Kinetic Properties and Structural Characterization of Highly Purified Acetyl-CoA Synthetase from Bovine Heart and Tissue Distribution of the Enzyme in Rat Tissues

ISHIKAWA, M., FUJINO, T., SAKASHITA, H., MORIKAWA, K. and YAMAMOTO, T. Kinetic Properties and Structural Characterization of Highly Purified Acetyl-CoA Synthetase from Bovine Heart and Tissue Distribution of the Enzyme in Rat Tissues. Tohoku J. Exp. Med., 1995, 175 (1), 55-67-Acetyl-CoA synthetase from bovine heart has been purified to homogeneity and been crystallized. The purification procedure involves ammonium sulfate precipitation and subsequent column chromatography on DEAE-Sepharose, Blue-Sepharose, CoA-Agarose and Superose 6. The purified enzyme has a specific activity of 45units/mg protein, and its molecular weight estimated by sodium dodecyl sulfate polyacrylamide gel electrophoresis is approximately 72, 000. The purified enzyme specifically utilizes acetate, ATP and CoA. Apparent Km values of the purified enzyme for acetate, CoA, and ATP were 0.16mM, 0.14mM and 0.25mM, respectively. Limited digestion with trypsin, subtilisin BPN' and chymotrypsin revealed that the enzyme contains a 56k segment resistant to these proteases. Secondary structure contents of the purified enzyme and the 56k Cryptic fragment were analyzed by circular dichroism measurement. The intact molecule contains 30% α-helix and 30% β-structure, and trypsin digests α-helix rich regions more substantially. Western blot analysis of rat tissue homogenates by specific antibodies against the purified enzyme indicated that the 72k enzyme is present in a wide variety of tissues and is most abundant in heart and kidney.