Endothelin-1 (ET-1) is the most potent vasoactive peptide known to date. The peptide is initially synthesized as an inactive precursor (proET-l) which undergoes proteolysis at specific pairs of basic amino acids to yield bigET-1. Production of ET-! then proceeds by cleavage of bigET-1 by the endothelin converting enzyme (ECE). Here, we demonstrate that the in vitro cleavage of proET-1 by furin, a mammalian convertase involved in precursor processing, produced bigET-1. Upon further processing, bigET-1 was converted to biologically active ET-1. Furthermore, we demonstrate that the furin inhibitor, decanoyI-Arg-ValLys-Arg chloromethylketone, abolished production of ET-1 in endothelial cells.

We have cloned cDNA encoding bovine endothelin converting enzyme (ECE). The predicted amino acid sequence of bovine ECE consisted of 758 amino acid residues. Northern blot analysis revealed that ECE mRNA was abundantly expressed in lung. Co-expression of the cloned cDNA of bovine ECE with human preproET-1 cDNA in CHO-K1 cells resulted in the production of mature ET-1 and this production was inhibited by phosphoramidon.

A nitroalkane-oxidizing enzyme, which was inducibly formed by addition of nitroethane to the medium, was purified to homogeneity from an extract of Fusarium oxysporum (IFO 5942) with an overall yield of about 20%. The enzyme catalyzed the oxidative denitrification of 1-nitropropane as follows: CH2(N02)CH2CH3 + 02 + H20 + OHCCH2CH3 + HNO2 + H202. In addition to 1-nitropropane, 3-nitro-2-pentanoL 2-nitropropane, and nitrocyclohexane are good substrates; the enzyme is designated "nitroalkane oxidase" (EC class 1.7.3). The enzyme has a molecular weight of approximately 185,000 and consists of four subunits identical in molecular weight (47,000). Flavine adenine dinucleotide was required for the enzyme activity and could be replaced in part by riboflavine 5'phosphate. The maximum reactivity was found at about pH 8.0. The enzyme was inhibited significantly by HgCl2, KCN, p-chloromercuribenzoate, and Nethylmaleimide. The Michaelis constants are as follows: 1-nitropropane, 1.54 mM; 2-nitropropane, 7.40 mM; nitroethane, 1.00 mM; 3-nitro-2-pentanol, 3.08 mM; nitrocyclohexane, 0.90 mM; and flavine adenine dinucleotide, 1.33 uM.

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