The ATP-dependent concentration of calcium by a Golgi apparatus-rich fraction isolated from rat liver.

Abstract

Fractions rich in Golgi apparatus were isolated from female rat liver and incubated with different concentrations of MgATP and calcium. Calcium was sequestered by the fraction in the presence but not in the absence of ATP. The kinetics of the uptake process showed an exponential type accumulation to a maximum value which could be explained by a 'pumpleak' mechanism where Vmax was estimated at 6.7 nmol calcium/mg protein/min. Apparent Km for ATP was 1 mM and for calcium was 85 micron. The uptake is not inhibited in 5 mM azide, nor is it enhanced in 5 mM oxalate, suggesting that the sequestration is not caused by contamination from mitochondria or microsomes. Parallel experiments on a fraction rich in plasma membrane show at least a 10-fold reduction of activity over the Golgi apparatus-rich fraction, which is interpreted as evidence that plasma membrane contamination in the Golgi apparatus-rich fraction is not primarily responsible for the calcium sequestration activity. Morphometric analysis showed that about 85% of the recognizable membrane profiles in the fraction was associated with elements of the Golgi apparatus. Further evidence is given which suggests that at least 90% of the fraction was uniform in its ability to sequester calcium. In preliminary experiments it was not found possible to separate galactosyltransferase activity (a secific enzyme associated with the Golgi apparatus) from the calcium-sequestration mechanism. The stoichiometry of the hydrolysis of ATP associated with the uptake of calcium is the same as that shown by other isolated membrane systems.