Circadian oscillation of nitrate reductase activity in Gonyaulax polyedra is due to changes in cellular protein levels

• Published in: Plant physiology (Bethesda) 1995-01, Vol.107 (1), p.225-231
• Main Authors: Ramalho, C.B. (Universidade de Sao Paulo, Sao Paulo (Brazil)), Hastings, J.W, Colepicolo, P
• Format: Article
• Language: English
• Subjects: ACIDE AMINE; ACTIVIDAD ENZIMATICA; ACTIVITE ENZYMATIQUE; Amino acids; Amino Acids - analysis; AMINOACIDOS; Animals; Antibodies; Antibodies - pharmacology; Barley; Biological and medical sciences; Cell Biology and Signal Transduction; Cell extracts; Chemical composition; Circadian Rhythm; COMPOSICION QUIMICA; COMPOSITION CHIMIQUE; CONTENIDO PROTEICO; Dinoflagellida - enzymology; Dinoflagellida - physiology; Enzymes; Fundamental and applied biological sciences. Psychology; Gene expression regulation; LUMIERE; LUZ; MASTIGOPHORA; Messenger RNA; Metabolism; NITRATE REDUCTASE; Nitrate Reductases - antagonists & inhibitors; Nitrate Reductases - chemistry; Nitrate Reductases - isolation & purification; Nitrate Reductases - metabolism; Nitrates; NITRATO REDUCTASA; Nitrogen metabolism; Plant physiology and development; PROTEINAS; PROTEINE; Proteins - metabolism; PURIFICACION; PURIFICATION; Rabbits; RITMOS BIOLOGICOS; RYTHME BIOLOGIQUE; SINTESIS DE PROTEINAS; SYNTHESE PROTEIQUE; TENEUR EN PROTEINES
• ISSN: 0032-0889; 1532-2548
• DOI: 10.1104/pp.107.1.225

A circadian rhythm in the activity of nitrate reductase (NR; EC 1.6.6.1 ) isolated from the marine dinoflagellate Gonyaulax polyedra is shown to be attributable to the daily synthesis and destruction of the protein. The enzyme was purified in three steps: gel filtration on S-300 Sephacryl, an Affigel-Blue column, and a diethylaminoethyl ion-exchange column. Undenatured protein shows a molecular mass of about 310 kD; based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the enzyme appears to be composed of six possibly identical subunits. The amino acid composition of the G. polyedra NR is very similar to that reported for the NR of barley leaves, Chlorella vulgaris, and Ankistrodesmus braunii. The experiments reported indicate that the cellular expression of NR is under circadian control. In extracts of cells grown under either constant dim light or a light-dark cycle, the activity of NR exhibits a daily rhythm, peaking at midday phase, as does photosynthesis. Staining with affinity-purified polyclonal antibodies, raised in rabbits against purified NR, shows that the amount of protein changes by a factor of about 10, with the maximum occurring in midday phase