title: Characterization and partial purification of endothelin-converting enzyme in rat lung.
abstract: Endothelin-1 (ET-1) was originally identified in the culture supernatant of porcine aortic endothelial cells. From the deduced amino acid sequence, a biosynthetic pathway has been proposed that a prepro- form of porcine ET-1 is initially processed by dibasic pair proteolysis to a 39-amino acid intermediate form (big ET), which is then converted to ET-1 by specific proteolytic cleavage between Trp21 and Val22. We have identified an enzyme activity that converts human big endothelin[1-38] to endothelin[1-21] and a C-terminal fragment (CTF, 22-38) in a homogenate fraction from rat lung. The conversion activity was enriched threefold in a plasma membrane fraction. Metal ions activated the activity by about 1.5- to 2.5-fold, in the order of Mn2+ greater than Zn2+ = Ca2+ greater than Mg2+ greater than Ba2+. The conversion activity was optimal at pH 4.0, was inhibited by pepstatin-A (IC50 = 20 nmol), but not affected by TLCK, aprotinin, PMSF, E-64, bestatin, phosphoramidon, or thiorphan at 40 microM. The converting enzyme was partially purified from rat lung plasma membranes by sequential HPLC on Mono Q, Superose 12, and Mono P. The enzyme has an apparent molecular mass of 90 kDa as estimated by SDS-PAGE or gel filtration and appears to be a single peptide protein. The enzyme may exist as isozymes with isoelectric point (pI) values at 6.2 and 6.3.
authors.affiliation: Pharmaceutical Products Division, Abbott Laboratories, Abbott Park, Illinois 60064.
authors.forename: J R
authors.initials: JR
authors.lastname: Wu-Wong
authors.forename: E M
authors.initials: EM
authors.lastname: Devine
authors.forename: G P
authors.initials: GP
authors.lastname: Budzik
authors.forename: T J
authors.initials: TJ
authors.lastname: Opgenorth
mesh_terms: D000818:Animals; D000837:Anion Exchange Resins; D016282:Aspartic Acid Endopeptidases / Q000302:isolation & purification*; D002412:Cations; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D000072396:Endothelin-Converting Enzymes; D006863:Hydrogen-Ion Concentration; D007475:Ion Exchange Resins; D007525:Isoelectric Focusing; D007536:Isomerism; D008168:Lung / Q000201:enzymology*; D008666:Metalloendopeptidases; D008970:Molecular Weight; D051381:Rats; D012117:Resins, Synthetic; D012685:Sepharose; D013347:Subcellular Fractions / Q000201:enzymology
chemical_list: D000837:Anion Exchange Resins; D002412:Cations; D007475:Ion Exchange Resins; D012117:Resins, Synthetic; C042855:Mono Q; D012685:Sepharose; C049225:Superose 12; D016282:Aspartic Acid Endopeptidases; D008666:Metalloendopeptidases; D000072396:Endothelin-Converting Enzymes