Purification and characterization of a phosphoramidon-sensitive endothelin-converting enzyme in porcine aortic endothelium

K. Ohnaka, R. Takayanagi, M. Nishikawa, M. Haji, H. Nawata

研究成果: Contribution to journalArticle査読

120 被引用数 (Scopus)

抄録

An enzyme that catalyzes the conversion of big endothelin-1 to endothelin- 1, designated as endothelin-converting enzyme, was solubilized with Lubrol PX from the membrane fraction of porcine aortic endothelium and was purified by sequential chromatography on DEAE-agarose, Ricinus communis agglutinin 120- agarose, peanut agglutinin-agarose, Mono Q, and TSK G3000SW(XL) columns. Approximately 12,000-fold purification of the membrane fraction enzyme was achieved. The purified enzyme had a very narrow neutral pH optimum and was inhibited by EDTA, 1,10-phenanthroline, phosphoramidon, and low concentrations of some divalent cations (Cu2+, Zn2+, Co2+, Fe2+) but not by thiorphan. Addition of Zn2+ was most effective for the restoration of the EDTA-inactivated enzyme. The purified enzyme showed the highest affinity for big endothelin-1 among big endothelin isopeptides, and the K(m) for big endothelin-1 and the corresponding V(max) for endothelin-1 formation were 3.3 ± 0.3 μM and 0.41 ± 0.02 μmol/min/mg of protein, respectively. The carboxyl-terminal sequence from His27 to Gly34 and Trp21 was essential for recognition by this enzyme, while the presence of the amino- terminal loop structure reduced the hydrolysis rate. The purified enzyme showed an isoelectric point of 4.1. The molecular mass was estimated to be 131 kDa by sucrose density gradient centrifugation, and a value of 120 kDa was obtained by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, indicating that endothelin-converting enzyme is a monomeric glycoprotein.

本文言語英語
ページ(範囲)26759-26766
ページ数8
ジャーナルJournal of Biological Chemistry
268
35
出版ステータス出版済み - 1 1 1993

All Science Journal Classification (ASJC) codes

  • Biochemistry
  • Molecular Biology
  • Cell Biology

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