Purification and characterization of dihydroxyacetone phosphate reductase from immature seeds of Brassica campestris L.

Dihydroxyacetone phosphate reductase (DHAP reductase) was purified to appar ent electrophoretic homogeneity with about 24% recovery from immature seeds of Brassica campestris using (NH4)(2)SO4 fractionation, affinity chromatog raphy, gel filtration and adsorption chromatography. The purified enzyme wi th molecular mass of about 62 kDa was a dimer with subunit molecular mass o f 32 kDa. The enzyme exhibited maximum activity at pH 7.5 and was highly sp ecific for NADH and DHAP. Typical Michaelis-Menten kinetics was obtained fo r both the substrates with K-m values of 3.3 and 26.6 muM for NADH and DHAP , respectively. The enzyme did not require any metal ion for its activity. Rather, the activity was inhibited by Na+, K+, Mn2+ Mg2+ and Ca2+. ATP and fructose-1,6-P-2 inhibited the enzyme non-competitively with respect to DHA P with Ki values of 0.96 and 1.3 mM, respectively. Substrate interaction ki netics and product inhibition studies were consistent with compulsory-order ed bi-bi reaction mechanism with NADH being the first substrate to bind and NAD being the last product Co dissociate. Based on the properties discusse d here, it appears that the enzyme probably functions for the production of glycerol-3-P from DHAP. (C) 2001 Elsevier Science Ireland Ltd. Ail rights reserved.