Purification and biochemical characterization of glucose 6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase and glutathione reductase from rat lung and inhibition effects of some antibiotics

Two species of trypanosomatids, Crithidia desouzai and Herpetomonas anglusteri, were recently isolated from Diptera in Minas Gerais, Brazil. The Crithidia species was found to harbor bacterium-like endosymbionts in the cytoplasm. To biochemically characterize these two species of trypanosomatids, and to try to verify the evolutionary meaning of the presence of endosymbionts, an electrophoretic study was undertaken whereby the two species were compared with eight other species in the same family. Horizontal 12.5% starch gel electrophoresis was used to resolve the isozymes of eight enzyme systems: acid phosphatase, glucose-6-phosphate dehydrogenase, hexokinase, malate dehydrogenase, malic enzyme, 6-phosphogluconate dehydrogenase, phosphoglucose isomerase, and phosphoglucomutase. Ten other enzyme systems were assayed without yielding any reproducible activity. The isozymes observed were conservatively interpreted as being due to the activity of 44 different alleles. All species studied differed in at least one enzyme system. The phenetic (Jaccard similarity index, UPGMA grouping) analysis produced a tree in which the species of Crithidia and Herpetomonas clustered separately, forming monophyletic groupings. All the endosymbiont-bearing species formed a monophyletic cluster, indicating that the presence of bacterium-like endosymbionts may be a synapomorphy of that group, and may represent, therefore, a unique event in the evolution of the genus.

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Purification of rat kidney glucose 6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase, and glutathione reductase enzymes using 2′,5′-ADP Sepharose 4B affinity in a single chromatography step

Protein Expression and Purification ◽

10.1016/j.pep.2011.08.031 ◽

2012 ◽

Vol 81 (1) ◽

pp. 1-4 ◽

Cited By ~ 21

Author(s):

Sevki Adem ◽

Mehmet Ciftci

Keyword(s):

Glutathione Reductase ◽

Rat Kidney ◽

Phosphogluconate Dehydrogenase ◽

Glucose 6 Phosphate Dehydrogenase ◽

Sepharose 4B ◽

Chromatography Step

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Expression and biochemical characterization of human glucose-6- phosphate dehydrogenase in Escherichia coli: a system to analyze normal and mutant enzymes

Blood ◽

10.1182/blood.v83.5.1436.bloodjournal8351436 ◽

1994 ◽

Vol 83 (5) ◽

pp. 1436-1441 ◽

Cited By ~ 1

Author(s):

TK Tang ◽

CH Yeh ◽

CS Huang ◽

MJ Huang

Keyword(s):

Escherichia Coli ◽

Biochemical Characterization ◽

Fold Increase ◽

Binding Domain ◽

Occurrence Rate ◽

Substrate Analogues ◽

Normal Enzyme ◽

Glucose 6 Phosphate Dehydrogenase

We have developed a system to characterize normal and mutated glucose-6- phosphate dehydrogenase (G6PD) enzymes in vitro. Normal or mutant G6PD cDNA was subcloned into a pGEX-3X vector, which allowed production of a functional fusion protein in Escherichia coli. When we compared the recombinant normal enzyme with authentic human G6PD, indistinguishable Km values for glucose-6-phosphate (G6P) and NADP were obtained, and the utilization rates for two substrate analogues (2-deoxy G6P and deamino NADP) also showed no difference between the enzymes. This system was used to assay a biochemically uncharacterized variant, G6PD Taipei (493 A-->wG; 165 Asn-->Asp), plus two other known mutations (487 G-->A; 163 Gly-->Ser and 592 C-->T; 198 Arg-->Cys) that are located close to or within the putative G6P binding domain. Our results show that the G6PD activities of these three mutants were greatly reduced. No significant alteration in G6PD kinetics was observed for both 487 and 493 mutations. However, a drastic reduction in the Km for G6P (4-fold decrease) and tremendous increases in utilization rates of 2-deoxy G6P (32-fold increase) and deamino NADP (6-fold increase) were associated with the 592 mutation. This results suggests that arginine 198 in human G6PD, possibly located within the putative G6P binding domain, may play an important role in binding the substrate G6P. In addition, we and others have recently identified that at least nine different types of mutations are responsible for G6PD deficiency in Chinese. In this report, we also present the occurrence rate of each mutation present in the population of Taiwan.

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