Purification and Characteristics of Sorbitol-6-phosphate Dehydrogenase from Loquat Leaves

Abstract

To study the role of sorbitol-6-phosphate dehydrogenase in sorbitol synthesis in leaves of Rosaceous plants, properties of the enzyme and its presence in several plants in the family was investigated. The activity of the enzyme, which catalyzes an NADP-dependent oxidation of the substrate to glucose-6-phosphate, was detected in leaves of Prunus mume, Prunus persica, Rhaphiolepsis indica, Sorbus aucuparia, Cydonia oblonga, Photinia glabra, Sorbaria kirilowii and Spiraea thunbergii. The enzyme was purified .apprx. 60-fold from leaves of loquat (E. japonica) using affinity chromatography with Blue Sepharose. Neither mannitol-1-phosphate nor fructose-6-phosphate served as substrate. The MW of the enzyme was 65,000 at pH 8.0 by gel filtration. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed a peptide of 33,000 daltons; the enzyme was presumably a dimer at pH 8.0. Km values for sorbitol-6-phosphate, G-6-P, NADP and NADPH were 2.22 mM, 11.6 mM, 13.5 .mu.M and 1.61 .mu.M, respectively. Equilibrium constant for sorbitol-6-phosphate oxidation was 5.12 .times. 10-10. Optimal pH for sorbitol-6-phosphate oxidation was 9.8. The enzyme showed its maximum activity within a broad pH range between 7-9 for G-6-P reduction. The enzyme was more effective in the direction of G-6-P reduction than in the reverse direction at neutral pH. Evidently the enzyme catalyzes sorbitol synthesis from G-6-P during photosynthesis [for translocation] in leaves of Rosaceous plants.