title: Precipitation of kidney myosin IIA and IIB by freezing.
abstract: Actomyosin precipitation is a critical step in the purification of myosins. In this work, the objective was to precipitate rat kidney actomyosin and isolate myosin by freezing and thawing the soluble fraction. Kidney was homogenized in imidazole buffer, centrifuged at 45000 g for 30 min, and the supernatant was frozen at -20°C for 48 h. The supernatant was thawed at 4°C, centrifuged at 45000 g for 30 min and the precipitate washed twice with imidazole buffer pH 7.0 (with and without Triton X-100, respectively). The resulting precipitate presented a polypeptide profile in SDS/PAGE characteristic of actomyosin and expressed Mg- and K/EDTA-ATPase activity. The actomyosin complex was solubilized with ATP and Mg, and the main polypeptide, p200, was purified in a DEAE-Sepharose column. p200 was marked with anti-myosin II, co-sedimented with F-actin in the absence, but not in the presence, of ATP and was identified by MS/MS with a high Mascot score for myosin IIA. The analysis identified peptides exclusive of myosin IIB, but detected no peptides exclusive of myosin IIC.
authors.affiliation: Universidade Federal de Uberlndia, Instituto de Gentica e Bioqumica, Bairro Umuarama, Uberlndia, Minas Gerais, Brazil.
authors.forename: Decivaldo dos Santos
authors.initials: Ddos S
authors.lastname: Dias
authors.forename: Grabriel Costa Nunes
authors.initials: GC
authors.lastname: da Cruz
authors.forename: Marcelo Valle
authors.initials: MV
authors.lastname: de Sousa
authors.forename: Milton Vieira
authors.initials: MV
authors.lastname: Coelho
mesh_terms: D000199:Actins / Q000737:chemistry; D000251:Adenosine Triphosphatases / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000737:chemistry; D000818:Animals; D011232:Chemical Precipitation; D002852:Chromatography, Ion Exchange; D005615:Freezing; D007668:Kidney / Q000378:metabolism*; D008274:Magnesium / Q000737:chemistry; D024507:Nonmuscle Myosin Type IIA / Q000737:chemistry / Q000302:isolation & purification*; D024501:Nonmuscle Myosin Type IIB / Q000737:chemistry / Q000302:isolation & purification*; D051381:Rats; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D000199:Actins; D000255:Adenosine Triphosphate; D000251:Adenosine Triphosphatases; D024507:Nonmuscle Myosin Type IIA; D024501:Nonmuscle Myosin Type IIB; D008274:Magnesium