Age-dependent conversion of nitrate reductase to cytochrome c reductase species in barley leaf extracts

Abstract

The stability of nitrate reductase (NR) in extracts from 4-, 5- and 6-day-old primary leaves of barley was examined. The half-time of loss of NR activity was found to be 358, 107 and 70 min, respectively. Bovine serum albumin (BSA) and phenylmethylsulphonylfluoride (PMSF) stabilized NR in extracts from 5- and 6-day-old primary leaves, but BSA was much more effective. The increased instability of NR with age correlated with increased conversion of the MW 203 000 NR complex to smaller NADH cytochrome c reductase (CR) species of MW 163 000, 61 000 and 40 000. The MW 163 000 CR species also possessed NR activity. BSA prevented and PMSF retarded the conversion of NR to the smaller CR species. The increased instability of NR in extracts from older tissue may be due to increased conversion of NR to smaller CR species. The ability of PMSF and BSA to stabilize NR and inhibit conversion of NR to the smaller CR species indicates that these phenomena are probably due to proteolytic degradation of NR. This suggestion is supported by the observation that trypsin cleaved NR to 3–4 S CR species and that cleavage was retarded by the presence of BSA. Endogenous proteinase attack at specific sites between domains of the barley NR complex may generate the CR species seen in barley extracts. The MW 40 000 CR species probably carries at least the FAD domain.