Energy-dependent calcium sequestration activity in rat liver microsomes

MgATP-dependent calcium sequestering activity of rat liver microsomes has been characterized. This activity is linear over a 90-min period and specifically requires MgATP. Substitution of CTP, UTP, GTP, or ADP will not support calcium uptake. Oxalate, which serves as a trapping agent in calcium uptake of skeletal muscle microsomes, is required to maintain net accumulation of calcium. The reaction is temperature-dependent and has an apparent Vmax of 11.2 nmol/mg of protein/min. The apparent Km for calcium is 23.2 muM calculated from total calcium concentration, and approximately 4.6 muM based on free calcium concentration, and apparent Km for ATP is 1.8 mM. Calcium uptake activity normally measured in presence of 100 mM KCl is only slightly depressed if 100 mM NaCl is substituted and is considerably depressed when 200 mM sucrose replaces KCl. An appropriate hydrolysis of ATP is associated with the calcium uptake. Separation of smooth and rough endoplasmic reticulum on sucrose gradients indicates a considerably lower specific activity per mg of protein in the fraction enriched with rough endoplasmic reticulum. Azide, at a level which completely inhibits liver mitochondrial calcium sequestration, has no effect on the liver microsomal system. Oligomycin, which inhibits ATP-dependent calcium uptake of liver mitochondria, has a considerably lesser effect on calcium uptake of liver microsomes. p-Chloromercuribenzoate and mersalyl inhibit the liver microsomal calcium pump at levels as low as 10- minus 7 M. Calcium uptake activity is considerably reduced in adult female rats. Weanling rats, both male and female, have calcium uptake activities like that of the adult males. Because of the higher activity in the male rat, the fatty acid composition of the liver microsomal phospholipids was analyzed. The male rat had a higher percentage of linoleic and palmitic acids in the microsomal phospholipids. Endoplasmic reticulum and plasma membrane are postulated to play a role in regulation of the levels of free cytoplasmic calcium in the mammalian liver.