Skip to main content
SpringerLink
Log in

Structural analysis of glycerol-3-phosphate dehydrogenase from severalDrosophila species

  • Published:
Biochemical Genetics Aims and scope Submit manuscript

Abstract

This report describes preliminary protein structural studies of glycerol-3-phosphate dehydrogenase (α-GPDH) fromDrosophila spp. and an important innovative feature of our enzyme purification protocol. The scheme involves the coupling of substrate (α-glycerophosphate) elution from CM-Sephadex and cofactor (NADH) elution from Affi-Gel blue resin. Using this method a 32.7% yield and a 111-fold purification were obtained from aD. melanogaster line carrying the α-Gpdh S allele at the α-Gpdh locus. The product obtained from 0 to 3-day-old adult flies was electrophoretically homogeneous and consisted mainly of the adult α-GPDH-1 isozyme. The method was used to obtain α-GPDH protein fromD. melanogaster (two lines),D. hydei, D. immigrans, andD. mercatorum. Peptide mapping revealed structural differences among the enzymes from the different species, and amino acid sequencing showed many similarities betweenD. melanogaster α-GPDH and the rabbit muscle enzyme.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

References

  • Bailey, C. J., and Boulter, D. (1969). The subunit structure of jack-bean urease.Biochem. J. 113669.

    PubMed  CAS  Google Scholar 

  • Ballas, R. A., Garavelli, J. S., and White, H. B. (1984). Estimation of the rate of glycerol-3-phosphate dehydrogenase evolution in higher vertebrates.Evolution (in press)

  • Bewley, G. C., and Lucchesi, J. C. (1977). Origin of the α-glycerophosphate dehydrogenase isozymes inDrosophila melanogaster and their functional relationship in the α-glycerophosphate cycle.Biochem. Genet. 15235.

    Article  PubMed  CAS  Google Scholar 

  • Bewley, G. C., Rawls, J. M., and Lucchesi, J. C. (1974). α-Glycerophosphate dehydrogenase inDrosophila melanogaster: Kinetic and developmental differentiation of the larval and adult isozymes.J. Insect Physiol. 20153.

    Article  PubMed  CAS  Google Scholar 

  • Bienz, M., and Deak, I. I. (1978). Microelectrophoresis of α-glycerophosphate dehydrogenase isozymes in various tissues of wild-type and mutantDrosophila.Insect Biochem. 8449.

    Article  CAS  Google Scholar 

  • Chambers, G. K., Laver, W. G., Campbell, S., and Gibson, J. B. (1981). Structural analysis of an electrophoretically cryptic alcohol dehydrogenase mutant from an Australian population ofDrosophila melanogaster.Proc. Natl. Acad. Sci. USA 783103.

    PubMed  CAS  Google Scholar 

  • Collier, G. E. (1977). Inter- and intraspecific variation of α-glycerophosphate dehydrogenase among Drosophila.Isozyme Bull. 1058.

    Google Scholar 

  • Collier, G. E., and MacIntrye, R. J. (1977). Microcomplement fixation studies on the evolution of α-glycerophosphate dehydrogenase within the genusDrosophila.Proc. Natl. Acad. Sci. USA 74684.

    PubMed  CAS  Google Scholar 

  • Collier, G. E., Sullivan, D. T., and MacIntyre, R. J. (1976). Purification of α-glycerophosphate dehydrogenase fromDrosophila melanogaster.Biochim. Biophys. Acta 429316.

    CAS  Google Scholar 

  • Coyne, J. A., Eanes, W. F., Ramshaw, J. A. M., and Koehn, R. K. (1979). Electrophoretic heterogeneity of α-glycerophosphate dehydrogenase among many species ofDrosophila.Syst. Zool. 28164;Genetics 92:353.

    CAS  Google Scholar 

  • Crestfield, A. M., Moore, S., and Stein, W. M. (1963). Preparation and enzymatic hydrolysis of reduced and S-carboxymethylated proteins.J. Biol. Chem. 238622.

    PubMed  CAS  Google Scholar 

  • Fernandez-Sousa, J. M., Gavilanes, J. G., Paredes, J. A., and Perez-Aranda, A. (1979). L-Glycerol-3-phosphate dehydrogenase from the insectCeratitis capitata. Purification, physicochemical and enzymic properties.Insect Biochem. 9177.

    Article  CAS  Google Scholar 

  • Fuerst, P. A., and Ferrell, R. E. (1980). The stepwise mutation model: an experimental evaluation utilizing hemoglobin variants.Genetics 94185.

    CAS  PubMed  Google Scholar 

  • Geer, B. W., McKechnie, S. W., and Langevin, M. L. (1983). Regulation of sn-glycerol-3-phosphate dehydrogenase inDrosophila melanogaster larvae by dietry ethanol and sucrose.J. Nutr. 1131632.

    PubMed  CAS  Google Scholar 

  • Hartley, B. S. (1970). Strategy and tactics in protein sequencing.Biochem. J. 119805.

    PubMed  CAS  Google Scholar 

  • Keith, T. P. (1983). Frequency distribution of esterase-5 alleles in two populations ofDrosophila pseudoobscura.Genetics 105135.

    PubMed  Google Scholar 

  • Kozak, L. P. (1974). Purification and characterization of two allelic forms ofl-glycerol 3-phosphate dehydrogenase from inbred strains of mice.Biochem. Genet. 1269.

    Article  PubMed  CAS  Google Scholar 

  • Laemmeli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4.Nature 227680.

    Article  Google Scholar 

  • Lakovarra, S., Saura, A., and Lankinen, P. (1977). Evolution at the α-GPDH locus inDrosophila.Evolution 31319.

    Google Scholar 

  • Lee, C. Y., Charles, D., and Bronson, D. (1979). Biochemical analyses of natural and induced null variants ofDrosophila enzymes.J. Biol. Chem. 254375.

    Google Scholar 

  • Marquardt, R. R., and Brosemer, R. W. (1966). Insect extramitochondrial glycerophosphate dehydrogenase. I. Crystallization and physical properties of the enzyme from honeybee (Apis mellifera) thoraces.Biochim. Biophys. Acta 128454.

    Google Scholar 

  • McLoughlin, D. J., Terry, D., and MacQuarrie, R. (1980). Affinity chromatography of s-n-glycerol-3-phosphate dehydrogenase on immobilized NAD+.Prep. Biochem. 10549.

    PubMed  CAS  Google Scholar 

  • Miller, S., and Berger, E. (1979). The purification of α-glycerophosphate dehydrogenase formDrosophila melanogaster larvae and partial structural comparison with the form purified from adults.Insect Biochem. 9355.

    Article  CAS  Google Scholar 

  • Narise, S. (1980). Purification and biochemical properties of allelic forms of cytoplasmic glycerol-3-phosphate dehydrogenase fromDrosophila virilis.Biochim. Biophys. Acta 615289.

    PubMed  CAS  Google Scholar 

  • Niesel, D. W., Bewley, G. C., Miller, S. G., Armstrong, F. B., and Lee, C.-Y. (1980). Purification and structural analysis of the soluble sn-glycerol-3-phosphate dehydrogenase isozymes inDrosophila melanogaster.J. Biol. Chem. 2554073.

    PubMed  CAS  Google Scholar 

  • Niesel, D. W., Pan, Y.-C. E., Bewley, G. C., Armstrong, F. B., and Li, S. S.-L. (1982). Structural analysis of adult and larval isozymes of sn-glycerol-3-phosphate dehydrogenase ofDrosophila melanogaster.J. Biol. Chem. 257979.

    PubMed  CAS  Google Scholar 

  • O'Brien, S. J., and MacIntyre, R. J. (1972). The α-glycerophosphate cycle inDrosophila melanogaster. I. Biochemical and developmental aspects.Biochem. Genet. 7141.

    PubMed  Google Scholar 

  • Ostro, M. J., and Fondy, T. P. (1977). Isolation and characterization of the multiple molecular forms of cytosolic NAD+-linked glycerol-3-phosphate dehydrogenase from normal and neoplastic rabbit tissues.J. Biol. Chem. 2525575.

    PubMed  CAS  Google Scholar 

  • Otto, J., Argos, P., and Rossmann, M. G. (1980). Prediction of secondary structural elements in glycerol-3-phosphate dehydrogenase by comparison with other dehydrogenases.Eur. J. Biochem. 109325.

    Article  PubMed  CAS  Google Scholar 

  • Pryor, S. C., and Ferrel, R. E. (1981). Biochemical genetics of theCulex pipiens complex. III. α-Glycerophosphate dehydrogenase.Comp. Biochem. Physiol. B6923.

    Google Scholar 

  • Ramshaw, J. A. M., Coyne, J. A., and Lewontin, R. C. (1979). The sensitivity of gel electrophoresis is a detector of genetic variation.Genetics 931019.

    PubMed  CAS  Google Scholar 

  • Rechstein, M. C. (1970).Drosophila lactate dehydrogenase and α-glycerophosphate dehydrogenase: Distribution and change in activity during development.J. Insect. Physiol. 161179.

    Article  Google Scholar 

  • Retzios, A. D., and Thatcher, D. R. (1979). Chemical basis for the electrophoretic variation observed at the alcohol dehydrogenase locus ofDrosophila melanogaster.Biochimie 61701.

    PubMed  CAS  Google Scholar 

  • Sacktor, B. (1970). Regulation of intermediary metabolism with special reference to control mechanisms in insect flight muscle.Adv. Insect Physiol. 7267.

    Article  CAS  Google Scholar 

  • Sedmak, J. J., and Grossberg, S. E. (1977). A rapid, sensitive, and versitile assay for protein using coomassie brilliant blue G250.Anal. Biochem. 79544.

    Article  PubMed  CAS  Google Scholar 

  • Senkbeil, E., and White, H. B. (1978). Parallel evolution of pairs of dehydrogenase enzymes.J. Mol. Evol. 1157.

    Article  PubMed  CAS  Google Scholar 

  • Shaw, C. R., and Prasad, R. (1970). Starch gel electrophoresis of enzymes—a compilation of recipes.Biochem. Genet. 4297.

    Article  PubMed  CAS  Google Scholar 

  • Thatcher, D. R. (1980). The complete amino acid sequence of three alcohol dehydrogenase alleloenzymes (AdhN-11, AdhS, AdhUF) from the fruitflyDrosophila melanogaster.Biochem. J. 187875.

    PubMed  CAS  Google Scholar 

Download references

Author information

Author notes

  1. A. A. Felton

    Present address: Biogen, Inc., 241 Binney Street, 02142, Cambridge, Massachusetts

  2. J. A. M. Ramshaw

    Present address: CSIRO, Protein Chemistry Division, 343 Royal Parade, 3052, Melbourne, Victoria, Australia

  3. D. Larson Rigby

    Present address: Microchemical Facility, Biological Laboratories, Harvard University, 16 Divinity Avenue, 02138, Cambridge, Massachusetts

Authors and Affiliations

  1. Museum of Comparative Zoology, Harvard University, 26 Oxford Street, 02138, Cambridge, Massachusetts

    G. K. Chambers, A. A. Felton, J. A. M. Ramshaw, D. Larson Rigby & D. T. Sullivan

  2. Department of Biology, Syracuse University, 130 College Place, 13210, Syracuse, New York

    D. T. Sullivan

Authors
  1. G. K. Chambers
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. A. A. Felton
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. J. A. M. Ramshaw
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. D. Larson Rigby
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. D. T. Sullivan
    View author publications

    You can also search for this author in PubMed Google Scholar

Additional information

This project was supported by NIH Grant GM 21179 to R. C. Lewontin and NIH Grant GM 26830 to D. T. Sullivan.

The order of authors is alphabetical.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Chambers, G.K., Felton, A.A., Ramshaw, J.A.M. et al. Structural analysis of glycerol-3-phosphate dehydrogenase from severalDrosophila species. Biochem Genet 23, 801–814 (1985). https://doi.org/10.1007/BF02399410

Download citation

  • Received:

  • Revised:

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF02399410

Key words

Search

Navigation