Inhibition of NADH-nitrate reductase degradation in barley leaf extracts by leupeptin

Abstract

NADH-nitrate reductase (NR) is unstable in extracts prepared from 6-day-old primary leaves of barley and breaks down to NADH-cytochrome c reductase (CR) species of 3.1 S and 3.8 S. Leupeptin, an inhibitor of trypsinlike enzymes, both stabilises NADH-NR activity in extracts and prevents conversion to the 3.1 S and 3.8 S NADH-CR species. We conclude that the leupeptin inhibited thiol-dependent acid endoproteinase described by Huffaker and Miller [6,7] is mainly responsible for degradation of NADH NR to smaller NADH-CR species in barley primary leaf extracts and that it cleaves peptide bonds on the carboxyl side of arginine/lysine in the enzyme. We suggest that these susceptible peptide bonds are located in interdomain hinge regions.