Abstract
A protein spot corresponding to l-glycerol-3-phosphate dehydrogenase (α-GPDH, E.C. 1.1.1.8, NAD+ oxidoreductase) has been identified on a two-dimensional gel (isoelectric focusing-SDS gel) containing up to 150 stained protein spots from a crude Drosophila homogenate. Preliminary identification of the α-GPDH spot was made by including a suitable amount of purified Drosophila α-GPDH in crude fly homogenates prior to electrophoresis and observing an intensity enhancement of the corresponding protein spot on the gels. When three purified electrophoretic variants (slow, fast, and ultrafast) were mixed and analyzed by two-dimensional gel electrophoresis, horizontal displacements of the three protein spots were observed. Immunoprecipitation of the enzyme prior to electrophoresis and gene mapping further confirmed the identity of the α-GPDH protein spot. The α-GPDH spot can also be detected by autoradiography of a two-dimensional gel from a single fly extract, where it has been estimated to constitute 0.5–1% of the total soluble protein. Mutants which express no apparent α-GPDH activity were analyzed by two-dimensional gels and immunoelectrophoresis in an attempt to identify and characterize the inactive proteins. It is suggested that these techniques provide a powerful tool for the analysis of CRM+-null activity mutants of a specific gene-enzyme system.
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References
Anderson, L. and Anderson, N. G. (1977). High resolution two-dimensional electrophoresis of human plasma proteins. Proc. Natl. Acad. Sci. 745421.
Axelson, N. H., Kroll, J., and Weeke, B. (1973). A Manual of Quantitative Immunoelectrophoresis: Methods and Application Universitetsforlaget, Oslo.
Bewley, G. C., and Miller, S. G. (1979). Origin and differentiation of the soluble α-glycerolphosphate dehydrogenase isozymes in Drosophila melanogaster. In Rattazzi, M. C., Scandalios, J. G. and Whitt, G. S. (eds.), Isozymes: Current Topics in Biological and Medical Research. Alan R. Liss, Inc., New York. Vol. 3, p. 23.
Bewley, G. C., Niesel, D. W., and Miller, S. G. (1979). Purification and structural analysis of α-GPDH isozymes in Drosophila melanogaster. Isozyme Bull. 1226.
Bewley, G. C., Dezurik, J. M., and Pagelson, G. (1980). Analysis of l-glycerol-3- phosphate dehydrogenase mutants in Drosophila: Complementation for intracellular degradation of the mutant polypeptide. Mol. Gen. Genet. 178:301.
Charles, D., and Lee, C. -Y. (1979). Analyses of mouse and Drosophila proteins by two-dimensional gel electrophoresis. Fred. Proc. 381402.
Elliot, R. W. (1979). Mouse News Let. 5959.
Lee, C. -Y., Charles, D., and Bronson, D. (1978). Biochemical analyses of null variants of Drosophila enzymes. J. Biol. Chem. 2546375.
Leigh-Brown, A., and Langley, C. H. (1979). A reevaluation of the level of genetic heterozygocity in natural population of Drosophila melanogaster. Proc. Natl. Acad. Sci. 762253.
Lindsley, D. L., and Grell, E. H. (1967) Genetic variation in Drosophila melanogaster, Publ. Carnegie Inst. Wash., No. 627.
Niesel, D., Bewley, G., Miller, S. G., Armstrong, F., and Lee, C. -Y. (1980). Purification and structural analysis of the soluble l-glycerol-3-phosphate dehydrogenase isozymes in Drosophila melanogaster. J. Biol. Chem. 2554073.
O'Brien, S. J., and MacIntyre, R. J. (1972). The α-GPDH in Drosophila melanogaster. II. Genetic aspects. Genetics 71127.
O'Farrell, P. H. (1975). High resolution two-dimensional electrophoresis of proteins. J. Biol. Chem. 2504007.
Pagelson, G. (1979). Isolation and partial characterization of variants affecting the turnover of α-glycerolphosphate dehydrogenase in D. melanogaster. Master's degree thesis, North Carolina State University.
Racine, R., and Langley, C. H., and Voelker, R. A. (1980). Enzyme mutants induced by low-dose-rate γ-irradiation in Drosophila: Frequency and characterization. Envir. Mutagenesis 2167.
Sheir-Neiss, G., Lai, M. G., and Morris, N. R. (1978). Identification of a gene for -tubulin in Aspergillus nidulans. Cell 15639.
Sondermeijer, P. J. A., and Lubsen, N. H. (1978). Heat shock peptides in Drosophila hydei. Eur. J. Biochem. 88331.
Steinberg, R. A., O'Farrell, P. H., Friedrich, U., and Coffino, P. (1977). Mutations causing charge alterations in regulatory subunits of the cAMP-dependent protein kinase of cultured S49 lymphoma cells. Cell 10381.
Storti, R. V., Horovitch, S. H., Scott, M. P., Rich, A., and Pardu, M. L. (1978). Myogenesis in primary cell cultures from Drosophila melanogaster: Protein synthesis and actin heterogeneity during development. Cell 13589.
Voelker, R., Langley, C. H., Leigh-Brown, A. J., Ohnishi, S., Dickson, B., Montgomery, E., and Smith, S. C. (1980). Enzyme null alleles in natural populations of Drosophila melanogaster: Frequencies in a North Carolina populations. Proc. Natl. Acad. Sci. 77(21091.
Whalen, R. G., Butler-Browne, G. S., and Gros, F. (1978). Identification of a novel form of myosin light chain present in Embryonic muscle tissue and cultured master cells. J. Mol. Biol. 126415.
Wilson, D. L., Hall, M. E., Stone, M. E., and Rubin, R. W. (1977) Some improvements in two-dimensional gel electrophoresis of proteins. Anal. Biochem. 8333.
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This investigation was supported inpart by NIH Research Grant No. GM-23617 and AG-01739. Paper No. 6037 of the Journal Series of the North Carolina Agricultural Research Service, Raleigh, North Carolina 27650.
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Lee, CY., Niesel, D. & Bewley, G.C. Analyses of genetic variants of l-glycerol-3-phosphate dehydrogenase in Drosophila melanogaster by two-dimensional gel electrophoresis and immunoelectrophoresis. Biochem Genet 18, 1003–1018 (1980). https://doi.org/10.1007/BF00500131
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DOI: https://doi.org/10.1007/BF00500131