Abstract
Chlorotetracycline (CTC) fluorescence is shown to be a competent and quantitative measure of the free internal calcium concentration, [Ca2+]i, obtained by ATP supported active uptake by bovine cardiac sarcolemmal (SL) vesicles. The fluorescence response of CTC to [Ca2+]i is calibrated by pre-equilibrating the vesicles with known Ca2+ concentrations and then diluting into a Ca2+-free medium containing CTC. The experiments show that CTC comes into equilibrium with the internal Ca2+ more rapidly than the latter can passively leak from the vesicles. The amplitude of the fluorescence increase is proportional to the Ca2+ concentration with which the vesicles are pre-equilibrated. This constitutes a calibration procedure for the use of CTC fluorescence as a quantitative measure of the free internal Ca2+ concentrations achieved in active transport. This method is applied to the determination of the average free Ca2+ concentrations achieved in ATP-energized uptake with sarcolemmal vesicles. Under optimal conditions an initial rate of 13 mM/min (37 nmol/mg/min) is observed. Uptake reaches a maximum corresponding to 70 mM (179 nmol/mg). Half-maximal values are obtained after 5 min of reaction. The mechanism of the CTC response to free internal Ca2+ concentration is discussed and is compared with measurements of vesicle-associated 45Ca2+.
Highlights
Chlorotetracycline(CTC)fluorescenceis shown tobe crease as the result of active Ca2+accumulation in SR and a competent and quantitative measure of the free in- mitochondria
Any remaining accumulated calcium is located in sarcoplasmic reticulum vesicles
The most important observation made in thispaper is that the CTC fluorescence response, AFLc., is proportional to the free internal Ca2+ concentration, [Ca2+Ii.This result was suggested from our previous equilibrium and kinetic study in SR (Millman et al, 1980)
Summary
Any remaining accumulated calcium is located in sarcoplasmic reticulum vesicles This value is subtracted from the total accumulated calcium to give an accurate measure of intravesicular sarcolemmal calcium concentration. Active Uptake Measured with CTC-Bovine sarcolemmal vesicles. The degree of mitochondrial contamination was assessed by monitoring the activity of cytochrome c reductase by the method of King (1967) This assay uses a hypertonic buffer which ruptures anyintact vesicle, allowing the expression of total enzyme activity. This activity (units/mg of protein) of the SL preparation was not greater than 5%of the level for purified mitochondria (3 different SL preparations) establishing the upper limit of contamination by mitochondrial debris This contamination did not, contribute to measured uptakes since the magnitude of the response in both active uptake and passive equilibration experiments was not affected by the inclusion of oligotime found to be adequate for sucrose equilibration across the SL membrane, a t 4 'C.
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