ELECTRON-PARAMAGNETIC RESONANCE STUDIES OF MEMBRANE-PROTEINS IN HEPATIC MICROSOMES

Rat hepatic microsomal membranes, prepared under various conditions that yield either intact or disrupted microsomal vesicles, were labeled via the SH groups of intrinsic membrane proteins using nitroxide analogs of N-ethylmaleimide. EPR spectra revealed the presence of 2 dominant classes of bound label corresponding to differing degrees of immobilization, the ratio of which was quantitated using a parameter designated the W/S ratio. For latent microsomes, the value of this parameter was determined to be 0.65 .+-. 0.02, and was influenced by factors such as label/protein ratio, incubation period, nitroxide structure, temperature and pH. The W/S ratio was also sensitive to the degree of membrane integrity, as revealed by the latency of mannose 6-phosphate activity of glucose-6-phosphohydrolase. In addition, membrane disruption resulted in a corresponding decrease in the order parameter for nitroxide-labeled fatty acids intercalated within the lipid bilayer. The W/S ratio was dependent upon the method of microsome preparation, yielding values of 1.02 .+-. 0.02 for hypertonically disrupted vesicles and 1.28 .+-. 0.02 for mechanically disrupted vesicles. Microsomal marker enzymes, such as cytochrome P-450 and FAD-containing monooxygenase, retained significant levels of functionality following nitroxide incorporation.