A new member of the leucyl aminopeptidase family purified and identified from a marine unicellular algae.

Leucyl aminopeptidase (LAP; EC 3.4.11.1) activity was purified from crude extracts of the marine unicellular algae Gonyaulax polyedra by a combination of hydrophobic interaction with phenyl sepharose, DEAE-cellulose, and mono-Q HR5/5 ion-exchange chromatography. The undenaturated protein has a molecular mass of about 110 kD and based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the enzyme appears to be composed of two possibly identical subunits of 55 kD. The identity of the protein was confirmed by a cross-reaction of the purified protein with an antibody raised against a commercial LAP. Biochemical characterization showed that the Gonyaulax enzyme was similar to most of the previously described LAPs. Gonyaulax LAP is a metallo-enzyme since EDTA and 1,10-phenathroline significantly inhibited activity. Addition of the metal ions Zn(2+), Cu(2+) inhibited 80% of LAP activity, suggesting they are not the natural cofactors of the enzyme. Other metals, such as Ca(2+), Co(2+), Mn(2+), or Mg(2+) (concentrations up to 4 mM), caused no alteration in the total activity of Gonyaulax LAP.

Response of superoxide dismutase to pollutant metal stress in the marine dinoflagellate Gonyaulax polyedra.

The response of superoxide dismutase (SOD) activity in the marine dinoflagellate Gonyaulax polyedra to chronic (5.0 ppb Hg, 0.5 ppm Cd, 2.0 ppm Pb and 0.1 ppm Cu, during 30 days) and acute (10.0 ppb Hg, 1.0 ppm Cd, 5.0 ppm Pb and 0.25 ppm Cu, during 48 hours) exposure to metals was investigated. Under chronic exposure to Hg, Cd, Pb, and Cu, total SOD activity of metal-treated cells increased during the first day of exposure to plateau levels of 134, 148, 127, and 139% of control values respectively. Under acute metal exposure, SOD activity increases were of similar magnitude but much more rapid (within several hours) and of shorter duration. In addition, assays for oxidative damage to lipids revealed high levels of lipid peroxidation in cells kept in either chronic or acute exposure to metals reaching values 2-fold greater than the control group. Changes in SOD activity were dependent on the metal, its concentration, and the time of exposure. Non-denaturing polyacrylamide gels revealed induction of Fe-SOD and Mn-SOD but not Cu-Zn-SOD isoforms in cells kept under acute exposure to metals. These results suggest that oxidative stress may be an important mediator of metal toxicity in algal systems, with SOD providing antioxidant protection.

The activity of superoxide dismutase oscillates in the marine dinoflagellate Gonyaulax polyedra

Gonyaulax polyedra is a marine unicellular algae that, together with other dinoflagellates, is responsible for most light emission in the ocean. In G. polyedra numerous processes are clockcontrolled, such as bioluminescence, that occurs during night phase, cell aggregation, photosynthesis that peak during the day and cell division that occurs at the transition between night and day. The activity of superoxide dismutase (SOD) in cell-free extracts of G. polyedra, carried out at different times of day and night, was found to be three to four times higher during the day. This rhythm continued in cells kept in constant light, indicating that the regulation can be attributed to the cellular circadian clock. Nondenaturating polyacrylamide gels reveal several SOD isoenzymes in G. polyedra, including CuZnSOD. Cyanide inhibits over 50 per cent of total SOD activity when added to the reaction mixture. Furthermore, G. polyedra SOD cross-reacts with polyclonal antibodies raised against SOD purified from erythrocytes (AbSOD) and over 50 per cent of its activity is inhibited by 25 muL of AbSOD. Although they are distant groups, such results suggest that algae and erythrocytes maintain high SOD structural homology. Besides gene regulation by reactive oxygen species concentration, G.polyedra SOD expression seems to be also under control of the biological clock.

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

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.

A circadian rhythm in the activity of superoxide dismutase in the photosynthetic alga Gonyaulax polyedra.

The activity of superoxide dismutase in cell-free extracts of Gonyaulax made at different times of day and night was found to be three to four times higher during the day. This rhythm continued in cells kept in constant light, indicating that the regulation can be attributed to the cellular circadian clock.