A new method to precipitate myosin V from rat brain soluble fraction.
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 x g for 40 min and the supernatant was frozen at -20 degrees C. Forty-eight hours later, the supernatant was thawed, centrifuged at 45000 x 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 Mg(2+)-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 Mg(2+)-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 Mg(2+)-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.
Melo
Hugo Christiano Soares
HC
Universidade Federal de Uberlandia, Institute of Genetics and Biochemistry, Uberlândia - MG, Brazil.
Coelho
Milton Vieira
MV
D000199:Actins / Q000378:metabolism; D000251:Adenosine Triphosphatases / Q000378:metabolism; D000818:Animals; D001921:Brain / Q000378:metabolism*; D002147:Calmodulin / Q000378:metabolism; D002154:Calpain / Q000378:metabolism; D011232:Chemical Precipitation; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D024701:Myosin Type V / Q000302:isolation & purification* / Q000378:metabolism*; D051381:Rats
D000199:Actins; D002147:Calmodulin; D002154:Calpain; D000251:Adenosine Triphosphatases; D024701:Myosin Type V