The introduction of the acetoxymethyl ester of fura-2 (fura-2/AM) has made possible the measurement of the concentration of intracellular free calcium (free [Ca
2+]
i) in bacteria. We report here a study of intracellular calcium levels and exclusion mechanisms in
Streptococcus bovis using fura-2/AM and
45CaCl
2. The total [Ca
2+]
i in cells not energized with glucose increased in proportion to external Ca
2+ concentration over the range 0.1-0.5mM, the intra- and extracellular levels being approximately equal. In contrast, the free [Ca
2+]
i remained at 71.1± 1.9nM in the face of extracellular concentrations from 0.1 to 5mM. Energizing by the addition of glucose produced a dramatic increase in the intracellular concentration of ATP and a marked decrease in total [Ca
2+]
i, but had no effect on the free [Ca
2+]
i regardless of whether extracellular calcium was high or low. Total [Ca
2+]
i was affected little, if at all, by the metabolic inhibitors FCCP and DCCD, implying that neither proton motive force nor F
0F
1H
+-ATPase is necessary for calcium exclusion. In contrast, total [Ca
2+]
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.
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