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Genetic Variants in Drosophila Disguised by Cofactor and Subunit Binding (Electrophoresis/Polymorphism/Alcohol Dehydrogenase/NAD) GEORGE B

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Genetic Variants in Drosophila Disguised by Cofactor and Subunit Binding (Electrophoresis/Polymorphism/Alcohol Dehydrogenase/NAD) GEORGE B Proc. Nati. Acad. Sci. USA Vol. 75, No. 1, pp. 395-399, January 1978 Genetics Structural flexibility of isozyme variants: Genetic variants in Drosophila disguised by cofactor and subunit binding (electrophoresis/polymorphism/alcohol dehydrogenase/NAD) GEORGE B. JOHNSON Department of Biology, Washington University, St. Louis, Missouri 63130 Communicated by Peter H. Raven, September 16, 1977 ABSTRACT Wild populations of Drosophila mojavensis cause any factor that tends to stabilize tertiary structure will exhibit considerable conformational variation in the NAD+-free act to obscure the variation from detection by electrophoresis. form of alcohol dehydrogenase (alcohol:NAD+ oxidoreductase; Thus, enzymes that tightly bind cofactors or that aggregate into EC 1.1.1.1). The variation appears genetic, as it does not occur within an inbred strain. The NAD+-bound form of alcohol de- multimers may exhibit fewer conformational classes than might hydrogenase, present in the same individuals, does not exhibit have been detected in the absence of the stabilizing influences the variation, suggesting that the binding of NAD+ acts to sta- of cofactor or subunit binding. bilize conformation. Such cofactor binding to enzymes may thus This paper reports studies on the stabilizing influences of conceal considerable variation. A similar effect is suggested for cofactor binding and of subunit aggregation on enzyme shape binding of esterase subunits. in Drosophila. The results suggest that variation in protein In the decade since electrophoretic variation at enzyme loci was shape may indeed be masked by such stabilizing influences. first reported (1), extensive investigation of insect (2), plant (3), MATERIALS AND METHODS small vertebrate (4), and human (5) populations has confirmed the generality of widespread polymorphism. Recent studies Stocks. Stocks of the cactus fly Drosophila mojavensis were suggest even greater levels of variation than were first detected provided by William Heed. Eighteen of the 20 lines investi- (6-8) and raise important questions concerning ultimate gated were established from single-pair matings of flies elec- amounts of variation and its biological significance. trophoretically homozygous for alcohol dehydrogenase (alco- One such question concerns the role of protein shape. In hol:NAD+ oxidoreductase; EC 1.1.1.1); two other lines of studies of the electrophoretic behavior of proteins in gels of multiple founder stocks were >90% one allele. Sixteen of the various pore size, alleles were detected that appeared to differ lines were initially collected at Libertan and San Carlos, Sonora, only in shape, having identical charge (9); in subsequent studies, Mexico; the other four originated from Arizona. Two isolates most electrophoretically distinguishable alleles were shown to of each of the 10 Libertad lines were analyzed. differ in shape as well as in charge (7, 10). Control studies Stocks of D. pseudoobscura were provided by Richard Le- showed that known conformational changes in proteins may wontin, as part of a blind test of the ability of gel sieving analysis be easily detected on electrophoresis and that neither modified to detect genic variation at the esterase-5 locus (12). From 10 subunit exchange rates nor altered hydration shells could be to 20 flies were individually analyzed from each stock. invoked as alternative explanations to conformational differ- Electrophoresis. Individual flies were ground in 75 Al of 50 ence between variants. The shape of particular variants proved mM Tris-HCI buffer, pH 7.3, 5% wt/vol with respect to sucrose, highly reproducible: in analyses of highly inbred lines of Dro- and the homogenate was centrifuged briefly in a capillary sophila, between-individual estimates varied no more than centrifuge in the cold. Ten microliters of this sample was added replicate characterizations of the purified hemoglobin run in to each of six gels of different acrylamide concentrations (4, 5, the same gels. 6, 7, 8, and 9% acrylamide). Beef hemoglobin, horse ferritin, An unusual aspect of these studies of natural polymorphism and bromphenol blue were added to each gel to serve as internal in protein shapes is the anomalous behavior of heterozygotes: standards (13). Multiphasic disc gels were constructed and their low frequency suggests that some of the electrophoreti- Tris/glycine buffers prepared according to Ornstein (14). cally detected variants may reflect post-translational epigenetic Electrophoresis was at 10° and 200 V for an average of 140 phenomena (7, 11). When heterozygotes between differing min. variants of dimeric enzymes do occur, the characteristics of the Alcohol dehydrogenase was assayed in 0.1 M Tris-HCI buffer, hybrid dimer are of particular interest: where the variants differ pH 8.4, with ethanol as substrate (15). Assays were stopped in in shape, the hybrid dimer is rarely intermediate in shape, cold 25% propanol/10% acetic acid, and all gels were scanned usually assuming the conformation of one of the variants or, on a Gilford 240 spectrophotometer at 540 nm within 90 min. more rarely, a widely divergent shape (7). This suggests a rather Esterase was assayed by the procedures of Hubby and Lewontin unexpected flexibility in protein shape, one subunit's confor- (16), and all, gels were scanned as before. NAD+, when added mation being significantly altered by the binding of the to grinding and running buffers, was at a final concentration other. of 1 mM. Conformational.flexibility between variants of an enzyme Gel Sieving Analysis. Protein shape for an individual sample has important implications in terms of our ability to detect the was estimated by plotting the logarithm of band mobility rel- variants in surveys of enzyme polymorphism. This is true be- ative to bromphenol blue against % acrylamide and taking the slope of the resulting regression as an estimator of size/shape The costs of publication of this article were defrayed in part by the (7, 9, 12, 13). In these data, experimental error in slope was payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate Abbreviation: AdHj, AdH2, etc., electrophoretic forms of alcohol de- this fact. hydrogenase. 395 Downloaded by guest on October 2, 2021 396 Genetics: Johnson Proc. Natl. Acad. Sci. USA 75 (1978) Fe 0 Fe Hb AdH Hb AdH Fe Hb 0 AdH FIG. 1. An experiment demonstrating the NAD+-mediated interconversion of AdH3 and AdH5. Electrophoresis was performed on 8% acrylamide disc gels at 100 in Tris/glycine buffer, pH 8.9, at 200 V. Gels were scanned on a Gilford 240 spectrophotometer at 540 nm. Bovine hemoglobin (Hb) and horse spleen ferritin (Fe) were used as internal standards in each gel. uniformally less than 5% of the slope value. Sampling error was of Ad-H activity indeed represent forms interconvertible by estimated by comparison with the hemoglobin standard run NAD+ was verified by four lines of investigation: in the same gels: the distribution for a homogeneous sample of (i) Experiments were done in which sample preparation and an enzyme should be no greater than for the hemoglobin electrophoresis were both carried out in the presence of 1 mM standard. Such comparisons were done by plotting an estimator NAD+, while in parallel analyses of the same lines no exogenous of charge ("mid-Y") on the vertical axis and the corresponding NAD+ was added. In the absence of exogenous NAD+ both slope (KR) of the horizontal axis [the procedure has been de- bands are obtained, but in its presence only the faster of the two scribed in detail (7)]. bands is observed. This suggests that the slower form corre- sponds to AdH5 and the faster one to AdH3. RESULTS (ii) Experiments were done in which endogenous NAD+ was removed by passage through a 5-cm Sephadex-G25 column. Alcohol dehydrogenase In no case was the fast band (AdH3) removed by this treatment, Alcohol dehydrogenase occurs in five interconvertible elec- but the proportion of total alcohol dehydrogenase activity at- trophoretic forms in D. melanogaster (17). The most slowly tributable to the slower band (AdH5) was more than doubled migrating form (AdH5) may be converted into faster forms by by the passage through Sephadex. NAD+ in a manner elegantly investigated by Jacobson et al. (iii) The interconversion of AdH3 and AdH5 by NAD+ was (18). In adult Drosophila only two forms are usually observed verified by performing serial cofactor addition and removal (AdH5 and-AdH3), with the fastest form, ADH1; present in trace experiments on extracts prepared from single individuals. A amounts and forms AdH2 and AdH4 undetectable. NAD+-free typical result is presented in Fig. 1. In all cases the results were AdH5 may be converted NAD+-bound AdH3 in vitro under fully consistant with NADW-mediated conversion. low concentrations of NAD+ (<10 mM); at higher concentra- (iv) In line with a penetrating suggestion by Ornstein (14) tions of NAD+ (>10 mM), significant conversion to AdH1 oc- and Cann and Goad (20), experiments were carried out in curs. Importantly, the NAD+-bound forms of AdH are reported which the sample concentration was varied. Ornstein pointed to be significantly more heat-stable than the NAD+-free form out an interesting property of "stacking" gels such as used in (18). Preliminary investigations have suggested that confor- these studies: in stacking gels band focusing is obtained by a mational change is associated with the binding of NAD+ local electrical field created by the ions and charged proteins (19). and ithe local strength of that field depends upon the amount When AdH was analyzed in D. mojavensis, two bands were of charged material. Because most reversible aggregations (such uniformly obtained which differed substantially in charge as alcohol dehydrogenase with NAD+) dissociate in strong (mid-Y of 0.20 compared to mid-Y of 0.09). That the two bands electric fields, the degree of dissociation (proportion of total Downloaded by guest on October 2, 2021 Genetics: Johnson Proc.
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