Oxidation of exogenous NAD(P)H by isolated carrot protoplasts is catalysed by isoperoxidase

Abstract

A plasma membrane-associated system for the oxidation of exogenous NAD(P)H has been identified in maize root protoplasts (Lin, 1982a, b, 1984) and in protoplasts derived from cell suspensions of sugar cane (Thom & Manetski, 1985). Lin (19826, 1984) reported that this system could be released from intact protoplasts by treatment with trypsin and from his investigations proposed that NADH was oxidized by molecular oxygen via a plasma membrane redox system, which consisted of a quinone pool, cytochrome b and an oxidase. We have investigated the oxidation of exogenous NAD(P)H by protoplasts obtained from carrot cells grown in suspension culture and on the basis of our studies we propose that NAD(P)H is oxidized by isoperoxidase (isoenzymes of peroxidase) extrinsically associated with the plasma membrane of intact protoplasts. Protoplasts were prepared from cultured carrot cells as described by Slabas et a f . (1980). Estimates of viability and intactness were made as described by Widholm (1972). Protoplasts suspensions with greater than 95% viability were routinely used. Addition of 1 .O mM-NAD(P)H to protoplasts markedly increased oxygen consumption as measured with an oxygen electrode. The reaction medium consisted of 0.4 M-sorbitol, 0.5m~-CaCI, and I.OmM-Mes buffer, pH 6.5 at 25°C. The NAD(P)H oxidation was absent or negligible in freshly prepared protoplasts suspensions but became apparent and increased to a maximal value (approximately 75 nmol/l O6 protoplasts per min, i.e. 2.5-3 times basal respiration) 1.5-2 h after preparation. During this period the viability decreased by only 5 7 % . The oxidation of NAD(P)H monitored either by an oxygen electrode or spectrophotometrically (A34-A3,4) had the following characteristics: