ATP-DEPENDENT CALCIUM-RELEASE FROM BINDING-SITE IN STREPTOCOCCUS-BOVIS - BOUND VERSUS FREE POOLS

The introduction of the acetoxymethyl ester of fura-2 (fura-2/AM) has made possible the measurement of the concentration of intracellular fr ee calcium (free [Ca2+](i)) in bacteria, We report here a study of int racellular calcium levels and exclusion mechanisms in Streptococcus bo vis using fura-2/AM and (CaCl2)-Ca-45. The total [Ca2+](i) in cells no t energized with glucose increased in proportion to external Ca2+ conc entration over the range 0.1-0.5 mM, the intra- and extracellular leve ls being approximately equal. In contrast, the free [Ca2+](i) remained at 71.1+/-1.9nM in the face of extracellular concentrations from 0.1 to 5 mM. Energizing by the addition of glucose produced a dramatic inc rease in the intracellular concentration of ATP and a marked decrease in total [Ca2+](i), but had no effect on the free [Ca2+](i) regardless of whether extracellular calcium was high or low. Total [Ca2+](i) was affected little, if at all, by the metabolic inhibitors FCCP and DCCD , implying that neither proton motive force nor F0F1H+-ATPase is neces sary for calcium exclusion. In contrast, total [Ca2+](i) and ATP were both greatly reduced by iodoacetate. These results suggest that 1) S, bovis has a remarkable calcium buffering capacity, and 2) most of its intracellular calcium exists in a bound form which can be released by energizing via a mechanism coupled to ATP hydrolysis.