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Published on: May 7, 2018
A new method to precipitate myosin V from rat brain soluble fraction
Hugo Christiano Soares Melo1, Milton Vieira Coelho
1Universidade Federal de Uberlandia, Institute of Genetics and Biochemistry, Uberlândia - MG, Brazil.
Abstract:
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.
Insights
Researchers developed a novel method to precipitate myosin V from rat brain soluble fractions using freezing. This technique successfully isolated myosin V, which exhibited significant Mg(2+)-ATPase activity.
Area of Science:
- Biochemistry
- Molecular Biology
- Neuroscience
Background:
- Myosin V is a crucial motor protein involved in intracellular transport.
- Existing methods for myosin V precipitation can be complex or inefficient.
- Rat brain is a rich source of various myosin isoforms.
Purpose of the Study:
- To establish a new, simplified method for precipitating myosin V from rat brain soluble fractions.
- To characterize the biochemical properties of the precipitated myosin V.
- To identify associated proteins co-purifying with myosin V.
Main Methods:
- Homogenization of rat brains in a specialized buffer.
- Differential centrifugation to obtain soluble fractions.
- Precipitation of myosin V via freezing of the supernatant.
- Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) for polypeptide analysis.
- Assay of Mg(2+)-ATPase activity.
- Western blot analysis using a specific myosin V antibody.
- Calpain proteolysis assay.
Main Results:
- A novel precipitation method involving freezing of the soluble fraction was successfully developed.
- SDS-PAGE revealed four major polypeptides in the precipitate, including a 205 kDa polypeptide recognized as myosin V.
- The precipitate exhibited high Mg(2+)-ATPase activity, which co-purified with the p205 polypeptide.
- Myosin V was proteolysed by calpain into p130 and p90 fragments.
- A 45 kDa polypeptide was found to co-purify with rat brain myosin V.
Conclusions:
- Freezing of soluble fractions provides an effective method for precipitating rat brain myosin V.
- The precipitated myosin V possesses significant Mg(2+)-ATPase activity.
- The study identified a novel 45 kDa co-purifying protein with myosin V.
