Endothelin converting enzymes in guinea-pig lung membrane fractions: purifications and characterizations.
We have identified neutral proteolytic activities that convert big endothelin-1 (big ET-1) to ET-1 in guinea-pig lung membrane fraction. The active proteins have been solubilized and two distinct enzymes have been purified by combinations of sequential column chromatographies. One purified enzyme was a metalloenzyme based upon its sensitivity to chelating agent with a molecular mass of 108 kDa on SDS-PAGE. Another enzyme was also a metalloenzyme with a molecular mass of 162 kDa. Further investigations revealed that the 108 kDa enzyme was inhibited by phosphoramidon and also by thiorphan, and produced ET-1 with the Km value of 5.7 microM for big ET-1. The 162 kDa enzyme was also inhibited by phosphoramidon, but neither by thiorphan nor by captopril, and quantitatively produced ET-1 from big ET-1 with a Km value of 2.1 microM. These results indicate that the 108 kDa enzyme probably seems to be a neutral-endopeptidase (EC 3. 4. 24. 11.) or similar one, but the 162 kDa enzyme is a unique metalloprotease that converts big ET-1 to ET-1.
Shima
H
H
Research Laboratory of Applied Biochemistry, Tanabe Seiyaku Co. Ltd., Osaka, Japan.
Kawashima
Y
Y
Ohmori
K
K
Sugiura
M
M
Kawashima
K
K
D000818:Animals; D016282:Aspartic Acid Endopeptidases / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D002458:Cell Fractionation; D002614:Chelating Agents / Q000494:pharmacology; D019332:Endothelin-1; D000072396:Endothelin-Converting Enzymes; D016232:Endothelins / Q000378:metabolism*; D006168:Guinea Pigs; D006801:Humans; D007124:Immunoenzyme Techniques; D008168:Lung / Q000201:enzymology*; D008297:Male; D008666:Metalloendopeptidases; D051379:Mice; D015260:Neprilysin / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D011480:Protease Inhibitors / Q000494:pharmacology; D011498:Protein Precursors / Q000378:metabolism*
D002614:Chelating Agents; D019332:Endothelin-1; D016232:Endothelins; D011480:Protease Inhibitors; D011498:Protein Precursors; D016282:Aspartic Acid Endopeptidases; D008666:Metalloendopeptidases; D015260:Neprilysin; D000072396:Endothelin-Converting Enzymes