PLANT PHYSIOLOGY , Vol 107, Issue 1 225-231, Copyright © 1995 by American Society of Plant Biologists
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CELL BIOLOGY AND SIGNAL TRANSDUCTION |
Circadian Oscillation of Nitrate Reductase Activity in Gonyaulax polyedra Is Due to Changes in Cellular Protein Levels
C. B. Ramalho, J. W. Hastings and P. Colepicolo
Universidade de Sao Paulo, Instituto de Quimica, Departamento de Bioquimica, Sao Paulo, CP 20780, Brazil (C.B.R., P.C.)
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.
