Glenn C. Bewley

Origin of ?-glycerophosphate dehydrogenase isozymes in Drosophila melanogaster and their functional relationship in the ?-glycerophosphate cycle

The basis for the differentiation of l-glycerol-3-phosphate dehydrogenase (α-GPDH) into larval and adult isozymes in Drosophila melanogaster was investigated by the correlation of a lack of appearance of each isozyme during development within Drosophila bearing α-GPDH “null” alleles and by the study of a putative conversion factor. Conversion studies indicate the presence of a heat-labile RNase-resistant conversion factor present in crude larval extracts with the ability to convert GPDH-1 to GPDH-2 and GPDH-3 but not vice versa. In addition, “null” mutations at the Gpdh locus obliterate all isozymatic species of α-GPDH in all developmental stages. These observations suggest that all α-GPDH isozymes are the product of a single structural gene and that the multiple forms of this enzyme arise during successive developmental stages through an epigenetic modification of the primary Gpdh+ polypeptide. Finally, observations are reported which bear on the functional divergence of the α-glycerophosphate cycle in the adult and larval stage of development.

Characterization of catalase purified from Drosophila melanogaster by hydrophobic interaction chromatography

Abstract 1. 1. A rapid procedure for the purification of Drosophila melanogaster catalase (H 2 O 2 oxidoreductase EC 1.11.1.6) to apparent homogeneity has been developed and monospecific antibodies have been raised against the purified antigen. 2. 2. Biochemical properties of the purified enzyme with emphasis on the mechanism of inactivation by the irreversible inhibitor 3-amino-1,2,4-triazole, are reported and discussed with reference to the characteristics of catalase from other sources. 3. 3. Rocket immunoelectrophoretic techniques have been developed for the quantitative determination of catalase-specific cross-reacting-material (CRM) in strains of Drosophila which differ in their steady state levels of catalase activity.

Heat stability studies at the α-glycerophosphate dehydrogenase locus in populations of Drosophila melanogaster

The level of hidden variation in populations of Drosophila melanogaster at the Gpdh+ locus was determined by thermal stability studies of the protein. The results indicate a lack of variation using these methods both in and between the two common electrophoretic variants. It is suggested that α-GPDH is conserved in primary structure, which may be related to its critical role in flight muscle metabolism.

Naturally occurring genetic variation affecting the expression of sn-glycerol-3-phosphate dehydrogenase in Drosophila melanogaster

Genetic variation among second and third chromosomes from natural populations of Drosophila melanogaster affects the activity level of sn-glycerol-3-phosphate dehydrogenase (EC 1.1.1.8; GPDH) at both the larval and the adult stages. The genetic effects, represented by differences among chromosome substitution lines with coisogenic backgrounds, are very repeatable over time and are generally substantially larger than environmental and measurement error effects. Neither the GPDH allozyme, the geographic origin, nor the karyotype of the chromosome contributes significantly to GPDH activity variation. The strong relationship between GPDH activity level and GPDH-specific CRM level, as well as our failure to find any thermostability variation among the lines, indicates that most, if not all, of the activity variation is due to variation in the steady-state quantity of enzyme rather than in its catalytic properties. The lack of a strong relationship between adult and larval activity levels suggests the importance of stage- or isozyme-specific effects.

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

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.

Purification and characterization of the naturally occurring allelic variants of sn-glycerol-3-phosphate dehydrogenase in Drosophila Melanogaster

The naturally occurring electrophoretic variants of sn-glycerol-3-phosphate dehydrogenase and a heterodimeric form of the enzyme resulting from a genetic cross of two variant strains of Drosophila were purified to homogeneity by a combination of DEAE-cellulose chromatography and 8-(6-aminohexyl)-amino-ATP-Sepharose affinity chromatography. Each purified protein was compared with respect to a number of physicochemical and kinetic properties. All forms of the enzyme were found to be similar, except for pI differences associated with the electrophoretic variation observed.

[51] sn-Glycerol-3-phosphate dehydrogenase (soluble) from Drosophila melanogaster

Publisher Summary Soluble glycerol-3-phosphate dehydrogenase in Drosophila melanogaster represents a family of three distinct isozymes that are designated as glyceraldehyde 3-phosphate dehydrogenase (GPDH)-1, -2, and -3 in order of their decreasing mobility toward the anode during starch gel electrophoresis. The multiple forms of glycerol-3-phosphate dehydrogenase in Drosophila are the product of the same structural gene mapped to the left arm of chromosome II and arise by an epigenetic mechanism. This chapter describes the assay method and properties of sn -glycerol-3-phosphate dehydrogenase isolated from Drosophila melanogaster . Glyccrol-3-phosphatc dehydrogenase activity is determined spectrophotomctrically at 340 nm using glycerol 3-phosphate or dihydroxyacetone phosphate as a substrate and measuring the rate of nicotinamide adenine dinucleotide kinase (NAD + ) reduction or nicotinamide adenine dinucleotide dehydrogenase (NADH) oxidation. The assay utilizing glycerol 3-phosphate is convenient and is used routinely. The steps involved in the purification procedure of GPDH-1 and GPDH-3 are (1) ammonium sulfate fractionation, (2) diethylaminoethyl (DEAE)-column chromatography, and (3) adenosine tripjosphate (ATP)-Sepharose 4B chromatography.

Analysis of l-glycerol-3-phosphate dehydrogenase mutants in Drosophila melanogaster: complementation for intracellular degradation of the mutant polypeptide.

SummaryNull and low activity alleles at the genetic locus coding for L-Glycerol-3-phosphate dehydrogenase (α-GPDH, NAD+ oxidoreductase, E.C. 1.1.1.8) in Drosophila melanogaster have been analyzed by a combination of rocket immunoelectrophoresis, interallelic complementation, and two-dimensional gel electrophoresis. In addition to providing information on the molecular weight, charged state, and steady state level of CRM in each of these mutants, it is suggested that each mutation has resulted in a genetic lesion within the structural element, Gpdh+. CRM levels appear to be the result of a differential sensitivity to the normal intracellular degradative process and the CRM- mutants represent “hypersensitive” alleles, such that the mutant polypeptide does not accumulate in the intracellular environment.

Gene Duplication and Concerted Evolution of the GPDH Locus in Natural Populations of Drosophila melanogaster

The sn-glycerol-3-phosphate dehydrogenase (GPDH, EC 1, 1, 1, 8) locus of Drosophila melanogaster is polymorphic with respect to the number of tandemly duplicated genes in natural populations. The duplicated genes were cloned and the nucleotide sequences were determined. The duplication deletes both the first and second exons and has a size of 4500 b. p. The fact that there is no sequence variation at the junction point of the duplicated units among strains suggests a single origin for the duplication event. Comparison of the nucleotide sequences among the duplicates indicates that the frequent transfer of genetic information occurs from one to the other of the duplicates on the same chromosome either by gene conversion or by unequal crossing over. Because the GPDH duplication is partial and therefore a kind of pseudogene, the observed polymorphism of the number of tandemly duplicated GPDH genes appears to have been driven mainly by random genetic drift.