Isolation of NADH Oxidation System from the Plasmalemma of Corn Root Protoplasts

Abstract

A plasmalemma-bound NADH oxidation system (Lin 1982) in corn root protoplasts was isolated by a mild treatment of intact protoplasts with trypsin. The majority of NADH-stimulated O2 consumption activity of the protoplasts could be recovered in the supernatant isolated from the intact protoplasts which were treated with trypsin. The activation energy of NADH oxidation in the supernatant is similar to that of the intact protoplasts (8.7 vs. 9.4 kc/mol/degree). Unlike that of the intact protoplasts, an Arrhenius plot of the temperature response (from 5-25.degree. C) of the activity in the supernatant shows no transition suggestive of a dissociation of the enzyme from the membrane. Trypsin treatment did not affect K+ uptake into cell volume of the protoplast. The NADH-stimulated K+ uptake and the increase of cell volume were greatly reduced. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of trichloroacetic acid-precipitated protein from the supernatant showed 1 extra peptide band with .apprx. 42 kdalton MW.