Physical and enzymatic properties of myosin from porcine brain

Hobbs, Donna S.; Frederiksen, Dixie W.

doi:10.1016/s0006-3495(80)85011-9

Cited by 18 publications

(8 citation statements)

References 26 publications

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“…7b). These activities were lower than those of gizzard and platelet myosin II, as has been previously reported by others [Hobbs and Frederiksen, 1980;Malik et al, 1983;Matsumura et al, 1988].…”

Section: Rhodamine-labeled Non-muscle Myosin Iibsupporting

confidence: 86%

Fluorescent analogues of myosin II for tracking the behavior of different myosin isoforms in living cells

Kolega

1998

J. Cell. Biochem.

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Fluorescently labeled smooth muscle myosin II is often used to study myosin II dynamics in non-muscle cells. In order to provide more specific tools for tracking non-muscle myosin II in living cytoplasm, fluorescent analogues of non-muscle myosin IIA and IIB were prepared and characterized. In addition, smooth and non-muscle myosin II were labeled with both cy5 and rhodamine so that comparative, dynamic studies may be performed. Non-muscle myosin IIA was purified from bovine platelets, non-muscle myosin IIB from bovine brain, and smooth muscle myosin II from turkey gizzards. After being fluorescently labeled with tetramethylrhodamine-5-iodoacetamide or with a succinimidyl ester of cy5, they retained the following properties: (1) reversible assembly into thick filaments, (2) actin-activatable MgATPase, (3) phosphorylation by myosin light chain kinase, (4) increased MgATPase upon light-chain phosphorylation, (5) interconversion between 6S and 10S conformations, and (6) distribution into endogenous myosin II-containing structures when microinjected into cultured cells. These fluorescent analogues can be used to visualize isoform-specific dynamics of myosin II in living cells.

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Section: Rhodamine-labeled Non-muscle Myosin Iibsupporting

confidence: 86%

Fluorescent analogues of myosin II for tracking the behavior of different myosin isoforms in living cells

Kolega

1998

J. Cell. Biochem.

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“…rat brain (Sun and Chantler, 1991), human macrophage (Saez et al, 1991), chicken intestinal epithelium (Shohet et al, 1989). phopeptide obtained from the COOH-end of the heavy chain of bovine brain myosin is not found in smooth muscle myosin heavy chains [Nagai et al, 1989;Murakami et al, 19901. Also, brain myosin has properties that are clearly distinct from those of myosins purified from other nonmuscle tissues or from smooth muscle [Kuczmarski and Rosenbaum, 1979a;Hobbs and Fredriksen, 1980;Matsumura et al, 1985;Ikeda et al, 19901. Although several attempts have been made to localize myosin within primary culture cells from brains and neuronal cell lines by immunocytochemical methods [Roisen et al, 1978;Kuczmarski and Rosenbaum, 1979b;Letourneau, 1981;Shaw et al, 1981;Bridgman and Dailey, 19891, we are not aware of any reports in which antibodies against brain myosin have been used to localize myosin within brain sections. In a previous study, polyclonal anti-thymus myosin antibodies were used to stain rat brain sections, and it was found that blood vessels [Drenckhahn et al, 19831, the molecular layer and glomeruli of the cerebellum, as well as nerve cell bodies in the brain stem [Drenckhahn and Kaiser, 19831, were immunostained.…”

Section: Introductionmentioning

confidence: 99%

Immunohistochemical studies on the distribution of cellular myosin II isoforms in brain and aorta

Murakami

Elzinga

1992

Cell Motil. Cytoskeleton

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The distribution of nonmuscle myosin isoforms in brain and aorta was studied by using polyclonal antibodies against two synthetic peptides selected from a region near the carboxyl terminus of bovine brain (peptide IIB) and human macrophage (peptide IIA) myosin. Immunoblots of brain homogenates and purified myosin showed two major bands stained by anti-peptide IIB (MIIB1 and MIIB2) and a minor band stained by anti-peptide IIA (MIIA2). Polyclonal anti-human platelet myosin antibodies did not react with MIIB isoforms. In cryosections from bovine, rat, and mouse brains, anti-peptide IIB stained most neuronal cells. In bovine cryosections, glial staining was also observed. In contrast, anti-peptide IIA and anti-platelet myosin antibodies primarily stained blood vessels. In bovine aorta, the anti-peptide antibodies recognized four bands, MIIB3, MIIB4, MIIA1, and MIIA2. Only MIIA2 was recognized by anti-human platelet myosin antibodies. In bovine aorta cryosections, anti-peptide IIB stained smooth muscle cells in tunica intima and tunica media but did not stain endothelial cells. Anti-peptide IIA stained smooth muscle cells in the tunica media, and endothelial cells of vaso vasorum but not of aorta. Only polyclonal anti-platelet myosin antibodies stained the endothelial cells of aorta tunica intima. These results indicate that multiple isoforms of cellular myosins exist in mammals, that these isoforms are expressed in a cell specific manner, and that the major myosin isoforms isolated from whole brain originate from neurons and, at least in bovine brain, from glia, but not from blood vessels.

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“…In addition, these authors were unable to achieve any actin activation of the myosin ATPase. Myosin isolated from porcine (Hobbs and Frederiksin, 1980) and from mouse (Scordilis and Adelstein, 1977) brain was shown to consist of a heavy chain of M, 200,000 daltons and two light chains of M, 20,000-19,000 and 16,000-15,000 daltons. Blitz and Fine (1974) isolated myosin from rat cortical synaptosomes, but owing to poor contrast of their Abbreviations used: DTT, Dithiothreitol; PMSF, Phenylmethyl sulfonyl fluoride; SDS-PAGE, Sodium dodecyl sulfatepolyacrylamide gel electrophoresis; TEMED, N,N,N',N'-Tetramethylethylenediamine.…”

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confidence: 99%

Purification and Characterization of Myosin from Calf Brain

Malik

Fenko

Scotto

et al. 1983

Journal of Neurochemistry

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Actomyosin complex was extracted from the brain cortex in a medium consisting of low salt, ATP, and EDTA, in the presence of protease inhibitors, followed by ammonium sulfate fractionation. Myosin was then purified from the actomyosin. Myosin obtained according to the procedure used was significantly contaminated with actin high (greater than 200,000 dalton) and low molecular weight proteins. Therefore, an alternative method based on affinity chromatography (Blue Dextran/Sepharose) and gel filtration (Sepharose 4B) was developed to purify myosin. This procedure yielded myosin that was greater than 95% pure as judged by electron microscopy and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The subunit composition of purified brain myosin was monitored by sodium dodecyl sulfate-polyacrylamide gel also containing a urea gradient. A closely migrating triplet in the heavy chain and three light chains, LC1, LC2, and LC3, of Mr 21,000, 19,000, and 17,000, respectively, were observed. These findings raise the possibility of the existence of myosin isoenzymes in the brain. Brain myosin formed bipolar thick filaments in 0.075 M KCl and MgCl2. At low ionic strength, the Mg2+-ATPase activity of myosin was stimulated 3- to 3.5-fold in the presence of skeletal muscle f-actin. Brain myosin also hydrolyzed other nucleotides; the rate of hydrolysis was ITP greater than ATP approximately equal to CTP greater than GTP approximately equal to UTP. The substrate (ATP) saturation curve in the presence of 10 mM CaCl2 and 0.6 M KCl was complex and consisted of plateau regions. The Arrhenius plot of the Ca-ATPase data was linear, whereas with ITPase, it was biphasic with a break occurring around 20 degrees C.

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Physical and enzymatic properties of myosin from porcine brain

Cited by 18 publications

References 26 publications

Fluorescent analogues of myosin II for tracking the behavior of different myosin isoforms in living cells

Fluorescent analogues of myosin II for tracking the behavior of different myosin isoforms in living cells

Immunohistochemical studies on the distribution of cellular myosin II isoforms in brain and aorta

Purification and Characterization of Myosin from Calf Brain

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