Role of phospholipid and protein-protein associations in activation and stabilization of soluble Ca2+-ATPase of sarcoplasmic reticulum.
The effect of increasing concentrations of the nonionic detergent Triton X-100 on catalytic activity, stability, phospholipid content, and aggregational state of solubilized Ca2+ ion activated adenosinetriphosphatase (Ca2+-ATPase) of sarcoplasmic reticulum has been investigated. Increasing concentrations of Triton X-100 in the range 0.2-0.6% (w/v) inhibited ATP hydrolysis and p-nitrophenyl phosphate hydrolysis in parallel to the extent of 50% and 95%, respectively. Inactivation of p-nitrophenyl phosphate hydrolysis by preincubation in excess ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) at 25 degrees C was monophasic and first order at all concentrations of Triton X-100. The rate constant for inactivation increased sharply in the range 0.1-0.6% Triton X-100. At higher concentrations, the increase was less marked. Protein-protein associations of the solubilized ATPase were assessed by glutaraldehyde cross-linking and by ultracentrifugation in sucrose gradients. Both methods indicated a decrease in these associations in the 0.1-0.5% range. Cross-linking studies established that above 0.5% Triton X-100 the enzyme is greater than 90% monomeric. The amount of phospholipid associated with the ATPase, recovered from sucrose gradients, decreased from about 50 mol of phospholipid/mol of ATPase at 0.1% Triton X-100 to about 3 mol of phospholipid/mol of ATPase at 0.5% and higher concentrations. Monomeric ATPase and aggregated ATPase isolated from equilibrium mixtures of these components had similar phospholipid/protein ratios. The results indicated that with increasing Triton X-100 concentrations, inhibition of catalysis, destabilization, loss of protein-protein associations, and loss of phospholipid occur concurrently.(ABSTRACT TRUNCATED AT 250 WORDS)
D000818:Animals; D000252:Calcium-Transporting ATPases / Q000302:isolation & purification / Q000378:metabolism*; D003432:Cross-Linking Reagents; D003902:Detergents / Q000494:pharmacology; D005976:Glutaral / Q000494:pharmacology; D007700:Kinetics; D008563:Membrane Lipids / Q000502:physiology*; D009132:Muscles / Q000201:enzymology; D017830:Octoxynol; D010713:Phosphatidylcholines / Q000494:pharmacology; D010743:Phospholipids / Q000502:physiology*; D011092:Polyethylene Glycols / Q000494:pharmacology; D011485:Protein Binding; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*
D003432:Cross-Linking Reagents; D003902:Detergents; D008563:Membrane Lipids; D010713:Phosphatidylcholines; D010743:Phospholipids; D011092:Polyethylene Glycols; D017830:Octoxynol; D000252:Calcium-Transporting ATPases; D005976:Glutaral