Serum levels of human placental leucine aminopeptidase/oxytocinase (P-LAP) increase with gestation. cDNA cloning of P-LAP revealed that the enzyme is a type II membrane-bound protein containing the consensus HEXXH(X) 18 E motif found in the M1 family of zinc-metallopeptidase proteins. In this study, a recombinant soluble form of P-LAP found in maternal serum was expressed in Chinese hamster ovary cells, purified to homogeneity and then characterized. Although N-terminal sequencing revealed a four-amino-acid deletion, the purified enzyme was active and was shown to be a zinc-containing homodimeric protein with molecular mass of 280 kDa in solution. Using artificial substrates, it was shown that the enzyme has broad specificity and is inhibited by several compounds known as aminopeptidase inhibitors. Subsequently, sequential N-terminal aminoacid liberation of several peptide hormones by the enzyme was monitored and structures of the products were determined. Among the hormones having a cysteine residue at their N-terminal end and intramolecular disulfide bonds, it was found that vasopressin and oxytocin, but not calcitonin and endothelins, were cleaved by the enzyme. Because the molecular properties of oxytocinase so far reported often conflict, our results provide an initial biochemical and enzymatic characterization of moleculary defined P-LAP/oxytocinase.
The possible presence of an unknown prostacyclin synthesis inhibitory substance has been reported in some strains of rats. We purified the inhibitory substance from the heart of spontaneously hypertensive rats by collecting active fractions after gel-filtration column chromatography and two steps of reverse-phase high performance liquid chromatography. The amino acid composition and automated gas-phase sequencing of the full-length substance and fragments cleaved by AspN indicated that the prostacyclin-inhibitory peptide was identical to coupling factor 6. Recombinant rat coupling factor 6, which was synthesized using a cleavable fusion protein strategy, attenuated base-line and bradykinin (10 ؊6 M)-induced prostacyclin synthesis and
We demonstrated recently that coupling factor 6, an essential component of the energy-transducing stalk of mitochondrial ATP synthase, suppresses the synthesis of prostacyclin in vascular endothelial cells. Here, we tested the hypothesis that coupling factor 6 is present on the cell surface and is involved in the regulation of systemic circulation. This peptide is present on the surface of CRL-2222 vascular endothelial cells and is released by these cells into the medium. In vivo, the peptide circulates in the vascular system of the rat, and its gene expression and plasma concentration are higher in spontaneously hypertensive rats (SHRs) than in normotensive controls. Elevation of blood pressure with norepinephrine did not affect the plasma concentration of coupling factor 6. Intravenous injection of recombinant peptide increased blood pressure, apparently by suppressing prostacyclin synthesis, whereas a specific Ab to coupling factor 6 decreased systemic blood pressure concomitantly with an increase in plasma prostacyclin. Interestingly, the antibody's hypotensive effect could be abolished by treating with the cyclooxygenase inhibitor indomethacin. These findings indicate that mitochondrial coupling factor 6 functions as a potent endogenous vasoconstrictor in the fashion of a circulating hormone and may suggest a new mechanism for hypertension.
We demonstrated recently that coupling factor 6, an essential component of the energy-transducing stalk of mitochondrial ATP synthase, suppresses the synthesis of prostacyclin in vascular endothelial cells. Here, we tested the hypothesis that coupling factor 6 is present on the cell surface and is involved in the regulation of systemic circulation. This peptide is present on the surface of CRL-2222 vascular endothelial cells and is released by these cells into the medium. In vivo, the peptide circulates in the vascular system of the rat, and its gene expression and plasma concentration are higher in spontaneously hypertensive rats (SHRs) than in normotensive controls. Elevation of blood pressure with norepinephrine did not affect the plasma concentration of coupling factor 6. Intravenous injection of recombinant peptide increased blood pressure, apparently by suppressing prostacyclin synthesis, whereas a specific Ab to coupling factor 6 decreased systemic blood pressure concomitantly with an increase in plasma prostacyclin. Interestingly, the antibody's hypotensive effect could be abolished by treating with the cyclooxygenase inhibitor indomethacin. These findings indicate that mitochondrial coupling factor 6 functions as a potent endogenous vasoconstrictor in the fashion of a circulating hormone and may suggest a new mechanism for hypertension.
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