A simple, rapid extraction and assay procedure for NAD⁺-dependent sorbitol dehydrogenase (SDH) in peach.

Abstract

Sorbitol is the major photosynthetic product in peach (Prunus persica (L.) Batsch.). In sink tissues, sorbitol is converted to fructose via NAD⁺?dependent SDH. A new procedure is described that allows rapid, simple quantification of SDH activity in growing tissues. The procedure uses only 0.01 to 5 g of fresh tissue per sample, such that a single shoot tip, a single root tip, or »5 g of fruit flesh can be assayed for SDH activity. Storage of samples at 4 or -20°C overnight resulted in significant loss of enzyme activity. Thus, freshly harvested tissues were ground with sand in buffer at 2°C in a mortar and pestle, and the homogenate was centrifuged at 3000 g_n to remove particulate matter and sand. The supernatant was desalted on a Sephadex G-25 column, and the eluent was assayed for SDH activity immediately. Activity was determined by measuring the production of NADH per minute in the assay mixture using a spectrophotometer (340 nm). Tris buffer at pH 9.0 was the best for extraction of peach SDH. Activity of SDH was strongly inhibited by dithiothreitol (DTT) in the extraction mixture and by DTT, L-cysteine, or SDI-158 in the assay mixture, similar to results reported for SDH from mammalian tissues. Peach SDH has a K_m of 37.7 mM for sorbitol and a pH optimum of 9.5, similar to those reported for apple (Malus xdomestica Borkh.) SDH. Unlike older protocols for SDH activity in plant tissues, the new procedure features reduced sample size (1/10 to 1/100 of that which was previously used), smaller volumes of buffer, fewer buffer ingredients, greatly reduced time for sample preparation, yet comparable or higher values of SDH-specific activity. Following the same procedure, SDH activity was also measured in Prunus fremontii Wats., Prunus ilicifolia (Nutt.) Walp., and Marianna 2624 plum (P. cerasifera Ehrh. x P. munsoniana Wight & Hedr.).

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