconds and was reversible upon wash-out. It was almost completely abolished by the PAF receptor antagonist BN 50730. Intracellular infusion of atrial cells with guanine 5′-(β-thio)diphosphate (GDPS) or pretreatment of cells with pertussis toxin abolished the PAF dependent reduction of the currents. Neither extracellularly applied isoproterenol nor intracellularly applied adenosine 3′,5′-cyclic monophosphate (cyclic AMP) attenuated the PAF effect. In multicellular preparations of auricles, PAF (10 nM) induced arrhythmias. The arrhythmogenic activity was also reduced by BN 50730. The data indicate that activated PAF receptors inhibit inwardly rectifying K+ currents via a pertussis toxin sensitive G-protein without involvement of a cyclic AMP-dependent step. Since IK1 is a major component in stabilizing the resting membrane potential, the observed inhibition of this type of channel could play an important role in PAF dependent arrhythmogenesis in guinea-pig heart.

Introduction
Platelet-activating factor (PAF, 1-O-alkyl-2-(R)acetyl-sn-glyceryl-3-phosphocholine) is released from endothelial cells and several types of blood ceils in acute allergic and inflammatory reactions ".(for a review see Snyderl). In the whole heart PAF is released under ischaemic conditions. 2 When administered to isolated perfused guinea-pig hearts, PAF reduced the coronary blood flow and exerted negative inotropism. [3][4][5] The negative inotropic effect was also seen in isolated papillary muscles 6 and auricular preparations of guinea-pig heart. 7 PAF has also been reported to induce arrhyth- 3 mias in isolated Langendorff hearts and in isolated guinea-pig papillary muscles. 9 In guinea-pig ventricular cardiomyocytes PAF has been shown to inhibit single inwardly rectifying K + channels. 1 Since the current through this channel type is assumed to stabilize the resting membrane potential and excitability of cardiac cells, the inhibition of inwardly rectifying K + channels induced by PAF has been suggested to contribute to the genesis of (C) 1994 Rapid Communications of Oxford Ltd arrhythmias. The recent recognition of the inhibitory effect of PAF on ventricular inwardly rectifying K + channels prompted us to examine the inhibitory effects of PAF on these currents in cardiomyocytes from various regions of the guinea-pig heart and the possible involvement of G-proteins and cyclic AMP in this process.

Materials and Methods
Single cardiomyocytes: Single cardiomyocytes were prepared by the method previously described by Trube and Hescheler. In brief, adult guinea-pigs of either sex (200-250 g) were anaesthetized with ether. The chest was opened, the heart was rapidly removed and washed twice in Tyrode's solution. 1'12 Retrograde perfusion through the aorta was performed at 37C using a Langendorff apparatus. The elapsed time from excision of the heart to cannulation and perfusion was less than 1.5 min.
The heart was perfused with nominally calcium-free added to the calcium-free Tyrode's solution and was recirculated through the heart for 10-20 min. The heart was then removed from the cannula, the atria were separated from the ventricles by cutting, and the auricles were dissected from the atria. Then the auricles, the atria (atria without auricles) and the ventricles were placed into separate flasks and cut into small pieces. Incubation in the collagenasecontaining solution proceeded for an additional 5-10min, during which gentle agitation was provided using a 10 ml pipette. The resulting cell suspensions were filtered through a nylon mesh and centrifuged. The cell pellets were resuspended in 'KB '1, recentrifuged and washed twice in this solution.
Cells were kept at 6-8C for 1 to  If not stated otherwise, recording of currents was usually started 5min after disruption of the membrane patch for intracellular dialysis with the pipette solution. Patch electrodes had an average resistance of 1-2 Mr/ when filled with (in mM)" K-aspartate 80, KC1 50, MgCI2 1, Mg-ATP 3, EGTA 10, K-HEPES 10 (pH 7.4). For intracellular infusion of cardiomyocytes with guanine 5'-(3thio)diphosphate (GDP/S), the pipette solution contained additionally 100 #M GDPflS. 13's Quasi steady-state current-voltage relations of inwardly rectifying K + currents were measured using linear voltage-ramp pulses 16 depolarizing the membrane within 3s from--100mV to -40mV (rate of pulses 0.2 Hz). The method of measurement of steady-state currents through inwardly rectifying K + channels is subject to small errors which might arise as a result of the existence of overlapping time dependent currents.
For the electrophysiological experiments, only quiescent relaxed auricular, atrial and ventricular cells with clear striations were used. Significant alterations in the slope conductances of the inwardly rectifying K + currents and in the -adrenergic stimulation of Ca 2+ currents during storage of the cells, were not observed. Isolated auricular muscle" Auricular muscles dissected from left atria were prepared from guinea-pigs anaesthetized with ether as described previously. 7 The muscles were superfused with gassed (95% O2 and 5% CO2) Tyrode's solution composed of (in mM)" NaC1 136.9, KC1 2.7, NaHCO3 22.4, CaCI.
1.8, MgCI2 1, NaHePO4 0.5, glucose 5.6 (pH 7.3-7.4 at 37C). The flow rate was 7 ml/min. The muscles were paced at 0.5 Hz by square wave pulses (duration 1 ms; voltage was adjusted to 1.5-fold about threshold) and isometric contractions were measured using a force transducer type 316 with RCA-5734 (Hugo Sachs Elektronik, Freiburg, Germany), as described previously. 7 Data were analysed off-line using a program written by D. Lewinsohn.
In order to demonstrate the involvement of PAF receptors in the inhibition of the currents, the specific PAF receptor antagonist BN 50730 was used. [20][21][22] Whereas external application of BN 50730 (1/M) did not affect control currents of atrial cells, the inhibitory effect of PAF (10 nM) on the current was almost completely abolished in the presence of BN 50730 (1 #M) (Fig. 4). In five cells the mean attenuation of inwardly rectifying currents by PAF (10 nM) plus BN 50730 (1 was --3 _--t-3%. As demonstrated in Fig. 4 (points d-f in panel B), the blockage of the PAF (10 nM) effect by BN 50730 (1 #M) occurred quickly. To block the effect of 1 #M PAF, BN 50730 had to be administered at a concentration of 10/M.
-66___5mV(n=4) and -53_6mV (n=3) in ventricular cells using 11 mM and 22 mM external K+, respectively. At 2 mM K + outside, the current reversed at a potential below -100 mV. The current was further characterized by its inward rectification; the slope conductance was large below the reversal and declined at more positive potentials. Currents could be blocked by Ba + (10 raM) or Cs + (5 raM) (see Fig. 2  Involvement of GTP-binding proteins" The PAF receptor interacts with its effectors through GTP-binding (G) proteins. Therefore, we tested whether the PAF effect on the K + current is sensitive to GDPflS, (100M), a GDP analogue which stabilizes all G-proteins in their inactive form. Intracellular infusion of GDPflS for 2-3 min via the patch-pipette only marginally affected the PAF dependent inhibition of the current (Fig. 5). A significant reduction of the PAF effect was seen after longer periods of cell dialysis with GDP/gS. After 5-7 min cell dialysis PAF induced a current inhibition of 11 _--F 9% (n 5); after 10-15 min the current was inhibited by 10 8% (n--3, Fig. 5). cyclase. 24 In endothelial cells and platelets it has been demonstrated that PAF receptors inhibit the adenylyl cyclase. 2s '26 To test whether such a mechanism is involved in the inhibition of inwardly rectifying K + currents, a possible involvement of adenosine 3',5'-cyclic monophosphate (cyclic AMP) in the regulation of the current was examined. It was found that direct intracellular application of cyclic AMP (100/.tM in the pipette solution) had no effect on the K + current during 5-10 min infusion and did not prevent the PAF dependent reduction of the current in atrial cells (n 4, see Figs 3 and 5). Arrhythmogenic activity of PAF" In isolated left guinea-pig auricle muscles, contractile responses were observed for about 10 min prior to each experiment. During this period, no extrasystoles were seen; the interval between electrically stimulated beats remained stable at 2 s; no extra beats were observed. PAF (10 nM, 10--15 min superfusion time) induced rhythm disturbances in nine out of 15 muscles studied. The rhythm disturbances induced by PAF occurred without detectable delay after PAF application and were equally distributed during the whole superfusion period of PAF. The interval from beat-to-beat in the muscles with PAF

Discussion
Evidence is presented that PAF inhibits inwardly rectifying K + currents (presumably of the I:l-type) in cardiomyocytes from various regions of guineapig heart. Since the effect of PAF occurred at low concentrations of PAF (nanomolar range) and was fully antagonized by BN 50730, presumably it is mediated by specific cardiac receptors for PAF. 1 As shown in other cellular systems the PAF receptor(s) couples to its effector via G-proteins. It was found that the inhibitory effect of PAF on the inwardly rectifying K + current was suppressed by GDP/S as well as pertussis toxin (PT). These results suggest the involvement of a PT sensitive G-protein in the receptor-induced reduction of the currents. In heart, G-protein dependent regulation of various types of K + channels has been described.
PT sensitive G:-proteins (Oil Oi2 and Oi3 are involved in the muscarinic acetylcholine receptor dependent activation of the KACh current in atrial ceils. The ATP sensitive K + channel (KA:rV) is directly activated by PT-sensitive Gi3 proteins; pacemaker K + channels (If) are activated by /-adrenoceptors via Gs-proteins and inhibited by muscarinic agonists via PT sensitive Go(i) proteins (for review see Brown and Birnbaumer23). The data from Wahler et al. 1 support the view that the PAF-induced inhibition of inwardly rectifying K + currents is mediated by intracellular messengers. These authors observed an inhibition of single inwardly rectifying K + channels (i:1) in cellattached patches following application of 1 nM PAF to the bath. Under these conditions the decrease of channel activity occurred with a delay of 7-10 min. In contrast, whole-cell inwardly rectifying K + currents were reduced within 10-20 s by PAF at 10 nM (present data); 1 nM PAF decreased the current after a delay of 0.5-1.5 min (M.G., unpublished). The faster effect of PAF on whole-cell currents may be explained by usage of the whole-cell clamp configuration which favours the ready diffusion of intracellular messengers from the PAF receptor to the channel protein.
Possible candidate messengers are products generated by phospholipase A 2 (PLA2) which is activated via PAF receptors in various cell types.
Whereas the hormonal activation of phospholipase C in most cell types, including cardiomyocytes, is insensitive to PT, activation of PLA2, e.g. by l-adrenoceptor stimulation, may be sensitive to the toxin. 27 The stimulation of 0q-adrenoceptors causes a PT sensitive decrease of inwardly rectifying K + current (I:1) in canine Purkinje fibres. 28 Nakajima et al. 29 reported a PT sensitive mechanism for activation of guinea-pig atrial Kach channels by PAF at micromolar concentrations probably being mediated by arachidonic acid metabolites (under GTP conditions). An activation of muscarinic K + current by PAF (>0.2/M) was also observed in bullfrog atrial myocytes. 3 The authors, however, found that inhibition of 5and 12-1ipoxygenases by eicosatetranoic acid did not prevent the PAF-mediated increase in muscarinic K + current. Therefore, Ramos-Franco et al.3 suggested that the mechanism of PAF action on bullfrog muscarinic K + current is analogous to acetylcholine in that PAF binds to its (low-affinity) receptor, which is coupled to a G-protein (possibly which in turn activates KAch channels. It has been suggested that subtypes of PAF receptors exist. Both high-affinity (Kd in the lower nM range) and low-affinity (Kd, 10-500 nM or more) receptors for PAF have been described. The involvement of low-affinity binding sites in PAF dependent activation of muscarinic K + current was suggested by the high PAF concentrations (micromolar range) used in the studies of Nakajima et al.29 and Ramos-Franco et al. 3 PAF at concentrations below 0.2 #M did not increase KACh .29 '30 The presence of PAF receptors with high-affinity binding sites in cardiomyocytes mediating the inhibition of inwardly rectifying K + currents can be suggested by the PAF concentrations (nanomolar range) used in this and in the study of Wahler et al. High concentrations of PAF (micromolar range) also induced an inhibition of the current. Therefore, one might suggest that the inhibition of cardiac inwardly rectifying K + (i:) currents is mediated by a PAF receptor subtype different from that stimulating atrial KAch channels.
The initiation of cardiac excitation in working myocardium depends on the threshold to generate an action potential, which by itself is determined by I:. 3 A large I: implies that the resting potential is 'stabilized', i.e. stronger depolarizing currents are necessary to trigger an action potential. 3 Data obtained with low concentrations of Ba 2+ suggest that the inhibition of inwardly rectifying K + channels is causally related to arrhythmogenesis in ventricular fibres. 32 By analogy, the PAF dependent inhibition of inwardly rectifying K + currents may contribute to the genesis of observed arrhythmias and automaticity in multicellular cardiac preparations. Indeed, PAF induces rhythm disturbances in auricular multicellular preparations and also inhibits inwardly rectifying K + currents in cardiomyocytes isolated from supraventricular regions of the heart at the same range of concentration.
With regard to the previous literature, the observed inhibition of inwardly rectifying K + currents is certainly not the only effect of PAF on cardiomyocytes. At low concentrations (nanomolar range) PAF augments Ca 2 + currents in multicellular guinea-pig preparations. 6'7'33 At high concentrations (micromolar range) PAF has been demonstrated to decrease intracellular Na + activity, 34 to reduce Ca 2 + currents and to stimulate an outward current presumably through delayed outward K + channels in frog atrial trabeculae. Although these mechanisms might also play a role in PAF induced arrhythmogenesis, they would act synergistically to cause arrhythmias in multicellular preparations.
Numerous questions about the processes involved in PAF induced arrhythmogenesis in heart muscle still remain (modulation of other ion transport systems, influence of endothelium etc.). Nevertheless, it is felt confidently that the hypotheses on the involvement of PAF inhibited Ii( in arrhythmogenesis will be useful to guide this work.


extfieldsofstudy: Biology Medicine

text:
An end in sight: tropomodulin

TIN filaments are required for cell locomotion and numerous intracellular activities in nonmuscle cells, and are a major component of the contractile apparatus in muscle cells. An understanding of the dynamics involved in the assembly and disassembly of actin filaments is fundamental to understanding many cellular functions. In this issue of The Journal of Cell Biology, Weber et al. (1994b) provide important new insights as to how actin assembly and disassembly is regulated in the cell with their demonstration of tropomodulin as a capping protein specific for the kinetically less active end of an actin filament. Filament formation is a dynamic and polarized process (for review see Pollard and Cooper, 1986). In the absence of other proteins, actin monomers self-assemble in vitro into filaments reaching many microns in length. Assembly begins with the formation of nuclei containing three or four actin monomers. Elongation of the nuclei proceeds with the addition of monomers, but net growth at each end is not to the same extent. Monomers add to the kinetically more active ("barbed" or "preferred") end 10 times as quickly as to the less active ("pointed" or "nonpreferred") end. At equilibrium, the filaments are not static and monomers are both adding and dissociating from both ends of the filaments, albeit at different rates. In vivo a number of actin accessory proteins exists which regulate filament assembly, for example, by associating with monomers thereby sequestering them from participating in assembly, or by capping the ends of actin filaments thereby preventing monomer exchange at that end (for a review on actin-binding proteins, see Hartwig and Kwiatkowski, 1991). Capping results in regulation of filament length and stabilization against disassembly. Proteins such as gelsolin (Yin and Stossel, 1979; Wang and Bryan, 1981) and villin (Bretscher and Weber, 1980; Glenney et al., 1981) are calcium-sensitive actin-binding proteins which both sever actin filaments and cap the barbed ends of actin filaments thereby preventing monomer exchange. Other capping proteins such as macrophage-capping protein (Southwick and DiNubile, 1986) or gcap39 (Yu et al., 1990) also cap the barbed ends of actin filaments in a calcium-sensitive manner, but do not sever actin filaments. Capping protein from Acanthamoeba (Isenberg et al., 1980) and capZ from muscle (Caldwell et al., 1989) bind to the barbed ends of actin ilia-

TIN filaments are required for cell locomotion and numerous intracellular activities in nonmuscle cells, and are a major component of the contractile apparatus in muscle cells. An understanding of the dynamics involved in the assembly and disassembly of actin filaments is fundamental to understanding many cellular functions. In this issue of The Journal of Cell Biology, Weber et al. (1994b) provide important new insights as to how actin assembly and disassembly is regulated in the cell with their demonstration of tropomodulin as a capping protein specific for the kinetically less active end of an actin filament.
Filament formation is a dynamic and polarized process (for review see Pollard and Cooper, 1986). In the absence of other proteins, actin monomers self-assemble in vitro into filaments reaching many microns in length. Assembly begins with the formation of nuclei containing three or four actin monomers. Elongation of the nuclei proceeds with the addition of monomers, but net growth at each end is not to the same extent. Monomers add to the kinetically more active ("barbed" or "preferred") end 10 times as quickly as to the less active ("pointed" or "nonpreferred") end. At equilibrium, the filaments are not static and monomers are both adding and dissociating from both ends of the filaments, albeit at different rates.
In vivo a number of actin accessory proteins exists which regulate filament assembly, for example, by associating with monomers thereby sequestering them from participating in assembly, or by capping the ends of actin filaments thereby preventing monomer exchange at that end (for a review on actin-binding proteins, see Hartwig and Kwiatkowski, 1991). Capping results in regulation of filament length and stabilization against disassembly. Proteins such as gelsolin (Yin and Stossel, 1979;Wang and Bryan, 1981) and villin (Bretscher and Weber, 1980;Glenney et al., 1981) are calcium-sensitive actin-binding proteins which both sever actin filaments and cap the barbed ends of actin filaments thereby preventing monomer exchange. Other capping proteins such as macrophage-capping protein (Southwick and DiNubile, 1986) or gcap39 (Yu et al., 1990) also cap the barbed ends of actin filaments in a calcium-sensitive manner, but do not sever actin filaments. Capping protein from Acanthamoeba (Isenberg et al., 1980) and capZ from muscle (Caldwell et al., 1989) bind to the barbed ends of actin ilia-ments, but do not sever actin filaments and do not require Ca 2÷ for activity. Although over the years candidates for pointed end-capping proteins have been proposed, until now, none has been confirmed with the exception of DNaseI (Podolski and Steck, 1988;Weber et al., 1994a), whose relevance to actin assembly is unclear since it is not known to be cytoplasmic.
Several pieces of information indicate that the pointed end of actin filaments is capped in cells. For example, experiments with cytochalasin E indicate that actin monomers do not assemble onto the pointed ends of protofilaments in the erythrocyte (Pinder et al., 1986). Similarly, actin monomers will not add to the pointed ends of thin filaments in isolated myofibrils (Sanger et al., 1984;Ishiwata and Funatsu, 1985). Moreover, since the lengths of actin filaments in biological systems including muscle (Huxley, 1960), microvilli (Mooseker and Tilnq¢, 1975), red blood cells (Byers and Branton, 1985), and stere, ocilia (Tilney et al., 1992) are uniform, one possibility is that length is partially determined by the existence of pointed end-capping proteins.
Identification of pointed end-capping proteins has been elusive not only because these ends of actin filaments are ki-neticaUy less active, but because of the limitations of the methods available to assay assembly (for a review of methods used to analyze actin assembly, see Gaertner et al., 1989). Most techniques including viscometry, light scattering, and flow birefringence measure bulk assembly which is dominated by growth at the preferred end because of the inherent polarity of the actin filament. Development of electron microscopy methods, in which actin assembly onto preformed nuclei such as microvillar core bundles or acrosomal bundles is monitored, has allowed for direct examination of activity at each end of the actin filament (Woodrum et al., 1975;Pollard and Mooseker, 1981;Tilney, 1983, 1984;Bonder et al., 1983), but these assays are not trivial to perform. The use of fluorescence probes such as pyrene iodoacetamide to label actin (Kouyama and Mihashi, 1981) has allowed for simple, quantitative analysis of actin polymerization and the interaction of actin accessory proteins with actin filaments; fluorescence intensity increases substantially when actin polymerizes. To distinguish events at the pointed ends of the actin filaments with this method, activity at the barbed ends must be squelched. Previously, illaments nucleated and capped at the barbed ends by villin (Northrop et al., 1986) have been used in pyrenyl-actin fluorescence assays designed to examine the effects of tropomyosin on actin assembly (Broschat et al., 1989;Broschat, 1990). Using a similar strategy, Weber et al. (1994b, this  issue) assay the effects of tropomodulin on the addition of pyrene-labeled actin monomers to filaments nucleated, and thereby capped at the preferred ends, by gelsolin (Bryan and Coluccio, 1985). The changes in fluorescence intensity therefore reflect activity at the nonpreferred ends of the actin filaments.
Previously, Fowler (1987) identified a 40-kD tropomyosinbinding molecule associated with the red blood cell membrane; the molecule was subsequently named tropomodulin (Fowler, 1990). Immunofluorescence microscopy on skeletal muscle with anti-tropomodulin antibodies stained a doublet in the region of the A-bands, the portion of the sarcomere in which the pointed ends of the thin filaments reside (Fowler et al., 1993); the barbed ends of the muscle thin filaments are located at the Z-discs, the main component being capZ (CaseUa et al., 1987). Separation of this doublet upon stretching of the myofibrils suggested, but did not prove, that tropomodulin was associated with the pointed ends of the thin filaments (Fowler et al., 1993). In this new report, Fowler and colleagues (Weber et al., 1994b; this issue) now provide compelling biochemical evidence to demonstrate that tropomodulin binds to the pointed ends of actin filaments and is directly responsible for altering the rate of monomer assembly and disassembly at that end (Fig. 1).
The basis for this conclusion is founded in a number of experimental observations. In the presence of tropomodulin, assembly and disassembly of actin monomers at the pointed ends of actin filaments are only partially blocked; however, when tropomodulin is added to tropomyosin-coated actin filaments, both assembly and disassembly at the pointed ends are completely prevented. This would suggest that the affinity for tropomodulin to the end of a filament increases when tropomyosin is present. Tropomyosin, an elongated molecule that lies along the length of actin filaments, is found in the thin filaments of muscle where in association with troponin, it regulates muscle contraction. In nonmuscle cells, a variety of tropomyosin isoforms exists where they are proposed to stabilize actin filaments (for a review on the various isoforms of tropomyosin, see Pittenger et al., 1994). Previous studies have shown that although tropomyosin stabilizes actin filaments from disassembly, elongation is not blocked (Broschat et al., 1990). Thus, the cooperation of tropomyosin and tropomodulin completely blocks activity at the pointed end.
Tropomyosin molecules associate head to tail along the length of the thin filaments in muscle. In the erythrocyte, the limited length of the actin filaments can accommodate only two tropomyosin molecules, one on each side of a short actin filament (Fowler, 1987). The amounts of tropomodulin relative to actin in both muscle and red blood cells indicate that in both cases each actin filament would be associated with one or two tropomodulin molecules (Fowler et al., 1993).
The initial identification of tropomodulin in red blood cells (Fowler, 1987) and its recognition as the elusive pointed end-capping protein long expected to be present in skeletal muscle (Fowler, 1990;Weber et al., 1994b, this issue) demonstrates once again how the simple paradigm of the red blood cell can provide information about the organization of the cytoskeleton in more complex cells (see Bennett, 1985). Expression of the erythrocyte membrane skeletal proteins spectrin, ankyrin, protein 4.1, and the adducins (or closely related proteins) in other nonmuscle cells are earlier exampies. In addition, tropomodulin joins a long list of cytoskeletai proteins including actin, myosin II, tropomyosin (Matsumura et al., 1983;Fowler, 1987), ~actinin (Maruyama and Ebashi, 1965;Burridge and Feramisco, 1981;Duhaiman and Bamburg, 1984), and capping proteins (Casella et al., 1987;Isenberg et al., 1980) common to both skeletal muscle and nonmuscle cells.
Future studies will determine whether short-capped illaments resembling those present in erythrocytes exist in other nonmuscle cells. One example might be provided by the dynactin complex whose major component is an actin-related protein that forms a short filament in association with capping protein at the barbed end of the filament and a potential tropomodulin homologue at the pointed end (Schafer et al., 1994). Equally exciting will be how to reconcile the prospect of cytoplasmic actin filaments stabilized at both ends with capping proteins with the dramatic and rapid reorganization of the actin cytoskeleton that accompanies cell locomotion or cytokinesis (for reviews see Wang, 1991;Satterwhite and Pollard, 1992). Microinjection experiments have indicated that preexisting actin filaments are recruited for cleavage formation, whereas drug studies have indicated that the illaments involved in the contractile ring are dynamic. The completion of cleavage is characterized by the quick disassembly of these filaments. One explanation relies on the reversibility of these capping proteins. Members of the gelsolin family including gelsolin (Yin and Stossel, 1979), villin (Bretscher and Weber, 1980), and macrophage-capping protein (Southwick and DiNubile, 1986) exhibit calcium-sensitive properties; moreover, the interaction of actin with some accessory proteins including gelsolin (Janmey and Stossel, 1987), villin (Janmey and Matsudaira, 1988), gcap39 (Yu et al., 1990), and capZ (Heiss and Cooper, 1991) is regulated in vitro by polyphosphoinositides. Determining how binding of tropomodulin to actin and tropomyosin is regulated will yield important clues as to the precise mechanism of actin assembly and disassembly in ceils.
The author is grateful to J. Cooper, W. Sale, D. Reines, and H. Joshi for helpful suggestions on the manuscript.
Received for publication 13 October 1994 and in revised form 21 October 1994.
The Journal of Cell Biology, Volume 127, 1994


extfieldsofstudy: Medicine Biology

text:
Autoantigens targeted in systemic lupus erythematosus are clustered in two populations of surface structures on apoptotic keratinocytes.

Systemic lupus erythematosus is a multisystem autoimmune disease in which the autoantibody response targets a variety of autoantigens of diverse subcellular location. We show here that these autoantigens are clustered in two distinct populations of blebs at the surface of apoptotic cells. The population of smaller blebs contains fragmented endoplasmic reticulum (ER) and ribosomes, as well as the ribonucleoprotein, Ro. The larger blebs (apoptotic bodies) contain nucleosomal DNA, Ro, La, and the small nuclear ribonucleoproteins. These autoantigen clusters have in common their proximity to the ER and nuclear membranes, sites of increased generation of reactive oxygen species in apoptotic cells. Oxidative modification at these sites may be a mechanism that unites this diverse group of molecules together as autoantigens.

S LE is a multisystem autoimmune disease that is characterized by pleiotropy in its clinical and laboratory features (1). The autoantibody response in SLE is both diverse and exuberant, and targets a variety of nucleoprotein particles, some containing DNA (nucleosomes), and others RNA. The latter include small nuclear ribonucleoproteins (snRNPs), 1 cytoplasmic RNPs (SS-A/Ro), and ribosomes (2). Antibodies are also directed against negatively charged phospholipids (3). A striking feature of the different autoantigens in lupus is their lack of restriction to any one subcellular location: they may be exclusively nuclear, cytosolic, or membrane associated, or distributed between the nudeus and cytosol under different conditions. The proposal that the total repertoire of autoantigens might be clustered on a limited number of distinct subceUular particles is an attractive one (4), but such a cluster has not been identified to date.
The evolution of the autoantibody response to nucleosomes in lupus has been studied during clinical flares, as well as in animal models of the disease (5,6). These studies have strongly indicated that the autoantibody response in lupus is antigen driven and T cell dependent. Thus, autoantibodies undergo isotype switching, somatic mutation, and affinity maturation that are characteristic of rechallenge of a primed immune system by antigen (for a review see reference 7). Furthermore, the presence of immune complexes containing double-stranded DNA (dsDNA) during acute flares (8) suggests that endog-1 Abbreviations used in this paper: dsDNA, double-stranded DNA; ER, endoplasmic reticulum; PI, propidium iodide; snRNP, small nuclear ribonudeoprotein; TMG, trimethylguanosine; TdT, terminal deoxynucleotidyl transferase. enous nucleosomes might be one of the antigens driving the immune response in this disease (5,6).
A potential source of these antigenic components is the abundant generation of nucleosomes in cells undergoing apoptosis, as internudeosomal cleavage of DNA is a well-defined biochemical feature of this mode of cell death (9). The morphologic sequence of apoptosis has been well-defined, and follows an orderly process of nuclear condensation, surface blebbing, cytoplasmic contraction, and packaging of cellular components within membranes before their budding from the cell as apoptotic bodies (9). This reorganization is striking, as membrane, cytoplasmic, and nuclear components all appear at the surface of the dying cell. Apoptosis occurs in different cell types in a wide variety of organisms (9)(10)(11)(12). It may occur in response to many different stimuli, including DNA damage (13,14), growth factor deficiency (15), ligation of a family of cell surface receptors (16,17), heat shock (18), and bacterial or viral infection (19,20). Many of these stimuli generate reactive oxygen species, with consequent oxidative damage to multiple cellular targets (21). Furthermore, exposure to H202 (which generates -OH radicals) also results in cell death with an apoptotic morphology, suggesting that oxidant stresses might function in a final common pathway leading to cell death (21). Bcl-2, a member of an interacting family of homologous proteins that modulate apoptoffs (22,23), has recently been shown to function in an antioxidant pathway that prevents apoptosis (21,24).
The skin is a prominent target in patients with lupus, and the characteristic photosensitivity has been proposed to be a consequence of UV-driven autoantigen exposure with resultant antibody-dependent cellular cytotoxicity (25). Indeed, others have shown that UV irradiation ofcuhured monolayers of human keratinocytes changes the subceUular location of the ribonucleoprotein autoantigens Ko, La, Sin, and U1-RNP (26,27). These antigens stained in a punctate pattern at the cell surface of irradiated cells, a site at which they were never present in unirradiated cells. Although UV irradiation induces apoptosis of keratinocytes (28), the correlation between altered autoantigen location and apoptosis has not previously been addressed.
In these studies, we have shown that autoantigens are clustered in two discrete populations of blebs at the surface of apoptotic cells. A population of small, abundant KNAcontaining surface blebs stained intensely for Ro, and contained fragmented endoplasmic reticulum (ELL) and ribosomes. The other population of larger blebs was less numerous and contained DNA (often nicked), surrounded by a rim of ribonucleoprotein antigens including Ko, La, and the snKNPs. These studies indicate that the clusters of autoantigens in lupus are confined to a cellular domain characterized by its proximity to ER and nuclear membranes. Study of these translocated autoantigen particles, and their ultimate fate, may provide a powerful system to explore the exposure and modification of autoantigens targeted in autoimmune disease.

Materials and Methods
Cell Culture and Irradiation. Human keratinocytes were obtained from neonatal foreskins, and cultured in Keratinocyte Growth Medium (KGM) (Clonetics Corp., San Diego, CA). All experiments were performed on confluent secondary monolayers. A panel of eight sunlamp bulbs (model FS40; Westinghouse, NJ) was used as a source of UVB. The UVB output, measured using a research radiometer (model I1700; International Light Inc., Newburyport, MA) and a cosine corrected probe with a SCS 280 filter, was 0.5 mW/cm 2 at a distance of 19 cm. To perform irradiations, cells were washed twice with PBS (2.7 mM KC1, 1.5 mM KHzPO4, 137 mM NaCI, and 8 mM NazHPO4) and irradiated in PBS. The cells were then incubated in KGM in a 5% CO2 humidified incubator at 37~ Initial dose-response and time course studies established that irradiation with 1,650 J/m 2 generated apoptotic cells within 6 h (data not shown). These conditions were used in all subsequent experiments.
DNA Extraction and Gels. DNA from whole cell populations was extracted, processed, and electrophoresed on 1.5% agarose gels as described (29).
lmmunofluorescence and Confocal Microscopy. Immunofluorescence microscopy was performed on keratinocytes that were grown on No. 1 glass coverslips. Unless otherwise specified in the figure legends, cells were fixed with 4% paraformaldehyde (5 rain, 4~ before permeabilization with acetone (30 s, 4~ and antibody staining. Ro, La, and U1-RNP were stained with human sen of defined specificities (diluted 1:160 in PBS) or athnity-purified antibodies, and were visualized with fluorescein-conjugated goat anti-human F(ab')2 (Organon Teknika Corp./Cappel, Durham, NC). The 5'-trimethylguanosine (TMG) cap structures of snRNAs were labeled using a mouse mAb to TMG (Oncogene Science Inc., Uniondale, NY) according to the manufacturer's directions. Visualization was with biotinylated goat anti-mouse IgG (Southern Biotechnology Associates, Birmingham, AL) followed by BODIFY FL-2-conjugated streptavidin (Molecular Probes, Inc., Eugene, OK). BiP (GRP78) was stained using a mouse mAb (StressGen, Victoria, Canada), followed by incubation with biotinylated goat anti-mouse Ig (Southern Biotechnology Associates) and visualization with Texas red-conjugated streptavidin (Pierce, Rockford, IL). A mAb to ribosomal $1 was a generous gift from M. Schmidt-Zachmann (German Cancer Research Center, Heidelberg, FKG). c~-mannopyranosyl and o~-glucopyranosyl residues were stained with 0.1 mg/ml fluorescein-conjugated Con A (Molecular Probes, Inc.) either before or after permeab'rlization with acetone. Coverslips were mounted on glass slides with Permount | (Lipshaw, Pittsburgh, PA), before viewing and photography on a microscope (Axiophot; Carl Zeiss, Inc., Thomwood, NY) equipped with phase contrast and epifinorescence optics. Confocal microscopy was performed on a scanning confocal microscopy system (model MRC600; Bio-Rad Laboratories, Richmond, CA).
Nick Labeling of Apoptotic DNA. Nick labeling of apoptotic DNA in individual cells was performed as described (29), with the following modifications. Keratinocytes grown on coverslips were fixed with paraformaldehyde and acetone (see above) before incubation in terminal deoxynucleotidyl transferase (TdT) buffer. The endogenous peroxidase inactivation and proteinase K treatments were omitted. Horseradish peroxidase-coupled st~tavidin (Southern Biotechnology Associates) was used at a 1:500 dilution before staining with 3-amino-9 ethyl carbazole (AEC) and visualization by light microscopy.
Electron Microsco~. Cells were grown on 35-ram tissue culture dishes, and irradiated according to standard procedures. 6 h after irradiation, monolayers were washed very gently with PBS, and fixed in 1% glutaraldebyde in 0.1 M sodium cacodylate, pH 7.4. Postfixation was in reduced osmium tetroxide. Monolayers were stained en-bloc with 1% uranyl acetate, before dehydration in ethanol, and embedding in Epon. Thin-sections were cut, and some were further stained with lead citrate before viewing and photography in an electron microscope (model TEM type 1A; Zeiss). Microwave fixation was performed as described (30), using a domestic microwave (General Electric, Louisville, KY) at full power for 20 s, such that a final fixative temperature of 50~ was obtained. Antibody Screening Procedures. Reactivity of human autoimmune and control sera with saline soluble extracts of human spleen and rabbit thymus was determined by Ouchterlony double immunodiffusion using standard reference sera to Ro, La, Sin, and RNP (31).

A~nity Purification of Antibodies From Human Sera.
Keratinocytes were lysed in Buffer A (1% NP-40, 20 raM T.ris, pH 7.4, 150 mM NaC1, and 1 raM EDTA containing the following protease inhibitors: pepstatin A, lenpeptin, ant/pain, chymostatin, and PMSF), and electrophoresed on 15% SDS-PAGE. 0.087% bisaerylamide was used to optimally separate the Ro and La proteins (32). The electrophoresed proteins were transferred to Immobiion | (Millipore Corp., Bedford, MA) (33) and used to affinity purify antibodies to 60-kD Ro, 48-kD La, and 70 kD U1-RNP proteins from human sera as described (34). The specificity of the at~inity-purified anti-60-kD Ko was further conhaned by hemagghtination with a purified Ko-RBC conjugate (Hemagen, Waltham, MA). When we used the same procedure (34) to affinity purify antibodies to 52-kD Ro (three different sera were used in three separate attempts), we were unable to dissociate the bound antibodies from the Immobilon | with 0.2 M glycine, pH 2.8. Subsequent attempts to elute bound antibody with 3 M ammonium thiocyanate (35) resulted in a complete loss of antigen binding. We therefore used a patient serum that was monospecitic for 52-kD Ro in immunoblotting and immunoftuorescence (see Figs. 4 and 5, respectively).

UV Irradiation Induces Apoptosis in Keratinocytes.
Keratinocyte monolayers were irradiated with UVB in order to induce apoptotic cell death. Within 6 h of irradiation, the asynchronous onset of apoptosis was detectable by phase contrast microscopy. Apoptotic cells were rounded up, pulled apart from their neighbors, and showed prominent surface blebbing ( Fig. 1 B [ 37]). Apoptosis was confirmed by detection of internucleosomal cleavage of DNA in irradiated populations ( Fig. 1 A, lane 2), in contrast to the DNA from unitradiated populations which remained intact ( Fig. 1 A, lane 1). The correhtion of morphologic changes with multiple internucleosomal nicks was assessed by introducing biotinylated dUTP at nicked sites with TdT (29). The labeled chromatin appeared condensed, and was found in morphologically apoptotic cells. In cells where the nucleus had already fragmented, the multiple apoptotic bodies formed were also strongly labeled ( Fig. 1 B). No labeling was obtained in irradiated cells when TdT was omitted, and labded cells were extremely rare in unirradiated keratinocytes (data not shown). Electron microscopy of irradiated cells demonstrated condensation ofchromatin and fragmentation of the nucleus (Fig. 1 C). Cytoplasmic contraction was accompanied by fragmentation ofintracellular membranes, whereas mitochondria appeared normal. These ultrastructural features are consistent with the morphology of apoptosis (9).

Small Surface Blebs on Apoptotic Keratinocytes Contain RNA.
In our initial studies, we visualized chromatin condensation in apoptotic cells with PI staining after RNase digestion. When RNase treatment was serendipitously omitted, we noted that the small surface blebs on apoptotic cells were intensely labeled by PI, indicating that they contained RNA. To define the nudeic acid contents of the small apoptotic blebs more precisely, we examined their sensitivity to various nudease digestions. In the absence of nudease treatment, surface blebs stained strongly with PI, whereas the cytoplasm stained only weakly. Nuclear staining was prominent, with  conspicuous nucleoli (Fig. 2 A). In contrast, unirradiated keratinocytes showed diffuse cytoplasmic staining, as well as prominent nuclear and nucleolar staining (see Fig. 5 E). When irradiated cells were pretreated with RNase, the small surface blebs remained dearly visible by phase contrast microscopy, but were no longer stained with PI ( Fig. 2 B, arrowheads). Condensed nuclear chromatin was obvious in these blebbed cells, and often showed the breaking up of the nucleus into several discrete fragments. Occasionally, these condensed nuclear fragments were seen at the cell surface in larger blebs (Fig. 2 B, arrows). These larger blebs also stained with the DNA-specific stain, 4',6-diamidino-2-phenylindole, dihydrochloride (DAH) (data not shown), and could be nick labded in the TdT assay ( Fig. 1 B). Pretreatment with DNase did not affect the staining of small surface blebs, which stained strongly with PI under these conditions (Fig. 2 C). In these cells, the nuclear matrix staining had completely disappeared, making the nucleoli even more prominent. No PI staining was seen when irradiated cells were pretreated with both DNase and RNase (Fig. 2 D).
We further defined the RNA-containing surface blebs with confocal microscopy. Accumulation of PI was only visible at the cell surface, and we were never able to visualize similar aggregates of PI within the cytosol (Fig. 3 A). Indeed, cytosolic PI staining in apoptotic cells was diminished when compared with nonapoptotic cells (see and compare Fig. 5, B and E). Furthermore, Z-series and vertical sectioning showed that the blebs were restricted to the apical surface (Fig. 3 B). The small surface blebs were 1.3 _+ 0.3/zm (mean _ SD, n = 54) in size, and were a common feature in apoptotic cells (both polarized and nonpolarized), including human fibroblasts, A431 cells, and Hep2 cells (data not shown). Blebs were always restricted to the nonadherent cell surface, and were present regardless of the initiating stimulus for apoptosis, which included irradiation, heat shock, or incubation in H202 (data not shown). Blebs were numerous in apoptotic keratinocytes with an intact, condensed nucleus, and were more prominent in these cells than in those where nuclear fragmentation was advanced. PI staining was heterogeneous in different blebs: some stained strongly and diffusely with PI, others appeared speckled, and still others contained small central areas that did not stain with H. These R.NAcontaining surface blebs could readily be distinguished from apoptotic bodies since the latter (a) tended to be larger (mean diameter 2.7 _+ 0.7/zm), (b) were fewer in number (3-10/ce11), (c) contained DNA, (at) were present when nuclear fragmentation was advanced, and (e) were detected within the cytoplasm as well as in surface blebs. Both populations of surface blebs could be seen undergoing phagocytosis by surrounding nonapoptotic keratinocytes (data not shown).

Ribonucleoprotein Autoantigens Redistribute in Apoptotic Cells.
We used antibodies to the ribonucleoprotcin autoantigens  to evaluate their dism~ution in apoptotic cells. In initial studies, 18 out of 21 sen positive for Ro by immunodiffusion showed intense staining of the RNA-containing surface blebs (data not shown). No staining of apoptotic surface structures was seen using eight different sera obtained from healthy adults (data not shown). For subsequent studies, we used a monospedfic human serum to the 52-kD Ro protein, and affinity-purified antibodies to the 60-kD Ro, the 48-kD La, and the 70-kD U1-RNP proteins to examine the distribution of these antigens in apoptotic cells. Before use in immunofluorescence microscopy, these reagents were characterized by two-dimensional immunoblotting to confirm that they each recognized a single protein of the predicted size and charge (Fig. 4) 08). In each case, the immunodominant protein focused as a series ofisoforms with estimated isodec-tric points ranging from 7.0-7.5 (60-kD Ro), 6.2-6.7 (52-kD Ro), 6.5-7.2 (48-kD La), and 7.2-7.6 (70 kD U1-RNP).
When these reagents were used in immunofluorescence studies of unirradiated keratinocytes, antibodies to Ro stained the cytoplasm ditfu~y and stained the nucleus with a speckled pattern that excluded nucleoli (Fig. 5 D). A similar pattern was obtained with affmity-puritied antibodies to La, although the cytophsmic staining was less prominent (data not shown). PI, used without RNase, stained the nucleus, with prominent nucleoli, as well as the cytoplasm (Fig. 5 E). In apoptotic keratinocytes, the small surface bhbs stained intensely with antibodies to Ro (Fig. 5, A and G) and with PI (Fig.  5 B), whereas the cytoplasmic st~i-i-g of both was conspicuously decreased (Fig. 5, A-C, G). go st~ini,,g was e~luded from areas of condensed chromatin, but stained intensdy around these areas (Fig. 5 A). Where apoptotic bodies were seen at the ceU surface, a characteristic rim of R_o staining could often be seen around a core of nucleic acid (data not shown). Neither La nor UI-RNP was detected in the small ILblA-containing surface blebs (Fig. 5, H-J). Instead, their staining was exclusively nuclear in the early stages of apoptosis, and was visualized as a bright eccentric rim of ribonucleoproteins around a core of nucleic acid (Fig. 5, I and J).
In the later stages of apoptosis, La and U1-KNP staining was detected at the cell surface surrounding large blebs containing nucleic acid (Pig. 5 H). The staining pattern obtained with antibodies to the 70-kD protein component of U1-RNP was confirmed and extended by staining apoptotic cells with mAbs to TMG (39). These antibodies recognize the unique 5'-modification of the small RblA components of the spllceosome, multiple components of which are targeted by the immune system in lupus. TMG did not stain the small surface blebs (Pig. 5 K), but stained as a bright rim around fragmented nuclear material, either still within the cell (Fig. 5 L), or at the surface (Fig. 5 K). Colocalization studies with DAPI or nick labeling confirmed that the La-and TMG-stained structures contained DNA in their core (data not shown).
Small Surface Blebs on Apoptotic Cells Contain Fragmented ER and Ribosomes. The enrichment of RNA in small surface blebs was accompanied by a marked decrease in its cytoplasmic staining (compare Fig. 5, B and E). This suggested that the blebs might contain ribosomes, as >70% of cytophsmic RNA is comprised of ribosomal RNA. Furthermore, since the ER fragments and moves outwards early in apoptoffs (9), we wondered whether the apicaUy directed movement of PdqA represented a membrane-based translocation. To address this, we used lectin staining (FITC-Con A) to probe for any glycoproteins within surface blebs. In apoptotic cells that were incubated with FITC-Con A before permeab'dization with acetone and subsequent staining with PI, a thin rim of FITC-Con A was visible around, but not within, areas that stained strongly with PI (Fig. 6, A and B). This was consistent with lectin binding to plasma membrane glycoproteins. In apoptotic cells that were permeabilized before incubation with FITC-Con A, clear substructure within the blebs was evident (Fig. 6, C and D): blebs appeared to be composed of multiple small, round, vesicular structures, defined by FITC-Con A staining around a central unstained area. This heterogeneous staining within the blebs was similar to, but much better defined than the heterogeneity previously seen with PI or Ro (Fig. 3 A and Fig. 5, A and B).
We further investigated the contents of the small surface blebs by using mAbs to BiP (GRP78) (Fig. 7, A and B) or ribosomal protein $1 (Fig. 7, C and D) in immunofluorescence studies. Antibodies to BiP (40), a resident ER lumenal protein, stained strongly and specifically in unirradiated keratinocytes; staining was reticular in pattern, and was most intense in the perinudear region (Fig. 7 A). In contrast, there was strong staining within the surface blebs of apoptotic cells, with diminished staining towards the perinudear region (Fig.  7 B). Ribosomal protein $1 stained with a similar reticular pattern in unirradiated cells (Fig. 7 C), and also demonstrated prominent nucleolar staining as previously described (41). Apoptotic cells showed prominent staining within their surface blebs, with decreased staining towards the perinuclear region ( Fig. 7/9). Isotype-matched, irrelevant mAbs did not stain the structures seen with the anti-BiP and anti-ribosomal $1 antibodies (data not shown).
To further resolve the internal structure of small surface blebs, irradiated keratinocytes were fixed in glutaraldehyde and processed for electron microscopy. Thin sections through the apical portion of irradiated cells were examined for sur-face blebs with a size of about 1.5/zm. Although such structures could be identified, the blebs appeared to contain a granular substance that could not be further resolved using this method of fixation. Since our confocal microscopy data had strongly suggested the presence of multiple small membrane-bound vesicular structures within the surface blebs ( Fig.  6 C), we subsequently used a microwave fixation protocol that is reported to improve preservation of membranes, perhaps by optimizing osmication (42)(43)(44). Numerous surface blebs of the expected size were identified in apoptotic cells that had been fixed using this technique (Fig. 8 A). Consistent with our confocal studies, these structures were most prominent at the apical surface of irradiated keratinocytes. Thus, many sections that optimally demonstrated the surface blebs did not also traverse the nucleus (Fig. 8 A). However, other micrographs dearly showed both structures (Fig.  8 B), and confirmed the impression obtained from confocal microscopy that surface blebbing was prominent before nuclear changes of apoptosis became pronounced. The surface blebs were membrane bound, and uranyl acetate staining demonstrated the bilayer structure of the limiting membrane. Many sections showed the blebs either forming at the plasma membrane (Fig. 8, B and C), or cut in a plane that made Figure 7. St~inlng of keratinocytes with antibodies to BiP (A and B) or antiribosomal protein $1 (C and D). Control (A and C) and irradiated (/3 and D) keratinocytes were stained with mAbs to BiP (A and/3) or ribosomal protein $1 (C and D), and visualized by confocal fluorescence microscopy. The cells were fixed in methanol and acetone (41) before staining with antiribosomal $1. Identical results were obtained in two separate experiments performed with each antibody. Bar, 10 ~m. them appear separate from the cell body (Fig. 8, A and B). Most surface blebs were round, with a mean diameter of 1.3 _ 0.23/zm (mean + SD, n --70). The limiting plasma membrane of these blebs enclosed multiple round membranebound vesicles with a diameter of 100 +_ 13.7 nm (mean _ SD, n ~-267) (Fig. 8, D and E). The vesicles were dear, contained minimal ground substance, and were surrounded by a bilayer as confirmed by uranyl acetate staining (Fig. 8  F). Granular structures that appeared to be ribosomes were seen in many blebs (Fig. 8, D and E). In areas of the cell underlying blebs, rough ER could be seen just beneath the plasma membrane (Fig. 8 C and data not shown). Numerous bundles of cytokeratins were prominent in many sections (Fig.  8, A and B). When normal keratinocytes were microwave fixed and processed identically, very few surface blebs with internal vesicles could be found. The few that were present were smaller (<0.5/zm diameter) than in irradiated cells (data not shown).

Autoantigens Are Clustered in the Su~ce Blebs of Apoptotic
Cells. These studies have defined a population of RNAcontaining blebs at the surface of apoptotic cells, that had previously been visualized by phase-and scanning dectronmicroscopy (37). Blebs are numerous, •1.4 ~m in diameter, and are restricted to the apical surface of apoptotic keratinocytes. Blebs are bounded by a membrane bilayer that is continuous with the plasma membrane, are filled with multiple membranous vesicles of uniform size (about 100 nm in diameter), and contain BiP (an ER hmenal marker), ribosomes, and the ribonudeoprotdn autoantigen, Ro. Both Ro and ribosomes are prominent targets of the immune system in lupus, and it is particularly striking that other components of the ER have also been demonstrated to act as autoantigens in this disease. These include the lumenal ER protein, calreticulin (45), the signal recognition particle (46,47), and acidic phospholipids (for a review see reference 3). These small surface blebs therefore constitute a novel duster of autoantigens that appear to rise from rough ER, but have a surface, rather than an internal location.
Although Ro binds to a family of small cytophsmic RNAs, it is not restricted to the cytoplasm (48), and is also found in the apoptotic nudens. Much of this nuclear Ro is reorganized during apoptosis. Ro is excluded from the nudeoplasm as the chromatin becomes condensed, and becomes concentrated around the rim of apoptotic bodies after nuclear fragmentation. La and the snRNPs are similarly rearranged. After forming within the call, the apoptotic bodies move to the surface where they give rise to a population of larger surface structures containing fragmented DNA. These apoptotic bodies also contain a distinct duster of autoantigens that are targeted in patients with lupus, including nudeosomes (5,6,49), Ro (50), La (51), and snRNPs (52). We propose that the majority of recognized autoantigens in lupus are located within these two distinct types of surface structures.
The conspicuous movement of cytoplasmic Ro to small surface blebs early in apoptosis implies that cytoplasmic Ro has an affinity for the other components in these blebs, and suggests that Ro has a function that is performed at the site of ER-ribosome interaction. Such functions might include, but are not limited to, a role in protein synthesis, control of translation, and protein translocation into the ER. Indeed, such roles have previously been proposed (50,53). Further study of the interaction of Ro with the various subcomponents of this particle may shed light on the function and interactions of Ro.
ER and Nuclear Membranes Are Sites of Free Radical Generation in Apoptotic Cells. After an apoptotic signal, cells sustain progressive lipid peroxidation, reflecting the generation within apoptotic cells of lipid-diffusible reactive oxygen species (21). In the ER, induction of lipid peroxidation results in membrane fragmentation into smaU resides (54), with some thickening of the bilayer (55). These vesicular fragments strongly resemble the 100-nm vesicles within membrane blebs (Fig. 8) that stain with specific ER lumenal markers (Fig.  7). It is therefore possible that these vesicles arise as a consequence of unopposed lipid peroxidation in the Elk membranes of apoptotic cells. The presence of peroxidated lipids in the small vesicles remains to be directly demonstrated.
The major sites of free radical generation in cells include mitochondria, EtL, and nuclear membranes (21,56,57). The free radicals produced at these sites are highly reactive species, and their damaging activities are focused at sites of their generation, where macromolecules may sustain oxidative damage (58). It is highly significant therefore that the two populations of autoantigens targeted in patients with lupus are topologically restricted to sites of free radical generation in apoptotic cells, that is, to areas of vesiculating ER (Ro, ribosomes, and EIK proteins), and to the rim of apoptotic bodies that are surrounded by nudear membrane (Ro, La, dsDNA, and snRNPs). It is also of interest that autoantibodies to mitochondrial components (e.g., cytochrome c) have been observed in patients with lupus and related diseases (59). A common feature of self-antigens at these sites might be their vulnerability to oxidative damage.
It has been proposed that some autoreactive T cells escape tolerance induction because they are directed against minor (cryptic) determinants on stir-antigens that are not efferently generated during antigen processing (60). These autoreactire cells may become pathogenic if that cryptic determinant is subsequently revealed (61)(62)(63). A number of free radical-induced modifications of proteins have been described that may reveal potentially cryptic determinants. These include fragmentation (64,65), amino acid modifications (e.g., trypto- phan loss) (64), and novel sensitivity to protease attack (66). Our recent identification of a specific cleavage of the 70-kD U1-RNP antigen in apoptotic cells (L. Casciola-Rosen, and A. Rosen, unpublished results) supports the hypothesis that site-specific mod_ification of proteins may generate unique peptide fragments. The strong association of particular autoantibody responses with specific MHC class II molecules in patients with SLE may reflect the ability of these class II molecules to capture and present self-peptides to T cells (67,68).
Apoptosis in Murine Models of Lupus. MILL mice spontaneously develop late onset autoimmune disease (69), and develop autoantibodies with a specificity very similar to that seen in human disease, including antibodies to dsDNA and Sm (70). When MILL mice are, in addition, homozygous for the lpr mutation, they develop massive lymphadenopathy, and have marked acceleration of autoimmune disease (69). The lpr mutation (71) results from integration of the retrotransposon, ETn into an intron in thefas gene, with diminished fas expression (72,73) and fas-mediated apoptosis. The abnormality in apoptosis has been proposed to be responsible for defective negative sdection of stir-reactive T lymphocytes in the thymus (71). Apoptosis in the thymus is not only a mechanism for the deletion of self-reactive clones, but is also an abundant source of apoptotic autoantigens to which the organism must become tolerant. The failure of fas-mediated T cell apoptosis within the thymus of devdoping 1pr/lpr mice may not only facilitate the persistence of autoreactive clones, but may also deprive the organism of the opportunity to generate a full repertoire of"apoptotic" autoantigens to which to become tolerant. The specificity of the immune response to a restricted, but common group of autoantigens in mouse and human disease is therefore particularly relevant.
Autoantigen Clusters in Lupus: A Model. In these studies, we have shown that apoptosis induces the physical clustering of autoantigens in two distinct populations of cell surface structures. As these blebs contain ER (small blebs) or nudear membrane (apoptotic bodies), sites of unbuffered generation of reactive oxygen species in apoptotic cells, we propose that the contained autoantigens have in common a vulnerability to oxidative modification, targeted by metal binding or active sites (58,66,74). In a genetically susceptible individual, at high rates of generation of novel fragments (e.g., irradiation, heat shock, bacterial or viral infection, or xenobiotic exposure), the appropriate MHC class II molecules may capture and present self-peptides that were previously cryptic (62,63,75,76). As in other systems, the immune response to this self-peptide may subsequently diversify to other areas on the self-molecule to which the organism was previously tolerant (60,77). Subsequent reexposure of the primed immune system to even low levels of"apoptotic" autoantigens (e.g., sun exposure) may lead to the clinical flares in this disease. The revelation of previously cryptic peptides to the immune system by oxidative modification of proteins may also be applicable to other autoimmune diseases.
We thank Carolyn Machamer for critical review of the manuscript, Hugh Rosen for numerous thoughtful suggestions, Alan Aderem and Joe Ahearn for helpful discussions, Ed Cluett for suggesting the microwave fixation and help in its execution, and Mike Delannoy for his invaluable assistance with confocal and electron microscopy. We also thank the members of the Dermatoimmunology laboratory for help with keratinocyte cultures and screening of sera.
This work was supported in part by National Institutes of Health (NIH) grants AR-32490 and AR-40018 (G. Anhalt). A. Rosen was supported by the NIH Physician-Scientist Program, and by American Cancer Society Institutional Research