ATP-dependent calcium accumulation in brain microsomes. Enhancement by phosphate and oxalate

Abstract

1. 1. ATP-dependent calcium uptake by a rabbit brain vesicular fraction (microsomes) was studied in the presence of phosphate or oxalate. These anions, which are known to form insoluble calcium salts, increased the rate of calcium uptake and the capacity of the vesicles for calcium accumulation. 2. 2. The degree of activation depended on the concentration of phosphate or oxalate. Under optimal conditions, phosphate promoted a 5-fold increase in the amount of calcium stored at steady state. This level was 200–250 nmol Ca 2+/mg protein. 3. 3. Initial rate of calcium uptake followed Michaelis-Menten kinetics with an apparent K m for calcium of 6.7 · 10 −5 and a V of 44 nmol/min per mg protein. Optimal pH was 7.0. With 2 mM ATP, optimal Mg 2+ concentration was 2 mM. 4. 4. Dinitrophenol and NaN 3 inhibited calcium uptake in a mitochondria-enriched fraction but not in the microsomal fraction. 5. 5. Calcium uptake activity was compared in the six subfractions prepared from the whole microsomal fraction by means of a sucrose density gradient fractionation. 6. 6. The Mg 2+-dependent ATPase activity of brain microsomes was activated by calcium. Maximal activation was attained with 100 μM CaCl 2. Greater calcium concentrations caused a progressive inhibition. 7. 7. The data suggest that the ATP-dependent calcium uptake in brain microsomes, as in muscle microsomes, is brought about by an active transport process, calcium being accumulated as a free ion inside the vesicles.