Kinetic properties of hexose-monophosphate dehydrogenases. I. Isolation and partial purification of glucose-6-phosphate dehydrogenase from rat liver and kidney cortex.
Glucose-6-phosphate dehydrogenase (G6PDH) from rat-liver and kidney-cortex cytosol has been partially purified and almost completely separated from 6-phosphogluconate dehydrogenase activity. The purification and isolation procedures included high-speed centrifugation, 40-55% ammonium sulphate fractionation, by which both enzyme activities were separated, and finally, the application of the protein fraction to a column of Sephadex G-25 equilibrated with 10 mM Tris-EDTA-NADP buffer, pH 7.6, to eliminate any contaminating metabolites. The kinetic properties of isolated liver and renal G6PDH were examined. Both enzymes showed a typical Michaelis-Menten kinetic saturation curve with no evidence of co-operativity. The optimum pH of both liver and kidney cortex G6PDH was 9.4. The Km values for glucose-6-phosphate (G6P) and for NADP were 3.29 x 10(-4) M and 1.00 x 10(-4) M respectively. The specific activity measured at 37 degrees C and optimum pH was 327.1 mU/ mg of protein. NADPH caused a competitive inhibition with a Ki of 10 microM. The Km values for the G6P and NADP of kidney-cortex G6PDH were 2.06 x 10(-4) and 0.25 x 10(-4) M respectively. The specific activity at pH 9.4 and 37 degrees C was 76.55 mU/mg of protein. The Ki value for NADPH inhibition was 4 microM. This work describes an easy, rapid and reliable method for the separation of the two dehydrogenases involved in the hexose-monophosphate shunt in animal tissues.
Corpas
F J
FJ
Department of Biochemistry and Molecular Biology, University of Granada, Spain.
García-Salguero
L
L
Peragón
J
J
Lupiáñez
J A
JA
D000818:Animals; D002852:Chromatography, Ion Exchange; D005954:Glucosephosphate Dehydrogenase / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007672:Kidney Cortex / Q000201:enzymology*; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D008297:Male; D009249:NADP / Q000494:pharmacology; D051381:Rats
D009249:NADP; D005954:Glucosephosphate Dehydrogenase