Use this URL to cite or link to this record in EThOS:
Title: Reversible inactivation of nitrate reductase from algae
Author: Al-Bassam, B. A. R.
Awarding Body: University College of Swansea
Current Institution: Swansea University
Date of Award: 1982
Availability of Full Text:
Access from EThOS:
The reversible inactivation of nitrate reductase (NR) has been studied in unicellular green algae. The addition of ammonium to nitrate-grown cultures did not result in a rapid loss of NR activity. Losses of NR activity from nitrate-grown cultures that received ammonium were similar to losses that occurred when such cultures were nitrogen starved. Similar results were obtained when either methylamine or arsenate were added to nitrate-grown cultures. Nitrate reductase activity was not inactivated in vitro after preincubation (100 min) with either NAD(P)H or NAD(P)H and ADP, added in equimolar concentrations (0.3mM). The addition of micromolar amounts of cyanide to cell-free extracts from wild-type marine and freshwater algae (11 strains) resulted in a rapid, and almost complete, inactivation of NR activity when NAD(P)H was also included in preincubation mixtures. Cyanide-inactivated enzymes were reactivated after oxidation with ferricyanide. Nitrate had a protective effect against reversible cyanide-inactivation. Nitrate reductase was almost fully active in freshly prepared extracts from nitrate-grown cultures of most species. Nitrate reductase activity in freshly prepared extracts from nitrate-grown cultures of Chlorella vulgaris and Chlorella variegata, were activated substantially after preincubation with terricyanide. Nitrate reductase from the nit A mutant of Chlamydomonas reinhardii 17/4 was not inactivated after preincubation with NAD(P)H and cyanide; reversible cyanide-inactivation of the mutant enzyme did occur in extracts preincubated with cyanide and, either reduced flavin mono-nucleotide or reduced benzyl viologen. The cyanide inactivated, mutant enzyme was reactivated by ferricyanide. The pyridine nucleotide specificity of NR from eleven algal strains was investigated. Only five strains contained NADH-NR and NADPH-NR activities; none contained NADPH-NR activity only. Further studies of the pyridine nucleotide specificity of algal NR were carried out by (i) using pyridine nucleotide analogues (ii) investigating the effectiveness of NADH and NADPH as protectants against p-chloromercuribenzoate (p-CMB) inactivation and (iii) comparing NADH and NADPH as effectors in reversible cyanide inactivation. The bispecific, NAD(P)H-NR of Chlorella variegata was purified by Blue Sepharose (CL-6B) affinity chromatography. Apparent Km values for NAD(P)H and nitrate were obtained and other properties of the enzyme were studied.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available