Precipitation of kidney myosin IIA and IIB by freezing Decivaldo dos Santos Dias*, Grabriel Costa Nunes da Cruz { , Marcelo Valle de Sousa { and Milton Vieira Coelho 1 * * Universidade Federal de Uberla ˆ ndia, Instituto de Gene ´tica e Bioquı´mica. Av. Para ´ 1720, Bloco 2E39b, Bairro Umuarama, Uberla ˆ ndia, Minas Gerais, Brazil { Universidade de Brası´lia, Instituto de Cie ˆ ncias Biolo ´ gicas, Departamento de Biologia Celular, Laborato ´rio de Bioquı´mica e Quı´mica de Proteı´nas – LBQP, Bloco C, Sub-solo, Mo ´dulo 2, Brası´lia, Distrito Federal, Brazil 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 220uC for 48 h. The supernatant was thawed at 4uC, 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. Keywords: actomyosin; ATPase; kidney; myosin IIA; precipitation; rat 1. Introduction Non-muscle myosin II form filaments at relatively low ionic strength and share a number of biological properties with skeletal, cardiac and smooth muscle myosin II (Sellers, 1999). They are hexamers composed of a pair of heavy chains of ,200 kDa and two pairs of light chains, ,20 and ,17 kDa. The heavy chain consists of a globular N-terminal region (termed the head) that binds to actin and hydrolyses ATP and an extended C-terminal region (termed tail) that coils together with another heavy chain tail to form a rigid rod- like structure (Sellers, 2000). The domain where light chains bind is located between the head domain and the tail domain. Non-muscle myosin II is widely expressed in eukaryotic cells, and it is involved in various cellular processes such as cytokinesis (Robinson and Spudich, 2004; Matsumura, 2005), cell adhesion (Mu ¨ sch et al., 1997) and vesicular traffic (Togo and Steinhardt, 2004). To date, three isoforms of the myosin II heavy chain, termed myosins IIA, IIB and IIC, have been identified in vertebrates (Katsuragawa et al., 1989; Shohet et al., 1989; Takahashi et al., 1992; Golomb et al., 2004). Though most cells express all myosin II isoforms, some cells, such as platelets, only express the protein myosin IIA (Maupin et al., 1994; Toren et al., 2000). In the human kidney, the gene that encodes myosin IIA heavy chain is expressed in both fetal and mature tissues (Arrondel et al., 2002), and mutation in this gene is associated with different syndromes with or without associated kidney disease (Kelley et al., 2000; Seri et al., 2000; Kunishima et al., 2001, 2008; Freedman et al., 2009). Myosin IIA is mostly expressed by the podocyte cells, and it constitutes the major component of the actin–myosin contractile apparatus in the podocyte foot process (Arrondel et al., 2002). The kidney myosin II isoform has not yet been characterized, and the characterization of non-muscle myosin II isoforms, in general, was carried out with part of the protein expressed in recombinant systems (Pato et al., 1996; Kova ´ cs et al., 2003; Wang et al., 2003; Golomb et al., 2004), since there is no effective method for purification of these proteins. As the native, full-length, myosin IIs are difficult to make in current recombinant systems, the purification of the native kidney myosin IIA molecule may contribute to a better understanding of the structure–function relationships of this molecular motor. Actomyosin precipitation is a common stage in myosin purification methods. The brain myosin V is precipitated selectively by treating the soluble fraction of brain with 600 mM NaCl (Cheney et al., 1993; Coelho and Larson, 1993), while brain myosin II is precipitated by dialysing the brain soluble fraction against low-salt-containing buffer (Espindola et al., 1992). The freezing of soluble fraction from brain (Melo and Coelho, 2007), testicle and muscle (Dias and Coelho, 2007) also causes actomyosin precipitation. In this work, we isolated myosin IIA starting from actomyosin obtained by freezing rat kidney soluble fraction. 2. Materials and methods 2.1. Materials Alkaline phosphatase conjugated to goat anti-rabbit IgG (Fc), NBT (nitrotetrazolium blue), BCIP (bromochloroindolyl phosphate), EDTA, EGTA, SDS, DTT (dithiothreitol), TFA (trifluoroacetic acid), ATP, NH 4 HCO 3 (ammonium bicarbonate), Ponceau, buffers, 1 To whom correspondence should be addressed (email mvcoelho@ufu.br). Abbreviations: DTT, dithiothreitol; NH 4 HCO 3 , ammonium bicarbonate; P i , inorganic phosphate; PMF, peptide mass fingerprinting; S1, supernatant; TFA, trifluoroacetic acid. Cell Biol. Int. (2011) 35, 259–266 (Printed in Great Britain) Research Article E The Author(s) Journal compilation E 2011 Portland Press Limited Volume 35 (3) N pages 259–266 N doi:10.1042/CBI20090397 N www.cellbiolint.org 259