N-ethylmaleimide differentiates endothelin converting activity by two types of metalloproteinases derived from vascular endothelial cells.
We have recently found that cultured vascular endothelial cells (ECs) contain two types of metalloproteinases which convert big endothelin-1 (big ET-1) to endothelin-1 (ET-1) via a single cleavage between Trp21 and Val22. In the present study, two enzymes were clearly differentiated by using sulfhydryl blocking reagents and anion-exchange HPLC. As reported, the converting activity of the membrane fraction of ECs was specifically inhibited by phosphoramidon. N-ethylmaleimide (NEM) markedly enhanced the apparent converting activity of the membrane fraction. This enhancement was not due to the direct action on the converting enzyme, but rather to inhibition of the degradation of big ET-1 and/or ET-1. In contrast, the converting activity of the cytosolic fraction was abolished by NEM treatment. Effects of phosphoramidon and NEM on converting activities of both fractions were confirmed after anion-exchange HPLC of each fraction, using a COSMOGEL QA column. Our results provide new information on two types of metalloproteinases which convert big ET-1 to ET-1, in vascular ECs.
Matsumura
Y
Y
Department of Pharmacology, Osaka University of Pharmaceutical Sciences, Japan.
Ikegawa
R
R
Tsukahara
Y
Y
Takaoka
M
M
Morimoto
S
S
D000818:Animals; D001013:Aorta, Thoracic; D002462:Cell Membrane / Q000201:enzymology; D002478:Cells, Cultured; D002851:Chromatography, High Pressure Liquid; D003600:Cytosol / Q000201:enzymology; D019332:Endothelin-1; D016232:Endothelins / Q000096:biosynthesis* / Q000235:genetics / Q000378:metabolism*; D005033:Ethylmaleimide / Q000494:pharmacology*; D007527:Isoenzymes / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D008666:Metalloendopeptidases / Q000302:isolation & purification / Q000378:metabolism*; D009131:Muscle, Smooth, Vascular / Q000201:enzymology*; D011498:Protein Precursors / Q000235:genetics / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D013552:Swine
D019332:Endothelin-1; D016232:Endothelins; D007527:Isoenzymes; D011498:Protein Precursors; D008666:Metalloendopeptidases; D005033:Ethylmaleimide