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 FiveTM insect cells in suspension culture. Both the monomeric (85kDa) and dimeric (170kDa) 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 11mg of ECE-1a monomer and 6mg of dimer were obtained from l 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 500mM, 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 2.2µM and 1.6min-1 respectively, while those of the dimer were 1.4µM and 4.9min-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.
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Conference Article|
September 01 2002
Expression, purification and characterization of the monomeric and dimeric forms of soluble bovine endothelin converting enzyme-1a
Raviraj KULATHILA
;
Raviraj KULATHILA
*
Novartis Institute for Biomedical Research, 556 Morris Avenue, Summit, NJ 07901, U.S.A.
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Kirk CLARK
;
Kirk CLARK
*
Novartis Institute for Biomedical Research, 556 Morris Avenue, Summit, NJ 07901, U.S.A.
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Paula SAVAGE
;
Paula SAVAGE
*
Novartis Institute for Biomedical Research, 556 Morris Avenue, Summit, NJ 07901, U.S.A.
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Benjamin R. BOWEN
;
Benjamin R. BOWEN
*
Novartis Institute for Biomedical Research, 556 Morris Avenue, Summit, NJ 07901, U.S.A.
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Noriaki EMOTO
;
Noriaki EMOTO
†
Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, Dallas, TX 75253, U.S.A.
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Masashi YANAGISAWA
;
Masashi YANAGISAWA
†
Howard Hughes Medical Institute, University of Texas Southwestern Medical Center, Dallas, TX 75253, U.S.A.
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Arco Y. JENG
*
Novartis Institute for Biomedical Research, 556 Morris Avenue, Summit, NJ 07901, U.S.A.
Dr A.Y. Jeng, Bldg. 130, Room 2227, Novartis Pharmaceuticals Corporation, 556 Morris Avenue, Summit, NJ 07901, U.S.A. (e-mail arco.jeng@pharma.novartis.com).
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Clin Sci (Lond) (2002) 103 (s2002): 94S-97S.
Citation
Raviraj KULATHILA, Kirk CLARK, Paula SAVAGE, Benjamin R. BOWEN, Noriaki EMOTO, Masashi YANAGISAWA, Arco Y. JENG; Expression, purification and characterization of the monomeric and dimeric forms of soluble bovine endothelin converting enzyme-1a. Clin Sci (Lond) 1 September 2002; 103 (s2002): 94S–97S. doi: https://doi.org/10.1042/CS103S094S
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