Mutation of an EF-hand Ca(2+)-binding motif in phospholipase C of Dictyostelium discoideum: inhibition of activity but no effect on Ca(2+)-dependence.

Phosphoinositide-specific phospholipase C (PLC) is dependent on Ca2+ ions for substrate hydrolysis. The role of an EF-hand Ca(2+)-binding motif in Ca(2+)-dependent PLC activity was investigated by site-directed mutagenesis of the Dictyostelium discoideum PLC enzyme. Amino acid residues with oxygen-containing side chains at co-ordinates x, y, z, -x and -z of the putative Ca(2+)-binding-loop sequence were replaced by isoleucine (x), valine (y) or alanine (z, -x and -z). The mutated proteins were expressed in a Dictyostelium cell line with a disrupted plc gene displaying no endogenous PLC activity, and PLC activity was measured in cell lysates at different Ca2+ concentrations. Replacement of aspartate at position x, which is considered to play an essential role in Ca2+ binding, had little effect on Ca2+ affinity and maximal enzyme activity. A mutant with substitutions at both aspartate residues in position x and y also showed no decrease in Ca2+ affinity, whereas the maximal PLC activity was reduced by 60%. Introduction of additional mutations in the EF-hand revealed that the Ca2+ concentration giving half-maximal activity was unaltered, but PLC activity levels at saturating Ca2+ concentrations were markedly decreased. The results demonstrate that, although the EF-hand domain is required for enzyme activity, it is not the site that regulates the Ca(2+)-dependence of the PLC reaction

Seasonal and spatial changes in water and sediment quality variables in Bafa Lake

The brackish Bafa Lake located in the southwestern part of Turkey is under stress because of both natural and untreated wastewater effluents. The purpose of this research is to determine spatiotemporal distributions of some physicochemical variables in water column (temperature, salinity, pH, conductivity, dissolved oxygen, NH4-N, NO2-N, NO3-N, oPO(4)-P, TPO4-P, chlorophyll-a, total suspended solids) and sediment (TN, TC, TOC, TP) and their relationships at coastal stations. In the water column, nitrate and phosphate concentrations showed seasonal variations with high values recorded in winter period. Ammonium was determined as a main source of TIN component. During summer period, a large amount of total phosphorus was found as dissolved organic form. However, in the winter period, inorganic phosphate levels increased at sampling stations. N limitation was a common feature throughout the lake where P-limitation was only observed in summer period. The total phosphorus levels which showed hypereutrophic condition at the western part of the lake changed between 1.55 and 4.99 mu M and did not remain in the range for uncontaminated condition. In the lake sediment, a strong relationship was found between TOC and TC levels. Generally, the mean TOC concentrations constitute small amount of TC values in the sampling stations. The results also indicated that a strong correlation exists between TOC and TN values, and TN was greatly regulated by organic sources. In the lake, TOC: TN ratios changed between 5 and 13; the ratio greater than 10 could be an indicator of algal and land plant sources mixing as an organic matter