CHARACTERIZATION OF NAD-DEPENDENT MANNITOL DEHYDROGENASE FROM CELERY AS AFFECTED BY IONS, CHELATORS, REDUCING AGENTS AND METABOLITES

NAD-dependent mannitol dehydrogenase (MTD) from celery (Apium graveole ns L. var. dulce (Mill.) Pers.) provides the initial step by which man nitol is committed to central metabolism and plays a critical role in regulating mannitol concentration in the plant. The pH optimum for man nitol oxidation occurs at pH 9.5 whereas the optimum for mannose reduc tion occurs at pH 6.5. Michaelis-Menten kinetics were exhibited for ma nnitol and NAD with K-m values of 64 and 0.14 mM, respectively at pH 9 .5. The K-m for mannose and NADH were 745 mM and 1.27 mu M, respective ly at pH 6.5. The high K-m for mannose is consistent with a reaction i n situ favoring mannitol oxidation rather than mannose reduction. The observed down-regulation of MTD in salt stressed celery is not due to a direct inhibition by NaCl or macronutrients. Inhibition by the chela tor 1,10-phenanthroline suggests that zinc is required for MTD activit y. Reducing agents DTT, DTE and beta-mercaptoethanol inactivated MTD r eversibly. At pH 7.0, ADP and to a lesser extend AMP and ATP were comp etitive inhibitors, with respect to NAD, having apparent K-i's of 0.24 , 0.64 and 1.10 mM, respectively. (C) 1998 Elsevier Science Ireland Lt d.