Effect of Enzyme Dilution on the Relative Electrophoretic Mobility of ...

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Author(s): Jose M. Hernandez-Juviel, David J. Morafka, Imelda Delgado, Gary D. Scott, Robert. W. Murphy. Source: Copeia, Vol. 1992, No. 4 (Dec. 18, 1992), pp.
Effect of Enzyme Dilution on the Relative Electrophoretic Mobility of Glutamate Dehydrogenase Isozymes in the Prairie Rattlesnake, Crotalus viridis viridis Author(s): Jose M. Hernandez-Juviel, David J. Morafka, Imelda Delgado, Gary D. Scott, Robert W. Murphy Source: Copeia, Vol. 1992, No. 4 (Dec. 18, 1992), pp. 1117-1119 Published by: American Society of Ichthyologists and Herpetologists Stable URL: http://www.jstor.org/stable/1446652 . Accessed: 04/02/2011 09:17 Your use of the JSTOR archive indicates your acceptance of JSTOR's Terms and Conditions of Use, available at . http://www.jstor.org/page/info/about/policies/terms.jsp. JSTOR's Terms and Conditions of Use provides, in part, that unless you have obtained prior permission, you may not download an entire issue of a journal or multiple copies of articles, and you may use content in the JSTOR archive only for your personal, non-commercial use. Please contact the publisher regarding any further use of this work. Publisher contact information may be obtained at . http://www.jstor.org/action/showPublisher?publisherCode=asih. . Each copy of any part of a JSTOR transmission must contain the same copyright notice that appears on the screen or printed page of such transmission. JSTOR is a not-for-profit service that helps scholars, researchers, and students discover, use, and build upon a wide range of content in a trusted digital archive. We use information technology and tools to increase productivity and facilitate new forms of scholarship. For more information about JSTOR, please contact [email protected].

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SHORTER CONTRIBUTIONS: GOOD,D. A. 1988. Phylogenetic relationships among gerrhonotine lizards, an analysis of external morphology. Univ. California Press 121:1-139. MARTIN DEL CAMPO, R. 1939.

Contribuci6n

al cono-

cimiento de los gerrhonoti mexicanos, con la presentaci6n de una nueva forma. An. Inst. Biol. Univ. Mexico 10:353-361. TIHEN, J. A. 1949a. A review of the lizard genus Barisia. Univ. Kansas Sci. Bull. 33:217-256. . 1949b. The genera of gerrhonotine lizards. Am. Mid. Nat. 41:580-601. WEIGMANN,A. F. 1828. Beytrage zur Amphibien Kunde. Isis 1828:364-383. . 1834. Herpetologica Mexicana, pt. 1, saurorum species. Luderlitz, Berlin, Germany. AND STANLEY F. Fox, Department of Zoology,OklahomaState University,Stillwater, Oklahoma 74078. Accepted 30 Sept. 1991.

J. KELLY McCoY

Copeia, 1992(4), pp. 1117-1119 ? 1992 by the American Society of Ichthyologists and Herpetologists

EFFECT OF ENZYME DILUTION ON THE RELATIVE ELECTROPHORETIC MOBILITY OF GLUTAMATE DEHYDROGENASE ISOZYMES IN THE PRAIRIE RATTLESNAKE, CROTALUSVIRIDISVIRIDIS.-Starch gel electrophoresis of enzymatic and nonenzymatic proteins is a broadly applied method in the study of genic variability and genealogical relationships. These studies assume that allozymes expressing the same relative mobility are homologous, and, in general, those that do not have the same relative mobility are not homologous. In the latter case, the relative mobility of homologous isozymes may be altered by posttranslational modifications (e.g., McGovern and Tracy, 1981; Murphy and Crabtree, 1985; Murphy et al., 1990), resulting in the false conclusion that the encoding gene loci are different. Recently, we studied the effects of starvation on enzyme concentration for several enzyme systems among a variety of tissues from prairie rattlesnakes, Crotalus viridis viridis (Murphy, Aguirre, and Scott, unpubl.). We observed that, for glutamate dehydrogenase (GTDH: E.C. 1.4.1.2) isozymes, the relative migration rate may be affected by enzyme concentration. This finding could have led to the misinterpretation of isozyme patterns on gels. Herein we report the nature of our observations.

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Methods.-Horizontal starch gel electrophoresis and histochemical staining (Murphy et al., 1990) were used to separate the GTDH isozymes. Approximately 0.3 g of liver tissue was dissected from each of 23 rattlesnakes collected at Campo, Colorado. Each tissue sample was weighed to the nearest 0.001 g, diluted with two parts (g:ml) of distilled, deionized water, homogenized for 20 sec using a Janke and Kunkel tissue homogenizer, and frozen at -20 C for 24-48 h. Thawed homogenates were centrifuged at 2000 x g for 25 min. The supernatant fraction was isolated and sequentially doubly diluted (Klebe, 1975; Kettler and Whitt, 1986; Kettler et al., 1986) producing the following seven dilutions: 1:3, 1:6, 1:12, 1:24, 1:48, 1:96, 1:192. Sample wicks (4 mm x 9 mm Whatman #3 filter paper) were saturated with 10 Adof liver extract. We varied the electrophoretic conditions in order to investigate the nature of the rate of migration change as a function of enzyme concentration and prepared fresh extracts using 0.85% phosphate buffered saline pH 7.2 both with and without 2-mercaptoethanol as additional grinding solutions. Two electrophoresis buffer combinations were used: discontinuous borate and lithium hydroxide (Murphy et al., 1990). Results and discussion.-Following staining, we observed that, on the lithium hydroxide buffer system, in all individuals the relative mobility of the Gtdh-A isozymes increased (anodally) with increasing dilution (Fig. 1). The changes in isozyme mobility were sufficiently conspicuous to potentially produce a misinterpretation of what constituted homologous gene products, especially if the enzyme concentrations varied by more than 4 x. Electrophoresis of samples homogenized in 2-mercaptoethanol failed to alter our observation of increased mobility with dilution, and it resulted in decreased enzyme activity but did not alter shifts in mobility. A shift in electrophoretic mobility with increasing dilution was not observed on the discontinuous borate buffer system. It has been shown that bovine liver Gtdh-A dissociates into four subunits of equal size upon dilution (Frieden, 1958). In addition, bovine liver Gtdh-A (the GIDH of Frieden) undergoes a reversible dissociation reaction induced by the binding of purine nucleotides to Gtdh-A in the presence of coenzyme (Frieden, 1959a, 1959b). The purine nucleotides cause conformational changes in the Gtdh-A molecule by inhibiting

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Fig. 1. Zymogramof glutamatedehydrogenaseexpressionof the prairierattlesnake,Crotalusviridisviridis.

Liver tissue dilutions (g tissue: ml H20) are as follows: 1 = 1:3, 2 = 1:6, 3 = 1:12, 4 = 1:24, 5 = 1:48, 6 =

1:96. More dilute concentrationsof enzyme results in relativelyhigher mobilityof isozymes.This zymogram is one of four on the same gel; the dilution series from all four individualsnakes showed the same pattern.

or inducing the dissociation reaction. Coenzyme concentration and the kind of coenzyme used are critical factors in determining the degree of dissociation that the Gtdh-A molecule undergoes (Frieden, 1963). Thus, electrophoresis under such conditions would result in an increased mobility that would vary with concentration. Thus, Frieden's data support our conclusions that similar reactions take place in rattlesnake Gtdh-A as well. Among the enzyme systems and specific loci surveyed [Acp-A, ADH, ALP, Ck-A, EST (general), Est-D, GCDH, GPI, G3PDH, GTDH, ICDH, IDDH, LDH, MAN, MDH, MDHP (ME), PCDH, PGDH, PGM, SOD, and XDH; see Murphy and Crabtree (1985) for number of loci resolved in multilocus systems], only Gtdh-A showed any change in mobility with enzyme concentration. This suggests that enzyme concentration has no significant effect on isozyme mobility for most enzyme systems, at least in rattlesnakes. For Gtdh-A, however, this phenomenon may be widespread among squamate

reptiles because intraspecific surveys of numerous divergent taxa (e.g., Murphy, 1983; Sites and Murphy, 1991) frequently show minor variation in the relative mobility of Gtdh-A isozymes (Murphy, pers. obs.), although this is not true for at least some ambystomatid salamanders and hylid frogs. Even precise diluting of tissues for homogenization would not completely eliminate these minor shifts because of the in vivo tissue-specific variance in the concentration of Gtdh-A among specimens sampled. Suspected effects of dilution on isozyme migration during electrophoresis can be examined using three methods. First, samples showing minor differences in electrophoretic mobility can be mixed together and rerun on a gel; a single tight band, as opposed to a broader isozyme, would lead to the conclusion that the two samples are homoallelic. Further experimentation could include either serial dilutions as we have done in this study, or reordering of all samples on a gel by relative mobility and observing whether the patterns are repeated following

SHORTER CONTRIBUTIONS: electrophoresis. Of these three approaches, the first would provide more efficient and conclusive evidence about the number of alleles involved. The second approach would help to form an explanation for the observed variation. Finally, the third approach would be least preferable because it would provide no explanation of the observed phenomenon. Thus, caution should be exercised when interpreting minor differences in the mobility of Gtdh-A and potentially other enzyme products, because these may reflect artifacts of electrophoresis and not heritable differences. In particular, enzyme concentrations may vary between individuals due to state of health, seasonality of activity (effects of temperature, brumation, etc.), and time interval elapsed since last feeding. Squamates, as poikilotherms, may be particularly vulnerable in light of their episodic activities and greater tendencies to be physiological conformers in contrast to homeotherms. Even cooling and the concomitant lethargy and short-term (15-day) "starvation" were sufficient to lower concentrations in an extensive suite of digestive enzymes assayed from the lacertid lizard, Gallotia galloti (Gonzalez et al., 1988). Animals having deteriorating health have been suggested as sources of tissues for electrophoretic comparisons. These animals are often donated by zoos or sold at discounts by supply houses. Such specimens, as well as those subject to long transit from date of capture to date of euthanization and individuals newly emergent from brumation, would be particularly subject to variance in isozyme concentration that might differences deprovide the electromorph scribed. Acknowledgments.-This research was supported by grants from the National Institutes of HealthNational Institutes of General Medical Sciences (NIH-NIGMS) to DJM (5 SO4 GM08156-14) and the Natural Sciences and Engineering Research Council (NSERC) of Canada to RWM (A-3148). B. J. Bangs generously provided the rattlesnakes. K. Ditz and L. A. Lowcock assisted with preparation of the manuscript and M. Rouse prepared the figure.

LITERATURE

CITED

FRIEDEN,C. 1958. The dissociationof glutamic de-

hydrogenaseby reduced diphosphopyridinenucleotide (DPNH).Biochim.Biophys.Acta 27:431-432. . 1959a. Glutamicdehydrogenase. I. The ef-

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fect of coenzymeon the sedimentationvelocityand kinetic behavior.J. Biol. Chem. 234:809-814. . 1959b. Glutamicdehydrogenase.II. The effect of various nucleotides on the association-dissociationand kineticproperties.Ibid.234:815-820. . 1963. Glutamicdehydrogenase.V. The relation of enzyme structureto the catalyticfunction. Ibid. 238:3286-3299. GONZALEZ, C., J. L. RODRIGUEZ-FERNANDEZ,H. CA-

J. A. PEREZ, AND J. M. and othSIVERIO.1988. Fructose-2,6-bisphosphate

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er metabolitesand enzymes in the process of coldinduced lethargy and starvation in lizard liver. Comp. Biochem. Physiol. 89B:131-135. KETTLER, M. K., AND G. S. WHITT. 1986. An apparent progressiveand recurrent evolutionaryrestriction in tissue expression of a gene, the lactate dehydrogenase-Cgene, within a familyof bony fish (Salmoniformes:Umbridae).J. Mol. Evol. 23:95107. , A. W. GHENT, AND G. S. WHITT. 1986. A comparisonof phylogeniesbased on structuraland tissue-expressionaldifferencesof enzymesin a family of teleostfishes(Salmoniformes:Umbridae).Mol. Biol. Evol. 3:485-498. KLEBE,R. J. 1975. A simple method for the quantificationof isozymepatterns. Biochem. Genet. 13: 805-812. McGOVERN, M., AND C. R. TRACY. 1981. Phenotypic variation in electromorphs previously considered Oecoto be genetic markersin Microtusochrogaster. logia 51:276-280. R. W. 1983. Paleobiogeographyand geMURPHY, netic differentiationof the Baja Californiaherpetofauna. Occ. Pap. CaliforniaAcad. Sci. 137:1-48. , ANDC. B. CRABTREE.1985. Evolutionaryaspects of isozyme patterns, number of loci, and tissue-specificgene expression in the prairie rattlesnake,Crotalusviridisviridis.Herpetologica41:451470. ,J. W. SITES, JR., D. G. BUTH,ANDC. H. HAUFLER.1990. Proteins I: isozyme electrophoresis,p. 45-125. In: Molecularsystematics.D. M. Hillis and C. Moritz (eds.). Sinauer Assoc. Inc., Sunderland, Massachusetts. SITES, J. W., JR., AND R. W. MURPHY.1991. Isozyme evidence for independently derived, duplicate G3PDH loci among squamatereptiles. Can.J. Zool. 69:In press. DAVIDJ. MORAFJOSEM. HERNANDEZ-JUVIEL, KA, IMELDA DELGADO,

GARY D. SCOTT,

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partment of Biology, California State University DominguezHills, 1000 E. VictoriaStreet,Carson, California 90747; and ROBERT W. MURPHY, Departmentof Ichthyologyand Herpetology,Royal Ontario Museum, 100 Queen's Park, Toronto, Ontario M5S 2C6 Canada (to whom correspondence and reprint requests should be sent). Accepted 29 Oct. 1991.