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Quantitative analysis of myocardial adrenergic receptors: False-positive cooperative interactions secondary to endogenous catecholamines
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CREATINEKINASE SOLUBILIZATION FROMHEARTMITOCHONDRIALINNER MEMBRANE ROLE OF SH GROUPSREAGENTS.B. Font, C. Vial and D. Goldschmidt. Laboratoire de Biologie et de Technologie des membranes, Universite Claude Bernard, 69622 Villeurbanne, France. We have previously shown that modifications like increased inorganic phosphate concentration and mitochondrial swelling which occur during myocardial ischemia are able to induce mitochondrial creatine kinase (CK) solubilization. CK release can also occur in isotonic KC1 when mitochondrial swelling is induced by addition of pHMBwhich allows Cl- and K+ penetration (Hunter ti &, 1969). In this study we show that organic mercurials (pHMB, pCMS, mersalyl) are able to induce CK solubilization even in the absence of KC1 (mitochondria suspended in distilled water), whereas disulfides (DTNB, CPDS, DTP) are ineffective. Furthermore our results show that CK solubilization by pHMB can be prevented or reversed by addition of an excess of thiol (DTT) and that a charged negative group is required to induce enzyme solubilization, since uncharged mercurials like phenylmercury chloride do not promote CK release. Our results also show that CK solubilization which is low (about 5 %> when only 3 nmoles of 14C pCMB are bound per mg protein, is maximal (75 %) when 8 nmoles SH/mg prot. are titrated by 14C pCMB. We conclude from our results that SH groups which could belong either to the enzyme or to the mitochondrial membrane could be implicated in the association of creatine kinase with heart mitochondria.
QUANTITATIVE ANALYSIS OF MYOCARDIAL ADRENERGIC RECEPTORS: FALSEPOSITIVE COOPERATIVE INTERACTIONS SECONDARY TO ENDOGENOUS CATECHOLAMINES. Forfar, J.C., Riemersma, R.A. and Oliver, M.F. Cardiovascular Research Unit, University of Edinburgh, George Square, EDINBURGH, Eli8 9XF, SCOTLAND. Adrenergic receptor modification may be an important mechanism of altered sympathetic responsiveness in several pathological states. A high affinity, high specific activity ligand (1251) Iodohydroxybenzylpindolol ((195X IHYP) was used to assess beta-adrenergic receptor populations in a crude ventricular membrane preparation from rat 5 and 50pM, myocardium. At concentrations of ( 1951) IBYP between specific ligand binding did not increase as expected, producing a sigmoid saturation isotherm suggesting positive cooperativity between receptor sites. The reduction in expected specific binding was closely related to the endogenous catecholamine concentration of the membrane fraction. Following depletion of endogenous catecholamines either by --in vivo or --in vitro techniques, a conventional single beta-receptor population of uniform affinity was demonstrated. At low ligand concentrations, endogenous hormones may interfere with receptor analysis and suggest false receptor site interactions.
CREATINEKINASE SOLUBILIZATION FROMHEARTMITOCHONDRIALINNER MEMBRANE ROLE OF SH GROUPSREAGENTS.B. Font, C. Vial and D. Goldschmidt. Laboratoire de Biologie et de Technologie des membranes, Universite Claude Bernard, 69622 Villeurbanne, France. We have previously shown that modifications like increased inorganic phosphate concentration and mitochondrial swelling which occur during myocardial ischemia are able to induce mitochondrial creatine kinase (CK) solubilization. CK release can also occur in isotonic KC1 when mitochondrial swelling is induced by addition of pHMBwhich allows Cl- and K+ penetration (Hunter ti &, 1969). In this study we show that organic mercurials (pHMB, pCMS, mersalyl) are able to induce CK solubilization even in the absence of KC1 (mitochondria suspended in distilled water), whereas disulfides (DTNB, CPDS, DTP) are ineffective. Furthermore our results show that CK solubilization by pHMB can be prevented or reversed by addition of an excess of thiol (DTT) and that a charged negative group is required to induce enzyme solubilization, since uncharged mercurials like phenylmercury chloride do not promote CK release. Our results also show that CK solubilization which is low (about 5 %> when only 3 nmoles of 14C pCMB are bound per mg protein, is maximal (75 %) when 8 nmoles SH/mg prot. are titrated by 14C pCMB. We conclude from our results that SH groups which could belong either to the enzyme or to the mitochondrial membrane could be implicated in the association of creatine kinase with heart mitochondria.
QUANTITATIVE ANALYSIS OF MYOCARDIAL ADRENERGIC RECEPTORS: FALSEPOSITIVE COOPERATIVE INTERACTIONS SECONDARY TO ENDOGENOUS CATECHOLAMINES. Forfar, J.C., Riemersma, R.A. and Oliver, M.F. Cardiovascular Research Unit, University of Edinburgh, George Square, EDINBURGH, Eli8 9XF, SCOTLAND. Adrenergic receptor modification may be an important mechanism of altered sympathetic responsiveness in several pathological states. A high affinity, high specific activity ligand (1251) Iodohydroxybenzylpindolol ((195X IHYP) was used to assess beta-adrenergic receptor populations in a crude ventricular membrane preparation from rat 5 and 50pM, myocardium. At concentrations of ( 1951) IBYP between specific ligand binding did not increase as expected, producing a sigmoid saturation isotherm suggesting positive cooperativity between receptor sites. The reduction in expected specific binding was closely related to the endogenous catecholamine concentration of the membrane fraction. Following depletion of endogenous catecholamines either by --in vivo or --in vitro techniques, a conventional single beta-receptor population of uniform affinity was demonstrated. At low ligand concentrations, endogenous hormones may interfere with receptor analysis and suggest false receptor site interactions.