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Reductase (Alditol-6-Phosphate:NADP 1-Oxidoreductase) from Apple Leaves",2 Received for Publication January 22, 1980 and in Revised Form August 21, 1980

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Reductase (Alditol-6-Phosphate:NADP 1-Oxidoreductase) from Apple Leaves Plant Physiol. (1981) 67, 139-142 0032-0889/8 1/67/0139/04/$00.50/0 Characterization and Partial Purification of Aldose-6-phosphate Reductase (Alditol-6-Phosphate:NADP 1-Oxidoreductase) from Apple Leaves",2 Received for publication January 22, 1980 and in revised form August 21, 1980 FAYEK B. NEGM3 AND WAYNE H. LOESCHER Department ofHorticulture and Landscape Architecture, Washington State University, Pullman, Washington 99164 ABSTRACT a sorbitol dehydrogenase (L-iditol:NAD oxidoreductase, EC 1.1.1.14) from apple callus tissues (13). Since then, we have also Aldose--phosphate reductase (alditol6-phosphate:NADP 1-oxidoreduc- detected and purified this enzyme from pear callus tissue, apple tase) was isolated and characterized from mature apple leaves (Malms and pear seeds, apple seedlings, young leaves, and fruits (unpub- domestica cv. Starkrimson). The enzyme was purified 79-fold. The enzyme lished data). In fully expanded apple leaves, however, another catalyzed the following reversible reaction: D-glucose 6-phosphate + sorbitol enzyme was present which oxidized S6P4 and reduced NADPH + H+ = D-sorbitol 6-phosphate + NADP+. No activity was G6P and D-galactose-6-P. A similar enzyme was recently reported detected when NAD+ was substituted for NADP+ or when NADH was in loquat fruit (9). Here, we consider some characteristics of this substituted for NADPH. The enzyme reduced D-galactose 6-phosphate at enzyme, an aldose-6-P reductase (alditol-6-P:NADP I-oxidore- a higher rate than D-glucose 6-phosphate. D-Mannose 6-phosphate and 2- ductase), and its occurrence and possible role in carbon metabo- deoxy-D-glucose 6-phosphate were reduced at low rates. D-Glucose 1- lism in leaves of apple and several other species in the Rosaceae. phosphate, D-fructose 6-phosphate, D-ribose 5-phosphate, D-glucose, and sorbitol did not serve as substrates. The pH optimum for both n-sorbitol 6-phosphate oxidation and D-glucose 6-phosphate reduction was 9.5. The MATERIALS AND METHODS Km values for n-sorbitol 6-phosphate oxidation and D-glucose 6-phosphate Chemicals. All chemicals were purchased from Sigma. D-Sor- reduction were 3.9 and 20 millimolar, respectively. AgNO3 (0.1 millimolar) bitol-6-P barium salt was converted to potassium salt before use and p-chloromercuribenzoate (1.0 millimolar) completely inhibited the (18). Affinity support media (Affi-Gel blue, 100-200 mesh) and enzyme. bovine y-globulin were obtained from Bio-Rad Laboratories. Aldose-6phosphate reductase activity was also detected in mature Partial Purification of Aldose-6P Reductase. Malus domestica leaves from Golden Delicious and Antonovka apples (Malus domestica), cv. Starkrimson fully expanded leaves on new growth were used Conference and Bartlett pears (Pyrus communis), Redhaven peach (Prunus for enzyme extraction. Leaves were washed in distilled H20 and persca), and Perfection apricot (Prunus armeniaca). This suggests that the petioles and midribs were discarded. Homogenization and all enzyme has a wide distribution and plays an important role in sorbitol subsequent steps were carried out in a cold room at 2 C. synthesis. In a typical preparation, 60 g leaves were homogenized in 480 ml 100 mm Tris-HCl buffer (pH 8) containing 1 mm DTT and 24 g insoluble polyvinylpolypyrrolidone. The polyvinylpolypyrroli- done was prewetted with one-half the volume of buffer 1 h prior to homogenization. Homogenization was performed with a Sorval Omni-Mixer for three 10-s bursts at full speed, followed with a Polytron tissue homogenizer for three additional 10-s bursts at full Sorbitol (D-glucitol) is a hexitol, one of several acyclic polyols speed. The homogenate was squeezed through a polypropylene or sugar alcohols found in higher plants (11). Sorbitol is the cloth and the filtrate was centrifuged at 2,000g for 20 min. The primary product of photosynthesis (3, 5, 7), the main translocated 2,000g supernatant was centrifuged at 25,400g for 30 min and the form of carbon (17), and a common constituent of fruits in many pellet was discarded. Solid (NH4)2SO4 was added slowly with species of the Rosaceae (14). The synthetic pathway for sorbitol constant stirring to the 25,400g supernatant of the crude extract to and other polyols in higher plants has not been established. In reach 40%o saturation (24.3 g/100 ml). The suspension was stirred other organisms, however, the synthesis and/or utilization of slowly for 30 min and centrifuged at 25,400g for 30 min, and the polyols is initiated by one of the following reactions (16): (a) pellet was discarded. The resulting supernatant was brought to oxidation to the ketose; (b) oxidation to the aldose; or (c) phos- 60%o saturation with (NH4)2SO4 (13.2 g/100 ml). The suspension phorylation to the corresponding polyol phosphate, which then was stirred for 30 min and centrifuged at 25,400g for 30 min. The can be converted to either the ketose phosphate (12) or the aldose pellet was dissolved in a minimum volume ofthe extracting buffer phosphate (8). Recently, we detected and partially characterized and dialyzed overnight against two changes of 2 liters 10 mM Tris- HCI buffer (pH 8) containing 100 ,lM DTT and 1 mm 2-mercap- ' This work was supported in part by the Washington State Tree Fruit toethanol. This dialysate was centrifuged at 27,000g for 20 mi. Research Commission. An aliquot (16.5 ml) of the dialyzed 40 to 60%1o fraction was 2This is Scientific Paper No. 5702 and Project No. 0322 from the applied to a Sephadex G-200 column (2.5 x 68 cm) equilibrated College of Agriculture, Washington State University, Pullman. 3Present address: Department of Floriculture and Ornamental Horti- 4Abbreviations: G6P, D-glucose 6-phosphate; F6P, D-fructose 6-phos- culture, 20 Plant Science Bldg., Cornell University, Ithaca, NY 14853. phate; S6P, D-sorbitol 6-phosphate. 139 140 NEGM AND LOESCHER Plant Physiol. Vol. 67, 1981 with 20 mi Tris-HCl buffer (pH 8) containing 0.2 mm DTT. The NADP+ or NADH at 100 gM was substituted for NADPH. enzyme was eluted with the column buffer using a flow rate of 7.8 The influence of pH on enzyme activity was studied in Tris- ml/h. Active fractions were pooled and concentrated to 6.1 ml HCI, glycine-NaOH, and 2-amino-2-methyl- 1,3-propanediol/HCl using an Amicon PM- 10 ultrafiltration membrane (Amicon Corp., buffers over the pH range of7.5 to 10.6 (Fig. 1). Maximum activity Lexington, MA). Five ml PM-10 concentrate were applied to an for both G6P reduction and S6P oxidation was at pH 9.5. Tem- Affi-Gel blue column (1 x 5 cm) previously equilibrated with 20 perature optimum for G6P reduction was 35 C and S6P oxidation mm Tris-HCl buffer (pH 8) containing 0.2 mm DTT. The column about 30 to 35 C. No activity was observed at 50 C. was washed with buffer (flow rate, 4.5 ml/h) until no protein was Kinetic constants for S6P oxidation and G6P reduction were detected in the eluate. The column then was washed with 15 ml determined in 100 pm' Tris-HCl (pH 9) at 25 C (Figs. 2 and 3). NAD+ (1 mm in buffer) followed by buffer (15 ml) and 15 ml The Km values for S6P and G6P were 3.9 and 20 mm, respectively. NADPH (100 ,UM in buffer). After an additional buffer wash (15 Both curves appear to indicate standard Michaelis-Menten kinet- ml), the enzyme was eluted with 1 M NaCl in buffer. Tubes ics. containing the highest specific activity were dialyzed overnight The following relative rates were obtained when substrates were against 1 liter 10 mm Tris-HCl (pH 8) containing 100 IM DTT. tested at 28 mm in the presence ofNADPH: G6P, 100; D-galactose- Unless otherwise indicated, this fraction was used throughout for 6-P, 120; D-mannose-6-P, 3; and 2-deoxy-D-glucose-6-P, 7. No all enzyme assays. activity was observed with D-glucose, D-glucose- I-P or D-ribose-5- Enzyme Assays. All experiments reported here were repeated at P. D-Fructose-6-P did not serve as a substrate for the enzyme in least twice. NAD+ and NADP+ were dissolved in glass-redistilled the presence of either NADH or NADPH. Concentrates of the 40 H20, and NADH and NADPH were dissolved in 1% NaHCO3 to 60%o (NH4)2SO4 and PM-10 gel filtration fractions, however, and kept on ice. Aldose-6-P reductase activity was assayed by showed nonlinear initial reaction rates with F6P. Incubating these following either the reduction of NADP+ in the presence of S6P fractions with F6P for 15 min prior to adding NADPH resulted in or the oxidation of NADPH in the presence of G6P at 340 nm a reaction rate that was nearly equivalent to that obtained using using a Beckman DB-GT spectrophotometer equipped with a G6P as a substrate. These results suggest the presence of a hexose- constant temperature cuvette compartment and a Heath recorder. P isomerase in these fractions and that the enzyme is specific for Routine assays for aldose-6-P reductase (Affi-Gel blue fraction) aldose-P. were performed at 25 C in a reaction mixture (1 ml) containing 1 Both forward and reverse reactions were completely inhibited mM NADP', 82.5 mm Tris-HCl (pH 9), 25 ,ul enzyme, and 10 mM by AgNO3 (100 Am) and p-chloromercuribenzoate (1.0 mM). S6P. For the reverse reaction, 0.1 1 mm NADPH, 85 mm Tris-HCl ZnSO4 (1.25 mM) completely inhibited S6P oxidation and caused (pH 9), 25 ,ul enzyme, and 50 mm G6P were used in a total volume 80%o inhibition of G6P reduction.
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