Effect of Metal Ions on the Cytosolic Nadp+- Dependent Isocitrate Dehydrogenase from Germinating Blackgram, Vigna Mungo (l.) Hepper

The cytosolic NADP+-dependent isocitrate dehydrogenase from germinating blackgram shows an absolute requirement of a divalent metal ion such as Mn2+ or Mg2+ for expression of its full activity. Other metal ions like Co2+, Zn2+ and Cu2+ could also activate the enzyme, though the degree of activation was lower than that of either Mn2+ or Mg2+ at the same concentration. In contrast, three metal ions Sn2+, Ba2+ and Ca2+ were found to be inhibitory. The kinetics analysis with Mn2+ shows sigmoidal response suggestive of a positive co-operativity in metal ion binding. The A0.5 was 3.3 μM as deduced from a Hill plot with a Hill coefficient value of 2.1. * Present address: Department of Veterinary Biochemistry, College of Veterinary Sciences and A.H., CAU, Selesih, Aizawl 796 014 (MIZORAM). INTRODUCT ION The enzyme NADP+dependent isocitrate dehydrogenase [threo-Ds-isocitrate : NADP+ oxidoreductase (decarboxylating), EC 1.1.1.42] (NADP+-IDH) is found to occur in both prokaryotes and eukaryotes. The enzyme catalyses the oxidative decarboxylation of isoc i t rate to yie ld a-ketog lutarate with concomitant reduction of NADP+ to NADPH and requires metal ion (Mn2+ or Mg2+) for catalysis. The enzyme is more active in the presence of Mn2+ than in presence of Mg2+ which seems to be a common feature for all NADP+-IDHs studied so far, independent of their origin (Curry and Ting, 1976; Kaur et al., 1996). NADP+-IDH is mainly cytosolic enzyme (Henson et al., 1986), with only small proportion of the enzyme normally residing in chloroplast (Randal l and Givan, 1981), mitochondria (Curry and Ting, 1976) and peroxisomes (Randall and Givan, 1981). In higher plants NADP+-IDH has been purified and characterized from some plant tissues such as green pea leaves and roots (Chen et al., 1988), castor bean seeds (Satoh, 1972), maize seeds (Curry and Ting, 1976), pigeonpea root nodules (Kaur et al., 1996) and germinating pea seeds (Srivastava and Singh, 2001). The present paper describes the effect of some divalent metal ions on the cytosolic NADP+IDH from germinat ing seeds of blackgram, Vigna mungo (L.) Hepper cv. yellowish-green seed coat as a prerequisite in mak ing an a t t empt to e luc i da te the physiological and metabolic functions of the metal ions on the enzyme. MATERIAL AND METHODS B la ckg r am seed : The seeds of blackgram, Vigna mungo (L.) Hepper cv. yellowish-green were purchased from local market. Chemicals: DLIsocitric acid, NADP+ (sodium salt), DEAE-cellulose, Sephadex G200, and blue dextran were purchased from Sigma Chemical Company, St. Louis, MO, USA., Tris (hydroxymethyl) aminomethane (T r i s ) was purchased f rom Hi Med ia Laboratories Pvt. Ltd., Bombay; citric acid and 2-mercaptoethanol were from Loba Chemie, Bombay; Ammonium sulphate, polyvinyl pyrrolidone, magnesium chloride, manganese Indian J. Agric. Res., 40 (4) : 267 271, 2006