Form or Function?

Potter, William Gray

doi:10.1300/j123v06n01_12

Cited by 6 publications

(6 citation statements)

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“…Bovine heart ventricles were used for the purification of all of the nonrecombinant (native) contractile proteins. TnC, TnI, and TnT were purified according to the method of Potter (44) and stored at Ϫ70°C in 50 mM Tris-HCl (pH 8.0) containing 6 M urea, 1 mM EDTA, and 15 mM 2-mercaptoethanol. Tm was prepared by the method of Stull and Buss (45) and F-actin by the procedures of Pardee and Spudich (46).…”

Section: Methodsmentioning

confidence: 99%

“…Phosphorylation of Wild-type TnI and Mutants-Prior to phosphorylation, all TnI preparations were dialyzed against dialysis buffer containing 10 mM Tris-HCl (pH 7.5) and 1 mM DTT with sequential KCl concentrations of 1, 0.7, and 0.3 M, according to the method of Potter (44). The conditions for phosphorylation by PKC and/or PKA were essentially the same as described previously (29,(31)(32)(33).…”

Section: Methodsmentioning

confidence: 99%

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Cardiac Troponin I Mutants

Noland

Guo

Raynor

et al. 1995

Journal of Biological Chemistry

157

173

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2؉sensitivity of the myofilament.It is suggested that Ser-43/Ser-45 and Ser-23/Ser-24 in cardiac TnI are important for normal Ca 2؉ sensitivity of the myofilament, and that phosphorylation of Ser-43/ Ser-45 and Ser-23/Ser-24 is primarily involved in the protein kinase C regulation of the activity and Ca 2؉ sensitivity, respectively, of actomyosin S-1 MgATPase.In cardiac myocytes, the activation of several types of receptors, such as ␣ 1 -adrenergic (1-5), muscarinic (1, 6), and purinergic (6) dynorphin A (7), endothelin-1 (8, 9), and angiotensin (14 -18). However, the complex molecular events mediated by PKC (or more precisely, by the individual isozymes) that are responsible for cardiac contractility regulation, for example, remain largely unclear. It has been reported that phenylephrine (␣ 1 -adrenergic receptor agonist) elicited transient negative inotropy followed by sustained positive inotropy (3, 19 -21), endothelin-1 caused monotonic positive inotropy (22), whereas dynorphin A (-opioid receptor agonist) induced negative inotropy (7). All three of these distinct receptor agonists are believed to act, at least in part, through PKC activation. Furthermore, phorbol esters (such as TPA), potent and long-acting PKC activators, produced predominantly negative inotropic effects in various cardiac preparations (23-27). These seemingly paradoxical observations might reflect certain opposing factors of PKC activation which include the net effects of intracellular pH change, the size of intracellular Ca 2ϩ transient, and the states and species of cellular proteins being phosphorylated.One target for PKC in the heart is the contractile apparatus itself. Cardiac TnI and TnT from the thin filament have been shown to be effective substrates for PKC (28), and some of the phosphorylation sites in these proteins have been determined (29,30). Phosphorylation of TnI and/or TnT by PKC resulted in an inhibition of Ca 2ϩ -stimulated MgATPase of the reconstituted actomyosin complex (31-33) or in native myofibril preparations (32, 34), an effect associated with altered interactions among the contractile protein components (32,33). PKC also phosphorylated MLC2 (34 -36) and C-protein (34 -36) in myofibrillar and thick filament preparations. Phosphorylation of

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Cardiac Troponin I Mutants

Noland

Guo

Raynor

et al. 1995

Journal of Biological Chemistry

157

173

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“…Bovine heart was collected from a local slaughterhouse and stored at -20 °C. Crude troponin was extracted from tissues essentially as described by Potter (1982) to the stage of a 30-50% ammonium sulfate precipitation. This precipitate was dissolved in low-salt column buffer A and dialyzed overnight in 100 vol of buffer A.…”

Section: Methodsmentioning

confidence: 99%

Cloning and Expression of Salmon Cardiac Troponin C: Titration of the Low-Affinity Ca²⁺-Binding Site Using a Tryptophan Mutant

Cd¹,

Borgford²,

Leblanc³

et al. 1996

Biochemistry

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Activation of cardiac actomyosin ATPase requires the occupation of the single low-affinity Ca(2+)-binding site of troponin C (cTnC). Previously, we demonstrated pronounced differences between mammals and cold-water salmonid fish in the Ca2+ sensitivity of cardiac preparations, particularly in relation to temperature [Churcotte, C., Moyes, C. D., Baldwin, K., Bressler, B., & Tibbits, G. F. (1994) Am. J. Physiol. 267, R62-R70]. In this study, we examine the extent to which cTnC structure could account for the observed differences in myofibrillar Ca2+ sensitivity. Salmonid (Oncorhynchus mykiss) cTnC was cloned, sequenced, and expressed in Escherichia coli as a maltose-binding protein fusion. The coding region has 87% homology with human cTnC cDNA and differs in 13 of 161 amino acid residues from the human/bovine/porcine isoform. The sequence corresponding to the single regulatory Ca(2+)-binding site II is completely homologous to that of mammals. The protein expressed exhibits optical properties similar (circular dichroism, intrinsic fluorescence) to those of cTnC purified from salmonid (Salmo salar) and bovine ventricle. A single tryptophan residue was introduced into the inactive Ca(2+)-binding site I (ScTnC-FW27) to facilitate Ca2+ titration. The Ca(2+)-binding constant (K1/2 = 5.33 pCa units) was within the range reported for the low-affinity sites of mammalian cTnC. Although differences in TnC primary structure are striking, Ca2+ affinity of intact cardiac myofibrils is likely influenced by interactions with other troponin proteins.

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“…Crude troponin was initially prepared from bovine cardiac and rabbit back skeletal muscle ether powder preparations (Potter, 1982). Final purification of cardiac and skeletal troponin subunits was performed by ion exchange column chromatography as detailed by Potter (1982).…”

Section: Preparationsmentioning

confidence: 99%

Isoform Specific Interactions of Troponin I and Troponin C Determine pH Sensitivity of Myofibrillar Ca2+ Activation

Ball¹,

Johnson²,

Solaro³

1994

Biochemistry

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We investigated whether differences in isoforms of troponin I (TnI) and troponin C (TnC) can account for the greater inhibition of Ca(2+)-dependent MgATPase activity by acidic pH in cardiac (c) than in fast skeletal (fs) myofilaments. We studied fast skeletal myofibrils from which whole Tn was extracted by displacement with excess fsTnT (the tropomyosin binding subunit of Tn) followed by reconstitution with TnC-TnI. Exchange of fsTnI with cTnI did not alter the effect of a drop in pH from 7.0 to 6.5 on the relation between pCa (-log[Ca2+]) and MgATPase activity of fast skeletal myofibrils. Exchange of fsTnC with cTnC did, however, induce an increase in the effect of this same pH change on Ca2+ activation. Yet, the pH sensitivity of Ca2+ activation of fast skeletal myofibrils containing cTnC was not as great as that of native cardiac myofibrils. However, when both fsTnC and fsTnI of fast skeletal myofibrils were replaced by cTnC-cTnI, there was a pH-induced shift in Ca2+ sensitivity similar to that of cardiac myofibrils. In studies using fluorescent probes, both pure fsTnC and pure cTnC showed decreased Ca2+ binding as pH was lowered. This decrease was potentiated in the fsTnC-fsTnI and cTnC-cTnI complexes. However, the effect of acidic pH was the same in fsTnC and the hybrid complex, fsTnC-cTnI, and in cTnC and the hybrid complex, cTnC-fsTnI. Thus, isoform specific interactions between TnI and TnC appear important in the differential response of skeletal and cardiac myofilaments to acidosis.

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Form or Function?

Cited by 6 publications

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Cardiac Troponin I Mutants

Cardiac Troponin I Mutants

Cloning and Expression of Salmon Cardiac Troponin C: Titration of the Low-Affinity Ca²⁺-Binding Site Using a Tryptophan Mutant

Isoform Specific Interactions of Troponin I and Troponin C Determine pH Sensitivity of Myofibrillar Ca2+ Activation

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Form or Function?

Cited by 6 publications

References 0 publications

Cardiac Troponin I Mutants

Cardiac Troponin I Mutants

Cloning and Expression of Salmon Cardiac Troponin C: Titration of the Low-Affinity Ca2+-Binding Site Using a Tryptophan Mutant

Isoform Specific Interactions of Troponin I and Troponin C Determine pH Sensitivity of Myofibrillar Ca2+ Activation

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Cloning and Expression of Salmon Cardiac Troponin C: Titration of the Low-Affinity Ca²⁺-Binding Site Using a Tryptophan Mutant