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Characterization and Partial Purification of Endothelin-Converting Enzyme in Rat Lung
- Source :
- Journal of Cardiovascular Pharmacology. 17:S20-25
- Publication Year :
- 1991
- Publisher :
- Ovid Technologies (Wolters Kluwer Health), 1991.
-
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.
- Subjects :
- Proteolysis
Endothelin-Converting Enzymes
Biology
Cleavage (embryo)
Isomerism
Cations
medicine
Animals
Aspartic Acid Endopeptidases
Lung
Peptide sequence
Anion Exchange Resins
Chromatography, High Pressure Liquid
Pharmacology
chemistry.chemical_classification
Dibasic acid
medicine.diagnostic_test
Sepharose
Metalloendopeptidases
Hydrogen-Ion Concentration
Endothelin 1
Molecular biology
Enzyme assay
Rats
Molecular Weight
Resins, Synthetic
Enzyme
chemistry
Biochemistry
Chromatography, Gel
biology.protein
Electrophoresis, Polyacrylamide Gel
Ion Exchange Resins
Isoelectric Focusing
Cardiology and Cardiovascular Medicine
Endothelin receptor
Subcellular Fractions
Subjects
Details
- ISSN :
- 01602446
- Volume :
- 17
- Database :
- OpenAIRE
- Journal :
- Journal of Cardiovascular Pharmacology
- Accession number :
- edsair.doi.dedup.....d2bf48170a7e10645535c5f526defad8