KINETIC-PROPERTIES OF HEXOSE-MONOPHOSPHATE DEHYDROGENASES .1. 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 optimun pH was 327.1 mU/ mg of protein. NADPH caused a competitive inhibition with a K-i of 10 mu M. The K-m 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 K-i value for NADPH inhibition was 4 mu M. 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.