Expression, purification and characterization of the monomeric and dimeric forms of soluble bovine endothelin converting enzyme-1a

Raviraj Kulathila, Kirk Clark, Paula Savage, Benjamin R. Bowen, Noriaki Emoto, Masashi Yanagisawa, Arco Y. Jeng

Research output: Contribution to journalArticlepeer-review

3 Scopus citations

Abstract

In this study, the catalytic domain of bovine endothelin converting enzyme-1a (ECE-1a) was cloned into a baculovirus transfer vector behind the human alkaline phosphatase signal sequence. The recombinant baculovirus was then used to infect High Five™ insect cells in suspension culture. Both the monomeric (85 kDa) and dimeric (170 kDa) forms of soluble ECE-1a were purified to electrophoretic homogeneity from concentrated culture media following sequential concanavalin A, SP-Sepharose, Mono Q and gel filtration column chromatography. Typically, approximately 11 mg of ECE-1a monomer and 6 mg of dimer were obtained from 1 litre of culture medium. No interconversion of the two forms was detected after purification. Both forms of ECE-1a had a pH optimum of 7.0, were maximally stimulated by NaCl at a concentration of 500 mM, and were inhibited to the same extent by metalloprotease inhibitors such as phosphoramidon and EDTA. However, in kinetic studies using big endothelin-1 (ET-1) as a substrate, the Km and kcat values for the monomer were 2.2 μM and 1.6 min-1 respectively, while those of the dimer were 1.4 μM and 4.9 min-1 respectively. These results show that, although the two forms of ECE-1a behave similarly in many aspects, the dimeric enzyme is more efficient in catalysing the conversion of big ET-1 to ET-1. The present protocol can be utilized to prepare large quantities of both forms of ECE-1a for further biochemical and structural characterization.

Original languageEnglish (US)
Pages (from-to)94S-97S
JournalClinical science
Volume103
Issue numberSUPPL. 48
StatePublished - Aug 1 2002

Keywords

  • Dimer
  • ECE-1a
  • Endothelin converting enzyme-1a
  • Expression
  • Monomer

ASJC Scopus subject areas

  • Medicine(all)

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