Characterization and Partial Purification of Aldose-6-phosphate Reductase (Alditol-6-Phosphate:NADP 1-Oxidoreductase) from Apple Leaves

Abstract

Aldose--phosphate reductase (alditol6-phosphate:NADP 1-oxidoreductase) was isolated and characterized from mature apple leaves (Malms domestica cv. Starkrimson). The enzyme was purified 79-fold. The Sorbitol (D-glucitol) is a hexitol, one of several acyclic polyols or sugar alcohols found in higher plants (11). Sorbitol is the primary product of photosynthesis (3, 5, 7), the main translocated form of carbon (17), and a common constituent of fruits in many species of the Rosaceae (14). The synthetic pathway for sorbitol and other polyols in higher plants has not been established. In other organisms, however, the synthesis and/or utilization of polyols is initiated by one of the following reactions (16): (a) oxidation to the ketose; (b) oxidation to the aldose; or (c) phosphorylation to the corresponding polyol phosphate, which then can be converted to either the ketose phosphate (12) a sorbitol dehydrogenase (L-iditol:NAD oxidoreductase, EC 1.1.1.14) from apple callus tissues (13). Since then, we have also detected and purified this enzyme from pear callus tissue, apple and pear seeds, apple seedlings, young leaves, and fruits (unpublished data). In fully expanded apple leaves, however, another sorbitol enzyme was present which oxidized S6P4 and reduced G6P and D-galactose-6-P. A similar enzyme was recently reported in loquat fruit (9). Here, we consider some characteristics of this enzyme, an aldose-6-P reductase (alditol-6-P:NADP I-oxidoreductase), and its occurrence and possible role in carbon metabolism in leaves of apple and several other species in the Rosaceae. MATERIALS AND METHODSChemicals. All chemicals were purchased from Sigma. D-Sorbitol-6-P barium salt was converted to potassium salt before use (18). Affinity support media (Affi-Gel blue, 100-200 mesh) and bovine y-globulin were obtained from Bio-Rad Laboratories.Partial Purification of Aldose-6P Reductase. Malus domestica cv. Starkrimson fully expanded leaves on new growth were used for enzyme extraction. Leaves were washed in distilled H20 and petioles and midribs were discarded. Homogenization and all subsequent steps were carried out in a cold room at 2 C. 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 speed. The homogenate was squeezed through a polypropylene cloth and the filtrate was centrifuged at 2,000g for 20 min. The 2,000g supernatant was centrifuged at 25,400g for 30 min and the pellet was discarded. Solid (NH4)2SO4 was added slowly with constant stirring to the 25,400g supernatant of the crude extract to reach 40%o saturation (24.3 g/100 ml). The suspension was stirred slowly for 30 min and centrifuged at 25,400g for 30 min, and the ...

Studies of Sugars and Sorbitol in Developing Corn Kernels

“…Furthermore, no sorbitol was detected in the shank upon analysis with the gas chromatograph, and none was found earlier in photosynthesizing corn leaves (5). An NADspecific enzyme from leaves of rosaceous plants converts glucose-6-P into sorbitol-6-P, and an NAD-specific enzyme from nonphotosynthetic apple tissue produces fructose from sorbitol (2,15,16). There is no information yet whether these enzymes occur in corn, or how sorbitol production and utilization fit into the overall conversion of sucrose to reserve materials in the developing seeds.…”

Analyses of Expressed Sequence Tags from Apple

“…In source leaves, sorbitol is derived from the same hexose phosphate pool as Suc. The enzyme aldose 6-P reductase (EC 1.1.1.200; 11 NR sequences) is the rate-limiting step for the conversion to sorbitol from the hexose phosphate pool (Negm and Loescher, 1981), synthesizing sorbitol 6-P from Glc 6-P. Antisense suppression of the aldose Figure 1. Microsatellite (SSR) positions relative to predicted coding regions of the apple NR sequences.…”

Carbon Fluxes in Mature Peach Leaves

“…It should be noted that glucose-6-P, the substrate of aldose-6-P reductase, i.e. the precursor for sorbitol (10,18,20), is an activator of sucrose-phosphate synthetase (24), a key enzyme of the sucrose pathway. (14).…”

“…celery (5,13), less is known about carbon partitioning in sorbitol-synthesizing species. Since the labeling study of Redgwell and Bieleski (20) and the purification of aldose-6-P reductase (EC 1.1.1.200) from loquat (10) and apple leaves (18), the precursor for sorbitol biosynthesis in mature leaves is known to be glucose-6-P. The catabolic pathways of sorbitol involve sorbitol dehydrogenase (NADdependent, EC 1.1.1.14 [17] or NADP-dependent [29]) or sorbitol oxidase (28) in sink tissues.…”