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Biochimica et Biophysica Acta, 543 (1978) 1 0 6 - - 1 1 5 © E l s e v i e r / N o r t h - H o l l a n d Biomedical Press
BBA 28655
E X T R A C T I O N OF M I T O C H O N D R I A L PROTEINS BY VOLATILE ANESTHETICS
J.J. R I C H T E R *, E. S U N D E R L A N D , U. J U H L and S. K O R N G U T H **
University of Wisconsin Medical Center, Departments of Anesthesiology, Neurology and Physiological Chemistry, Madison, Wisc. 53706 (U.S.A.) (Received F e b r u a r y 1 6 t h , 1978)
Summary Heavy beef heart mitochondria were exposed to controlled concentrations of several volatile anesthetics including halothane, enflurane and chloroform. These anesthetics caused a concentration-dependent release of protein from mitochondria with maximal release occurring at 17.5% halothane and less release at lower and higher concentrations. The proteins released into the supernatants were analyzed by electrophoresis on slab gels containing a 6--20% gradient of acrylamide. The anesthetics caused the release of several polypeptides from mitochondria into the incubation medium; the major polypeptides released had molecular weights of 78 000; 48 000; 47 000; 43 000; 32 000 and 22 000. Two of these were identified by enzyme analysis and by co-electrophoresis with crystalline enzymes as the subunits of aspartate aminotransferase (43 000 daltons; EC 2.6.1.1) and malate dehydrogenase (32 000 daltons; EC 1.1.1.37}. Mitochondria exposed to saturated halothane vapors were similar ultrastructurally to controls except that the halothane mitochondria appeared uncoupled. Similar results were obtained with 02 or N: as carrier gas.
Introduction Polyhalogenated volatile anesthetics, such as halothane, have a variety of effects on membranes. They interact with biological or artificial membranes to increase the membrane volume [1,2] and decrease membrane density [3]. NMR spectra [4] indicate that such anesthetics fluidize and expand membranes * Current address: D e p a r t m e n t o f A n e s t h e s i o l o g y , Milton S. Hershey Medical Center, Pennsylvania State University, Hershey. Pa. 17033, U.S.A. ** To w h o m c o r r e s p o n d e n c e s h o u l d be addressed. Abbreviations: TEMED, N,N,N~,Nr-tetramethyl-ethylenediamine; SDS, s o d i u m d o d e c y l sulfate.
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and ESR studies [5] reveal that halothane induced a phase transition in phospholipid vesicles. Because anesthetics cause these physical-chemical changes in membranes, we are studying their effects on the composition and orientation of membrane proteins. In this laboratory, we have recently described some features of protein composition and topography in synaptic mad mitochondrial membranes [6,7]. Heavy beef heart mitochondria were selected for the studies in this report for several reasons. They may be highly purified and their protein composition and organization has been well described. Analysis of mitochondria also will simplify further studies of synaptic membranes because mitochondria are present both within synaptosomal particles and as free organelles in such preparations [8]. The data presented here indicate that certain volatile anesthetics can extract selected proteins from mitochondrial membranes. Materials and Methods Beef heart mitochondria (heavy), prepared by the method of Crane et al. [9], were obtained from the laboratory of Dr. David Green at the University of Wisconsin Institute for Enzyme Research. The anesthetics were halothane (2-bromo-2-chloro-l,l,l-trifluoroethane from Ayerst); enflurane (2~chloro-l,l,2-trifluoroethyl
Biochimica et Biophysica Acta, 543 (1978) 1 0 6 - - 1 1 5 © E l s e v i e r / N o r t h - H o l l a n d Biomedical Press
BBA 28655
E X T R A C T I O N OF M I T O C H O N D R I A L PROTEINS BY VOLATILE ANESTHETICS
J.J. R I C H T E R *, E. S U N D E R L A N D , U. J U H L and S. K O R N G U T H **
University of Wisconsin Medical Center, Departments of Anesthesiology, Neurology and Physiological Chemistry, Madison, Wisc. 53706 (U.S.A.) (Received F e b r u a r y 1 6 t h , 1978)
Summary Heavy beef heart mitochondria were exposed to controlled concentrations of several volatile anesthetics including halothane, enflurane and chloroform. These anesthetics caused a concentration-dependent release of protein from mitochondria with maximal release occurring at 17.5% halothane and less release at lower and higher concentrations. The proteins released into the supernatants were analyzed by electrophoresis on slab gels containing a 6--20% gradient of acrylamide. The anesthetics caused the release of several polypeptides from mitochondria into the incubation medium; the major polypeptides released had molecular weights of 78 000; 48 000; 47 000; 43 000; 32 000 and 22 000. Two of these were identified by enzyme analysis and by co-electrophoresis with crystalline enzymes as the subunits of aspartate aminotransferase (43 000 daltons; EC 2.6.1.1) and malate dehydrogenase (32 000 daltons; EC 1.1.1.37}. Mitochondria exposed to saturated halothane vapors were similar ultrastructurally to controls except that the halothane mitochondria appeared uncoupled. Similar results were obtained with 02 or N: as carrier gas.
Introduction Polyhalogenated volatile anesthetics, such as halothane, have a variety of effects on membranes. They interact with biological or artificial membranes to increase the membrane volume [1,2] and decrease membrane density [3]. NMR spectra [4] indicate that such anesthetics fluidize and expand membranes * Current address: D e p a r t m e n t o f A n e s t h e s i o l o g y , Milton S. Hershey Medical Center, Pennsylvania State University, Hershey. Pa. 17033, U.S.A. ** To w h o m c o r r e s p o n d e n c e s h o u l d be addressed. Abbreviations: TEMED, N,N,N~,Nr-tetramethyl-ethylenediamine; SDS, s o d i u m d o d e c y l sulfate.
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and ESR studies [5] reveal that halothane induced a phase transition in phospholipid vesicles. Because anesthetics cause these physical-chemical changes in membranes, we are studying their effects on the composition and orientation of membrane proteins. In this laboratory, we have recently described some features of protein composition and topography in synaptic mad mitochondrial membranes [6,7]. Heavy beef heart mitochondria were selected for the studies in this report for several reasons. They may be highly purified and their protein composition and organization has been well described. Analysis of mitochondria also will simplify further studies of synaptic membranes because mitochondria are present both within synaptosomal particles and as free organelles in such preparations [8]. The data presented here indicate that certain volatile anesthetics can extract selected proteins from mitochondrial membranes. Materials and Methods Beef heart mitochondria (heavy), prepared by the method of Crane et al. [9], were obtained from the laboratory of Dr. David Green at the University of Wisconsin Institute for Enzyme Research. The anesthetics were halothane (2-bromo-2-chloro-l,l,l-trifluoroethane from Ayerst); enflurane (2~chloro-l,l,2-trifluoroethyl
