Erythrocyte glucose-6-phosphate dehydrogenase from Brazilian opossum Didelphis marsupialis.
In a comparative study of erythrocyte metabolism of vertebrates, the specific activity of glucose-6-phosphate dehydrogenase (G6PD) of the Brazilian opossum Didelphis marsupialis in a hemolysate was shown to be high, 207 +/- 38 IU g-1 Hb-1 min-1 at 37 degrees C, compared to the human erythrocyte activity of 12 +/- 2 IU g-1 Hb-1 min-1 at 37 degrees C. The apparent high specific activity of the mixture led us to investigate the physicochemical properties of the opossum enzyme. We report that reduced glutathione (GSH) in the erythrocytes was only 50% higher than in human erythrocytes, a value lower than expected from the high G6PD activity since GSH is maintained in a reduced state by G6PD activity. The molecular mass, determined by G-200 Sephadex column chromatography at pH 8.0, was 265 kDa, which is essentially the same as that of human G6PD (260 kDa). The Michaelis-Menten constants (Km: 55 microM) for glucose-6-phosphate and nicotinamide adenine dinucleotide phosphate (Km: 3.3 microM) were similar to those of the human enzyme (Km: 50-70 and Km: 2.9-4.4, respectively). A 450-fold purification of the opossum enzyme was achieved and the specific activity of the purified enzyme, 90 IU/mg protein, was actually lower than the 150 IU/mg protein observed for human G6PD. We conclude that G6PD after purification from the hemolysate of D. marsupialis does not have a high specific activity. Thus, it is quite probable that the red cell hyperactivity reported may be explained by increased synthesis of G6PD molecules per unit of hemoglobin or to reduced inactivation in the RBC hemolysate.
Barretto
O C de O
OC
LIM-23, Faculdade de Medicina, Universidade de São Paulo, São Paulo, SP, Brasil. ocdobarr@usp.br
Oshiro
M
M
Oliveira
R A G
RA
Fedullo
J D L
JD
Nonoyama
K
K
D000818:Animals; D001938:Brazil; D002845:Chromatography; D048293:Didelphis / Q000097:blood*; D004912:Erythrocytes / Q000737:chemistry / Q000201:enzymology*; D005954:Glucosephosphate Dehydrogenase / Q000097:blood* / Q000302:isolation & purification; D005978:Glutathione / Q000378:metabolism*; D010084:Oxidation-Reduction
D005954:Glucosephosphate Dehydrogenase; D005978:Glutathione