A specific method for the preparation of pure myosin

Pinset-Härström, I.; Ehrlich, Eva

doi:10.1016/0014-5793(73)80799-9

Cited by 8 publications

(4 citation statements)

References 16 publications

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“…This component was previously shown to react immunologically with the M line (30). Bands of molecular weight similar to that of all of these components have also been noted on SDS gels of partially purified myosin preparations (45,39). Our results suggest that, when A-and M-band components are extracted from Triton-extracted, protease-free fibrils, no proteins which migrate between C protein (approximately 140,000 daltons) and 55,000 daltons are seen in this A-band extract.…”

Section: Length (Pro)mentioning

confidence: 64%

“…Examination of glycerinated muscle labeled with antisera to purified C protein reveals antigenic sites in the A band which are distributed as nine transverse strips spaced 420-440 A apart on each half of the A band (37). Several additional proteins have also been noted on electrophoretograms of myosin preparations (45,9,39). The relationship of any of these proteins to muscle contraction or myofibrillogenesis is, at most, poorly understood.…”

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confidence: 99%

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Compositional studies of myofibrils from rabbit striated muscle.

Etlinger¹,

Zak²,

Fischman³

1976

The Journal of Cell Biology

163

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The localization of high-molecular-weight (80,000-200,000-daltons) proteins in the sarcomere of striated muscle has been studied by coordinated electron-microscopic and sodium dodecyl sulfate (SDS) gel electrophoretic analysis of native myofilaments and extracted and digested myofibrils. Methods were developed for the isolation of thick and thin filaments and of uncontracted myofibrils which are devoid of endoproteases and membrane fragments. Treatment of crude myofibrils with 0.5% Triton X-100 results in the release of a 110,000-dalton component without affecting the myofibrillar structure. Extraction of uncontracted myofibrils with a relaxing solution of high ionic strength results in the complete disappearance of the A band and M line. In this extract, five other protein bands in addition to myosin are resolved on SDS gels: bands M 1 (190,000 daltons) and M 2 (170,000 daltons), which are suggested to be components of the M line; M 3 (150,000 daltons), a degradation product; and a doublet M 4, M 5 (140,000 daltons), thickfilament protein having the same mobility as C protein. Extraction of myofibrils with 0.15% deoxycholate, previously shown to remove Z-line density, releases a doublet Z 1, Z 2 (90,000 daltons) with the same mobility as a-actinin, as well as proteins of 60,000 daltons and less, and small amounts of M 1, M 2, M 4, and M 5; these proteins were not extracted with 0.5% Triton X-100. The C, M-line, and Zline proteins and/or their binding to myofibrils are very sensitive to tryptic digestion, whereas the M 3 (150,000 daltons) component and an additional band at 110,000 daltons are products of proteolysis. Gentle treatment of myofibrils with an ATP relaxing solution results in the release of thick and thin myofilaments which can be pelleted by 100,000-g centrifugation. These myofilaments lack Mand Z-line structure when examined with the electron microscope, and their electrophoretograms are devoid of the M 1, M 2, Z 1, and Z 2 bands. The M 4, M 5 (C-protein doublet), and M 3 bands, however, remain associated with the filaments.Striated muscle offers some unique advantages for studies of the synthesis and assembly of organelles, due to its predominant content of a highly ordered arrangement of myofibrillar proteins (12, 13). To gain a better understanding of the mechanisms underlying myofibrillogenesis and of the assembly and disassembly steps which must occur during protein turnover in the adult, one must establish

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Section: Length (Pro)mentioning

confidence: 64%

mentioning

confidence: 99%

Compositional studies of myofibrils from rabbit striated muscle.

Etlinger¹,

Zak²,

Fischman³

1976

The Journal of Cell Biology

163

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“…It has been clearly shown by SDS polyacrylamide gel electrophoresis that myosin prepared in the usual manner contains small amounts of several other proteins of intermediate molecular weight. 4,5) As shown in plate I-a, myosin preparation in this experiment also contains small amounts of several other proteins of intermediate molecular weight. However, there is no contamination of actin in this preparation.…”

Section: Resultsmentioning

confidence: 99%

The Effect of the Modification of Amino Groups with β-Naphthoquinones-4-sulfonic Acid on Heat Denaturation of Myosin

Ito

Ando

1975

Agricultural and Biological Chemistry

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The heat denaturation process of myosin and the effect of the modification of amino groups of myosin with ti-naphthoquinone-4-sulfonic acid (NQS) on heat denaturation reaction were studied by using SDS polyacrylamide gel electrophoresis.Heavy chain and one of the light chains, g2, disappeared in the supernatant after centrifugation of heat treated myosin during two hours' heating, whereas two sorts of the light chains, gl and gs, were still found in the supernatant. In addition, a new peptide fraction appeared during heating.Heat denaturation of myosin was significantly suppressed by the modification of amino groups of myosin with NQS, where only heavy chain's electrophoretic pattern was influenced by NQS-modification.

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“…Myosin was prepared from human vastus lateralis muscle as described [29] but further purified by column chromatography on Sepharose 2B (Pharmacia) [30].…”

Section: Column-purified Myosinmentioning

confidence: 99%

Analysis of Myosin Light and Heavy Chain Types in Single Human Skeletal Muscle Fibers

et al. 1981

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In this study, myosin types in human skeletal muscle fibers were investigated with electrophoretic techniques. Single fibers were dissected out of lyophilized surgical biopsies and typed by staining for myofibrillar ATPase after preincubation in acid or alkaline buffers. After I4C-labeling of the fiber proteins in v i m by reductive methylation, the myosin light chain pattern was analysed on two-dimensional gels and the myosin heavy chains were investigated by one-dimensional peptide mapping. Surprisingly, human type I fibers, which contained only the slow heavy chain, were found to contain variable amounts of fast myosin light chains in addition to the two slow light chains LCls and LC2s. The majority of the type I fibers in normal human muscle showed the pattern LCls, LC2s and LClf. Further evidence for the existence in human muscle of a hybrid myosin composed of a slow heavy chain with fast and slow light chains comes from the analysis of purified human myosin in the native state by pyrophosphate gel electrophoresis. With this method, a single band corresponding to slow myosin was obtained ; this slow myosin had the light chain composition LCls, LC2s and LClf.Type IIA and IIB fibers, on the other hand, revealed identical light chain patterns consisting of only the fast light chains LClf, LC2f and LC3f but were found to have different myosin heavy chains.On the basis of the results presented, we suggest that thc histochemical ATPase normally used for fibre typing is determined by the myosin heavy chain type (and not by the light chains). Thus, in normal human muscle a number of 'hybrid' myosins were found to occur, namely two extreme forms of fast myosins which have the same light chains but different heavy chains (IIA and IIB) and a continuum of slow forms consisting of the same heavy chain and slow light chains with a variable Fast light chain composition. This is consistent with the different physiological roles these fibers are thought to have in muscle contraction.

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A specific method for the preparation of pure myosin

Cited by 8 publications

References 16 publications

Compositional studies of myofibrils from rabbit striated muscle.

Compositional studies of myofibrils from rabbit striated muscle.

The Effect of the Modification of Amino Groups with β-Naphthoquinones-4-sulfonic Acid on Heat Denaturation of Myosin

Analysis of Myosin Light and Heavy Chain Types in Single Human Skeletal Muscle Fibers

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