Isolation and characterization of an alkali metal ion-sensitive NAD +-dependent glyceraldehyde 3-phosphate dehydrogenase from germinating green gram ( Phaseolus aurieus)

Abstract

An alkali metal ion-sensitive NAD +-specific glyceraldehyde 3-phosphate dehydrogenase has been purified 250-fold from germinating green gram ( Phaseolus aurieus). The purified enzyme shows a single protein band on gel electrophoresis. It has been shown to be a tetrameric protein (molecular weight 160,000) made up of apparently identical monomers (subunit molecular weight 40,000). It shows an A 280 A 260 ratio equal to 1.38, which is not changed on treatment with animal charcoal or cellulosic ion exchangers. Direct estimation shows less than 0.07 mol bound NAD +/mol enzyme. Green gram glyceraldehyde 3-phosphate dehydrogenase is inhibited fairly strongly at physiological concentrations of Na + ions. The inhibition is stronger at higher pH and lower protein concentration. Deproteinated extract, cysteine, and reduced glutathione reverse the Na + ion inhibition. The effect of deproteinated extract is attributable to the presence of some SH-containing compounds. Potassium and rubidium ions have a mild activating effect at lower concentration (below 100 m m) and are inhibitory at higher, nonphysiological, concentrations. Ammonium and lithium ions have no effect. The inhibition due to Na + ions is noncompetitive with respect to NAD + and phosphate ions but competitive with respect to glyceraldehyde 3-phosphate, with K i about 60 m m. Sodium ions protect the enzyme against proteolysis with trypsin. It is suggested that Na + ions and the small molecular weight SH-compounds may possibly be involved in regulation of the overall rate of glycolysis via modulation of glyceraldehyde 3-phosphate dehydrogenase activity.