Studies on the 'in vitro' degradation of barley nitrate reductase

Abstract

The effects of haem deficiency, as produced by treatment with laevulinic acid, on synthesis/assembly of NR in barley leaves was investigated. Interpretation of results was complicated by instability of NR in vitro therefore the age-dependent stability of NR was investigated. The half-time of loss of NR activity in 4, 5 and 6 day old leaves of barley was found to be 358, 107 and 70 min respectively. BSA, PMSF, and 1,10-phenanthroline stabilised NR in extracts from 5 and 6 day old primary leaves, but BSA was most effective. The increased instability of NR with age of leaf correlated with increased conversion of the MW 203 000 NR complex to small NADH-CR species of MW l63 000, 6l 000 and 4-0 000. The MW 163 000 CR species also possessed NADH-NR activity. BSA prevented and PMSF and 1,10-phenanthroline retarded the conversion of NR to the smaller OR species. The ability of BSA and the proteinase inhibitors to stabilise NR and inhibit conversion of NR to the small CR species indicates that the age-dependent in vitro stability of NR may be due to proteolytic degradation of NR. This suggestion is supported by the observation that trypsin cleaves purified NR into CR species sedimenting with the same coefficients as those observed in crude extracts. In addition, tryptic cleavage was retarded by the presence of BSA. Semi-purified preparations of maize root and barley leaf inactivating factors failed, however, to generate 3-4-S CR species, the maize root inactivator appearing to be active against the 3-4-S CR species in addition to NR. It was concluded that neither factor was responsible for generating the small CR species observed in crude extracts. The 4-0 000 MW CR species was purified and its MW confirmed using the method of Siegal and Monty (I966). The species was shown to possess NET reductase activity but attempts to characterise the species with respect to haem content proved unsuccessful. Antibodies raised against the 40 000 MW CR species were found to inhibit all partial activities of NR and it was therefore concluded that this species, at least, was likely to be derived from NR. Antibodies raised against purified barley NR were found to inhibit all NR activities to a similar extent. Pre-immune serum was found to stimulate NR activity and this was found not to be due to the presence of serum albumin. A protection of inhibition assay was developed for estimating NR-CRM. Negligible NR-CRM was detected in nitrate-less leaf extracts while substantial NR-CRM was found in ammonium-grown plant extracts. Pre-treatment of purified NR with NADH, but not with nitrate, was shown to preserve the enzyme, to some extent, from antibody inhibition. In the General Discussion, evidence available regarding the in vitro breakdown of NR is reviewed and a model for the structure of higher plant NR is presented. In addition the probable route of genetic evolution of NRs is discussed. Evidence for the role of proteolysis in regulation of NR activity in vivo is also analysed.