Purification and Properties of NADP-Dependent Sorbitol-6-Phosphate Dehydrogenase from Apple Seedlings

NADP-dependent sorbitol-6-phosphate dehydrogenase (S6PDH) waspurified from apple (Malus domestica) seedlings by a purificationprocedure that included two fractionations by affinity chromatography.The purified enzyme was a homogeneous protein that migratedas a single polypeptide chain with an apparent relative massof 36,000 during SDS-polyacrylamide gel electrophoresis andthe native enzyme was a homodimer of the polypeptide. The maximumvelocity of the reduction of glucose-6-phosphate (G6P) was muchhigher than that of the oxidation of sorbitol-6-phosphate (S6P)and the enzyme had high G6P-reducing activity over the pH rangefrom 7 to 11 even though the oxidation of S6P proceeded veryslowly at neutral pH. These results are consistent with thehypothesis that S6PDH plays a major role in the biosynthesisof sorbitol in vivo. The reduction of G6P to S6P was inhibitedby the addition of nucleotide di- or triphosphates. ATP, thestrongest inhibitor, and ADP inhibited the reduction of G6Pin a competitive manner with respect to NADPH and the K_i valueswere 0.18 mM for ATP and 0.30 mM for ADP. (Received March 24, 1992; Accepted May 25, 1993)