Ca2+ uptake in bovine adrenocortical microsomes: formation of phosphorylated intermediate of Ca2+ dependent ATPase.
Bovine adrenocortical microsomes were prepared and partially purified by discontinuous sucrose density gradient. Light fractions of the microsomes at the interface between 15 and 30% sucrose solution, exhibited ATP dependent Ca2+ uptake. The Ca2+ uptake was dependent on temperature and stimulated by free Ca2+ (the concentration for half maximal activation = 1.0 microM) and Mg2+. The Ca2+ uptake was inhibited by ADP but not affected by 10 mM NaN3 or 0.5 mM ouabain. Calcium release from the microsomes was accelerated by a Ca2+ ionophore, A23187, but not by a Ca2+ antagonist, diltiazem. A microsomal protein with a molecular weight of 100-110 kDa was phosphorylated by [gamma-32P]ATP in the presence of Ca2+, and the Ca2+ dependency was over the same range as the Ca2+ uptake (the concentration for half maximal activation = 3.0 microM). The phosphorylated protein (EP) was stable at acidic pH but labile at alkaline pH and sensitive to hydroxylamine. The rate of EP formation at 0 degrees C in the presence of 1 microM ATP and 10 microM Ca2+ (half time = 0.2 s) was less than that in the sarcoplasmic reticulum (SR) of rabbit skeletal muscle (half time = 0.1 s). The rate of EP decomposition at 0 degrees C after adding EGTA was about 6.7 times slower (rate constant: kd = 4.3 X 10(-3) s-1) than that of SR. It was suggested that adrenocortical microsomes contain a Ca2+ dependent ATPase which function as a Ca2+ pump with similar properties to that of SR.
Sumida
M
M
Second Department of Medical Biochemistry, Ehime University School of Medicine.
Hamada
M
M
Shimowake
A
A
Morimoto
C
C
Okuda
H
H
D000244:Adenosine Diphosphate / Q000494:pharmacology; D000302:Adrenal Cortex / Q000378:metabolism*; D000818:Animals; D001693:Biological Transport, Active / Q000187:drug effects; D000001:Calcimycin / Q000494:pharmacology; D002118:Calcium / Q000493:pharmacokinetics*; D000252:Calcium-Transporting ATPases / Q000378:metabolism*; D002417:Cattle; D008274:Magnesium / Q000494:pharmacology; D008861:Microsomes / Q000378:metabolism*; D010750:Phosphoproteins / Q000302:isolation & purification / Q000378:metabolism; D013696:Temperature; D014638:Vanadates / Q000494:pharmacology
D010750:Phosphoproteins; D000001:Calcimycin; D014638:Vanadates; D000244:Adenosine Diphosphate; D000252:Calcium-Transporting ATPases; D008274:Magnesium; D002118:Calcium