Partial characterization of a metalloendopeptidase activity produced by cultured endothelial cells that removes the COOH-terminal tripeptide from 125I-atrial natriuretic factor.
The presence of the COOH-terminal region of human atrial natriuretic factor-(99-126) (hANF) is necessary for the full expression of its biological activity. Here, we report on the partial characterization of a proteolytic activity in the conditioned medium from cultured bovine aortic endothelial cells that cleaves the Ser123-Phe124 bond of 125I-hANF generating the COOH-terminal tripeptide. The concentrated conditioned medium was fractionated by gel filtration high performance liquid chromatography and fractions were assayed for the ability to generate the COOH-terminal tripeptide from 125I-hANF. This analysis indicated that the protein responsible for this activity had an approximate molecular weight of 200,000 daltons. Of 16 protease inhibitors tested, only 1,10 phenanthroline, EDTA, EGTA and N-ethylmaleimide significantly inhibited the endopeptidase activity. Thus, we conclude that cultured bovine aortic endothelial cells produce a potentially novel phosphoramidon-insensitive metalloendopeptidase that removes the COOH-terminal tripeptide from 125I-hANF.
Johnson
G R
GR
Department of Pharmacology, Schering-Plough Research, Bloomfield, NJ 07003.
Foster
C J
CJ
D000818:Animals; D009320:Atrial Natriuretic Factor / Q000378:metabolism*; D002417:Cattle; D002478:Cells, Cultured; D002850:Chromatography, Gel; D004730:Endothelium, Vascular / Q000166:cytology / Q000201:enzymology* / Q000378:metabolism; D006868:Hydrolysis; D007457:Iodine Radioisotopes; D008666:Metalloendopeptidases / Q000378:metabolism*; D008970:Molecular Weight; D011480:Protease Inhibitors
D007457:Iodine Radioisotopes; D011480:Protease Inhibitors; D009320:Atrial Natriuretic Factor; D008666:Metalloendopeptidases