scholarly journals Actin in Xenopus oocytes

1978 ◽  
Vol 77 (2) ◽  
pp. 427-438 ◽  
Author(s):  
TG Clark ◽  
RW Merriam
Keyword(s):  
Skeletal Muscle ◽  
Molecular Weight ◽  
High Speed ◽  
Xenopus Oocytes ◽  
Supernatant Fraction ◽  
Skeletal Muscle Myosin ◽  
Prominent Component ◽  
High Molecular Weight Component ◽  
High Concentrations ◽  
Muscle Myosin

It has been found that a high-speed supernatant fraction from Xenopus oocytes extracted in the cold will form a clear, solid gel upon warming. Gel formation occurs within 60 min at 18 degrees-40 degrees C, and is, at least initially, temperature reversible. Gelation is strictly dependent upon the addition of sucrose to the extraction medium. When isolated in the presence of ATP, the gel consists principally of a 43,000-dalton protein which co-migrates with Xenopus skeletal muscle actin on SDS-polyacrylamide gels, and a prominent high molecular weight component of approx. 250,000 daltons. At least two minor components of intermediate molecular weight are also found associated with the gel in variable quantities. Actin has been identified as the major consituent of the gel by ultrastructural and immunological techniques, and comprises roughly 47% of protein in the complex. With time, the gel spontaneously contracts to form a small dense aggregate. Contraction requires ATP. In the absence of exogenous ATP, a polypeptide which co-migrates with the heavy chain of Xenopus skeletal muscle myosin becomes a prominent component of the gel. This polypeptide is virtually absent from gels which have contracted in ATP-containing extracts. It has also been found that Ca++ is required for gelation in oocyte extracts. At both low and high concentrations of Ca++ (defined as a ratio of Ca++/EGTA in the extraction medium), gelation is inhibited.

Effect of β-adrenoceptor activation on [Ca2+]i regulation in murine skeletal myotubes

1999 ◽  
Vol 276 (5) ◽  
pp. C1038-C1045 ◽  
Author(s):  
Y. S. Prakash ◽  
H. F. M. van der Heijden ◽  
E. M. Gallant ◽  
G. C. Sieck
Keyword(s):  
Skeletal Muscle ◽  
Ryanodine Receptors ◽  
Immunocytochemical Staining ◽  
Skeletal Muscle Myosin ◽  
Low Concentrations ◽  
Adrenoceptor Agonist ◽  
Intracellular Ca ◽  
Mouse Limb ◽  
High Concentrations ◽  
Muscle Myosin

The present study used real-time confocal microscopy to examine the effects of the β2-adrenoceptor agonist salbutamol on regulation of intracellular Ca2+ concentration ([Ca2+]i) in myotubes derived from neonatal mouse limb muscles. Immunocytochemical staining for ryanodine receptors and skeletal muscle myosin confirmed the presence of sarcomeres. The myotubes displayed both spontaneous and ACh-induced rapid (<2-ms rise time) [Ca2+]itransients. The [Ca2+]itransients were frequency modulated by both low and high concentrations of salbutamol. Exposure to α-bungarotoxin and tetrodotoxin inhibited ACh-induced [Ca2+]itransients and the response to low concentrations of salbutamol but not the response to higher concentrations. Preexposure to caffeine inhibited the subsequent [Ca2+]iresponse to lower concentrations of salbutamol and significantly blunted the response to higher concentrations. Preexposure to salbutamol diminished the [Ca2+]iresponse to caffeine. Inhibition of dihydropyridine-sensitive Ca2+ channels with nifedipine or PN-200-110 did not prevent [Ca2+]ielevations induced by higher concentrations of salbutamol. The effects of salbutamol were mimicked by the membrane-permeant analog dibutyryl adenosine 3′,5′-cyclic monophosphate. These data indicate that salbutamol effects in skeletal muscle predominantly involve enhanced sarcoplasmic reticulum Ca2+ release.

scholarly journals Chick brain actin and myosin. Isolation and characterization.

1979 ◽  
Vol 80 (2) ◽  
pp. 341-355 ◽  
Author(s):  
E R Kuczmarski ◽  
J L Rosenbaum
Keyword(s):  
Skeletal Muscle ◽  
Molecular Weight ◽  
Atpase Activity ◽  
Molecular Sieve ◽  
Acetone Powder ◽  
Chick Brain ◽  
Skeletal Muscle Myosin ◽  
Isolation And Characterization ◽  
Muscle Myosin ◽  
The Brain

Brain actin extracted from an acetone powder of chick brains was purified by a cycle of polymerization-depolymerization followed by molecular sieve chromatography. The brain actin had a subunit molecular weight of 42,000 daltons as determined by co-electrophoresis with muscle actin. It underwent salt-dependent g to f transformation to form double helical actin filaments which could be "decorated" by muscle myosin subfragment 1. A critical concentration for polymerization of 1.3 microM was determined by measuring either the change in viscosity or absorbance at 232 nm. Brain actin was also capable of stimulating the ATPase activity of muscle myosin. Brain myosin was isolated from whole chick brain by a procedure involving high salt extraction, ammonium sulfate fractionation and molecular sieve chromatography. The purified myosin was composed of a 200,000-dalton heavy chain and three lower molecular weight light chains. In 0.6 M KCl the brain myosin had ATPase activity which was inhibited by Mg++, stimulated by Ca++, and maximally activated by EDTA. When dialyzed against 0.1 M KCl, the brain myosin self-assembled into short bipolar filaments. The bipolar filaments associated with each other to form long concatamers, and this association was enhanced by high concentrations of Mg++ ion. The brain myosin did not interact with chicken skeletal muscle myosin to form hybrid filaments. Furthermore, antibody recognition studies demonstrated that myosins from chicken brain, skeletal muscle, and smooth muscle were unique.

scholarly journals Electron microscopy of synthetic myosin filaments. Evidence for cross-bridge. Flexibility and copolymer formation.

1975 ◽  
Vol 67 (1) ◽  
pp. 93-104 ◽  
Author(s):  
T D Pollard
Keyword(s):  
Skeletal Muscle ◽  
Smooth Muscle ◽  
Structural Features ◽  
Length Frequency ◽  
Single Population ◽  
Skeletal Muscle Myosin ◽  
Distinctive Features ◽  
Myosin Filaments ◽  
The Mean ◽  
Muscle Myosin

Electron micrographs of negatively stained synthetic myosin filaments reveal that surface projections, believed to be the heads of the constituent myosin molecules, can exist in two configurations. Some filaments have the projections disposed close to the filament backbone. Other filaments have all of their projections widely spread, tethered to the backbone by slender threads. Filaments formed from the myosins of skeletal muscle, smooth muscle, and platelets each have distinctive features, particularly their lengths. Soluble mixtures of skeletal muscle myosin with either smooth muscle myosin or platelet myosin were dialyzed against 0.1 M KC1 at pH 7 to determine whether the simultaneous presence of two types of myosin would influence the properties of the filaments formed. In every case, a single population of filaments formed from the mixtures. The resulting filaments are thought to be copolymers of the two types of myosin, for several reasons: (a) their length-frequency distribution is unimodal and differs from that predicted for a simple mixture of two types of myosin filaments; (b) their mean length is intermediate between the mean lengths of the filaments formed separately from the two myosins in the mixture; (c) each of the filaments has structural features characteristic of both of the myosins in the mixture; and (d) their size and shape are determined by the proportion of the two myosins in the mixture.

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