extfieldsofstudy: Medicine Biology

s2fieldsofstudy: Biology Chemistry

text:
Strand displacement synthesis in the central polypurine tract region of HIV-1 promotes DNA to DNA strand transfer recombination.

Two distinct plus strand initiation sites have been identified in human immunodeficiency virus (HIV), the central polypurine tract (cPPT) and the polypurine tract located just upstream of the U3 region (U3-PPT). When synthesis from the U3-PPT reaches the cPPT, the elongating primer causes limited strand displacement of the product created from the cPPT. We examined whether reverse transcriptase (RT) catalyzed strand transfer recombination is promoted by this process. Using a substrate having the viral sequence of the displaced region, we measured transfer of an elongating DNA primer from a donor DNA to an acceptor DNA. Strand transfer synthesis was only efficient when RT was performing strand displacement synthesis. Transfer efficiency was directly related to acceptor concentration but independent of the reaction time. Transfer could occur to acceptors containing 80, 40, or 20 nucleotides of homology with the template DNA. Using different acceptors, we found that DNA to DNA transfer occurred at positions throughout the donor template, except near the 5′ end. This shows that a number of the sequences downstream of the cPPT region can promote transfer, but once synthesis has progressed to the point where the downstream segment is completely displaced transfer is not allowed. When the DNA to DNA transfer reactions were performed using a template containing nonviral sequences, the transfer efficiency dropped significantly. This indicates that transfer efficiency is determined by the sequences of the templates used. HIV-RT RNase H-dependent strand transfer between RNA templates is well documented. We propose a quite different mechanism for DNA to DNA transfer, consistent with the ability of RNase H minus RT to perform this reaction. If these DNA to DNA transfer events occur in vivo, they will result in plus strand recombination.

Genetic variability is a characteristic of the retrovirus family. Variability of HIV 1 results mainly from recombination events and from misincorporation by the error prone viral reverse transcriptase (RT). Recombination during reverse tran-scription is possible because each HIV virion contains two genomic RNA molecules that are often different. Two models of retroviral recombination have been proposed, the forced copy choice (1) and the strand displacement/assimilation model (2,3). The forced copy choice model proposes that recombination occurs during synthesis of the first strand of DNA by a process that involves the transfer of the newly synthesized DNA from one viral RNA to the second viral RNA. The strand displacement/assimilation model proposes that recombination occurs during synthesis of the second strand of DNA. According to this latter model, when a piece of downstream DNA is displaced by synthesis from an upstream primer, this unannealed fragment of DNA can bind a second copy of minus strand DNA and there serve as a primer (Fig. 1A).
The strand displacement/assimilation model assumes that each copy of the viral RNA is converted into DNA and that plus strand DNA is initiated at multiple sites. Two distinct plus strand initiation sites have been identified, the central polypurine tract (cPPT) and the polypurine tract located just upstream of the U3 region (U3-PPT) (4). When synthesis from the U3-PPT reaches synthesis from the cPPT, elongation of the U3-PPT causes limited displacement of the product created from the cPPT (5). Strand displacement is limited to approximately 100 nucleotides by the presence of very strong pause sites within this region, which are referred to as the central termination sequences (5). The final plus strand DNA product consists of two segments containing an approximately 100-base pair DNA overlap (5). Presumably the two DNA segments are repaired by cellular enzymes after the proviral DNA has been transported into the nucleus. This paper deals with the ability of HIV-RT to transfer an elongating DNA primer from its DNA template to a homologous DNA. DNA to DNA transfer has been observed previously by us and others (6,7). In vivo, DNA to DNA transfer events can lead to the formation of plus strand recombination products by strand displacement and assimilation (2,3). Our proposed mechanism is different from the strand displacement/assimilation model in that it is the upstream primer rather than the displaced downstream strand that transfers and serves as a primer on the new template (Fig. 1B).
The ability of HIV-RT to perform strand transfer reactions between RNA templates is well documented. During reverse transcription an RNA to RNA strand transfer reaction is an obligatory part of each replication cycle (for a review see Ref. 8). Additionally, RNA to RNA transfer can occur in other areas of the genome, and this process can lead to minus strand recombination products via a mechanism similar to that proposed in the forced copy choice model (9,10).
Here we analyzed the conditions that promote DNA to DNA transfer, and the extent of homology necessary for this event to occur. The template used was 101 nucleotides long and con-tained the viral sequences found downstream of the central polypurine tract. Using this model template, DNA to DNA transfer was only efficient when the enzyme was performing strand displacement synthesis. Transfer efficiency was directly related to acceptor concentration, but it was independent of the reaction time.

Materials
HIV-RT with native primary structure was provided by the Genetics Institute (Cambridge, MA). The enzyme had a specific activity of 40,000 units/mg. One unit is defined as the amount required to incorporate 1 nmol of dTTP into nucleic acid product in 10 min at 37°C using poly(rA)-oligo(dT) 16 as template primer. The enzyme was divided into aliquots and stored at Ϫ70°C, and a fresh aliquot was used for each experiment. An HIV-RT lacking Mg 2ϩ -dependent RNase H, (p66 EϾQ / p51), was obtained through the AIDS Research and Reference Reagent Program from Dr. Stuart Le Grice (11,12). T4 polynucleotide kinase, T4 DNA polymerase, dNTPs, and Quick Spin columns (G-25 Sephadex) were purchased from Boehringer Mannheim. The Sequenase version 2.0 DNA sequencing kit was obtained from U. S. Biochemical Corp.
[␥-32 P]ATP (3000 Ci/mmol) was obtained from New England Nuclear. The DNA substrates were obtained from Genosys, Inc. All other chemicals were from Sigma.

Methods
Preparation of Substrates-The DNA oligonucleotides used in the experiments were synthesized by Genosys, Inc. The upstream primer was 5Ј end-labeled with 32 P using T4 polynucleotide kinase according to the manufacturer's instructions and purified through G-25 Sephadex Quick Spin columns. The hybrids were prepared by mixing the primers and the template at a 5 to 1 molar ratio in 50 mM Tris-HCl (pH 8.0), 0.1 mM EDTA and 80 mM KCl. The mixture was heated at 90°C for 10 min and then cooled slowly to room temperature. In most of the experiments the upstream and downstream primers were mixed together with the template; however, in some experiments the downstream primer was omitted.
HIV-RT Reactions-Approximately 1.5 nM of substrate was pre-incubated with the enzyme for 3 min in 50 mM Tris-HCl (pH 8.0), 32 mM KCl, 1 mM dithiothreitol, 0.1 mM EDTA. The reactions were started by the addition of 6 mM MgCl 2 , 50 M of each deoxynucleoside triphosphate, and, in the experiments indicated, the acceptor template. The reactions were performed in a final volume of 12.5 l at 37°C for 30 min unless otherwise specified. The amount of enzyme and the concentration of acceptor used in each experiment is specified in each figure legend. The reactions were stopped by adding an equal volume of a 2 ϫ loading buffer (90% formamide (v/v), 10 mM EDTA (pH 8), 0.1% xylene cyanole, 0.1% bromphenol blue).
Gel Electrophoresis-Unless otherwise specified, samples were separated using 8% denaturing sequencing gels containing 7 M urea (13). The gels were dried and analyzed by autoradiography. All quantitations were performed in the PhosphorImager (Molecular Dynamics).

RESULTS
The substrates used in this study are shown in Fig. 2. The substrate sequences are found in the middle of the HIV genome, downstream of the central polypurine tract, in the integrase coding region. The nucleotide sequence of donor template 1 is complementary to the plus strand sequence from nucleotides 4387 to 4487 of the HIV-1 provirus (14). This region was chosen because it experiences strand displacement synthesis in vivo and because it contains sites that cause prominent pausing of the RT, which are designated the central termination sequences (5,15). The donor template and downstream primer are 101 and 80 nucleotides long, respectively. The reaction is initiated from a 16-nucleotide primer that binds two nucleotides in from the 3Ј end of the template. The strand transfer acceptor templates are of various lengths, and their regions of homology with the template DNA are shown in Fig. 2. Fulllength synthesis over the donor template produces a 99-nucleotide-long product, whereas strand transfer synthesis produces a 119 -139-nucleotide-long product, depending on the acceptor template used.
An Elongating DNA Primer Can Transfer to an Acceptor Template during Strand Displacement Synthesis- Fig. 3A shows a reaction in which the primer-donor template, having or lacking a downstream primer, was subjected to HIV-RT-directed DNA synthesis in the presence of increasing concentrations of acceptor A. The first 80 bases of the acceptor A are homologous to the last 80 bases of the donor template. The strong pause sites that the elongating primer encounters at the end of the donor template correspond to the positions of the central termination sequences (indicated by an arrow in Fig.  3A). Significant amounts of transfer products were detected only in the presence of a downstream primer annealed to the  (2). Panel B shows a model for the strand displacement/assimilation event as it occurs in our system. The latter model differs from the former in that it is the upstream primer rather than the downstream primer that transfers and continues synthesis on the new template. Solid lines represent synthesis from DNA template 1, and hatched lines represent synthesis from DNA template 2.
donor template (Fig. 3A, lanes 9 -13). A faint transfer product was occasionally observed in the absence of a downstream primer after long exposure times (data not shown). We calculated transfer efficiency using a PhosphorImager. Transfer efficiency is defined as the amount of full-length transfer product divided by the sum of the amounts of full-length donor template-directed synthesis and the amount of full-length transfer product (16). In general, the DNA to DNA transfer efficiency increased with increasing concentrations of acceptor template. This phenomenon has been previously observed for RNA to RNA transfer (7). In the absence of a downstream primer, the highest DNA to DNA strand transfer efficiency observed was 2.1%. In the presence of a downstream primer, this value increased to 18.6% (Fig. 3B). At the highest acceptor concentration used, DNA synthesis and strand transfer were inhibited. This could result from trapping of the enzyme by the large excess of DNA in the reaction. The DNA to DNA transfer reactions shown in Fig. 3A were also measured over a time course (Fig. 3C). Again, significant transfer was only detected in the presence of a downstream primer. Transfer products were observed after 10 min. From this point on, the total amount of transfer product increased with time, but the transfer efficiency remained constant at approximately 18%. The homologous sequences between donor and acceptor extend to the very 5Ј end of the donor template, so that homologous strand transfer could have occurred after complete synthesis on the donor. The constant efficiency suggests that DNA to DNA strand transfer is an event that takes place actively during strand displacement synthesis, rather than after full-length donor template-directed synthesis has been achieved. On the other hand, RNA to RNA transfer efficiency, with substrates allowing homologous transfer from the end of the donor tem-plate, increases with the reaction time (7). This observation indicates that the rate-limiting factor for the RNA transfer reaction is the formation of a full-length donor template-directed product (7), from which transfer product then derives.
The DNA to DNA transfer product was undetectable in the absence of a downstream primer ( Fig. 3A and data not shown). Therefore, unless otherwise specified, the rest of the experiments presented in this paper will use the substrate containing a downstream primer.
The Mechanism of HIV-RT-directed DNA to DNA Strand Transfer Is Different Than That for RNA to RNA Strand Transfer-RNA to RNA transfer is dependent on the ability of the RNase H activity of HIV-RT to cleave the template RNA (7,16,17). Although RNA to RNA transfer is relatively efficient, DNA to DNA transfer is inefficient unless the enzyme is performing strand displacement synthesis (Figs. 3 and 7). These observations indicate that DNA to DNA transfer must be occurring by a completely different mechanism. This conclusion is verified by the ability of an RNase H minus HIV-RT to perform DNA to DNA transfer (Fig. 4). We hypothesize that strand displacement synthesis is the mechanistic key to DNA to DNA transfer. When the elongating primer is performing strand displacement synthesis, the displaced primer will compete with the elongating upstream primer for binding to the template. During this process, the downstream primer can transiently bind the template beneath the upstream primer and dislodge the 3Ј end of the elongating primer. The 3Ј end of the upstream primer is then available to bind to the acceptor template.
Defining of the Degree of Homology Needed for Transfer-To determine the degree of homology necessary for transfer to occur, we measured transfer to acceptors containing 80, 40, or 20 nucleotides of homology with the donor template DNA (ac-  9 -13). The acceptor was present at a 2.5-, 5-, 10-, or 20-fold excess over the template. B, the effect of acceptor concentration on transfer efficiency is shown. Strand transfer efficiency was assessed by quantitating the products of synthesis over the donor and acceptor templates using a PhosphorImager. Strand transfer efficiency was calculated as described under "Results." C, primer-donor template 1 in the absence (lanes 1-6) or the presence (lanes 7-12) of a downstream primer was incubated with 4 units of HIV-RT. A 10:1 molar ratio of acceptor to donor template was used. The reactions in lanes 1-6 and 7-12 were stopped after 2.5, 5, 10, 20, 30, or 60 min. Full-length donor-directed products and transfer products are indicated by the letters F and T, respectively. ceptors shown in Fig. 2). Transfer occurred to both acceptors B and C, each containing 40 nucleotides of homology with the donor template DNA (Fig. 5, B and C). Transfer occurred to acceptor E but not to acceptor D, both containing 20 nucleotides of homology with the donor template DNA (Fig. 5, D and E). Because transfer to acceptor E was efficient, transfer can occur to acceptor templates containing a homologous region of 20 or even possibly fewer nucleotides. The fact that transfer to acceptor D was not observed indicates that most transfers occur during strand displacement synthesis, rather than near the 5Ј end of the donor template after the displaced strand is completely dissociated. These results suggest that DNA transfer can occur anywhere throughout the donor template, as long as the downstream strand is only partially displaced.
We cannot interpret moderate differences in transfer efficiency measured with different acceptor templates, because this process involves synthesis through different sequences. For example, primer elongation after transfer to acceptors A and C involves synthesis through the same strong pause sites encountered at the end of the donor template DNA (indicated by an arrow on Fig. 3A). However, elongation on acceptors B and E may not involve synthesis through any strong pause sites, because these templates have large 5Ј regions that are not homologous to the donor template. This situation would cause an increase in transfer efficiency in experiments with acceptor B and E (Fig. 5, B and E). Interestingly, transfer efficiency did not drop when the homology in the acceptor template was reduced from 80 to 40 nucleotides.
At What Positions on the Sequence Does Transfer Take Place?-In order to determine the positions of transfer, we created an acceptor having a 2-nucleotide deletion every 18 nucleotides (acceptor F, Fig. 2). Using acceptor F, the length of the transfer product could be 113, 115, 117, or 119 nucleotides depending on the point of primer transfer. Transfer experiments were performed using acceptors A and F. The reaction products were separated on a 5% polyacrylamide gel (Fig. 6). To determine the precise size of the products, sequencing reactions were run in the same gel. Full-length donor-directed products were 99 and 100 nucleotides long. The 99-nucleotidelong product corresponds to the full-length product, and the 100-nucleotide-long product is generated by the addition of a nontemplate-directed nucleotide by HIV-RT (17,18). In the presence of acceptor A, we observed the expected 119 and 120 nucleotide long products. The 120-nucleotide-long product corresponds to the nontemplate-directed addition of a nucleotide by HIV-RT before or after transfer. In the presence of acceptor F, we observed products 113-118 nucleotides long. These products correspond to the expected 113-, 115-, and 117-nucleotidelong transfer products, plus the corresponding set of products one nucleotide longer caused by nontemplate-directed nucleotide addition. Using acceptor F, none of the transfer products were 119 nucleotides long. The strongest transfer bands using acceptor F were at positions 115 and 117 rather than at position 113. This indicates that transfer is not uniquely enhanced at the position where the upstream primer initiates strand displacement synthesis. Furthermore, these results confirm that DNA to DNA transfer can occur anywhere throughout the template, except near the end.
The Efficiency of DNA to DNA Strand Transfer Depends on the Overall Sequence of the Templates Used-To determine the effect of the overall template sequence in the DNA to DNA transfer event, we tested whether DNA transfer could take place using substrate 2. The donor template was 104 nucleotides long, and the downstream primer was 79 nucleotides long (see Fig. 2). Thus, the strand displacement region in substrate 2 was approximately the same length as the one in substrate 1. The acceptor was 99 nucleotides long, and the first 79 bases of the acceptor were homologous to the last 79 bases of the donor template. Although the sequences of substrate 2 are not encountered in the virus, they also contain strong pause sites. Fig. 7 shows a reaction in which the primer-donor template 2, having or lacking a downstream primer, was incubated with increasing concentrations of HIV-RT in the presence of acceptor 2. Transfer products were detected only in the presence of a downstream primer, and the transfer efficiency ranged from 6 -7.8%. This result indicates that the efficiency of DNA to DNA transfer is influenced by the overall sequences of the templates used in the reaction.

DISCUSSION
Two distinct plus strand initiation sites have been identified in HIV, the cPPT and the U3-PPT (4). When synthesis from the U3-PPT reaches the cPPT, the elongating primer causes limited strand displacement of the product created from the cPPT (5). Because this region is involved in strand displacement in vivo, we considered that it could be a site for efficient recombination via the strand displacement/assimilation model or a similar mechanism. HIV-RT catalyzed DNA strand transfer was found to occur efficiently in vitro, when measured on substrates having the viral sequences involved in displacement HIV-RT can catalyze the transfer of a DNA primer from a donor template DNA to an acceptor DNA. We show that this reaction requires strand displacement synthesis and is influenced by the acceptor concentration. The length, position, and sequence of the homology between the acceptor DNA and the template DNA are also important influences. Likewise, the overall sequences of the templates used to measure DNA to DNA transfer have a major impact on the efficiency of the process.
The experiments shown here were carried out with a 101nucleotide-long template containing viral sequences downstream of the central polypurine tract. Using these sequences and other sequences of similar length, efficient DNA to DNA transfer was only observed when the enzyme was performing strand displacement synthesis. We have shown previously, us- ing a 667-nucleotide-long DNA template containing the sequences of the HIV long terminal repeats, that HIV-RT can catalyze strand transfer synthesis independent of the downstream primer (6). We proposed that with the long terminal repeat substrate, DNA to DNA transfer could be independent of the downstream primer because the template can form hairpin structures that resemble the strand displacement substrate. Displacement would then have to occur periodically as the primer was elongated. This interpretation was supported by results showing that the reaction was further stimulated when the presence of a downstream primer necessitated continuous strand displacement synthesis.
Strand transfer from an RNA template requires the action of the RNase H of the RT (7,16,17). It is thought to occur because RNase H-directed degradation of the RNA template frees the primer to bind to a new template. In this case, sequences and structures of the donor template that cause pausing of synthesis promote transfer. They do so by allowing the RNase H a longer opportunity to degrade the RNA template while the RT is paused. We hypothesize that strand displacement synthesis promotes primer transfer from a DNA template by an entirely different mechanism. The displaced downstream primer can compete with the extending upstream primer for binding to the template. The downstream primer could transiently bind the template beneath the upstream primer, dislodging a region of the upstream primer near its 3Ј end. This region would then be available to bind to the acceptor template. The ability of the displaced downstream primer to compete with the upstream primer may be similar at all points throughout the reaction, because both primers are always equivalently complementary to the template at any position. This should tend to make the probability of transfer similar throughout the template. However, other factors, discussed below, could alter transfer efficiency at each position. The DNA to DNA transfer mechanism is not unique to HIV-RT, because Sequenase, which is capable of strand displacement synthesis, can also perform this reaction (data not shown).
The probability of transfer is not likely to be absolutely uniform along the donor template. Some sequences may allow either more rapid equilibration between the two competing primers or a longer region of displacement. Also it is possible that sequences that promote dissociation of the RT allow more primer equilibration. In this way, pause site sequences may still influence transfer but for a completely different reason than for transfer from RNA templates. In support of a lack of uniformity, we have shown that transfer from DNA templates is influenced by the overall sequence of the templates used in the reaction. The long terminal repeat and the sequences downstream of the central polypurine tract are the two areas of the viral genome known to be involved in strand displacement in vivo. Higher transfer efficiencies were observed using both of these viral sequences (Ref. 6; Fig. 3) than with the nonviral substrate 2 sequence (Fig. 7). This observation suggests that particular areas of the viral genome involved in strand displacement synthesis could form structures that promote DNA to DNA transfer.
In our experiments, transfer efficiency was directly related to acceptor concentration, but independent of the reaction time. As explained above, this observation suggests that transfer takes place actively during strand displacement synthesis and not after a full-length product has been synthesized. Furthermore, our results using acceptor templates with homologies to different regions of the donor template indicate that transfer can occur at more than one position in the homologous region. In particular, use of an acceptor template with periodic two nucleotide deletions allowed determination of the positions of transfer based on the final length distribution of transfer products. This experiment further showed that transfer occurred throughout the homologous region, except near the 5Ј end of the donor template. Overall, these results show that once strand displacement is initiated, strand transfer can occur with high efficiency. The point at which the upstream primer on the donor template encounters the displaceable downstream primer is not a particularly hot spot for transfer. Similarly, it is not clear whether prominent pause sites for synthesis over the donor template are exceptionally effective at promoting transfer. Transfer does not occur near the 5Ј end of the donor template, presumably because once synthesis has progressed to the point where the downstream primer is completely displaced transfer is not allowed. All of these observations support the hypothesis that competition for binding the template between the extending upstream primer and the partly displaced downstream primer forces transient dissociation of the 3Ј end of the upstream primer, driving the transfer reaction.
In vivo, DNA to DNA transfer events could lead to the formation of plus strand recombination products via a mechanism similar to the strand displacement/assimilation model (Fig.  1B). The only direct evidence supporting the displacement/ assimilation model of plus strand recombination is the observation of branched structures during replication using electron microscopy (2). The reactions discussed in this paper can give rise to the same phenotype. We are currently sequencing transfer products to investigate whether the DNA to DNA transfer process promotes mutagenesis.


extfieldsofstudy: Chemistry Medicine

text:
Deglycosylated products of endogenous digoxin-like immunoreactive factor in mammalian tissue.

Digoxin-like immunoreactive factor (DLIF) from adrenal cortex is an endogenous molecule with structural features remarkably similar to those of digoxin, a plant-derived cardiac glycoside (Shaikh, I. M., Lau, B. W. C., Siegfried, B. A., and Valdes, R., Jr.(1991) J. Biol. Chem. 266, 13672-13678). Two characteristic structural and functional features of digoxin are a lactone ring and three digitoxose sugars attached to a steroid nucleus. Digoxin is known to undergo deglycosylation during metabolism in humans. We now demonstrate the existence of several naturally occurring deglycosylated components of DLIF in human serum. The components are identified as DLIF-genin, DLIF-mono, and DLIF-bis, corresponding to the aglycone, and the aglycone with one and two sugars, respectively. Similar components are produced by acid-induced deglycosylation of DLIF isolated from bovine adrenal cortex. The elution pattern and sequence of DLIF-deglycosylation was identical to that of digoxin suggesting identical sugar stoichiometry. However, analysis of these newly discovered congeners by reverse-phase chromatography, spectrophotometry, antibody reactivity, and kinetics of deglycosylation, demonstrates that subtle structural and physical differences do exist when compared to digoxin. DLIF was chromatographically distinct from digoxin, and interestingly, the mobility of the DLIF-genin was shifted toward increased polarity relative to digoxigenin. DLIF and DLIF-bis, -mono, and -genin congeners have absorbance maxima at 216 nm, whereas digoxin and its congeners absorb at 220 nm. Reaction with specific antibodies directed at the lactone portion of these molecules shows DLIF and its deglycosylated congeners to be 103-fold less reactive than digoxin. Kinetics of sugar removal suggests that DLIF is 8-fold more susceptible to deglycosylation than is digoxin. Two less polar DLIF components produced from the DLIF-genin have at 196 nm and are 4-fold less immunoreactive than DLIF. Our data suggest that subtle structural differences exist between DLIF and digoxin at or near the lactone ring as well as in the nature of the sugars. The presence of deglycosylated congeners of DLIF in human serum, including the less polar components, suggests in vivo deglycosylation of these factors. This is the first demonstration of the existence of naturally occurring deglycosylated derivatives of DLIF and establishes the likelihood of active metabolism of DLIF in mammals.

Endogenous digoxin-like immunoreactive factors (DLIFs) 1 present in mammalian blood (1,2) were discovered in part as a consequence of their cross-reactivity with antidigoxin antibodies. Considerable evidence suggests that endogenous DLIF (3) and ouabain-like factors (OLF) (4) are produced in the adrenal glands and may be linked to modulation of Na,K-ATPase activity in a manner analogous to the family of cardiac glycosides, digoxin or ouabain (5). DLIF, a steroid-like molecule (780 Da), has structural and molecular properties remarkably similar to those of the cardiac glycoside digoxin (3). These properties include a characteristic five-ring structure (aglycone) to which is attached three linearly linked sugar residues (digitoxoses, in case of digoxin) extending from the aglycone C-3 position (Fig. 1). The aglycone portion of the glycoside consists of a steroid skeleton with an unsaturated lactone ring attached at position C-17. The drug digoxin undergoes removal of its digitoxose sugars during metabolism, forming several deglycosylated species (6). The metabolic products of digoxin are important because they interact in a very structure-specific manner with sodium pump isoforms (7) as well as with highly specific antibodies raised against digoxin (8). However, to date no study has identified the presence of naturally occurring metabolic products of DLIF. With improved chromatographic techniques we recently identified the presence of a DLIF with a chemically reduced lactone ring, dihydro-DLIF, analogous to the metabolic product dihydrodigoxin (9).
The ␣ subunit of the sodium pump is the only known functional receptor for digitalis compounds. The stoichiometry of the sugars at the C-3 position on digoxin (8) or of steroids (e.g. progesterone) (10) affect their binding to this receptor. The individual isoforms of Na,K-ATPase have been shown to possess differing affinities for binding of digitalis, ouabain, and their respective deglycosylated derivatives (11,12). The importance of characterizing the molecular and physical properties of DLIF is further underscored by the linkage observed between increased DLIF concentrations in blood and clinical or pathological conditions associated with altered ion-transport homeostasis such as: renal failure (13,14), hepatic failure (15), pregnancy (16), neonatal development (17,18), diabetes-and exercise-induced stress (19,20), and hypertension (21)(22)(23). The source of digitalis-like factors in a hormone-secreting tissue such as the adrenal, presence in blood, and the distribution of likely receptors in target tissues suggest the likely possibility of a new hormonal axis linking the adrenal cortex, endogenous digitalis-or ouabain-like factors, and sodium pump activity (5,24).
In this study we demonstrate the existence of several deglycosylated congeners of DLIF in human serum and show that similar derivatives can be produced in vitro by acid-induced deglycosylation of DLIF isolated from bovine adrenal cortex. These congeners are analogous to the formation of bis, mono, and genin components of digoxin by sequential removal of the three sugars at the C-3 position of the aglycone and suggest the likelihood of metabolism of DLIF in mammals. We also describe a technique for purifying these newly discovered DLIF congeners and investigate several physical properties indicating structural differences between DLIF and digoxin at both the sugars and the aglycone portions of these molecules.

EXPERIMENTAL PROCEDURES
Chemicals and Reagents-All chemicals used were reagent grade. 5-Sulfosalicylic acid (SSA), calcium carbonate (CaCO 3 ), and bovine serum albumin were obtained from Sigma. Digoxin was purchased from Sigma and its related congeners (digoxigenin, mono-and bis-digitoxosides) were donated by Burroughs Wellcome Co. (Research Triangle Park, NC). Pure DLIF (quantitated as dig-equivalent concentration) isolated either by the current or our previous procedures (3) was used as a standard to observe the formation of DLIF-deglycosylated congeners. The most common steroids in the adrenal cortex (cortisone, corticosterone, progesterone, and cortisol) were obtained from Sigma and used as standards to define their chromatographic mobility and cross-reactivity. Acetonitrile (CH 3 CN) was HPLC grade and obtained from Aldrich.
Equipment and Materials-We used a Polytron PT-3000 (Brinkmann, Westbury, NY) for homogenizing adrenocortical tissue and an Orion pH/SE meter model 710A (Orion, Cambridge, MA) for pH measurements. The Sep-Pak reverse-phase C-18 cartridges were obtained from Waters Associates. The HPLC fractions were dried in a Savant SpeedVac Plus SC210A (Savant, Farmingdale, NY) connected to a Refrigerated Condensation Trap RT 400 and a Virtis Freeze Mobile 12 Lyophilizer (Virtis, Gardiner, NY). A Beckman JA-2 centrifuge (Beckman) was used for centrifugation in the isolation procedures.
Tissue Preparation-Bovine adrenal glands were obtained from Pel-Freeze Biologicals (Rogers, AR) and kept at Ϫ80°C until dissected. Tissues were maintained at 4°C during the isolation procedure (dissection, extraction, and centrifugation), while other steps were performed at 22-25°C. Briefly, after isolating the glands from any sur-rounding connective tissues and fats, the cortex was separated from the medulla based on the physical appearance of the outer cortical ring. Each gland weighed approximately 15 g and contained twice as much cortex as medulla. The cortical tissue was sliced (3-mm thick sections), chopped, and homogenized in 2 ml of dH 2 O/g of cortex. The homogenate was then centrifuged three times at 34,000 ϫ g for 30 min at 4°C in which the pellet from each cycle was resuspended in dH 2 O (1 ml/g cortex). The pooled supernatants were stored at Ϫ80°C until further use.
Protein Precipitation-The thawed extracts of adrenal cortical tissue were re-centrifuged at 34,000 ϫ g for 30 min at 4°C to remove any remaining cellular debris. Protein was precipitated by incubating the supernatant with 1% SSA (1 g of sulfosalicylic acid/100 ml supernatant) at room temperature for 60 s followed immediately by adding an excess amount of CaCO 3 until the pH increased and stabilized at 5.2 (the pH of the normal adrenal cortex starting homogenate). This extract was then centrifuged at 80,000 ϫ g for 10 min at 4°C followed by vacuum filtration of the supernatant using two layers of Whatman #1 filter paper.
Solid Phase Extraction (C-18 Cartridge Column)-C-18 Waters reversed-phase Sep-Pak solid-phase extraction column (Vac 3 cc) was primed with 1 volume of CH 3 CN and rinsed with 2 volumes of dH 2 O. The protein-free supernatant was passed through the column twice at a rate of 1 ml/min which was then washed with 2-3 volumes (typically 10 ml) of dH 2 O and eluted with 20 ml of 60% CH 3 CN. To remove the CH 3 CN, the eluates were evaporated to dryness, reconstituted in H 2 O, and filtered through 0.22-m Whatman PVDF in preparation for HPLC. Reconstitution was made to achieve average yields of immunoreactivity of approximately 10,000 pg/ml by the RIA.
HPLC Reverse-phase Chromatography-We used a C-18 reversephase micro-Bondapak column (3.9 ϫ 300 mm, 10-micron particle size) connected with a Waters 600E System Controller and a Waters 966 Photodiode Array Detector to monitor the HPLC output eluent for both UV absorbance and spectral analyses. The eluate was fractionated on HPLC using a linear 20 to 80% CH 3 CN in H 2 O elution gradient. Unless otherwise indicated, typically, 1-ml (1 min) fractions were collected during each chromatographic run over 40 min using a Waters Fraction Collector from Millipore Corp. (Milford, MA). The time delay between detector and the fraction collector was typically 18 s. All fractions were evaporated to dryness, reconstituted in 1 ml of H 2 O, and measured by dig-radioimmunoassay. Steroids co-elute with the fractions of interest (at 15, 19, 23, and 25 min representing DLIF-genin, DLIF-mono, DLIFbis, and DLIF, respectively), were separated by re-injected the fractions individually on selected isocratic mobile phase modes (18,25,30, and 39% CH 3 CN, respectively) for 20 min. Radioimmunoassay measurements showed a single chromatographic peak for each species with immunoreactivity eluted at fraction 6.00 min for DLIF, 8.30 min for DLIF-bis, 12.30 min for DLIF-mono, and 16.30 min for DLIF-genin. The final yield of DLIF and its related deglycosylated congeners were measured by uv spectra and their digoxin equivalent molar absorptivity were determined.
Comparative chromatographic mobility studies between DLIF (and its deglycosylated congeners) and its counterparts digoxin (and its deglycosylated congeners) were conducted. Pure DLIF and digoxin were mixed and co-injected on selected an isocratic mode of 25% CH 3 CN mobile phase for 60 min. Similarly, the genin, the mono-and the bisderivatives of the two parents DLIF and digoxin, were run on 15, 20, and 25% CH 3 CN mobile phase, respectively.
Purification of DLIF from Human Serum-Normal dig-free freshfrozen human serum obtained from the American Red Cross, Louisville, KY, was treated with SSA (final concentration 1% SSA), vortexed, and centrifuged for 2 min at 9,500 ϫ g. Immediately after centrifugation, we applied the entire supernatant to a small primed C-18 reversed-phase Sep-Pak solid-phase extraction cartridge. The column was then washed with 3 ml of H 2 O and eluted with 2 ml of 100% CH 3 CN. For serum samples not treated with SSA, we applied the whole 1-ml sample directly to the Sep-Pak cartridge. In both cases, to remove the CH 3 CN, we evaporated the eluates to dryness, dissolved the residue in 1 ml of dH 2 O, and filtered the solution through a Whatman 0.22-m polyvinylidine difluoride filter in preparation for HPLC.
Molar Absorptivity and Concentration of DLIF-The molar absorptivity of digoxin was determined experimentally by injecting known weighed amounts of digoxin on HPLC and the measured peak area was monitored. At their respective maximum absorbance wavelengths, we assumed comparable molar absorptivity between digoxin and DLIF. Measured amounts of DLIF and its related congeners (by dig-radioimmunoassay equivalence) were injected on HPLC and the apparent molar-immunoreactive concentration of DLIF (or the respective congeners)

FIG. 1. Structure of digoxin and its deglycosylated congeners.
Digoxin has a characteristic five-ring structure (aglycone) to which are attached three sugar (digitoxoses) at the C-3 position. The aglycone consists of a steroid nucleus with an unsaturated lactone ring attached at the C-17 position. Note that digitoxose residues are sequentially removed to form the bis, mono, and genin derivatives. was determined. From the above data the percentage cross-reactivity of DLIF and its congeners (competitive displacement of 125 I-digoxin from polyclonal digoxin-specific antibodies) were obtained.
Protein Assay-Protein concentrations were measured at different steps of the purification procedure using a Pierce BCA protein assay reagent (Rockford, IL). Absorbances were measured at 562 nm on a Beckman DU-64 Spectrophotometer (Beckman) and protein concentration was estimated using bovine serum albumin as standard.
Digoxin-like Immunoreactivity-DLIF was measured using competitive displacement of 125 I-digoxin of polyclonal digoxin-specific antibodies relative to digoxin standards as described elsewhere (25). The standards consisted of digoxin dissolved in human serum and ranged in concentration from 25 to 2,000 pg/ml. The zero standard had no digoxin. The polyclonal antisera and components for the assay were obtained from DuPont NEN (Wilmington, DE). All radioimmunoassay measurements were done in duplicate.

RESULTS
Acid Hydrolysis of Adrenocortical DLIF-The retention times of a mixture of pure digoxin and several deglycosylated digoxin congeners as standards are shown in (Fig. 2, panel A). A similar HPLC pattern (elution time and order) was observed when digoxin was treated with SSA ( Fig. 2, panel C). Incuba-tion of bovine adrenocortical DLIF with SSA (Fig. 2, panel B) resulted in immunoreactive fractions with identical retention times as those of the digoxin standards or the digoxin treated with the same acid. Based on the known sequential removal of sugars by acid hydrolysis (26) and the similarity in the retention times observed for the new DLIF fractions and those of the digoxin congeners (digoxigenin, mono-, and bis-digitoxosides), we named the DLIF fractions eluting at 15, 19, 23, and 25 min as DLIF-genin, DLIF-bis, DLIF-mono, and DLIF, respectively. In addition, note the additional immunoreactive DLIFs appearing in fractions corresponding to less polar products of digoxin.
Acid Hydrolysis of Human Serum DLIF-DLIF was extracted from human serum (subject not taking digoxin) as described under "Experimental Procedures." The initial HPLC data shows a small peak of endogenous DLIF as seen in Fig. 3A (open bar) with a retention time (25 min) similar to that of digoxin or of adrenocortical DLIF. When similar serum was treated with SSA (final concentration 1%) for 60 s, the major part of the 25-min fraction was converted to products with retention times similar to those after acid exposure of digoxin or of DLIF from bovine adrenal cortex, including the less polar product at 31 min (Fig. 3A, solid bar). Fig. 3B shows HPLC analyses of serum drawn from three human subjects (out of 22 human subjects not taking digoxin) but processed without acid treatment. The HPLC elution pattern shows evidence of several naturally occurring DLIF species consistent with the deglycosylated congeners of DLIF. This may indicate in vivo metabolism of DLIF by the cleavage of the sugar moieties attached at the C-3 position of the steroid nucleus (deglycosylation pathway). The remaining 19 subjects showed only one DLIF component at fraction 25, suggesting possible inter-individual differences in metabolism of DLIF. Chromatographic Mobility Study-Physical properties related to molecular polarity and solubility can be distinguished by HPLC analysis. Fig. 4, panel A, demonstrates the baseline resolving capacity of the HPLC system used to separate various deglycosylated DLIF congeners. After DLIF and its related deglycosylated congeners were purified and separated by HPLC chromatography, they were mixed and re-chromatographed on HPLC using the gradient described in Fig. 4, panel A. Absorbance at 216 nm is used to monitor elution of the various species and demonstrate the capability of isolating the individual deglycosylated congeners of DLIF by HPLC. Pure DLIF and digoxin were mixed and co-injected on HPLC using an isocratic 25% CH 3 CN mobile phase. The two molecules separated by a minimum of 2-3 min and absorbance was monitored at 220 or 216 nm (Fig. 4, panel B). Similarly, the genin compounds (aglycone without sugar molecules) of both parent compounds were clearly separated by 1.5-2.0 min on an isocratic 15% CH 3 CN mobile phase (Fig. 4, panel C). Interestingly, the elution order of the genin compounds (DLIF-genin and dig-genin) were reversed from the parent compounds (DLIF and dig). Similar separation results to the parent molecules (DLIF and digoxin) were obtained when DLIF-bis and DLIFmono were mixed with their counterparts (dig-bis and digmono) on selected various isocratic mobile phase (25 and 20% CH 3 CN mobile phase, respectively) (data not shown).
Spectrophotometric Analysis of DLIF Congeners- Fig. 5 shows the absorbance spectra of the DLIF, DLIF-genin, digoxin, and digoxigenin. The results show that DLIF congeners (like the parent DLIF) have the same characteristic absorbance maxima at 216 nm compared to that of digoxin and its congeners at 220 nm (see Table I). The absorbance spectra of digoxin, digoxigenin, and cortisone are included for comparison. The absorbance at 250 nm is a characteristic feature of steroids. The less polar species (digoxin-and DLIF-31) has a uv shift with an absorbance maximum at approximately 196 nm. This suggests some structural modification at or near the lactone ring on the fraction 31 compound.
Molar Immunoreactivity of DLIF and Its Congeners-We investigated the relative molar immunoreactivity based on displacement of 125 I-labeled digoxin by purified DLIF and its deglycosylated congeners from the antiserum to digoxin. We applied the assumption of comparable molar absorptivities of DLIF (at 216 nm) and digoxin (at 220 nm) to calculate the picomole of actual DLIF congeners per pmol of digoxin equivalent immunoreactivity. Note that deglycosylated DLIF derivatives all show a similar immunoreactive potency (approximately 1000-fold less than digoxin) identical to their parent compound, DLIF. On the other hand, DLIF-31 is approximately 4,000-fold less immunoreactive than is digoxin (Fig. 6 and Table I).
Kinetics of DLIF Species Formation-To study the kinetics of formation of the individual DLIF congeners, we compared the time course for production of both DLIF and digoxin congeners by acid hydrolysis. Fig. 7 shows the production of the deglyco- Fig. 2A when the linear gradient (20 -80% CH 3 CN over 35 min) was used. The injected amounts of DLIF and its congeners measured as digoxin equivalent by radioimmunoassay were: DLIF, 2.5 ng; DLIF-bis, 1.5 ng; DLIF-mono, 1.5 ng; DLIFgenin, 2.0 ng. Panel B represents a mixture of pure amounts of DLIF (2.4 ng) and digoxin (10 g) co-injected and run on 25% CH 3 CN over 60 min. Note the separation of the two molecules. Panel C, co-injection of DLIF-genin (2 ng) with its counterpart digoxigenin (20 g) on 15% CH 3 CN over 60 min clearly shows two distinct chromatographic peaks. sylated congeners as a result of incubating DLIF or digoxin with 7.6% SSA. The reaction was terminated (by neutralization) at zero time (no acid) plus 9 time points over the course of 24 h. After each neutralization at the times indicated the contents of the reaction were processed through Sep-Pak and then fractionated by HPLC chromatography. Having identified the HPLC fractions above (Fig. 2), we constructed a fractional molar distribution versus time graph (Fig. 7). This graph provides the relative amount of each species present in the reaction mixture at each time point. Note that acid hydrolysis of both digoxin and DLIF produces a less polar immunoreactive product (F-31). To confirm that digoxigenin (and DLIF-genin by analogy) was being converted to fraction 31, we incubated digoxigenin with 1% SSA at 22°C and monitored the formation of fraction 31 by HPLC. Fig. 8 shows the disappearance of digoxigenin (monitored by absorbance at 220 nm) and the simultaneous appearance of fraction 31 (monitored at 196 nm). DISCUSSION Since the original discovery of DLIF in mammals (1, 2) little has been learned about the metabolism of these endogenous steroid-like factors. We have recently identified the existence of a naturally occurring reduced lactone ring form of digoxin-like immunoreactive factor (dihydro-DLIF, analogous to that of dihydrodigoxin) and showed that dihydrodigoxin is converted to a digoxin-like immunoreactive compound by microsomal cytochrome p450 mediated activity (9). In this study, we demonstrate the existence of several naturally occurring deglycosy-lated species of DLIF in human serum. We show that these derivatives can also be produced in vitro by acid-induced deglycosylation of DLIF isolated from bovine adrenal cortex. The various DLIF species (DLIF-genin, DLIF-mono, DLIF-bis, and DLIF, by analogy to the cardenolide counterparts, digoxin and its deglycosylated congeners) correspond to the factor with no sugars (aglycone), one sugar, two sugars, and three sugars (DLIF), respectively. These findings provide a working model for characterizing two important structural epitopes found on these endogenous factors and also establish a basis for characterizing metabolic pathways of synthesis of endogenous DLIF in mammals.

FIG. 4. Chromatographic analysis of a mixture of pure DLIF and its deglycosylated congeners (DLIF-bis, DLIF-mono, DLIFgenin). Panel A, note identical retention times of the DLIF congeners compared to the digoxin standards in
We used several techniques, each specific for probing structural differences on different portions of the cardiac glycosidelike molecules. Reverse-phase HPLC is sensitive to modifications affecting molecular polarity and solubility; UV-spectral analysis is sensitive to structural features of or in close proximity to the lactone ring; kinetics of deglycosylation of the sugars detects differences in sugar stoichiometry and their binding to the aglycone; and, immunoreactivity is sensitive to molecular modifications near the C-and D-rings of the sterol and the lactone-ring portion of DLIF or digoxin.
By optimizing HPLC elution conditions we demonstrate the ability to separate DLIF from digoxin and their respective aglycones from each other, including two here-to-fore unknown DLIF species. The HPLC elution pattern of deglycosylated adrenocortical DLIF was identical to that of digoxin using the linear elution gradient (Fig. 2, B and C). The DLIF fractions eluted in fractions correspond to the standards of digoxin and its deglycosylated congeners ( Fig. 2A). A similar chromatographic pattern was observed after acid-deglycosylation of DLIF isolated from human serum (Fig. 3A) and, in fact, the immunoreactive fragments were found naturally in human serum (3 of 22 subjects) without acid treatment (Fig. 3B). These results are consistent with our recent hypothesis suggesting molecular similarity between DLIF extracted from bovine adrenal cortex and that from human serum (27).
Additional HPLC studies using mobile phases clearly showed relative mobility shifts when comparing DLIF and digoxin (Fig. 4B) and also for their respective aglycones (Fig.  4C). Interestingly, the mobility of DLIF-genin was shifted toward increased polarity relative to digoxigenin when compared to the opposite relative mobilities of the parent compounds. The genin compounds are aglycone sugar-free molecules (see Fig.  1), hence, these findings are the first reported observation indicating that structural differences between DLIF and
b Measured as digoxin equivalents by digoxin radioimmunoassay. c Molar immunoreactivity ratio of DLIF and its related congeners was calculated by dividing the Abs/measured ratio of each species by the Abs/true ratio for the standard compound (digoxin). This ratio represents the molar concentration of DLIF (or any of its congeners) needed to compete with a unit molar concentration of digoxin, with its antibody. d ND, not determined.  Table I). digoxin are in the aglycone portions of these compounds. Both the chromatographic mobility shifts and the immunoreactive potency differences observed for the two genin compounds of DLIF and digoxin (Table I and Fig. 6) suggests structural differences in/or near the lactone ring for the aglycones. This is supported by the spectral analysis showing a uv shift in the maximum absorbance for DLIF (216 nm) compared to digoxin (220 nm) (Fig. 5). However, the kinetics of acid-induced deglycosylation studied by monitoring the rate of formation of different DLIF species showed that DLIF-deglycosylation is considerably faster (approximately 8-fold) than that observed for digoxin (see Fig. 7). This is an indication that the sugars on DLIF are different than those on digoxin and thus in the strength of the bond to each other in the linear linkage and/or to the DLIF-aglycone at position C-3 (Fig. 1).
Characterizing the structural features of endogenous digoxin-or ouabain-like counterparts of the cardenolides has been difficult because of their low concentrations in tissues and difficulty in extracting sufficient quantities for detailed structural analysis. For example, initial data suggested that OLF (ouabain-like factor) was structurally identical to ouabain using mass spectroscopy and other related techniques (28,29). However, more recent studies using an exciton-coupled circular dichroism (CD) technique show that OLF and HIF (an isomer of ouabain from bovine hypothalamus) are structurally different from ouabain (30). The initial suggestion of a structural similarity between OLF and ouabain was based on the similarity in their chromatographic mobility, mass spectral data, and their unit immunoreactivity with antibodies to ouabain. However, in the case of DLIF we now have evidence of chromatographic, spectral, and molar immunoreactivity differences which suggests that DLIF is more structurally distinct from digoxin than OLF is to ouabain. However, sufficient quantities of these compounds will still need to be harvested to fully permit the identification of fine structure.
Of interest is the discovery of a new as yet undefined product of DLIF and of digoxin (fraction 31, less polar by HPLC elution than DLIF or digoxin) produced by acid-induced deglycosylation of these molecules and also existing naturally in vivo in human serum. Our data are consistent with the hypothesis that DLIF-31 (or digoxin-31) is formed preferentially from the genin derivatives (Fig. 8). The far uv shift to 196 nm observed for the DLIF-31 immunoreactive component is, however, consistent with and characteristic of a structural modification at or near the lactone ring. A similar uv shift is observed after formation of dihydrodigoxin (196 nm) from digoxin (220 nm) (31) or after modification by a double bond saturation on the Aor B-ring of the steroid (32). However, the presence of a double bond in the A-or B-ring structures of the DLIF aglycone is not suspected because that structural feature is usually associated with a characteristic absorption band at 250 nm (see Fig. 5, e.g. cortisone). DLIF-31, therefore, is a new species and since it is detected naturally in some human sera, may be a metabolic FIG. 7. Apparent molar fractional distribution and time course of formation of DLIF and digoxin congeners after incubation with acid. Conditions are as described under "Experimental Procedures." Panel A shows the relative production of DLIF congeners from DLIF isolated from bovine adrenal cortex (starting with 10 ng of digoxin equivalent DLIF) after treatment with 7.6% SSA (7.6 g of SSA for each 100-ml homogenate). Panel B shows a comparable fractional distribution profile for digoxin congeners produced (starting with 50 ng of digoxin) under similar experimental conditions. Note: time scales are different and that DLIF deglycosylates more rapidly than digoxin. product of DLIF. The 4-fold decrease in immunoreactivity of DLIF-31 compared to DLIF and relative to digoxin ( Fig. 6 and Table I) is again consistent with structural changes at or near the lactone ring as is the spectral shift toward the uv (31).
Three principal routes of metabolism have been reported for digoxin in mammals: sequential deglycosylation by the stepwise removal of sugar molecules (with formation of bis-, mono-, and digoxigenin) resulting primarily from acid hydrolysis in the stomach; conversion of digoxin to polar metabolites like glucuronide and sulfate conjugates of 3-epi-digoxigenin (6,33); and conversion of digoxin to dihydrodigoxin by chemical reduction of the lactone ring occurring within the gastrointestinal tract by bacterial action of Eubacterium lentum (34). Extensive digoxin biotransformation has been reported in some patients with polar metabolites being predominant (35). To date, however, little is known about the metabolism of DLIF in mammals. Our data suggest that the route of metabolism of DLIF may be similar to that of digoxin. The recent discovery of a dihydro-DLIF species existing in the adrenal establishes a basis for synthesis of DLIF in the adrenal gland (9).
The DLIF deglycosylated congeners reported here may explain some of the discrepancies reported in other studies isolating digoxin-like compounds (36). In fact, some of these deglycosylated congeners (generated during isolation procedures and/or those found naturally) may have been identified as different kinds of digoxin-like immunoreactive compounds. DLIF is considered as one of the most interesting and detrimental interferant to most commonly used digoxin assays. The variable cross-reactivity and the different amounts or ratios of the DLIF congeners in serum may explain the wide variation in detection sensitivities for endogenous DLIF reported by digoxin immunoassays (37). Recognizing the presence of these newly discovered congeners and understanding their relative immunoreactivity with the antibodies used will provide a more accurate means of therapeutic digoxin monitoring for digoxin (38). DLIF is known to exist bound tightly to a 22-36-kDa protein in serum (25,39). The stoichiometry of DLIF-glycosylation may also affect this protein binding and thus influence the detection of these factors in serum when using different digoxin immunoassays. This may well explain changes in the extent of protein binding of DLIF observed in some clinical conditions, e.g. pregnancy induced hypertension (40) or renal failure (2).
Digitalis cardiac glycoside molecules have two important structural features linked to their ability to functionally inhibit Na,K-ATPase: the lactone ring and the digitoxose sugar residues. Studies of the molecular structure of the ␣ (catalytic) subunit of the sodium pump (Na,K-ATPase) has revealed the involvement of specific regions of the polypeptide in binding the sugar portion of the cardiac glycosides (41)(42)(43)(44). Our data taken together are consistent with subtle but important structural differences existing between DLIF and digoxin. Differences are evident on all three major portions of the molecule: sugars, lactone ring, and sterol nucleus.


A reliable microplate assay for determination of B-galactosidase activity in Neurospora crassa strains bearing lacZ fusions

We have been using lacZ as a reporter gene in N. crassa. The standard -galactosidase assay can be labor intensive and time consuming when large numbers of strains are assayed simultaneously. We sought a technique to simplify the pipetting steps involved in assay preparation and in optical density reading. High reproducibility and rapid processing was obtained by adapting the standard test tube method to a microassay performed in a 96-well microplate. Creative Commons License This work is licensed under a Creative Commons Attribution-Share Alike 4.0 License. This regular paper is available in Fungal Genetics Reports: http://newprairiepress.org/fgr/vol43/iss1/15 A reliable microplate assay for determination of B-galactosidase activity in Neurospora crassa strains bearing lacZ fusions Kwangwon Lee and Daniel J. Ebbole, Department of Plant Pathology and Microbiology, Texas A&M University, College Station, TX 77843. We have been using lacZ as a reporter gene in N. crassa. The standard -galactosidase assay can be labor intensive and time consuming when large numbers of strains are assayed simultaneously. We sought a technique to simplify the pipetting steps involved in assay preparation and in optical density reading. High reproducibility and rapid processing was obtained by adapting the standard test tube method to a microassay performed in a 96-well microplate. The protocol described below is a relatively straightforward adaptation of the standard assay to a microplate format. 1. Harvest mycelial samples in a Buchner funnel on 70 mm diameter Whatman filter paper. Conidial samples are harvested on 24 mm diameter glass fiber filters (Whatman). Briefly remove the excess water from the samples by pressing the sample between paper towels. Wrap the samples in aluminum foil, freeze the sample in liquid nitrogen and save until assay. 2. A 2 ml screw cap tube is filled with 1 g of glass beads (0.5 mm dia.) and 1 ml ice-cold Z buffer (Sambrook et al., 1989, Molecular Cloning, page 17.35). Add 10 ul of 100 mM PMSF (phenylmethyl sulfonyl fluoride; Sambrook et al., 1989, Molecular Cloning, page 16.66) just before the first bead beating. 3. Place 70 to 100 mg of frozen cells into the tube and break in a mini bead beater (Biospec Products, Bartlesville, OK), twice for 30 sec each, with cooling on ice between bead-beating. 4. Microcentrifuge for 5 min at maximum rpm to pellet debris. Transfer the supernatant to new tubes. Centrifuge again if debris is present. 5. For protein assay, we adapted the manufacturer’s instructions (Bio-Rad Protein Assay, Biorad, Richmond, CA) for the microassay using a microplate dish to measure the OD. 250 u l of dye + extract was transferred to each well of a microplate. The microplate reader (EmaxTM Precision microplate reader, Molecular Devices Corporation, Menlo Park, CA) was set to read with wavelength of 595 nm. 6. Each well of a microplate is filled with Z buffer + extract (total of 150 l). We perform duplicate assays of two different amounts of extract. A control well is also included for each amount of extract (i.e., 6 wells for each mycelial extract is required). The amount of extract can be adjusted depending on the specific activity of the extract. Reaction times of 15-60 min are ideal. Published by New Prairie Press, 2017 7. Add 70 l of 1 M Na2CO3 and 30 l of ONPG (o-Nitrophenyl-D-galactopyranoside) to each control well of a dish. It is important to add Na2CO3 first. Place the dish on a support (test tube rack) in a 28 C water bath. 8. Add 30 l of ONPG to the test wells at 10 sec intervals. Note the time of addition. Solutions are mixed with pipetting 2-3 times. 9. When a reaction shows a yellow color, the reaction can be stopped by adding 70 l of Na2CO3. Note the time of addition to so that the total reaction time can be determined. 10. Water adhering to the bottom of the microplate dish is removed completely with a Kimwipe. The lid of the microplate is removed (if present) and transferred to the microplate reader. 11. The OD value of each sample is determined by subtracting the OD value of the control from that of the sample. The assay values are calculated by the formula below: U= { OD x 380 } / Protein (mg) x Time (min) Table 1. Comparison of microplate assay and the standard assay. Strains Published Specific Activity 2 Microplate Assay activity 1 Value 3 ________________________________________________________________________ DE3 not tested 2 8 (1) DE1559 6(1) 6 19 (1) DE778 16(1) 14 41 (2) DE353 128(1) 111 442 (6) DE236 225(36) 98 389 (5) _________________________________________________________________________ Published values from Corrachano et al., Dev. Biol. 167:190-200, 1995. The numbers in parentheses show standard deviation. The values are means of two duplicate samples of each strain. The values are means of five duplicate samples of each strain. The numbers in parentheses show the standard deviation of the five assays. Strains presented in Table 1 have a single con-10/lacZ translational fusion construct at the his-3 locus. DE3 is a strain containing pDE3, which has no activation elements in the 5’ end of lacZ. Other strains contain a translational fusion construct that is under control of different sized fragments of the con-10 promoter (Corrochano et al., Dev. Biol. 167:190-200, 1995). The microplate assay values correlate well with the specific activities determined by the standard assay. However, the microplate assay gives values for relative b-galactosidase activity that are 3 to 4-fold higher than the true specific activities (in nmole ONPG hydrolyzed/min/mg) of the extracts as determined by cuvette assay. In the microplate assay, the path length of the wells in a microplate dish differs from the path length of a standard cuvette. Also, the wavelenth used in http://newprairiepress.org/fgr/vol43/iss1/15 DOI: 10.4148/1941-4765.1311 the plate reader is 405 nm rather than 420 nm, as used in the standard assay. This could account for the different values between the two assay methods. However, we found the method to be reproducible and it provides measurements that are consistent with specific activities determined using the standard cuvette assay. We also noticed that strain DE236 behaved differently in our assays from the published value. The cultures reported by Corrachano et al. (ibid) were grown in constant darkness. In our experiments the cultures were grown in constant light. It is known that light influences the activity of the con-10 promoter and this is the likely cause of the activity difference. Last modified 7/25/96 KMC Published by New Prairie Press, 2017

Neurospora crassa strains bearing lacZ fusions
Kwangwon Lee and Daniel J. Ebbole, Department of Plant Pathology and Microbiology, Texas A&M University, College Station, TX 77843.
We have been using lacZ as a reporter gene in N. crassa. The standard -galactosidase assay can be labor intensive and time consuming when large numbers of strains are assayed simultaneously. We sought a technique to simplify the pipetting steps involved in assay preparation and in optical density reading. High reproducibility and rapid processing was obtained by adapting the standard test tube method to a microassay performed in a 96-well microplate.
The protocol described below is a relatively straightforward adaptation of the standard assay to a microplate format.
1. Harvest mycelial samples in a Buchner funnel on 70 mm diameter Whatman filter paper. Conidial samples are harvested on 24 mm diameter glass fiber filters (Whatman). Briefly remove the excess water from the samples by pressing the sample between paper towels. Wrap the samples in aluminum foil, freeze the sample in liquid nitrogen and save until assay.
3. Place 70 to 100 mg of frozen cells into the tube and break in a mini bead beater (Biospec Products, Bartlesville, OK), twice for 30 sec each, with cooling on ice between bead-beating. 4. Microcentrifuge for 5 min at maximum rpm to pellet debris. Transfer the supernatant to new tubes. Centrifuge again if debris is present. 5. For protein assay, we adapted the manufacturer's instructions (Bio-Rad Protein Assay, Biorad, Richmond, CA) for the microassay using a microplate dish to measure the OD. 250 u l of dye + extract was transferred to each well of a microplate. The microplate reader (EmaxTM Precision microplate reader, Molecular Devices Corporation, Menlo Park, CA) was set to read with wavelength of 595 nm.
6. Each well of a microplate is filled with Z buffer + extract (total of 150 l). We perform duplicate assays of two different amounts of extract. A control well is also included for each amount of extract (i.e., 6 wells for each mycelial extract is required). The amount of extract can be adjusted depending on the specific activity of the extract. Reaction times of 15-60 min are ideal.
7. Add 70 l of 1 M Na2CO3 and 30 l of ONPG (o-Nitrophenyl--D-galactopyranoside) to each control well of a dish. It is important to add Na2CO3 first. Place the dish on a support (test tube rack) in a 28 C water bath.
8. Add 30 l of ONPG to the test wells at 10 sec intervals. Note the time of addition. Solutions are mixed with pipetting 2-3 times.
9. When a reaction shows a yellow color, the reaction can be stopped by adding 70 l of Na2CO3. Note the time of addition to so that the total reaction time can be determined.   Corrachano et al., Dev. Biol. 167:190-200, 1995. The numbers in parentheses show standard deviation. 2 The values are means of two duplicate samples of each strain. 3 The values are means of five duplicate samples of each strain. The numbers in parentheses show the standard deviation of the five assays. Table 1 have a single con-10/lacZ translational fusion construct at the his-3 locus. DE3 is a strain containing pDE3, which has no activation elements in the 5' end of lacZ. Other strains contain a translational fusion construct that is under control of different sized fragments of the con-10 promoter (Corrochano et al., Dev. Biol. 167:190-200, 1995).

Strains presented in
The microplate assay values correlate well with the specific activities determined by the standard assay. However, the microplate assay gives values for relative b-galactosidase activity that are 3 to 4-fold higher than the true specific activities (in nmole ONPG hydrolyzed/min/mg) of the extracts as determined by cuvette assay. In the microplate assay, the path length of the wells in a microplate dish differs from the path length of a standard cuvette. Also, the wavelenth used in the plate reader is 405 nm rather than 420 nm, as used in the standard assay. This could account for the different values between the two assay methods. However, we found the method to be reproducible and it provides measurements that are consistent with specific activities determined using the standard cuvette assay.
We also noticed that strain DE236 behaved differently in our assays from the published value. The cultures reported by Corrachano et al. (ibid) were grown in constant darkness. In our experiments the cultures were grown in constant light. It is known that light influences the activity of the con-10 promoter and this is the likely cause of the activity difference.