title: Electron paramagnetic resonance studies of membrane proteins in hepatic microsomes.
abstract: Hepatic microsomal membranes, prepared under various conditions that yield either 'intact' or 'disrupted' microsomal vesicles, have been labeled via the sulfhydryl groups of intrinsic membrane proteins using nitroxide analogs of N-ethylmaleimide. Electron paramagnetic resonance spectra revealed the presence of two dominant classes of bound label corresponding to differing degrees of immobilization, the ratio of which were 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 observed to be 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.
mesh_terms: D000345:Affinity Labels / Q000378:metabolism; D000818:Animals; D003497:Cyclic N-Oxides / Q000378:metabolism; D004578:Electron Spin Resonance Spectroscopy; D006863:Hydrogen-Ion Concentration; D007425:Intracellular Membranes / Q000032:analysis; D008297:Male; D008565:Membrane Proteins; D008862:Microsomes, Liver / Q000201:enzymology / Q000648:ultrastructure*; D051381:Rats; D011919:Rats, Inbred Strains; D013696:Temperature
chemical_list: D000345:Affinity Labels; D003497:Cyclic N-Oxides; D008565:Membrane Proteins; C017480:4-maleimido-2,2,6,6-tetramethylpiperidinooxyl