Isoenzymes of glucose-6-phosphate dehydrogenase from tobacco cells

Abstract

Two anodic isoenzymes of glucose-6-phosphate dehydrogenase (G6PDH) were isolated from tobacco suspension culture WR-132, utilizing fractional ammonium sulfate precipitation and DEAE-cellulose chromatography. The pH optimum was 9.0 for isoenzyme G6PDH I and 8.0–8.3 for G6PDH IV. Isoenzyme G6PDH I exhibited Michaelis-Menten kinetics for both substrates, G6P and NADP +, with K m's of 0.22 mM and 0.06 mM, respectively. G6PDH IV exhibited Michaelis-Menten kinetics for G6P with a K m of 0.31 mM. The NADP + double reciprocal plot showed an abrupt transition between two linear sections. This transition corresponds to an abrupt increase in the apparent K m and V max values with increasing NADP +, denoting negative cooperativity. The two K m's for high and low NADP + concentrations were 0.06 mM and 0.015 mM, respectively. MWs of the isoenzymes as determined by SDS disc gel electrophoresis were 85 000–91 000 for G6PDH I and 54 000–59 000 for G6PDH IV. Gel filtration chromatography on Sephadex G-150 showed MW's of 91 000 for G6PDH I and 115 000 for G6PDH IV. A probable dimeric structure for IV is suggested, with two NADP + binding sites.