THE PURIFICATION AND CHARACTERIZATION OF MYOSIN FROM BOVINE BRAIN.
Item
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Title
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THE PURIFICATION AND CHARACTERIZATION OF MYOSIN FROM BOVINE BRAIN.
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Identifier
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AAI8023739
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identifier
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8023739
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Creator
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TOMA, MICHAEL JOHN.
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Contributor
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Soli Berl
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Date
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1980
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Language
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English
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Publisher
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City University of New York.
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Subject
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Biology, General
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Abstract
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Myosin is an ATPase and a structural protein involved in the contraction of muscle. The presence of myosin in non-muscle cells and brain has led to the consideration that this protein is involved in contractile events occurring within these non-muscle cells. The possibility that cell motility, transmitter release, and axonal and dendritic outgrowth involve some form of contractile mechanism warrants an investigation into the nature of myosin from brain.;A study was undertaken to investigate the ultrastructure and enzymology of myosin from bovine brain. A procedure utilized for the isolation of myosin from platelets (Pollard et.al., 1974), was modified and extended for the isolation of myosin from bovine brain. Briefly, this procedure involves extraction of bovine brain cortex with a high salt buffer, precipitation of the myosin component by dilution to low ionic strength, ammonium sulfate fractionation and column chromatography. The enzyme is located in column eluted fractions by virtue of its K('+)-EDTA activated ATPase activity and further purified by two cycles of precipitation with a concomitant increase in activity. The preparation was then examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and compared to myosins from skeletal and smooth muscle.;The ability of brain myosin to form filaments was compared to that of skeletal muscle and smooth muscle myosins, electron microscopically. A more detailed study of ultrastructure was accomplished by comparing subfragments of the myosin molecule prepared by proteolytic cleavage. The light meromyosin (LMM) subfragments of brain and smooth muscle myosin assemble in a similar fashion, whereas the LMM portion of skeletal muscle myosin assembles in a different fashion. The head portions, obtained by proteolytic cleavage of all three myosins were able to bind to actins from all three tissue sources with a similar periodicity of attachment.;The effect of pH on the substrate saturation kinetics of brain and skeletal muscle myosin was also studied. Both proteins show similar Vmax versus pH curves with a tendency of the Vmax values to increase with increasing pH. Plots of Km versus pH also show a tendency of the Km values for both myosins to increase with increasing pH. Plots of pKm versus pH reveal a possible ionization within the components of the system at/or between pH 7.5-8.0 for brain Ca('2+) - and K('+)-EDTA-ATPase, and skeletal muscle K('+)-EDTA-ATPase. Whereas for skeletal muscle myosin Ca('2+)-ATPase it appears that there are multiple ionizations occurring over the pH range.;The importance of the similarities and differences of the three myosins studied is discussed in terms of their functioning in a contractile mechanism.
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Type
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dissertation
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Source
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PQT Legacy CUNY.xlsx
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degree
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Ph.D.
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Program
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Biomedical Sciences
