Analyses of genetic variants of L-glycerol-3-phosphate dehydrogenase in Drosophila melanogaster by two-dimensional gel electrophoresis and immunoelectrophoresis

Abstract

A protein spot corresponding to L-glycerol-3-phosphate dehydrogenase (alpha-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 alpha-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 alpha GPDH protein spot. The alpha-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 alpha-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.