Evidence for the occurrence of, and possible physiological role for, Cyanbacterial calmodulin

Oscillatoria limnelica was isolated from Lake Opinicon, Ontario, by Karen Jones. Stocks were maintained at 25°C in medium modified by Daley and Brown (4) from Hughes et al. (6). Experimental cells were grown in half-strength medium bubbled with 0.5% C02-enriched air and harvested in mid-log phase (36 h after inoculation). Illumination of stock and experimental cells was provided by 3 Gro-Lux and I Warm White fluorescent tubes and was 110 E m2 s-'. Cells were harvested by centrifugation at 4000g for 5 min and washed in 5 mm Tris-H2S04 buffer (pH 8.0 at 25°C). The cells were then Pi-starved by resuspension in half-strength growth medium lacking Pi. After 4 h, cells were harvested, washed, and resuspended to an A660nm of 0.05 in 5 mm Tris-H2SO4 buffer. Initial rates of Pi uptake were determined using a light table described elsewhere (19) that provided illumination of 100 4E m-2 s_'. Aliquots of 25 ml of the cell suspension were put into 50-ml Erlenmeyer flasks on top of the light table, and bubbled with moist air. Studies on the kinetics of Pi uptake were initiated by the addition of 32Pi solutions to the flasks. Ca2+ (as CaCl2 was added 1 min prior to the Pi solutions. Uptake was terminated by filtering the cells from the medium using a vacuum filtration system and 0.4-,um Metricel filters (Gelman, Ann Arbor, MI), a procedure that took 3 to 5 s. The filters and samples were placed in low-K glass scintillation vials containing 15 ml of 5% (w/v) K2S208 (to decolorize the cells). The extent of Pi uptake was determined by measuring the Cerenkov radiation in a liquid scintillation counter.

Fluphenazine-HCI was added to the cells to a concentration of 50 gM 5 min prior to addition of the Pi solutions. The cells were collected after 1 min of exposure to the labeled Pi and Pi uptake was determined as above.

For the quantitation of the CaM-like protein, 0. limnetica cells were suspended in homogenization buffer (125 mm borate buffer with 75 mM NaCl and 1 mm EGTA, pH 8.4), and broken in a French pressure cell. The homogenate was then rapidly brought to a boil, quenched on ice, then centrifuged at l0,OOOg for 30 min. RIA was performed according to the manufacturers directions (Caabco). Briefly, aliquots of the supernatant along with CaM standards were mixed with assay buffer (homogenization buffer plus 0.02 g/l BSA), 125I CaM, and sheep anti-rat testis CaM antibodies, and incubated for 18 h at 25C. Next, staphylococcus-A cells were added to all tubes which were then mixed and incubated for 30 min at 25C. The tubes were centrifuged at lOOOg for 10 min. The activity of 1251I in each pellet was determined in a gamma counter and corrected for nonspecific binding. The values were divided by the activity in total bound samples (which contained no CaM) and expressed as a percentage of the total bound. A standard curve of CaM concentration was prepared by plotting the values of the per cent total bound of the CaM standards as a function of their CaM concentration on log/logit paper.

Chl a was determined by the method of Rigby (19), and protein was quantitated by the method of Lowry et al. (15), using BSA as the standard. The radioisotope 32Pi (carrier free as orthophosphoric acid in 20 mm HCI) was purchased from New England Nuclear. Fluphenazine-HCl was the kind gift of Dr. P. Abadie (Squibb Canada, Montreal). The CaM RIA kit was obtained from Caabco Inc. (Houston, Texas). Deionized H20 was prepared by passing distilled H20 through a Barnstead Ultrapure deionizer cartridge. The specific resistance was always greater than I megaohm/cm3. All other chemicals were analytical grade (J. T. Baker).