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

Momoyo Ishikawa, Takahiro Fujino, Hitoshi Sakashita, Kosuke Morikawa, Tokuo Yamamoto

Research output: Contribution to journalArticlepeer-review

13 Citations (Scopus)

Abstract

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 45 units/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 0.16 mM, 0.14 mM and 0.25 mM, respectively. Limited digestion with trypsin, subtilisin BPN' and chymotrypsin revealed that the enzyme contains a 56 k segment resistant to these proteases. Secondary structure contents of the purified enzyme and the 56 k 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 72 k enzyme is present in a wide variety of tissues and is most abundant in heart and kidney.

Original languageEnglish
Pages (from-to)55-67
Number of pages13
JournalTohoku Journal of Experimental Medicine
Volume175
Issue number1
DOIs
Publication statusPublished - 1995

Keywords

  • acetyl-CoA synthetase
  • bovine heart
  • crystallization
  • purification
  • tissue distribution

ASJC Scopus subject areas

  • Biochemistry, Genetics and Molecular Biology(all)

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