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 adenosinetri-phosphatase (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(β-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) at 25 °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 >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. However, 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. This suggests that associated phospholipid, and not protein-protein association, is the principle determinant of the activity and stability of Ca2+-ATPase in Triton X-100 solutions. We propose that mixtures of micelles containing one, two, or more ATPases are in slow equilibrium and each ATPase is equally unstable in excess EGTA. It is evident that 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. © 1985, American Chemical Society. All rights reserved.
