Characterization and daily variation of nitrate reductase in Gracilaria tenuistipitata (Rhodophyta).
A daily rhythm in the activity of nitrate reductase (NR: EC 1.6.6.1) isolated from the marine red algae Gracilaria tenuistipitata is shown to be attributable to changes in amounts of the protein. The enzyme was purified in four steps: ion exchange Q-Sepharose separation, ammonium sulfate precipitation, gel filtration on Sephacryl S-300, and affinity chromatography on Affigel-blue resin. This purification procedure yielded an active purified NR of about 500-fold with a recovery of 85%. The SDS-PAGE silver staining of purified NR revealed a 110 kDa single band. Non-denaturated protein showed a molecular mass of 440 kDa on gel filtration comparing with SDS-PAGE, the enzyme is apparently composed of four identical subunits. In extracts of algae grown under either constant dim light or a light-dark cycle, the activity of NR exhibited a daily rhythm, peaking at midday phase as does photosynthesis. Staining with monoclonal antibodies, raised against NR from Porphyra yezoensis, showed that the amount of protein changes by a factor of about 12, with a maximum occurring in the midday phase.
Lopes
Patricia Fátima
PF
Departamento de Bioquímica, Universidade de São Paulo, Instituto de Química, São Paulo, CP 20780, CEP 05599-970, Brazil.
de Cabral Oliveira
Mariana
M
Colepicolo
Pio
P
D000911:Antibodies, Monoclonal / Q000494:pharmacology; D002940:Circadian Rhythm; D004791:Enzyme Inhibitors / Q000494:pharmacology; D007700:Kinetics; D050901:Nitrate Reductase; D009565:Nitrate Reductases / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism*; D000461:Rhodophyta / Q000201:enzymology*
D000911:Antibodies, Monoclonal; D004791:Enzyme Inhibitors; D009565:Nitrate Reductases; D050901:Nitrate Reductase