Purification and characterization of putative endothelin converting enzyme in bovine adrenal medulla: evidence for a cathepsin D-like enzyme.
A specific and sensitive assay has been established for measurement of endothelin converting activity in a tissue extract. This assay is based on measuring endothelin-1 generated from big endothelin-1 by endothelin converting enzyme (ECE) with radioimmunoassay using an endothelin C-terminal specific antibody. By using this assay, we purified and characterized ECE in bovine adrenomedullary chromaffin granules ECE was purified over 3,000 times by a combination of DEAE, hydrophobic and gel filtration chromatography. A molecular weight of ECE was estimated to be approximately 30,000 by gel filtration. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that ECE had three major components with estimated molecular weights of 45,000, 30,000 and 15,000 like bovine spleen cathepsin D. ECE had a pH optimum at 3.5 and was inhibited by pepstatin. These results strongly suggest that ECE is a cathepsin D-like aspartic protease.
Sawamura
T
T
Department of Biochemistry, University of Tsukuba, Ibaraki, Japan.
Kimura
S
S
Shinmi
O
O
Sugita
Y
Y
Yanagisawa
M
M
Goto
K
K
Masaki
T
T
D000313:Adrenal Medulla / Q000201:enzymology*; D000818:Animals; D016282:Aspartic Acid Endopeptidases; D002402:Cathepsin D / Q000378:metabolism; D002417:Cattle; D002837:Chromaffin Granules / Q000201:enzymology; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases / Q000302:isolation & purification* / Q000378:metabolism; D000072396:Endothelin-Converting Enzymes; D006863:Hydrogen-Ion Concentration; D008666:Metalloendopeptidases; D008970:Molecular Weight; D010447:Peptide Hydrolases; D011863:Radioimmunoassay
D010450:Endopeptidases; D010447:Peptide Hydrolases; D016282:Aspartic Acid Endopeptidases; D002402:Cathepsin D; D008666:Metalloendopeptidases; D000072396:Endothelin-Converting Enzymes