ABSTRACT
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, a Blue-Affigel column, and a DEAE ion exchange column. Undenatured protein shows a molecular mass of about 310 kDa; based on SDS-PAGE, 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 mid day-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 mid-day phase.