Detection and Characterization of Sorbitol Dehydrogenase from Apple

Sorbitol dehydrogenase (L-iditol:NAD' oxidoreductase, EC 1.1.1.14) has been detected and characterzed from apple (Maims donestica cv. Granny Smith) mesocarp tissue cultures. The enzyme oxidized sorbitol, xylitol, L-arabitol, ribitol, and L-threitol in the presence of NAD. NADP could not replace NAD. Mannitol was slightly oxidized (8% of sorbitol). Other polyols that did not serve as substrate were galactitoL myo-inositoL D-arabitoL erythritol, and glycerol. The dehydrogenase oxidized NADH in the presence of n-fructose or L-sorbose. No detectable activity was observed with D-tagatose. NADPH could partially substitute for NADH. Maximum rate of NAD reduction in the presence of sorbitol occurred in tris(hydroxymethyl)aminomethane-HCI buffer (pH 9), or in 2-amino-2L methyl-1,3-propanediol buffer (pH 9.5). Maximum rates of NADH oxidation in the presence of fructose were observed between pH 5.7 and 7.0 with phosphate buffer. Reaction rates increased with increasing temperature up to 60 C. Ihe K. for sorbitol and xylitol oxidation were 86 millimolar and 37 millimlar, respectively. The K. for fructose reduction was 1.5 molar. Sorbitol oxidation was completely inhibited by heavy metal ions, iodoacetate, p-chloromercuribenzoate, and cysteine. ZnSO4 (0.25 millimolar) reversed the cysteine inhibition. It is suggested that apple sorbitol dehydrogenase contains sulfhydryl groups and requires a metal ion for full activity. Sorbitol (D-glucitol) is an alditol distributed widely in plants (14, 24). It is found mainly in the Rosaceae where it occurs in all genera of the tribes Spiraeoideae, Pomoideae, and Prunoideae (19), and it is common in many fruits (26). Information on sorbitol metabolism in plants is limited even though in apple and related species sorbitol is the major product of photosynthesis (5) and the principal transport material (2, 25). Sorbitol has also been implicated in many other roles in these plants (6). Sorbitol dehydrogenase (L-iditol:NAD' oxidoreductase, EC 1.1.1.14) catalyzes the reversible reaction D-sorbitol + NAD+ ;± D-fructose + NADH + H+. The enzyme was first partially purified by Blakley in 1951 from rat liver (4). Since then the enzyme has been found in the liver of a variety of mammalian species and it has been purified from other animal tissues (12, 21, 23, 27). There are very few reports on the isolation of polyol dehydrogenases from higher plants (8, 16), and only one report has 1 This work was supported in part by funds provided by the Washington State University Research and Arts Committee and by the Washington State Tree Fruit Research Commission. Scientific Paper SP 5241. Project 0322. College of Agriculture Research Center, Washington State University, Pullman. 2 preliminary report of this work was presented at the ASPP meeting in June 1978. provided evidence for the presence of a polyol dehydrogenase (13). Data from labeling studies suggest that sorbitol is synthesized in the leaf by way of fructose-6-P which is reduced to sorbitol-6P and subsequently dephosphorylated (3, 20). Others, however, have reported the conversion of D-glucose to sorbitol in apple ( 11) and plum leaves (1) when [1'CJglucose was used as a substrate. Studies of breakdown of ['4Clsorbitol in fruit and other tissues indicate that the polyol may be converted to a hexose (1), primarily fructose (9). Chong and Taper (6) were able to grow apple callus tissue using sorbitol as a carbon source. We have developed similar cultures grown on sorbitol on the assumption that these should provide a model system with high activity ofan enzyme or enzymes involved in sorbitol metabolism. The following paper is a description of some characteristics of an enzyme preparation from Malus tissue cultures which is capable of reducing NAD in the presence of sorbitol and oxidizing NADH in the presence of fructose. MATERIALS AND METHODS Chemicals. All chemicals were purchased from Sigma Chemical Co. Plant Material. Malus domestica cv. Granny Smith mesocarp tissues were excised from 10-week-old fruits in July, 1976. Intact fruits were washed in water and detergent (Alconox), surfacesterilized for 3 min in 70%/o ethanol followed by 15 min in 10%Yo Clorox containing 3 drops of Tween 20, rinsed three times in sterile H20, and the skin removed. Sections of mesocarp tissue were placed on a medium consisting of Murashige and Skoog inorganics (18) plus thiamine (0.5 mg/i), pyridoxin (0.1 mg/i), niacin (0.1 mg/1), inositol (100 mg/l), sucrose (3%), 2,4-D (2.5 mg/ 1), BA (5.0 mg/l), and Difco Bacto-agar (0.8%). The pH of the medium was adjusted to 5.7 and all components were autoclaved for 15 min at 121 C. The resulting callus was subsequently subcultured monthly on the same medium containing 1 mg/l 2,4D and 1 mg/l BA. One month prior to enzyme extraction, the callus was transferred to a medium containing D-sorbitol (2.5%) instead of sucrose. Enzyme Extraction. It was necessary to prepare extracts of fresh tissue prior to each experiment, since enzyme preparations were found to lose substantial activity with storage at 0 C overnight or with freezing and thawing. Homogenization and all subsequent steps were carried out at 0 to 2 C. In a typical preparation, approximately 60 g callus tissue were ground in a mortar and pestle with 120 ml 0.1 M Tris-HCl buffer (pH 8) containing 1 mnm DTT and 6 g insoluble PVP. The homogenate was transferred to a Sorval Omni-Mixer and homogenized for four 15-s bursts at full speed. The homogenate was squeezed through a polypropylene cloth and the filtrate was centrifuged at 1,100g for 10 min. The 1, 0Og supernatant was centrifuged at 20,000g for 20 min, and the pellet discarded. Solid (NH4)2SO4 was added slowly with constant