Starch-gel electrophoretic variants of erythrocyte 6-phosphogluconate dehydrogenase in asian macaques

Five alleles with eight electrophoretic phenotypes of 6-phosphogluconate dehydrogenase were found in 1,195 blood samples from fourteen populations of nine macaque species.Macaca fascicularis from Malaya showed the most polymorphism, with three Pgd alleles resulting in five phenotypes.Macaca mulatta, M. speciosa, M. nemestrina, andM. cyclopis had two alleles each (although the last two species showed a high percentage of homozygosity). The remaining four species (M. fuscata, M. radiata, M. maura, andM. nigra) were homozygous for the Pgd^a allele. The predominance of Pgd^a was observed in all macaque species, exceptM. speciosa which showed a high (57%) frequency of Pgd^d. The distinctive position ofM. speciosa with regard to 6PGD variants parallels observations that indicate that this species carries transferrin and carbonic anhydrase I alleles in different frequencies from those of the other macaque species. Other similarities between the patterns of transferrin and 6PGD variations include a tendency toward homozygosity at the Pgd locus in the insular macaque forms. However, in this case only the Pgd^a allele is involved, while some variation was found in the transferrin alleles fixed by the founder effect in the insular macaques.This research was supported by NSF grants GF 253, GB 7426, and GB 15060 of the U.S.-Japan Cooperative Science and Systematic Biology Programs.     

Expression of the human adenylate kinase isozymes,phosphopyruvate hydratase, 6-phosphogluconate dehydrogenase,and phosphoglucomutase-1 in man-rodent somatic cell hybrids

The expression of the adenylate kinase isozymes and of phosphopyruvate hydratase was studied in man-mouse and man-hamster hybrid clones. Concordant segregation of the loci coding for AK-2 and PPH was observed in 54 of 55 primary hybrid clones, and these loci were demonstrated to be synthetic with the loci specifying PGM-1 and PGD. The pattern of expression of the four enzymes in discordant clones suggests the gene order 1pter-(PGD,PPH)-AK-2-PGM-1-centromere. In addition, AK-1 was found to be expressed independently of AK-2.This work was supported by the NIH Grants 5 PO1 HB 06276-04, HD 04807-06, and HD 06285-04.     

Regulation of glucose 6-phosphate dehydrogenase expression in CHO-human fibroblast somatic cell hybrids

Human--hamster somatic cell hybrids have been obtained by fusion of a CHO line (NA31) doubly deficient in hypoxanthine guanine phosphoribosyltransferase and glucose 6-phosphate dehydrogenase (G6PD) with normal G6PD(+) human fibroblasts. Analysis of NA31 extracts has revealed that, although G6PD activity is nearly absent, significant activity can be detected with 2-deoxyglucose 6-phosphate as substrate, so that the mutant and normal forms of the enzyme can both be easily detected. The cell hybrids obtained express human G6PD. The human G6PD subunits are distributed in homodimeric molecules as well as in human--hamster heterodimeric molecules. However, whereas the amount of hamster G6PD subunits present in the hybrid is similar to that in the hamster parental cells, the amount of human G6PD subunits is decreased by 3- to 10-fold when compared to the human parental cell. These results indicate that either the expression of the G6PD gene or the stability of the gene product is altered in the hybrid. By mutagenesis and selection in diamide (a substance that oxidizes intracellular glutathione), we have isolated a clone with a 3- to 5-fold increase in human G6PD activity. This derivative may have an increased rate of expression of the human G6PD structural gene.     

Effectors of glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase of rat liver.

Summary The lower Vmax of 6PGDH with respect to G6PDH and its higher sensitivity to inhibition by NADPH, suggest the existence of an imbalance between the two dehydrogenases of the pentose phosphate pathway in rat liver. Possible modulators of these activities, particularly in relation with the inhibition by NADPH in physiological conditions, have been investigated. The results suggest that in both cases the inhibition by NADPH is strictly isosteric and that the relative affinities for the reduced and oxidized forms of the pyridine nucleotide are unaffected by glutathion, the intermediates of the pentose phosphate shunt or some divalent ions.Abbreviations G6PDH glucose-6-phosphate dehydrogenase (EC 1.1.1.49) - 6PGDH 6-phosphogluconate dehydrogenase (EC 1.1.1.44) On leave from the Instituto de Bioquímica, Facultad de Ciencias, Universidad Austral de Chile, Casilla 567, Valdivia, Chile.     

Primate red cell enzymes: glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase

Glucose-6-phosphate dehydrogenase (E. C.: 1.1.1.49) phenotypes and 6-phosphogluconate dehydrogenase (E. C.: 1.1.1.44) phenotypes were determined by starch-gel electrophoresis of red cell hemolysates of Galago crassicaudatus subspp., Propithecus verreauxi, Lemur spp., Hapalemur griseus, and Macaca mulatta. A single glucose-6-phosphate dehydrogenase (G6PD) phenotype was found in each species. A single 6-phosphogluconate dehydrogenase (6PGD) phenotype was found in Lemur spp., Hapalemur griseus, and Galago crassicaudatus argentatus. In a group of six Propithecus verreauxi, three 6PGD phenotypes, PGD A, PGD AB, and PGD B, were found. Three phenotypes, PGD A, PGD AB, and PGD B, were found in 38 G. c. crassicaudatus. The three phenotypes in each species are apparently the products of two codominant autosomal alleles, PGD^A and PGD^B. The frequency of PGD^A in G. c. crassicaudatus is 0.263. A population of 260 free-ranging macaques displays a polymorphism at the 6PGD locus. Three phenotypes, PGD A, PGD AB, and PGD B, were found. These also appear to be controlled by two codominant autosomal alleles, PGD^A and PGD^B the frequency of PGD^A = 0.913. Additional analysis of three well-defined troops within the macaque population indicated that there are no significant differences between the troops or within the population at the 6PGD locus.     

Glucose 6-phosphate dehydrogenase in Drosophila: A sex-influenced electrophoretic variant

A variant of glucose 6-phosphate dehydrogenase (G6PD) in Drosophila melanogaster shows different electrophoretic migration in males and females. In heterozygotes, the variant influences the migration of G6PD produced by both chromosomes. Mixing of homogenates of males and females changes migration of the female-produced enzyme, suggesting that a protein produced in males is capable of altering the variant G6PD molecule. The hypothetical protein is also present in pseudomales and intersexes produced by sex transformation genes.This research was supported by NIH grants # 5-T1-GM 216-06 and GM 12768-01 and NSF grants GB 4587 and GB 4824.     

Quantitation of messenger RNA levels for rat liver 6-phosphogluconate dehydrogenase

Liver poly(A)-containing RNA isolated from rats in different dietary states was translated in a cell free protein synthesizing system employing reticulocyte lysates. Immunoprecipitation of the cell-free reaction products with goat anti-6-phosphogluconate dehydrogenase followed by sodium dodecyl sulfate-urea-gel electrophoresis showed that the induction of this lipogenic enzyme was accompanied by a corresponding increase in the concentration of its specific translatable mRNA.     

Human mitochondrial NADP-dependent isocitrate dehydrogenase in man-mouse somatic cell hybrids.

Human mitochondrial NADP-dependent isocitrate dehydrogenase (IDH-2) is expressed in man-mouse somatic cell hybrids as a dimeric molecule. The gene specifing this enzyme was observed to be syntenic with the mannose phosphate isomerase locus in the 56 primary man-mouse clones in this series. The human IDH-2 locus, therefore, may be assigned to chromosome 15.     

Effects of insulin on the adaptation of glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase in rat adipose tissue

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1. The effects of insulin on adipose tissue glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase were studied.     

The Thai variant and the distribution of alleles of 6-phosphogluconate dehydrogenase and the distribution of glucose 6-phosphate dehydrogenase deficiency in Thailand

The samples were taken from 3185 subjects from ten provinces throughout Thailand. In 1577 males the frequency of glucose 6-phosphate dehydrogenase deficiency was 11.98%. In the far south the gene frequency was 2.83%; in the remainder of the country the frequency did not vary significantly about a mean of 13.76%. The deficiency is of a severe type. The G6PD of all of the nondeficient individuals had the electrophoretic mobility of type B. The mean frequency of the A/B electrophoretic phenotype of 6-phosphogluconate dehydrogenase is 8.47%. The maximum frequency was in central and southern Thailand with a decline to the north and northeast. A variant form of 6-PGD, referred to as the Thai variant, has been found in which two additional electrophoretic components migrate anodally to the normal A band, confirming that the molecule is at least a dimer. The hypothesis is advanced that erythrocyte 6-PGD is determined by two genetic loci, only one of which is translated in leukocytes.Supported by U.S. Army Contract DA-49-193 MD 2879, U.S.P.H.S. GM 09252, and U.S.P.H.S. 5-K3-GM-15325.     

Importance in catalysis of the 6-phosphate-binding site of 6-phosphogluconate in sheep liver 6-phosphogluconate dehydrogenase

The 6-phosphate of 6-phosphogluconate (6PG) is proposed to anchor the sugar phosphate in the active site and aid in orientating the substrate for catalysis. In order to test this hypothesis, alanine mutagenesis was used to probe the contribution of residues in the vicinity of the 6-phosphate to binding of 6PG and catalysis. The crystal structure of sheep liver 6-phosphogluconate dehydrogenase shows that Tyr-191, Lys-260, Thr-262, Arg-287, and Arg-446 contribute a mixture of ionic and hydrogen bonding interactions to the 6-phosphate, and these interactions are likely to provide the majority of the binding energy for 6PG. All mutant enzymes, with the exception of T262A, exhibit an increase in K(6PG) that ranges from 5- to 800-fold. There is also a less pronounced increase in K(NADP), ranging from 3- to 15-fold, with the exception of T262A. The R287A and R446A mutant enzymes exhibit a dramatic decrease in V/E(t) (600- and 300-fold, respectively) as well as in V/K(6PG)E(t) (10(5) - and 10(4)-fold), and therefore no further characterization was carried out with these two mutant enzymes. No change in V/E(t) was observed for the Y191A mutant enzyme, whereas 20- and 3-fold decreases were obtained for the K260A and T262A mutant enzymes, respectively, resulting in a decrease in V/K(6PG)E(t) range from 3- to 120-fold. All mutant enzymes also exhibit at least an order of magnitude increase in 13C-isotope effect -1, indicating that the decarboxylation step has become more rate-limiting. Data are consistent with significant roles for Tyr-191, Lys-260, Thr-262, Arg-287, and Arg-446 in providing the binding energy for 6PG. In addition, these residues also likely ensure proper orientation of 6PG for catalysis and aid in inducing the conformation change that precedes, and sets up the active site for, catalysis.     

Biochemical and genetic characterization of the lowell variant. A new phenotype of 6-phosphogluconate dehydrogenase

Summary A new electrophoretic variant of 6-phosphogluconate dehydrogenase (6PGD) has been detected in the Caucasian population in North Carolina, and family studies have shown this variant to be transmissible. This variant has been given the trivial name Lowell and the allele controlling its synthesis in conjunction with 6PGD ^A has been named 6PGD ^L . The banding pattern produced by the Lowell variant after electrophoresis is not affected by either NADP or 2-mercaptoethanol. Inhibition studies with urea and iodoacetate have shown that this variant is inhibited to approximately the same degree as the Richmond and Friendship variants. Thermostability studies have shown that the isozyme bands encoded by 6PGD ^A , 6PGD ^Elcho , 6PGD ^C , and 6PGD ^L have a relative thermal stability in the order 6PGD ^A >6PGD ^Elcho >6PGD ^C >6PGD ^L .