Stability and partial reactions of soluble and membrane-bound sarcoplasmic reticulum ATPase.
The Ca-ATPase of sarcoplasmic reticulum was solubilized at pH 6.5 and 30 degrees C using different nonionic detergents, Triton X-100, C12E8, Lubrol PX, or Tween 20. After full solubilization by any of these detergents, the enzyme was unstable (t1/2 = 2-3 min) in the absence of Ca2+. The soluble enzyme was stable in the presence of calcium, half-maximal protection being attained in the presence of 0.2 mM Ca2+. In the absence of Ca2+, stability was restored by addition of co-solvents dimethyl sulfoxide or glycerol. In the presence of 4 mM Ca2+, the progressive addition of nonionic detergents to a medium containing leaky vesicles promoted an increase, up to 3-fold, in the rate of ATP hydrolysis. This was not observed when ITP was used as substrate. The small amount of ADP accumulated in the medium during ATP hydrolysis was sufficient to inhibit the ATPase activity of the membrane-bound enzyme but had no effect on the soluble enzyme. Increasing concentrations of detergent promoted a progressive inhibition of the ATP----Pi exchange reaction. The ATP hydrolysis/synthesis ratio of soluble enzyme was 10 times higher than that of membranous enzyme. Addition of co-solvent restored this ratio to values similar to those obtained with membrane-bound Ca-ATPase. Soluble enzyme prepared from native sarcoplasmic reticulum vesicles was able to catalyze the net synthesis of ATP when phosphorylated by Pi in the presence of dimethyl sulfoxide and then diluted in a medium containing 10 mM CaCl2 and 2 mM ADP. This was not observed when the soluble enzyme was prepared from purified Ca-ATPase. The results suggest that some of the partial reactions of the catalytic cycle of Ca-ATPase are dependent on the hydrophobic environment found in the native membrane. This environment can be mimicked by co-solvents.
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D000244:Adenosine Diphosphate / Q000494:pharmacology; D000818:Animals; D000252:Calcium-Transporting ATPases / Q000378:metabolism*; D003902:Detergents / Q000494:pharmacology; D005990:Glycerol / Q000494:pharmacology; D007700:Kinetics; D008566:Membranes / Q000201:enzymology; D010766:Phosphorylation; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*; D012995:Solubility
D003902:Detergents; D000244:Adenosine Diphosphate; D000252:Calcium-Transporting ATPases; D005990:Glycerol