Purification and properties of NADP-dependent sorbitol-6-phosphate dehydrogenase from apple seedlings

NADP-dependent sorbitol-6-phosphate dehydrogenase (S6PDH) was purified from apple (Malus domestica) seedlings by a purification procedure that included two fractionations by affinity chromatography. The purified enzyme was a homogeneous protein that migrated as a single polypeptide chain with an apparent relative mass of 36,000 during SDS-polyacrylamide gel electrophoresis and the native enzyme was a homodimer of the polypeptide. The maximum velocity of the reduction of glucose-6-phosphate (G6P) was much higher than that of the oxidation of sorbitol-6-phosphate (S6P) and the enzyme had high G6P-reducing activity over the pH range from 7 to 11 even though the oxidation of S6P proceeded very slowly at neutral pH. These results are consistent with the hypothesis that S6PDH plays a major role in the biosynthesis of sorbitol in vivo. The reduction of G6P to S6P was inhibited by the addition of nucleotide di- or triphosphates. ATP, the strongest inhibitor, and ADP inhibited the reduction of G6P in a competitive manner with respect to NADPH and the K_i values were 0.18 mM for ATP and 0.30 mM for ADP.