A new method to precipitate myosin V from rat brain soluble fraction

• Autorzy: Melo H. C. S. , Coelho M.V.
• Języki publikacji: EN

Abstrakty

EN

Myosin can be precipitated from soluble fraction under different assay conditions. This paper describes a new method for precipitating myosin V from rat brain soluble fraction. Brains were homogenized in 50 mM imidazole/HCl buffer, pH 8.0, containing 10 mM EDTA/EGTA, 250 mM sucrose, 1 mM DTT and 1 mM benzamidine, centrifuged at 45000 × g for 40 min and the supernatant was frozen at -20 °C. Forty-eight hours later, the supernatant was thawed, centrifuged at 45000 × g for 40 min and the precipitate was washed in 20 mM imidazole buffer pH 8.0. SDS/PAGE analysis showed four polypeptides in the precipitate: 205, 150, 57 and 43 kDa. The precipitate presented high Mg2+-ATPase activity, which co-purifies with p205. This polypeptide was recognized by a specific myosin V antibody and was proteolised by calpain, generating two stable polypeptides: p130 and p90. The Mg2+-ATPase activity was not stimulated by calcium in both the absence and presence of exogenous calmodulin and the K+/EDTA-ATPase activity represented 25% of the Mg2+-ATPase activity. In this work, myosin V from rat brain was precipitated by freezing the soluble fraction and was co-purificated with a 45 kDa polypeptide.

Słowa kluczowe

EN

myosin V; brain; ATPase; new method; F-actin; rat; myosin; polypeptide

• Wydawca: -
• Czasopismo: Acta Biochimica Polonica
• Rocznik: 2007
• Tom: 54
• Numer: 3
• Opis fizyczny: p.575-581,fig.,ref.

Twórcy

autor

Melo H. C. S.

• Universidade Federal de Uberlandia, Av.Para, 1720, Campus Umuarama - Bloco 2E - Sala 39b, Uberlandia, MG, Brazil

autor

Coelho M.V.

Bibliografia

• Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye biding. Anal Biochem 72: 248–254.
• Brown ME, Bridgman PC (2004) Myosin function in nervous and sensory systems. J Neurobiol 58: 118–130.
• Cheney R (1998) Purification and assay of myosin V. Methods Enzymol 298: 3–18.
• Cheney R, O’shea MK, Heuser JE, Coelho MV, Wolenski JS, Espreafico EM, Forscher J, Larson RE, Mooseker MS (1993) Brain myosin-V is a two-headed unconventional myosin with motor activity. Cell 75: 13–23.
• Coelho M, Larson R (1993) Ca2+-dependent phosphorylation of the tail domain of myosin-V, a calmodulin-biding myosin in vertebrate brain. Braz J Med Biol Res 26: 465–472.
• De La Cruz EM, Wells AL, Rosenfeld SS, Ostap EM, Sweeney HL (1999) The kinetic mechanism of myosin V. Proc Natl Acad Sci USA 96: 13726–13731.
• De La Cruz EM, Ostap EM (2004) Relating biochemistry and function in the myosin superfamily. Curr Opin Cell Biol 16: 61–67.
• Dias DS, Coelho MV (2007) Purification and partial characterization of myosin II from rat testis. Int J Biol Macromol 41: 475–480.
• Espindola FS, Espreafico EM, Coelho MV, Martins AR, Costa FRC, Mooseker MS, Larson RE (1992) Biochemical and immunological characterization of p190-calmodulin complex from vertebrate brain: a novel calmodulin-binding myosin. J Cell Biol 118: 359–368.
• Espreafico EM, Cheney RE, Matteoli M, Nascimento PVC, Larson RE, Mooseker M (1992) Primary structure and cellular localization of chicken brain myosin-V (p190), an unconventional myosin with calmodulin light chains. J Cell Biol 119: 1541–1557.
• Foth BJ, Goedecke MC, Soldati D (2006) New insights into myosin evolution and classification. Proc Natl Acad Sci USA 103: 3681–3686.
• Gibson F, Walsh J, Mburu P, Varela A, Brown KA, Antonio M, Belsel KW, Steel KP, Brown SDM (1995) A type VII myosin encoded by the mouse deafness gene shaker-1. Nature 374: 62–64.
• Harlow E, Lane D (1988) Antibodies: a laboratory manual, Cold Spring Harbor Laboratory, pp. 305, Cold Spring, NY.
• Hasson T, Gillespie PG, Garcia JA, MacDonald RB, Zhao Y, Yee AG, Mooseker MS, Corey DP (1997) Unconventional myosins in inner-ear sensory epithelia. J Cell Biol 137: 1287–1307.
• Heinonen JK, Lahti RJ (1981) A New and convenient colorimetric determination of inorganic orthophosphate and its application to the assay of inorganic pyrophosphatase. Anal Biochem 113: 313–317.
• Hill KL, Catlett NL, Weisman LS (1996) Actin and myosin function in directed vacuole movement during cell division in Saccharomyces cerevisiae. J Cell Biol 135: 1535–1549.
• Johnston G, Prendergast JA, Singer R (1991) The Saccharomyces cerevisiae MY02 gene encodes an essential myosin for vectorial transport of vesicles. J Cell Biol 113: 539–551.
• Krementsov DN, Krementsova EB, Trybus KM (2004) Myosin V: regulation by calcium, calmodulin, and the tail domain. J Cell Biol 164: 877–886.
• Krendel M, Mooseker M (2005) Myosins: tails (and heads) of functional diversity. Physiology 20: 239–251.
• Laemmli UK, Favre M (1973) Maturation of the head of bacteriophage T4. I. DNA packaging events. J Mol Biol 80: 575–599.
• Larson RE, Espindola FS, Espreafico EM (1990) Calmodulin-regulated enzyme activities associated with brain actomyosin. J Neurochem 54: 1288–1294.
• Larson RE, Pitta DE, Ferro JA (1988) A novel 190 kDa calmodulin-binding protein associated with brain actomyosin. Braz J Med Biol Res 21: 213–217.
• Margossian SS, Lowey S (1982) Preparation of myosin and its subfragments from rabbit skeletal muscle. Methods Enzymol 85 (Pt B): 55–71.
• Mercer J, Seperack P, Strobel M, Copeland N, Jenkins N (1991) Novel myosin heavy chain encoded by murine dilute coat colour locus. Nature 349: 709–713.
• Müller RT, Honnert U, Reinhard J, Bähler M (1997) The rat myosin myr 5 is a GTPase-activating protein for Rho in vivo: essential role of arginine 1695. Mol Biol Cell 8: 2039–2053.
• Nascimento AAC, Cheney RE, Tauhata SBF, Larson RE, Mooseker MS (1996) Enzymatic characterization and functional domain mapping of brain myosin-V. J Biol Chem 271: 17561–17569.
• Nguyen H, Higuchi H (2005) Motility of myosin V regulated by the dissociation of single calmodulin. Nat Struct Mol Biol 12: 127–132.
• Novak KD, Titus MA (1997) Myosin I overexpression impairs cell migration. J Cell Biol 136: 633–647.
• O’Connell CB, Tyska MJ, Mooseker MS (2007) Myosin at work: motor adaptations for a variety of cellular functions. Biochim Biophys Acta 1773: 615–630.
• Perrin BF, Huttenlocher A (2002) Calpain. Int J Biochem Cell Biol 34: 722–725.
• Post PL, Bokoch GM, Mooseker MS (1998) Human myosin-IXb is a mechanochemically active motor and a GAP for rho. J Cell Sci 111: 941–950.
• Rechsteiner M, Rogers SW (1996) PEST sequences and regulation. Trends Biochem Sci 21: 267–271.
• Redowicz MJ (2007) Unconventional myosins in muscle. Eur J Cell Biol 86: 549–558.
• Rogers SL, Karcher RL, Roland JT, Minin AA, Steffen W, Gelfand VI (1999) Regulation of melanosome movement in the cell cycle by reversible association with myosin V. J Cell Biol 146: 1265–1276.
• Ruppel K, Spudich J (1996) Structure-function analysis of the motor domain of myosin. Annu Rev Cell Dev Biol 12: 543–573.
• Schietroma C, Yu HY, Wagner MC, Umbach JA, Bement WM, Gundersen CB (2007) A role for myosin 1E in cortical granule exocytosis in Xenopus oocytes. J Biol Chem 10.1074/jbc.M705825200.
• Studier FW (1973) Analysis of bacteriophage T7 early RNAs and proteins on slabgels. J Mol Biol 79: 237–248.
• Towbin H, Staehelin T, Gordon J (1979) Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications. Proc Natl Acad Sci USA 76: 4350–4354.
• Tuxworth RI, Wessels D, Addicks GC, Soll DR, Gerisch G, Titus MA (2001) A role for myosin VII in dynamic cell adhesion. Curr Biol 11: 318–329.
• Uyeda TQ, Nagasaki A (2004) Variations on a theme: the many modes of cytokinesis. Curr Opin Cell Biol 16: 1–6.
• VaradiV, Tsuboi T, Rutter GA (2005) Myosin Va transports dense core secretory vesicles in pancreatic MIN6-cells. Mol Biol Cell 16: 2670–2680.
• Wang F, Thirumurugan K, Stafford WF, Hammer JA, Knight PJ, Sellers JR (2004) Regulated conformation of myosin V. J Biol Chem 279: 2333–2336.
• Warrick H, Spudich J (1987) Myosin structure and function in cell motility. Ann Rev Cell Biol 3: 379–421.
• Werner M, Munro E, Glotzer M (2007) Astral signals spatially bias cortical myosin recruitment to break symmetry and promote cytokinesis. Curr Biol 17: 1286–1297.
• Wu X, Bowers B, Wei Q, Kocher Q, Hammer JA (1997) Myosin V associates with melanosomes in mouse melanocytes: evidence that myosin V is an organelle motor. J Cell Sci 110: 847–859.
• Zang J, Cavet C, Sabry JH, Wagner P, Moores SL, Spudich JA (1997) On the role of myosin-II in cytokinesis: division of dictyostelium cells under adhesive and nonadhesive conditions. Mol Biol Cell 8: 2617–2629.