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Electrophoretic differentiaion f myofibrillar proteins in the pig.

ABSTRACT: 1. Starch-gel electrophoretograms of myosin and tropomyosin preparations in 8m-urea, from longissimus dorsi and psoas muscles of the pig, were characterized by laser densitometry. 2. The typical pattern for freshly prepared myosin from both muscles was similar, there being five electrophoretically distinct components. 3. The number of electrophoretically distinct components in both muscles increased after freeze-drying, but the effect of freeze-drying was more marked in longissimus dorsi. 4. Extraction with 8m-urea containing 2% beta-mercaptoethanol decreased the number of major electrophoretically distinct components of the fresh myosin of both muscles to four. 5. Although there was also some simplification of the patterns after freeze-drying the greater susceptibility of the myosin from longissimus dorsi was still evident. 6. The typical pattern for freshly prepared tropomyosin in 8m-urea differed in the two muscles: in each case it was more complex than that of the corresponding myosins. 7. The pattern of tropomyosin from neither longissimus dorsi nor psoas was altered significantly after freeze-drying. 8. Electrophoretograms of pig longissimus dorsi tropomyosin in 8m-urea differed from those of longissimus dorsi tropomyosin from sheep, ox and rabbit. 9. Extraction of the tropomyosins in 8m-urea and 2% beta-mercaptoethanol simplified the electrophoretic pattern to two major components with samples from pig, sheep and ox, and to one major component with samples from rabbit. 10. It was concluded that classification of skeletal muscles as ;red' or ;white' is insufficient to account for the degree of functional specialization which the electrophoretograms suggest.

SUBMITTER: Parsons AL

PROVIDER: S-EPMC1187770 | biostudies-other | 1969 May

REPOSITORIES: biostudies-other

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Project description:Rabbit predominantly fast-twitch-fibre and predominantly slow-twitch-fibre skeletal muscles of the hind limbs, the psoas, the diaphragm and the masseter muscles were fibre-typed by one-dimensional polyacrylamide-gel electrophoresis of the myofibrillar proteins of chemically skinned single fibres. Investigation of the distribution of fast-twitch-fibre and slow-twitch-fibre isoforms of myosin light chains and the type of myosin heavy chains, based on peptide 'maps' published in Cleveland. Fischer, Kirschner & Laemmli [(1977) J. Biol. Chem. 252, 1102-1106], allowed a classification of muscle fibres into four classes, corresponding to histochemical types I, IIA, IIB and IIC. Type I fibres with a pure slow-twitch-type of myosin were found to be characterized by a unique set of isoforms of troponins I, C and T, in agreement with the immunological data of Dhoot & Perry [(1979) Nature (London) 278, 714-718], by predominance of the beta-tropomyosin subunit and by the presence of a small amount of an additional tropomyosin subunit, apparently dissimilar from fast-twitch-fibre alpha-tropomyosin subunit. The myofibrillar composition of type IIB fast-twitch white fibres was the mirror image of that found for slow-twitch fibres in that the fast-twitch-fibre isoforms only of the troponin subunits were present and the alpha-tropomyosin subunit predominated. Type IIA fast-twitch red fibres showed a troponin subunit composition identical with that of type IIB fast-twitch white fibres. On the other hand, a unique type of myosin heavy chains was found to be associated with type IIA fibres. Furthermore, the myosin light-chain composition of these fibres was invariably characterized by a small amount of LC3F light chain and by a pattern that was either a pure fast-twitch-fibre light-chain pattern or a hybrid LC1F/LC2F/LC3F/LC1Sb light-chain pattern. By these criteria type IIA fibres could be distinguished from type IIC intermediate fibres, which showed coexistence of fast-twitch-fibre and slow-twitch-fibre forms of myosin light chains and of troponin subunits.

Project description: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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Electrophoretic differentiaion f myofibrillar proteins in the pig.

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