Purification and characterization of angiotensin II degradation factor from porcine endothelial cells.
We investigated the degradation of angiotensin II by vascular endothelial cells and smooth muscle cells in vitro. When angiotensin II was incubated with confluent culture of endothelial cells or with serum free conditioned medium of the endothelial cells, angiotensin II was destroyed rapidly. When angiotensin II was incubated with cultured vascular smooth muscle cells or their serum free conditioned medium, degradation was not observed. To identify the angiotensin II degradation factor (ADF), we have purified ADF from the conditioned medium of endothelial cells, by column chromatographies, i.e., hydroxyapatite, ion exchange and gel filtration chromatography. The partially purified ADF had apparent molecular masses of 154 kDa on gel filtration chromatography. Its pH optimum was about 7.0. ADF was inhibited by p-chloromercuribenzoic acid, iodoacetamide and high concentration of EDTA, but not by diisopropyl fluorophosphate, bestatin, amastatin or pepstatin A. Of the synthetic substrates examined, ADF degrades human angiotensin II, [Val5]-angiotensin II and [Asn1, Val5]-angiotensin II. It did not degrade angiotensin I.
Fujioka
H
H
Department of Internal Medicine, Juntendo University School of Medicine, Tokyo.
Okabe
T
T
Yamaguchi
H
H
D000645:Ammonium Sulfate; D000804:Angiotensin II / Q000378:metabolism*; D000818:Animals; D001011:Aorta / Q000737:chemistry / Q000166:cytology / Q000201:enzymology; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D016895:Culture Media, Serum-Free; D003546:Cysteine Endopeptidases / Q000032:analysis / Q000302:isolation & purification* / Q000378:metabolism; D015853:Cysteine Proteinase Inhibitors; D004730:Endothelium, Vascular / Q000737:chemistry* / Q000166:cytology / Q000201:enzymology; D006863:Hydrogen-Ion Concentration; D013552:Swine
D016895:Culture Media, Serum-Free; D015853:Cysteine Proteinase Inhibitors; D000804:Angiotensin II; D003546:Cysteine Endopeptidases; C102008:angiotensin II degradation factor; D000645:Ammonium Sulfate