Role of phospholipid and protein-protein associations in activation and stabilization of soluble calcium-ATPase of sarcoplasmic reticulum

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Abstract
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 ATPase (Ca2+-ATPase) of sarcoplasmic reticulum was investigated. Increasing concentrations of Triton X-100 in the range 0.2-0.6% (wt/vol) 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.degree. C was monophasic and 1st 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 > 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. Evidently, with increasing Triton X-100 concentrations, inhibition of catalysis, destabilization, loss of protein-protein associations and loss of phospholipid occur concurrently. It was possible to maintain the monomeric state, reverse the inhibition of ATP and p-nitrophenyl phosphate hydrolysis, and restore stability by adding soybean phospholipid to the monomeric enzyme in 2% Triton X-100. Associated phospholipid, and not protein-protein association, is apparently the principle determinant of the activity and stability of Ca2+-ATPase in Triton X-100 solutions. Mixtures of micelles containing 1, 2 or more ATPases may be in slow equilibrium and each ATPase is equally unstable in excess EGTA. Evidently, while Triton X-100 can substitute for phospholipids in supporting catalytic activity, although at a slower rate especially with p-nitrophenyl phosphate as substrate, it cannot substitute for phospholipids in maintaining a stable native enzyme structure, and this suggests a specific phospholipid-ATPase interaction.