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

Home , Reductase, Sorbitol dehydrogenase

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 polyvinylpolypyrrolidone 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. At 10 mm, iodoacetate, of 1 ml. In every assay, the reference cuvette contained all reagents Na2EDTA, and cysteine inhibited S6P oxidation by 42, 35 and except the substrate. Reactions were initiated by the addition of 47%, respectively. G6P reduction was inhibited 19, 11, and 11%, substrate 2.5 min after incubating the enzyme with NADP+ or respectively. NADPH. When the enzyme was incubated with substrate and the Table II shows the effect of leaf age on the levels of both reaction was initiated with coenzyme, lower reaction rates were sorbitol dehydrogenase and aldose-6-P reductase. Sorbitol dehy- observed. Sorbitol dehydrogenase activity was assayed as previ- drogenase activity was detected only in leaf primordia, whereas ously described (13). aldose-6-P reductase activity was detected in fully expanded A unit of aldose-6-P reductase is defined as the amount of leaves. Sorbitol dehydrogenase activity was also inversely related enzyme catalyzing the oxidation of 1 ytmol NADPH/min at 25 C to Chl content. under standard assay conditions. Specific activity is expressed as Fully expanded mature leaves from several other species and units per mg protein. Protein was routinely estimated by the cultivars were utilized to determine the extent ofthe occurrence of method of Bradford (6) using bovine y-globulin as a standard. Chl aldose-6-P reductase. Table III lists the species and cultivars in was determined using the method of Arnon (2). the Rosaceae in which appreciable enzyme activity was detected. In none of these, however, was an attempt made to determine the effects of leaf age on enzyme activity. RESULTS The purification procedure for aldose-6-P reductase from fully DISCUSSION expanded apple leaves is given in Table I. The enzyme was purified 79-fold over-all. Neither NAD+ or NADP+ at 1 mm Here, we report on an enzyme isolated from fully expanded eluted the enzyme from the affinity column, and only slight apple leaves which catalyzed the following reaction: D-glucose-6- activity was eluted with 100 lM NADPH. The partially purified P + NADPH + H+ - D-sorbitol-6-P + NADP+. This enzyme enzyme was stable for 1 week at 1 C. reduced D-galactose-6-P but not F6P. The data on substrate Study of the cofactor requirements for the enzyme indicated no specificity also indicate a requirement for a free aldehyde group detectable activity when either NAD+ at 1 mm was substituted for at C1 since there is no reaction with D-glucose-l-P. In addition,

Table I. Summary ofPur!rication ofAldose-6-P Reductasefrom Apple Leaves The enzyme activity assay system used during purification contained 85 mm Tris-HCl (pH 9), 25 or 50 ,ul of each fraction, 0.1 mM NADPH, and 50 mM glucose-6-P in a final volume of I ml. The reaction was initiated by the addition of substrate. Specific Yield Step Volume Protein Activity Activity Purifica-tion

ml mg units unitslmg -fold % Crude extract 418 899.3 7.19 8 1 100 (NH4)2SO4 (40-60%) 19 415.6 6.65 16 2 92 Sephadex G-200 6.1 41.9 3.23 77 10 45 Affi-Gel blue 2.8 2.8 1.76 628 79 24 Plant Physiol. Vol. 67, 1981 APPLE LEAF ALDOSE-6-PHOSPHATE REDUCTASE 141

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, -t 4 XL 0 0 z z .7 75 E pH E [G-6-P]f mM FIG. 3. Lineweaver-Burk plot of aldose-6-P reductase activity as a FIG. 1. Response of aldose-6-P reductase to pH of assay mixture using function of D-glucose-6-P concentration. V = ,umol NADPH oxidized/mg D-sorbitol-6-P (closed symbols, - - -) or D-glucose-6-P (open symbols, protein min. Reaction mixtures were as described except that the D- -) as substrate. Reaction mixtures were as described. The following glucose-6-P concentration was varied as indicated. buffers were used at a concentration of 100 mM: (0, 0), Tris-HCl; (El, *), 2-amino-2-methyl-1,3-propanediol; and Table II. Distribution of Sorbitol Dehydrogenase and Aldose-6-P (A, A), glycine-NaOH. Reductase in Apple Leaves Leaves were homogenized as described in the text and the 40 to 60%o (NH4)2SO4 fractions were used for enzyme assays. Assays were carried out in l-ml cuvettes maintained at 25 C. Reference cuvettes contained all reagents except G6P or sorbitol. Reaction mixture for sorbitol dehydrogenase contained 80 mm Tris-HCl (pH 9), 1.5 mm NAD+, 50 1I enzyme, and 300 mm sorbitol. Reaction mixture for aldose-6-P reductase contained 85 mm Tris-HCl (pH 9), 0.11 mim NADPH, 50 Id enzyme, and 50 mm G6P. Specific Activity

Leaf Stage Chl Sorbitol Aldose- dehy- 6-P re- drogen- ductase ase -0.4 0 0.4 0.8 1.2 i.6 mgig units/mgprotein x fresh wt lo3 mM [S-6-P]' Leaf primordia, <1.5 cm 0.42 12.0 0 FIG. 2. Lineweaver-Burk plot of aldose-6-P reductase activity as a Expanding, one-half full size 1.17 1.5 4.4 function of D-sorbitol-6-P concentration. V (rate of enzyme activity) = Fully expanded leaf 1.99 0 16.0 ,umol NADP+ reduced/mg protein. min. Reaction mixtures were as de- Mature leaf, on 1-year-old spur 2.56 0 8.1 scribed except that the D-sorbitol-6-P concentration was varied as indicated. Table III. Distribution ofAldose-6-P Reductase in Rosaceae the hydroxyl group at C2 must be in the same configuration as Mature leaves were homogenized as described in the text and 40 to 60%o that of D-glucose since low activity was observed when this (NH4)2SO4 fractions were used for enzyme assays. Assays were carried out hydroxyl was reduced (2-deoxy-D-glucose-6-P) or inverted (D- in l-ml cuvettes maintained at 25 C and reference cuvettes contained all mannose-6-P). The apple enzyme is similar in substrate specificity reagents except G6P. Assay mixture contained 80 mm Tris-HCl (pH 9), and specific activity to sorbitol-6-P dehydrogenase of loquat fruit 0.11 mm NADPH, 50 IlI enzyme, and 50 mM G6P. (9) and polyhydric alcohol phosphate dehydrogenase of silkworm Plant Specific blood (8) but is quite distinct from the microbial NAD-dependent Activity sorbitol-6-P dehydrogenase (12). We suggest that a more appro- priate name for the enzyme is aldose-6-P reductase (alditol-6-P: units/mg protein NADP l-oxidoreductase). x 1O3 The detection of the enzyme aldose-6-P reductase in the leaves M. domestica cv. Golden Delicious 37.2 of apple and other members of the Rosaceae is consistent with M. domestica cv. Antonovka 17.0 reports on sorbitol synthesis using labeled substrates. In those P. communis cv. Bartlett 15.8 reports (1, 10), glucose to sorbitol interconversions have been P. communis cv. Conference 24.4 shown, even though the interconversions could equally well be P. persica cv. Redhaven 11.3 through a phosphate ester. Recently, Redgwell and Bieleski (15) P. armeniaca cv. Perfection 19.7 isolated S6P from apricot leaves and suggested the pathway of sorbitol synthesis may follow the sequence F6P -- S6P -+ sorbitol. partially purified extract, which is apparently free of a hexose-P The crude enzyme preparations of apple leaves, loquat fruit (9), isomerase, reduced only G6P. This indicates that the enzyme is and silkworm blood (8) reduced both F6P and G6P, but our specific for aldose-P. 142 NEGM AND)ILOESCHER Plant Physiol. Vol. 67, 1981 The lack of aldose-6-P reductase activity in very young apple 2. ARNON DI 1949 Copper enzymes in isolated chloroplasts. Polyphenoloxidase in Beta vulgaris. Plant Physiol 24: 1-15 leaves and its detection in mature leaves suggest some relationship 3. BiELSKI RL 1969 Accumulation and translocation of sorbitol in apple phloem. between chloroplast development, photosynthetic capacity, and Aust J Biol Sci 22: 611-620 sorbitol synthesis. In addition, Bieleski (4) has reported, on the 4. BIELEsKI RL 1977 Accumulation ofsorbitol and glucose by leafslices ofRosaceae. basis of pulse-labeling experiments, that sorbitol is poorly metab- Aust J Plant Physiol 4: 11-24 5. BIELSIU RL, RJ REDGWELL 1977 Synthesis of sorbitol in apricot leaves. Aust J olized in fully expanded pear leaves and suggested that actively Plant Physiol 4: 1-10 growing regions would use sorbitol readily and that maturing 6. BRADFORD MM 1976 A rapid and sensitive method for the quantitation of tissues would tend to lose this ability (3). Our results agree with microgram quantities of protein utilizing the principle of protein-dye binding. Bieleski's findings since sorbitol dehydrogenase activity was de- Anal Biochem 72: 248-254 7. CHONG C, CD TAPER 1971 Daily variation ofsorbitol and related carbohydrates tected in young meristematic leaves but not in mature leaves. in Malus leaves. Can J Bot 49: 173-177 Consequently, it appears that some tissues, e.g. fully expanded 8. FAULKNER PD 1956 Enzymatic reduction of sugar phosphates in insect blood. leaves, contain an aldose-6-P reductase and a phosphatase that Biochem J 64: 436-441 constitute a system involved in sorbitol synthesis, whereas other 9. HIRAI M 1979 Sorbitol-6-phosphate dehydrogenase from loquat fruit. Plant Physiol 63: 715-717 tissues, e.g. young meristematic leaves, contain sorbitol dehydro- 10. HUTCHINSON A, CD TAPER, GHN TowERs 1959 Studies of phloridzin in Malus. genase, which constitutes the enzyme responsible for sorbitol Can J Biochem Physiol 37: 901-910 oxidation and eventual utilization (13). A developing leaf would 11. LEWIs DH, DC SMITH 1967 Sugar alcohols (polyols) in fungi and green plants. represent a transitional step between these two situations. I. Distribution, physiology and metabolism. New Phytol 66: 143-184 aldose-6-P reductase in 12. Liss M, SB HORWITz, NO KAPLAN 1962 D-Mannitol 1-phosphate dehydrogenase Finally, the occurrence of loquat (9) and D-sorbitol 6-phosphate dehydrogenase in Aerobacter aerogenes. J Biol and in the species reported here, i.e. apple (M. domestica), peach Chem 237: 1342-1350 (Prunus persica), pear (Pyrus communis), and apricot (Prunus 13. NEGM FB, WH LOESCHER 1979 Detection and characterization of sorbitol armeniaca), indicates the ubiquity of this enzyme in species that dehydrogenase from apple callus tissue. Plant Physiol 64: 69-73 and translocate sorbitol (19). Questions still remain, 14. PLOUVIER V 1963 Distribution of aliphatic polyols and cyclitols. In T Swain, ed, synthesize Chemical Plant Taxonomy, Chap 11. Academic Press, New York, pp 313-336 however, as to the mechanisms regulating sorbitol synthesis and 15. REDGwELL RJ, RL BIELESKI 1978 Sorbitol 1-phosphate and sorbitol 6-phosphate transport, and what roles, ifany, sorbitol may play beyond serving in apricot leaves. Phytochemistry 17: 407-409 as a major transport material. 16. TouSTER 0 1974 The metabolism of polyols. In HL Sipple, KN McNutt, eds, Sugars in Nutrition, Chap 15. Academic Press, New York, pp 228-239 Acknowledgment-We thank G. Marlow for his assistance and helpful discussion. 17. WEBB K, JWA BuRLEY 1962 Sorbitol translocation in apple. Science 137: 766 18. WoLFF JB, NO KAPLAN 1956 D-Mannitol 1-phosphate dehydrogenase from LITERATURE CITED Escherichia coiL J Biol Chem 218: 849-869 19. ZIMRMAN MH, H ZIEGLER 1975 List ofsugars and sugar alcohols in sieve-tube 1. ANDERSON JD, P ANDREWS, L HOUGH 1962 The biosynthesis and metabolism of exudates. In MH Zimmerman, JA Milburn, eds, Encyclopedia of Plant Phys- polyols. II. The metabolism of "4C-labelled D-glucose, D-glucuronic acid, and iology, New Series Vol I Appendix III. Springer-Verlag New York, pp 480- D-glucitol (sorbitol) by plum leaves. Biochem J 84: 140-146 503