Electron spin resonance studies on the selective labeling of N-(1-oxyl-2,2,5,5-tetramethyl-3-pyrrolidinyl)maleimide of rat liver mitochondria.
N-(1-Oxyl-2,2,5,5-tetramethyl-3-pyrrolidinyl)maleimide (MSL) was incorporated into rat liver mitochondria and the nitroxide radical incorporated was found to decay considerably. The incorporation was blocked by a high concentration of NEM, but not by pCMB. Spin labeled fatty acid derivatives, 2-(3-carboxypropyl)-2-tridecyl-4,4-dimethyl-3-oxazolidinyloxyl (FSL1) and 2-(14-carboxytetradecyl)-2-ethyl-4,4-dimethyl-3-oxazolidinyloxyl (FSL2), were also incorporated and the nitroxide radical decayed. However, incorporation of FSL1 or FSL2 was not blocked by NEM or pCMB. The ESR spectrum of 3-carboxyl-2,2,5,5-tetramethyl-pyrroline-1-oxyl (CSL) did not change on reaction with the mitochondria. The labeled MSL exhibited an ESR spectrum composed of both strongly immobilized and weakly immobilized components. A similar reaction with FSL1 gave an ESR spectrum mainly composed of a strongly immobilized component, the weakly immobilized component was negligibly small, while FSL2 exhibited an ESR spectrum in which free-like signals of the nitroxide radical were predominant. The results suggest that MSL is labeled selectively in the mitochondrial membrane through those SH groups that are not reactive to pCMB, and the labeled nitroxide radical is reduced in situ. The mode of incorporation into the mitochondria differs between MSL and the other spin labeled reagents, and labeling of MSL at the binding site may precede reduction of the nitroxide radical. The incorporation of MSL was dependent on the concentration of MSL used. ADP-acceleration of mitochondrial oxygen uptake with succinate was inhibited by labeling the mitochondria with MSL without loss of the electron transferring activity.
D000818:Animals; D002729:Chloromercuribenzoates / Q000494:pharmacology; D003497:Cyclic N-Oxides; D004578:Electron Spin Resonance Spectroscopy; D005033:Ethylmaleimide / Q000494:pharmacology; D066298:In Vitro Techniques; D008930:Mitochondria, Liver / Q000378:metabolism*; D011506:Proteins / Q000378:metabolism; D051381:Rats; D013113:Spin Labels; D020245:p-Chloromercuribenzoic Acid
D002729:Chloromercuribenzoates; D003497:Cyclic N-Oxides; D011506:Proteins; D013113:Spin Labels; C024516:N-(1-oxyl-2,2,5,5-tetramethyl-3-pyrrolidinyl)maleimide; D020245:p-Chloromercuribenzoic Acid; D005033:Ethylmaleimide