Expression, purification and characterization of the monomeric and dimeric forms of soluble bovine endothelin converting enzyme-1a.
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(TM) 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 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 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 K(m) and k(cat) values for the monomer were 2.2 microM and 1.6 min(-1) respectively, while those of the dimer were 1.4 microM 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.
Kulathila
Raviraj
R
Novartis Institute for Biomedical Research, 556 Morris Avenue, Summit, NJ 07901, U.S.A.
Clark
Kirk
K
Savage
Paula
P
Bowen
Benjamin R
BR
Emoto
Noriaki
N
Yanagisawa
Masashi
M
Jeng
Arco Y
AY
D000469:Alkaline Phosphatase / Q000235:genetics; D000818:Animals; D016282:Aspartic Acid Endopeptidases / Q000302:isolation & purification* / Q000378:metabolism; D016367:Baculoviridae / Q000235:genetics; D002417:Cattle; D002460:Cell Line; D002845:Chromatography; D003001:Cloning, Molecular; D004492:Edetic Acid / Q000494:pharmacology; D019332:Endothelin-1 / Q000378:metabolism; D000072396:Endothelin-Converting Enzymes; D016232:Endothelins / Q000378:metabolism; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005822:Genetic Vectors; D006020:Glycopeptides / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007313:Insecta; D007527:Isoenzymes / Q000302:isolation & purification* / Q000378:metabolism; D008666:Metalloendopeptidases / Q000037:antagonists & inhibitors; D011498:Protein Precursors / Q000378:metabolism; D012965:Sodium Chloride / Q000494:pharmacology; D014162:Transfection
D019332:Endothelin-1; D016232:Endothelins; D004791:Enzyme Inhibitors; D006020:Glycopeptides; D007527:Isoenzymes; D011498:Protein Precursors; D012965:Sodium Chloride; D004492:Edetic Acid; D000469:Alkaline Phosphatase; D016282:Aspartic Acid Endopeptidases; D008666:Metalloendopeptidases; D000072396:Endothelin-Converting Enzymes; C008890:phosphoramidon