Characterization of myosin light chains from histochemically identified fibres of rabbit psoas muscle

Weeds, Alan G.; Hall, R. J. C.; Spurway, Neil

doi:10.1016/0014-5793(75)80776-9

Cited by 130 publications

(45 citation statements)

References 16 publications

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“…All SDS/polyacrylamide gels were composed of 12% bisacrylamide, 0.1 M Tris-bicine, pH 8.3, 0.2% SDS, 0.5% N,N,N:N'-tetramethylethylenediamine (TEMED) and 0.1 % ammonium persulphate [16]. All samples were boiled in 0.8% SDS and bromophenol blue/glycerol were added prior to loading onto the gel.…”

Section: Gel Electrophoresismentioning

confidence: 99%

Overexpression of Human Cardiac Troponin‐I and Troponin‐C in Escherichia coli and Their Purification and Characterisation

Al-Hillawi

Minchin

Trayer

1994

European Journal of Biochemistry

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We have overexpressed human cardiac troponin-I in Escherichia coli. Initially, protein expression was not detected in the bacterial cell extracts. Systematic deletion of the N-terminal region of the protein generated a series of truncated mutants which were expressed at varying levels in the bacteria. This allowed us to narrow the problem down to the first five codons in the gene sequence. In order to achieve expression at high levels, two base changes were required, in the second and the fourth codons of the cDNA sequence. The codon changes, (Ala2) GCG+GCC and (Gly4) GGG+ GGT, do not alter the coding potential of the DNA. We have also overexpressed the human cardiac isoform of troponin-C. Both proteins were purified using ion-exchange chromatography and have been proved to be biologically active. The recombinant troponin-I was able to bind to a troponin-C affinity column in the presence of 9 M urea in a calcium-dependent manner. The calcium-dependent troponin-I -troponin-C complex between both recombinant proteins was also demonstrated by alkaline-urea gel electrophoresis. In addition, troponin-I inhibited the acto-Sl MgATPase activity ; this inhibition was potentiated by the presence of tropomyosin and was reversed by the addition of troponin-C to the system. Biological activity was also demonstrated in vivo in that the recombinant proteins were able to restore the calcium-dependent force generation to calcium-insensitive skinned muscle fibres.Troponin and tropomyosin, located on the actin thin filament of vertebrate skeletal and cardiac muscles, are responsible for the calcium-dependent regulation of the actomyosin Mg-ATPase activity [l]. Troponin is a complex of three subunits ; troponin-C (Tn-C), the calcium-binding component, troponin-I (Tn-I), the inhibitory subunit, and troponin-T (Tn-T), which locates the troponin complex on the tropomyosin (TM) complex. Tn-C undergoes a conformational change upon the binding of calcium; this is transmitted through the troponin complex, weakening the Tn-I-actin interaction and causing TM to move away from the myosin-binding sites on actin [2]. The result is the formation of actomyosin crossbridges at these sites, the hydrolysis of Mg-ATP and force generation [3, 41. Cardiac and skeletal isoforms of Tn-I are highly similar in their sequences. The cardiac isoform differs from its skeletal counterpart in possessing a 30-33-amino-acid (speciesdependent) N-terminal extension [ 5 ] . This contains the cardiac-specific adjacent phosphorylation sites at Ser23-Ser24 in human and bovine sequences [6, 71. There is good evidence that this phosphorylation is involved in the modulaCorrespondence to I. P. Trayer,

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Section: Gel Electrophoresismentioning

confidence: 99%

Overexpression of Human Cardiac Troponin‐I and Troponin‐C in Escherichia coli and Their Purification and Characterisation

Al-Hillawi

Minchin

Trayer

1994

European Journal of Biochemistry

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“…Brooke & Kaiser (1974) The above studies have been performed largely by optical-microscopic observations on cross-sections of muscle tissue. An alternative approach has been to isolate single muscle fibres, either for enzymic analyses (Lowry et al, 1978) or for direct identification of proteins by SDS/polyacrylamide-gel electrophoresis (Weeds et al, 1975;Pette & Schnez, 1977a,b). This latter technique has had only limited success, partly because of the small amount of protein in a rabbit muscle fibre.…”

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confidence: 99%

Electrophoretic analysis of proteins from single bovine muscle fibres

Young¹,

Davey²

1981

Biochemical Journal

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A number of single fibres were isolated by dissection of four bovine masseter (ma) muscles, three rectus abdominis (ra) muscles and eight sternomandibularis (sm) muscles. By histochemical criteria these muscles contain respectively, solely slow fibres (often called type I), predominantly fast fibres (type II), and a mixture of fast and slow. The fibres were analysed by conventional sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and the gels stained with Coomassie Blue. Irrespective of the muscle, every fibre could be classed into one of two broad groups based on the mobility of proteins in the range 135000-170000 daltons. When zones containing myosin heavy chain were cut from the single-fibre gel tracks and ‘mapped’ [Cleveland, Fischer, Kirschner & Laemmli (1977) J. Biol. Chem. 252, 1102-1106] with Staphylococcus proteinase, it was found that one group always contained fast myosin heavy chain, whereas the second group always contained the slow form. Moreover, a relatively fast-migrating alpha-tropomyosin was associated with the fast myosin group and a slow-migrating form with the slow myosin group. All fibres also contained beta-tropomyosin; the coexistence of alpha- and beta-tropomyosin is at variance with evidence that alpha-tropomyosin is restricted to fast fibres [Dhoot & Perry (1979) Nature (London) 278, 714-718]. Fast fibres containing the expected fast light chains and troponins I and C fast were identified in the three ra muscles, but in only four sm muscles. In three other sm muscles, all the fast fibres contained two troponins I and an additional myosin light chain that was more typical of myosin light chain 1 slow. The remaining sm muscle contained a fast fibre type that was similar to the first type, except that its myosin light chain 1 was more typical of the slow polymorph. Troponin T was bimorphic in all fast fibres from a ra muscles and in at least some fast fibres from one sm muscle. Peptide ‘mapping’ revealed two forms of fast myosin heavy chain distributed among fast fibres. Each form was associated with certain other proteins. Slow myosin heavy chain was unvarying in three slow fibre types identified. Troponin I polymorphs were the principal indicator of slow fibre types. The myofibrillar polymorphs identified presumably contribute to contraction properties, but beyond cud chewing involving ma muscle, nothing is known of the conditions that gave rise to the variable fibre composites in sm and ra muscles.

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“…Several forms of the alkali light chains are often present in a given skeletal muscle cell type [17,18]. We have demonstrated that the P light chain exists in two forms, P1 and P2, in ventricular muscle from several species [ 19].…”

Section: Introductionmentioning

confidence: 78%