Identification of Possible Intermediates in in Vivo Degradation of Spinach Nitrate Reductase

Abstract

Degradation intermediates of nitrate reductase (NR) were extracted from fresh spinach leaves in the presence of protease inhibitors, and concentrated by chromatography on an anti-NR IgG-conjugated Sepharose-4B column. Seven polypeptides, 120 (that of the intact subunit), 110, 100, 76, 64, 62, and 44 kDa, were obtained. Their cross-reactivities to mono-specific polyclonal antibody and monoclonal antibody prepared against NR subunit were assessed by an immunoblotting method. All polypeptides were recognized with mono-specific polyclonal antibody. On the other hand, four of them, at 120, 110, 100, and 44 kDa, were recognized with monoclonal antibody which specifically binds to the NADH-ferricyanide reductase domain of NR. The possibility that these polypeptides were artifacts in preparation was ruled out by an internal tracer experiment. Thus, it is concluded that spinach leaf NR in vivo degraded via putative intermediates catabolic products of 110, 100, 76, 64, 62, and 44 kDa.