Phosphoramidon-sensitive endothelin-converting enzyme in vascular endothelial cells converts big endothelin-1 and big endothelin-3 to their mature form

AbstractIncubation of big endothelin-3 (big ET-31–41) with the membrane fraction obtained from cultured endothelial cells (ECs) resulted in an increase in immunoreactive-ET (IR-ET). This increasing activity was markedly suppressed by phosphoramidon, which is known to inhibit the conversion of big ET-11–39 to ET-11–21. Reverse-phase HPLC of the incubation mixture of the membrane fraction with big ET-3 revealed one major IR-ET component corresponding to the elution position of synthetic ET-31–21. When the cultured ECs were incubated with big ET-3, a conversion to the mature ET-3, as well as an endogenous ET-1 generation, was observed. Both responses were markedly suppressed by phosphoramidon. By the gel filtration of 0.5% CHAPS-solubilized fraction of membrane pellets or ECs, the molecular mass of the proteinase which converts big ET-1 and big ET-3 to their mature form was estimated to be 300–350 kDa. Phosphoramidon almost completely abolished both converting activities of the proteinase. We conclude that the above type of phosphoramidon-sensitive metalloproteinase functions as an ET-converting enzyme to generate the mature form from big ET-1 and big ET-3 in ECs