A Streamlined Method of Subfragment One Preparation from Myosin1

Okamoto, Yoh; Sekine, Toshimori

doi:10.1093/oxfordjournals.jbchem.a135365

Cited by 103 publications

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“…As observed previously for native S-1, the nucleotide trapping was less than stoichiometric. Although the occurrence of some nonspecific modification cannot be directly ruled out, it is more likely that the substoichiometry results from the presence of S-1 molecules with altered active sites, as has been reported for the trapping via the SH1-SH2 crosslink (12,26). Also, when the nucleotide employed was…”

Section: Resultsmentioning

confidence: 93%

Nucleotide trapping at the ATPase site of myosin subfragment 1 by a new interthiol crosslinking.

Chaussepied¹,

Mornet²,

Kassab³

1986

Proc. Natl. Acad. Sci. U.S.A.

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When myosin subfragment 1 derivatives in which the reactive sulfhydryl SH1 has been blocked react with N,N'-p-phenylenedimaleimide or 5,5'-dithiobis(2-nitrobenzoic acid), the reactive sulfhydryl group SH2 of the 20-kDa domain is crosslinked with a thiol of the 50-kDa domain of the heavy chain. The crosslink induces the stable trapping of a significant amount of Rabbit skeletal muscle actin was prepared as described by Eisenberg and Kielley (17), and its concentration was estimated by using A19i, = 11.0.Crosslinking Reactions. The reaction of 1.3-fold excess Ph(NMal)2 in 50 mM Hepes (pH 8.0) was done with 50 ,uM modified or native enzyme in the presence or absence of 2.5 mM Mg2+-ATP or Mg2+-ADP at 4°C. The crosslinking of S-1 with Nbs2 was performed under similar conditions, using a 5-fold excess of reagent. The reactions were quenched and the protein derivatives were purified by filtration over Sephadex G-50 (20 x 1.5 cm) in 50 mM Hepes, pH 7.0/200 mM KCl/0.5 mM NaN3 at 40C. NaDodSO4/PAGE. Tryptic fragments of modified S-1 were separated by electrophoresis in 0.1% NaDodSO4/polyacrylamide slab gels containing a 5-18% (wt/vol) gradient of acrylamide (18). The running buffer was 50 mM Tris/100 mM boric acid (pH 8.0) (19). The densitometric scanning of the gels was carried out with a computerized model CS930 Shimadzu (Kyoto, Japan) gel scanner. The following were used as molecular mass markers: S-1 heavy chain (95 kDa), S-1 light chain 1 (LC1, 25 kDa), S-1 light chain 3 (LC3, 17 kDa), the three fragments obtained by tryptic cleavage of S-1 (50 kDa, 27 kDa, and 20 kDa), and actin (42 kDa).Specific Modifications of S-1 Thiols. The specific fluorescence labeling of SH1 with 5-[2-(iodoacetyl)aminoethyl]aminonaphthalene-1-sulfonic acid (1,5-IAEDANS) was done according to the procedure of Takashi et al. (20). Under our conditions, 1.1 mol of the dye was incorporated per mol of S-1. Fluorescence gel scanning allowed us to quantify the labeling ratio as 0.2 in the light chains and 0.9 in the heavy chain.The specific chemical modifications of SH1 by 2,4-dinitrofluorobenzene (N2ph-F) and iodoacetamide were carried out as described by Reisler et al.

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Section: Resultsmentioning

confidence: 93%

Nucleotide trapping at the ATPase site of myosin subfragment 1 by a new interthiol crosslinking.

Chaussepied¹,

Mornet²,

Kassab³

1986

Proc. Natl. Acad. Sci. U.S.A.

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“…Rabbit skeletal muscle myosin was prepared by a procedure modified from [28], and HMM made from fresh myosin according to [29]. HMM was aliquoted, frozen in liquid nitrogen and stored at -70 °.…”

Section: In Vitro Motility Assaysmentioning

confidence: 99%

Calponin reduces shortening velocity in skinned taenia coli smooth muscle fibres

et al. 1995

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Calponin (4.1-5.9/.tM, pig stomach) inhibited maximal shortening velocity (Vm,x) by 20-25% with only minor influence on force in skinned smooth muscle from guinea-pig taenia coli activated at different Ca 2+ levels and with thiophosphorylation. Similar results were obtained with a fragment of the Nterminal 1-228 amino acids engineered using a mouse cDNA construct (5.4 pM). Both the native calponin and the fragment inhibited actin filament sliding in a graded manner in an in vitro motility assay. We conclude that calponin influences the kinetics of the actin-myosin interaction in the organised smooth muscle contractile system and that engineered fragments of calponin can be used to probe its action in muscle fibres. The effects can be due to an introduction of an internal load during filament sliding, possibly by decreasing the detachment rates and increasing the cross-bridge time spent in the attached state.

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“…Myosin was isolated from rabbit back muscle (24). Chymotryptic myosin subfragment 1 (CT-Si) (25) and papain/Mg2e subfragment 1 (P-Mg.Sl) (2) For binding to the rabbit actin-phalloidin complex, 0-2 p.M actin was used, while for binding to yeast actin, 0-7.9 p.M actin was used. Reaction mixtures were incubated 60 min on ice before centrifugation at 4°C for 10 min at 100,000 rpm.…”

mentioning

confidence: 99%

Yeast actin filaments display ATP-dependent sliding movement over surfaces coated with rabbit muscle myosin.

Kron

Drubin

Botstein

et al. 1992

Proc. Natl. Acad. Sci. U.S.A.

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The yeast Saccharomyces cerevisiae has been used to study the function of components of the actin cytoskeleton in vivo, mainly because It is easy to derive and characterize mutations affecting these proteins. In contrast, biochemical studies have generally used proteins derived from higher eukaryotes. We have devised a simple procedure to prepare, in high yield, homogeneous native actin from wild-type and act] mutant yeast. Using intensified video fluorescence microscopy, we found that actin filaments polymerized from these preparations exhibit ATP-dependent sliding movement over surfaces coated with rabbit skeletal muscle myosin. The rates of sliding movement of the wild-type and mutant yeast actins were each about half that of rabbit skeletal muscle actin under similar conditions. We conclude that over the large evolutionary distance between yeast and mammals there has been si iant conservation of actin function, specifically the ability to be moved by interaction with myosin.The interaction of the ubiquitous eukaryotic proteins actin and myosin couples the hydrolysis of ATP to force production, driving muscle contraction and many other types of cell motility. After nearly 50 years of intense biochemical and structural analysis of actin-myosin interaction, the mechanisms of both chemomechanical coupling and regulation of actomyosin activity remain obscure. To enhance structurefunction studies of proteins purified from mammalian muscle tissues, powerful biochemical techniques have been developed that provide true functional assays for actin-myosin interaction. The sliding movement of actin filaments over myosin-coated surfaces can be directly observed by fluorescence microscopy (1), reconstituting motility in a geometry where quantitative measurement of rates of movement (2) and magnitude of forces (3) 4466The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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A Streamlined Method of Subfragment One Preparation from Myosin1

Cited by 103 publications

References 0 publications

Nucleotide trapping at the ATPase site of myosin subfragment 1 by a new interthiol crosslinking.

Nucleotide trapping at the ATPase site of myosin subfragment 1 by a new interthiol crosslinking.

Calponin reduces shortening velocity in skinned taenia coli smooth muscle fibres

Yeast actin filaments display ATP-dependent sliding movement over surfaces coated with rabbit muscle myosin.

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