Detection of nitrate reductase activity in nitrate reductase deficient mutants of barley (Hordeum vulgare)

Abstract

Nitrate reductase deficient mutants of barley Az12 and Az13 showed 40-50% in vivo enzyme activity as compared with the Steptoe non-mutant under strictly anaerobic conditions of assay. However, in vitro nitrate reductase activity could not be detected in the mutant extracts prepared by different methods. The extracts did not contain any inhibitory factor as judged by effect of in vitro nitrate reductase activity in the Steptoe non-mutant. Defective functioning of the Mo cofactor in the mutants was indicated by the fact that reduced benzyl viologen was ineffective as an electron donor. Incorporation of Mo-cofactor obtained from the Steptoe non-mutant into the mutant extract significantly reconstituted nitrate reductase activity. The reconstituted enzyme was NADH specific, indicating that coenzyme specificity is not altered in the mutants. Thus association of Mo-cofactor with the apoenzyme appears to be defective in the mutants.