Purification and properties of NAD-dependent sorbitol dehydrogenase from apple fruit

This is the first report of the purification of NAD-dependent sorbitol dehydrogenase (NAD-SDH) from a plant source. The enzyme was extracted from apple (Malus domestica cv. Ourin) fruit and purified until it appeared as a single polypeptide chain on a gel after SDS-PAGE. From the apparent molecular mass of 62 kDa obtained by SDS-PAGE and that of 120 kDa by gel filtration, the enzyme appeared to be a homodimer. Maximum rates of oxidation of sorbitol and reduction of fructose were observed at pH 9.6 and pH 6.0, respectively. The K_m for oxidation of sorbitol was 40.3 mM and that for reduction of fructose was 215 mM. The maximum rate of oxidation of sorbitol was about 10 times higher than that of the reduction of fructose. The results of the kinetic analysis strongly suggest that in vivo the enzyme would favor the conversion of sorbitol to fructose over the reverse reaction. None of the divalent cations tested had any effect on the oxidation of sorbitol by NAD-SDH. The reaction catalyzed by NAD-SDH was not specific to sorbitol and other substrates could also be oxidized. Among the tested substrates, ethyl alcohol had a particularly high affinity for the enzyme.