Solubilization of sarcoplasmic reticulum with Triton X-100

The nonionic detergent Triton X-100 has been found to solubilize approximately 80% of the protein, and nearly all the phospholipid from sarcoplasmic reticulum membranes. The soluble fraction contains Ca²⁺ dependent ATPase, but no basic ATPase activity. In comparative experiments, Triton X-100 causes much less enzyme denaturation than ionic detergents. Analytical ultracentrifugation and disc electrophoresis indicate that the Triton-solubilized material consists of particles having a a molecular weight of approximately 80,000 daltons. These particles have a strong tendency to aggregate into size isomers. The aggregation proceeds at a faster rate in the presence of MgCl₂, and is temperature dependent with an optimum at 25°. When the solubilized fraction is centrifuged on a sucrose density gradient, [³²P]-labeled protein and phospholipid are separated, and ATPase activity is lost. If ATP is present in the density gradient during centrifugation, both ATPase inactivation and separation of protein from phospholipid are partially prevented. Gel electrophoresis of SR solubilized with sodium dodecylsulfate (SDS) shows that approximately 60% of the membrane protein migrates in a band corresponding to a molecular weight of 90,000 daltons. This fraction is absent in residues of SR previously extracted with Triton. Minor components are also separated in the SDS gels.