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 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.

scholarly journals THE POLYMERIZATION OF ACTIN: ITS ROLE IN THE GENERATION OF THE ACROSOMAL PROCESS OF CERTAIN ECHINODERM SPERM

1973 ◽  
Vol 59 (1) ◽  
pp. 109-126 ◽  
Author(s):  
Lewis G. Tilney ◽  
Sadashi Hatano ◽  
Harunori Ishikawa ◽  
Mark S. Mooseker
Keyword(s):  
Molecular Weight ◽  
Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Acetone Powder ◽  
Heavy Meromyosin ◽  
Skeletal Muscle Myosin ◽  
Sperm Extract ◽  
Triton X ◽  
Muscle Myosin

When Asterias or Thyone sperm come in contact with egg jelly, a long process which in Thyone measures up to 90 µm in length is formed from the acrosomal region. This process can be generated in less than 30 s. Within this process is a bundle of microfilaments. Water extracts prepared from acetone powders of Asterias sperm contain a protein which binds rabbit skeletal muscle myosin forming a complex whose viscosity is reduced by ATP. Within this extract is a protein with the same molecular weight as muscle actin. It can be purified either by collecting the pellet produced after the addition of Mg++ or by reextracting an acetone powder of actomyosin prepared by the addition of highly purified muscle myosin to the extract. The sperm actin can be polymerized and by electron microscopy the polymer is indistinguishable from muscle F-actin. The sperm actin was shown to be localized in the microfilaments in the acrosomal processes by: (a) heavy meromyosin binding in situ, (b) sodium dodecyl sulfate (SDS) gel electrophoresis of the isolated acrosomal processes and a comparison to gels of flagella which contain no band corresponding to the molecular weight of actin, and (c) SDS gel electrophoresis of the extract from isolated acrosomal caps. Since the precursor for the microfilaments in the unreacted sperm appears amorphous, we suspected that the force for the generation of the acrosomal process is brought about by the polymerization of the sperm actin. This supposition was confirmed, for when unreacted sperm were lysed with the detergent Triton X-100 and the state of the actin in the sperm extract was analyzed by centrifugation, we determined that at least 80% of the actin in the unreacted sperm was in the monomeric state.

scholarly journals The effects of caldesmon on the ATPase activities of rabbit skeletal-muscle myosin

1986 ◽  
Vol 238 (2) ◽  
pp. 523-530 ◽  
Author(s):  
M S Lim ◽  
M P Walsh
Keyword(s):  
Skeletal Muscle ◽  
Atpase Activity ◽  
Striated Muscle ◽  
Physiological Activity ◽  
Skeletal Muscle Myosin ◽  
Contractile State ◽  
Calmodulin Binding ◽  
Muscle Tissues ◽  
Troponin Complex ◽  
Muscle Myosin

We studied the effects of caldesmon, a major actin- and calmodulin-binding protein found in a variety of muscle and non-muscle tissues, on the various ATPase activities of skeletal-muscle myosin. Caldesmon inhibited the actin-activated myosin Mg2+-ATPase, and this inhibition was enhanced by tropomyosin. In the presence of the troponin complex and tropomyosin, caldesmon inhibited the Ca2+-dependent actomyosin Mg2+-ATPase; this inhibition could be partly overcome by Ca2+/calmodulin. Caldesmon, phosphorylated to the extent of approximately 4 mol of Pi/mol of caldesmon, inhibited the actin-activated myosin Mg2+-ATPase to the same extent as did non-phosphorylated caldesmon. Both inhibitions could be overcome by Ca2+/calmodulin. Caldesmon also inhibited the Mg2+-ATPase activity of skeletal-muscle myosin in the absence of actin; this inhibition also could be overcome by Ca2+/calmodulin. Caldesmon inhibited the Ca2+-ATPase activity of skeletal-muscle myosin in the presence or absence of actin, at both low (0.1 M-KCl) and high (0.3 M-KCl) ionic strength. Finally, caldesmon inhibited the skeletal-muscle myosin K+/EDTA-ATPase at 0.1 M-KCl, but not at 0.3 M-KCl. Addition of actin resulted in no inhibition of this ATPase by caldesmon at either 0.1 M- or 0.3 M-KCl. These observations suggest that caldesmon may function in the regulation of actin-myosin interactions in striated muscle and thereby modulate the contractile state of the muscle. The demonstration that caldesmon inhibits a variety of myosin ATPase activities in the absence of actin indicates a direct effect of caldesmon on myosin. The inhibition of the actin-activated Mg2+-ATPase activity of myosin (the physiological activity) may not be due therefore simply to the binding of caldesmon to the actin filament causing blockage of myosin-cross-bridge-actin interaction.

IV. The mechanism of contraction - Introductory remarks

1973 ◽  
Vol 265 (867) ◽  
pp. 167-167
Keyword(s):  
Skeletal Muscle ◽  
Smooth Muscle ◽  
Atpase Activity ◽  
Soluble Proteins ◽  
Smooth Muscle Myosin ◽  
Trypsin Treatment ◽  
Skeletal Muscle Myosin ◽  
Muscle Myosin

Our programme this afternoon is in two parts. We first welcome Professor Hamoir and Dr Kendrick-Jones to describe the several ways in which smooth muscle myosin differs from skeletal muscle myosin. It was in this biochemical field that my own work, with Dr Jennifer Williams, lay some twelve years ago. We were impressed at that time by the very low ATPase activity of the uterus actomyosin, and by the fact that on trypsin treatment meromyosins were obtained in some ways similar to those of skeletal muscle. Speakers this afternoon will have far more to tell us of the nature, behaviour and structure of the myosins concerned. We were also interested in the properties and possible function of certain soluble proteins, including tropomyosin, which figure so largely in smooth muscle constitution. This subject also will come up today.

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