Precipitation of kidney myosin IIA and IIB by freezing

Authors

  • Decivaldo dos Santos Dias,

    1. Universidade Federal de Uberlndia, Instituto de Gentica e Bioqumica. Av. Par 1720, Bloco 2E39b, Bairro Umuarama, Uberlndia, Minas Gerais, Brazil
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  • Grabriel Costa Nunes da Cruz,

    1. Universidade de Braslia, Instituto de Cincias Biolgicas, Departamento de Biologia Celular, Laboratrio de Bioqumica e Qumica de Protenas LBQP, Bloco C, Subsolo, Mdulo 2, Braslia, Distrito Federal, Brazil
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  • Marcelo Valle de Sousa,

    1. Universidade de Braslia, Instituto de Cincias Biolgicas, Departamento de Biologia Celular, Laboratrio de Bioqumica e Qumica de Protenas LBQP, Bloco C, Subsolo, Mdulo 2, Braslia, Distrito Federal, Brazil
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  • Milton Vieira Coelho

    Corresponding author
    1. Universidade Federal de Uberlndia, Instituto de Gentica e Bioqumica. Av. Par 1720, Bloco 2E39b, Bairro Umuarama, Uberlndia, Minas Gerais, Brazil
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To whom correspondence should be addressed (email mvcoelho@ufu.br).

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.

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