Triton solubilization of proteins from pig liver mitochondrial membranes.
Various aspects of membrane solubilization by the Triton X-series of nonionic detergents were examined in pig liver mitochondrial membranes. Binding of Triton X-100 to nonsolubilized membranes was saturable with increased concentrations of the detergent. Maximum binding occurred at concentrations exceeding 0.5% Triton X-100 (w/v). Solubilization of both protein and phospholipid increased with increasing Triton X-100 to a plateau which was dependent on the initial membrane protein concentration used. At low detergent concentrations (less than 0.087% Triton X-100, w/v), proteins were preferentially solubilized over phospholipids. At higher Triton X-100 concentrations the opposite was true. Using the well-defined Triton X-series of detergents, the optimal hydrophile-lipophile balance number (HLB) for solubilization of phosphatidylglycerophosphate synthase (EC 2.7.8.5) was 13.5, corresponding to Triton X-100. Activity was solubilized optimally at detergent concentrations between 0.1 and 0.2% (w/v). The optimal protein-to-detergent ratio for solubilization was 3 mg protein/mg Triton X-100. Solubilization of phosphatidylglycerophosphate synthase was generally better at low ionic strength, though total protein solubilization increased at high ionic strength. Solubilization was also dependent on pH. Significantly higher protein solubilization was observed at high pH (i.e., 8.5), as was phosphatidylglycerophosphate synthase solubilization. The manipulation of these variables in improving the recovery and specificity of membrane protein solubilization by detergents was examined.
D000818:Animals; D006863:Hydrogen-Ion Concentration; D008565:Membrane Proteins / Q000302:isolation & purification*; D008930:Mitochondria, Liver / Q000032:analysis*; D017830:Octoxynol; D009994:Osmolar Concentration; D010743:Phospholipids / Q000302:isolation & purification; D010770:Phosphotransferases / Q000302:isolation & purification; D011092:Polyethylene Glycols / Q000494:pharmacology*; D011755:Pyrophosphatases / Q000302:isolation & purification; D012995:Solubility; D013552:Swine; D017855:Transferases (Other Substituted Phosphate Groups)
D008565:Membrane Proteins; D010743:Phospholipids; D011092:Polyethylene Glycols; D017830:Octoxynol; D010770:Phosphotransferases; D017855:Transferases (Other Substituted Phosphate Groups); C020676:CDP-diacylglycerol-glycerol-3-phosphate 3-phosphatidyltransferase; D011755:Pyrophosphatases; C019855:CDPdiacylglycerol pyrophosphatase