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Abstract
This paper presents the first purification of the branched chain aminotransferase (EC 2.6.1.42) from rat heart mitochondria. The enzyme has been purified from the 100,000 x g supernatant obtained after sonication and ultracentrifugation of rat heart mitochondria. A combination of open column chromatography, high pressure liquid chromatography (HPLC), and discontinuous polyacrylamide disc gel electrophoresis was used. The key step in the procedure was hydrophobic interaction chromatography on HPLC. The final purification step was polyacrylamide disc gel electrophoresis where the enzyme appeared as a doublet. When electroeluted from the gel, each of these bands had the same specific activity demonstrating that there are two forms of the purified enzyme which differ slightly in electrical charge. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, these two enzyme forms appeared as a single band with a molecular mass of 43 kDa. Size exclusion chromatography on Sephacryl S-100 identified the enzyme as a 50-kDa protein. These experiments argue against the existence of a dimeric form of this enzyme. The ratio of enzyme activity with leucine (0.84), valine (0.88), or glutamate (0.66) as amino acid substrate versus isoleucine remained constant throughout the purification procedure. Specific activity of the final preparation was 66 units/mg of enzyme protein. Polyclonal antibodies against the purified enzyme were raised in rabbits. On an immunoblot the antiserum recognized a 43-kDa protein in the 100,000 x g supernatant from a rat heart mitochondrial sonicate but did not recognize any proteins in rat brain cytosol. Quantitative immunodot assay resulted in an estimated enzyme content of about 100 micrograms of branched chain aminotransferase protein/g of heart, wet weight. Finally, 97% of the heart branched chain aminotransferase activity could be neutralized by the antiserum, but the antiserum would not neutralize aminotransferase activity in brain cytosol. These data suggest that close sequence homology does not exist between the two proteins.
Highlights
From the Departments of Biochemistry and Medicine, Section of Rheumatology, Wake Forest University, Winston-Salem, North Carolina 27103
Purification-The data presented in Table I describe purification of branched chain aminotransferase isolated from rat heart mitochondria
SDS-PAGE of the purified enzyme when carried out both under reducing (5% mercaptoethanol) and nonreducing conditions yielded a protein band of 43 kDa (Fig. 5, lane E, nonreducing gel not shown). We concluded from these data that the branched chain aminotransferase isolated from rat heart mitochondria is a single polypeptide with a molecular mass of 43 kDa
Summary
Heart mitochondria were prepared as described bv LaNoue et al (12). The isolation medium contained 0.225 M mannitol, 0.075 M sucrose, 0.1 mM EDTA, 5 mM. For purification of the branched chain aminotransfer&e, i mM DIFP-was added to the 600 X g supernatant before
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