phosphoramidon and 1-10-phenanthroline

The presence of an extrahypothalamic gonadotropin releasing hormone (GnRH) in human placenta is well known and this decapeptide is presumed to play an important role in the regulation of the function and growth of human placenta. Immunohistochemistry showed that neutral endopeptidase 24.11 (NEP), a candidate of the responsible enzyme of GnRH degradation, is highly expressed on the cell surface of trophoblasts. Hydrolysis of GnRH by human villi was studied by measuring liberated amino acids using high performance liquid chromatography. The GnRH degrading activity was 1.53 times higher after incubation with the membrane fraction of first trimester villi than that after incubation with the membrane fraction of term villi. Phosphoramidon, a potent inhibitor of NEP, reduced the liberated amino acids to about a half, suggesting that NEP is a responsible enzyme for GnRH degradation. Ubenimex, which can inhibit several aminopeptidases, also reduced the liberated amino acids to about 50 per cent. O-phenanthroline, EDTA, and thiorphan could inhibit GnRH degradation but inhibitors of post proline endopeptidase could not. Furthermore, GnRH degrading activity of the membrane fraction was reduced remarkably after the membrane fraction was immunotitrated by anti NEP and anti placental leucine aminopeptidase (P-LAP) IgG. In conclusion, NEP and P-LAP are responsible enzymes for GnRH degradation in human villi.

Topics: Adult; Antibodies, Blocking; Chorionic Villi; Chromatography, High Pressure Liquid; Edetic Acid; Female; Glycopeptides; Gonadotropin-Releasing Hormone; Humans; Intracellular Membranes; Labor, Obstetric; Leucine; Leucyl Aminopeptidase; Neprilysin; Organ Culture Techniques; Phenanthrolines; Pregnancy; Pregnancy Trimester, First; Protease Inhibitors; Thiorphan

Proteolytic cleavage of the extracellular domain of CD44 from the surface of cells has been observed recently in different cell types. In cell culture supernatants of human melanoma cell lines a 70 kDa soluble CD44 protein (solCD44) was detected at concentrations of 250-300 ng/ml. Protease inhibitor studies revealed that serine proteases and metalloproteases are involved in the cleavage of CD44 from the surface of melanoma cells. To analyse a possible function of soluble CD44 a human malignant melanoma cell line was stably transfected with cDNAs encoding either wild type soluble CD44s or mutated forms with defective HA binding properties (CD44sR41A and CD44sR150A/R154A). Soluble CD44s almost completely inhibited hyaluronic acid binding by melanoma cells, whereas soluble CD44 mutated in the HA binding domain had no effect. When cultivated on hyaluronic acid, melanoma cell proliferation was induced by 30% for both the parental and the control transfected cells. This increase in proliferation was blocked completely in solCD44s-secreting transfectants, whereas solCD44sR41A and solCD44sR150A/R154A-secreting cells again showed hyaluronic acid-induced cell proliferation. These cell lines were subcutaneously injected into MF1 nu/nu mice to compare their growth as tumors in vivo. Compared to tumors derived from parental and control transfected cells, we observed a dramatic reduction of primary tumor growth with solCD44s expressing MM cells. Transfectants expressing solCD44s mutated in the HA binding domain in contrast developed fast-growing primary tumors. These results provide strong evidence that direct solCD44 interactions with hyaluronic acid interfere competitively with processes induced by hyaluronic acid binding to surface CD44. Autocrine, or drug-induced secretion of solCD44 by human melanoma cells may thus exert potent antitumoral effects in vivo.

Topics: Animals; Binding Sites; Binding, Competitive; Cell Adhesion; Cell Division; Culture Media; Glycopeptides; Humans; Hyaluronan Receptors; Hyaluronic Acid; Melanoma; Melanoma, Experimental; Metalloendopeptidases; Mice; Mice, Nude; Neoplasm Proteins; Pepstatins; Phenanthrolines; Protease Inhibitors; Protein Binding; Protein Structure, Tertiary; Recombinant Fusion Proteins; Sequence Deletion; Sulfones; Transfection; Tumor Cells, Cultured

Endothelin-1 (ET-1) levels are elevated in human breast tumours compared with normal and benign tissues, and in the presence of insulin-like growth factor 1 (IGF-I) ET-1 is a potent mitogen for human breast fibroblasts. In this study we have examined the ability of intact human breast cancer cell lines to process exogenously added big ET-1 (1-38) to the active mature ET-1 peptide by using a specific radioimmunometric assay. In both hormome-dependent (MCF-7, T47-D) and hormone-independent (MDA-MB-231) breast cancer cell lines the putative endothelin-converting enzyme (ECE) exhibited apparent Michaelis-Menten kinetics when converting added big ET-1 to ET-1. Both basal ET-1 production and exogenously added big ET-1 to ET-1 conversion were greatly reduced in all three cell lines in response to the metalloproteinase inhibitor phosphoramidon but were insensitive to other classes of protease inhibitors. Inhibition was also observed when cells were incubated in the presence of the divalent cation chelators 1,10-phenanthroline and EDTA. In MCF-7 cells the optimal pH for the ECE activity using a saponin cell permeabilisation procedure was found to residue within a narrow range of 6.2-7.26. Our results indicate that human breast cancer cells contain a neutral phosphoramidon-sensitive metalloproteinase which can process big ET-1 to ET-1. In the breast this conversion could contribute substantially to the local extracellular levels of this proposed paracrine breast fibroblast mitogen.

Topics: Aspartic Acid Endopeptidases; Breast; Breast Neoplasms; Chromatography, High Pressure Liquid; Culture Media; Edetic Acid; Endothelin-1; Endothelin-Converting Enzymes; Endothelins; Fibroblasts; Glycopeptides; Growth Substances; Humans; Hydrogen-Ion Concentration; Iron Chelating Agents; Kinetics; Metalloendopeptidases; Mitogens; Phenanthrolines; Protease Inhibitors; Protein Precursors; Sensitivity and Specificity; Tumor Cells, Cultured

Human proteoglycan was aggregated to an immobilized hyaluronan solid phase on a 96-well ELISA plate. This complex was then degraded by recombinant human stromelysin. The remaining proteoglycan fragments were detected using a monoclonal antibody probe directed against the chondroitin sulfate (CS) region of the core protein. Stromelysin degraded the aggregate in a time and dose dependent manner as reflected by the loss of the CS epitope. Assay sensitivity was 0.125 U/well with total loss of the CS epitope occurring at 4 U/well. o-phenanthroline (IC50 = 52 microM) and U24522 (IC50 = 9 microM) inhibited degradation, while phosphoramidon did not. Serine and cysteine protease inhibitors had no effect. A comparative analysis of this assay with a reference method, substance P assay, gave similar inhibitor profiles. The use of aggregated human proteoglycan (native conformation) as a substrate, may better reflect how stromelysin inhibitors behave in the presence of complex substrates such as cartilage matrix.

Topics: Animals; Antibodies, Monoclonal; Cartilage, Articular; Chondroitin Sulfates; Enzyme-Linked Immunosorbent Assay; Femur; Glycopeptides; Humans; Hyaluronic Acid; Matrix Metalloproteinase 3; Metalloendopeptidases; Mice; Phenanthrolines; Proteoglycans; Recombinant Proteins; Substance P

In porcine aortic endothelial cells, the 21-amino acid peptide endothelin-1 (ET-1) is formed from a 39-amino acid intermediate called "big endothelin-1" (big ET-1) by a putative ET-converting enzyme (ECE) that cleaves the 39-mer at the bond between Trp-21 and Val-22. Since big ET-1 has only 1/100-1/150th the contractile activity of ET-1, inhibition of ECE should effectively block the biological effects of ET-1. Big ET-1 injected intravenously into anesthetized rats produces a sustained pressor response that presumably is due to conversion of big ET-1 into ET-1 by ECE. We determined the type of protease activity responsible for this conversion by evaluating the effectiveness of protease inhibitors in blocking the pressor response to big ET-1 in ganglion-blocked anesthetized rats. The serine protease inhibitor leupeptin, the cysteinyl protease inhibitor E-64, and the metalloprotease inhibitors captopril and kelatorphan were all ineffective at blocking the pressor response to big ET-1. However, the metalloprotease inhibitors phosphoramidon and thiorphan dose-dependently inhibited the pressor response to big ET-1, although phosphoramidon was substantially more potent than thiorphan. None of the inhibitors blocked the pressor response to ET-1 and none had any effect on mean arterial pressure when administered alone. In a rabbit lung membrane preparation, ECE activity was identified that was blocked by the metalloprotease inhibitors phosphoramidon and 1,10-phenanthroline in a concentration-dependent manner. This enzyme converted big ET-1 to a species of ET that comigrated on HPLC with ET-1 and produced an ET-like contraction in isolated rat aortic rings. Our results suggest that the physiologically relevant ECE is a metalloprotease.

Topics: Animals; Aorta; Endothelin-1; Endothelins; Endothelium, Vascular; Glycopeptides; Humans; In Vitro Techniques; Male; Muscle, Smooth, Vascular; Phenanthrolines; Protease Inhibitors; Protein Precursors; Rats; Rats, Inbred Strains; Swine; Vasoconstriction

Incubation of big endothelin-1 (big ET-1(1-39] with either the cytosolic or membrane fraction obtained from cultured endothelial cells, resulted in an increase in immunoreactive-endothelin (IR-ET), which was markedly inhibited by metal chelators. Phosphoramidon, a metalloproteinase inhibitor, specifically suppressed the membrane fraction-induced increase in IR-ET, whereas the increase in IR-ET observed with the cytosolic fraction was not influenced by phosphoramidon. Reverse-phase (RP)-HPLC of the incubation mixture of big ET-1 with the cytosolic or membrane fraction revealed one major IR-ET component corresponding to the elution position of synthetic ET-1(1-21). Simultaneously, immunoreactivities like the C-terminal fragment (CTF22-39) of big ET-1 were present, as deduced from the RP-HPLC coupled with the radioimmunoassay for CTF. Our results indicate the presence of two types of metalloproteinases, which convert big ET-1 to ET-1 via a single cleavage between Trp21 and Val22, in vascular endothelial cells.

Topics: Animals; Aorta, Thoracic; Cell Membrane; Cells, Cultured; Chromatography, High Pressure Liquid; Cytosol; Edetic Acid; Endothelin-1; Endothelins; Endothelium, Vascular; Glycopeptides; Hydrogen-Ion Concentration; Metalloendopeptidases; Peptide Fragments; Phenanthrolines; Protein Precursors; Protein Processing, Post-Translational; Swine

The effect of peptidase inhibitors on the degradation of [3H]-bradykinin by rat hypothalamic slices was studied using HPLC to separate and identify the products. The degradation appears to be mainly mediated by an enzyme which cleaves the peptide at the Phe5-Ser6 bond and is inhibited by 1,10-phenanthroline, dynorphin(1-13) and carboxyphenylethyl-Ala-Ala-Phe-p-aminobenzoate. This suggest the involvement of a membrane bound variant of the soluble metalloendopeptidase (EC3.4.24.15) isolated from rat brain which degrades neurotensin, angiotensin and other neuropeptides as well as bradykinin.

Topics: 4-Aminobenzoic Acid; Animals; Bradykinin; Captopril; Chromatography, High Pressure Liquid; Dynorphins; Endopeptidases; Glycopeptides; Hypothalamus; Leucine; Male; Metalloendopeptidases; Neurotensin; Oligopeptides; para-Aminobenzoates; Peptide Fragments; Phenanthrolines; Rats; Rats, Inbred Strains

A preparation of closed membrane vesicles derived from the longitudinal and circular smooth muscle of pig small intestine was enriched eight-fold in the activity of 5'-nucleotidase and six-fold in the activity of peptidyl dipeptidase A relative to the tissue homogenate. The membrane vesicles specifically bound [3H]bradykinin and the concentration of bradykinin required to inhibit 50% binding was 0.76 +/- 0.05 nM. This concentration was not significantly different from the corresponding concentration of lysyl-bradykinin (0.45 +/- 0.13 nM) but was less (P less than 0.05) than the concentration of methionyl-lysyl-bradykinin (1.25 +/- 0.10 nM). The concentration of des-Arg9 bradykinin (7.5 microM) required for 50% inhibition was greater than 10(3) times less than bradykinin indicating the presence of a B2-type receptor. The membrane vesicles also degraded bradykinin and the principal metabolite was identified as bradykinin. Des-Arg1 bradykinin, des-Arg9 bradykinin and bradykinin were also formed in low yield. Cleavage of the Pro7-Phe8 bond was inhibited by phosphoramidon but not by enalapril or captopril indicating that proteolytic inactivation of bradykinin in the muscle layer of the intestine is mediated through endopeptidase 24.11 ("enkephalinase") but not through peptidyl dipeptidase A ("angiotensin-converting enzyme").

Topics: 5'-Nucleotidase; Animals; Binding Sites; Bradykinin; Captopril; Cell Membrane; Drug Interactions; Enalapril; Endopeptidases; Enzyme Repression; Glycopeptides; In Vitro Techniques; Muscle, Smooth; Nucleotidases; Phenanthrolines; Swine

Utilizing both thin-layer chromatography and high pressure liquid chromatography, it was determined that a vascular plasma membrane preparation contains a carboxypeptidase capable of converting kinins (B2 agonists) to des(Arg)kinins (B1 agonists) by hydrolysis of C-terminal Arg. The plasma membrane carboxypeptidase also converted Leu5-enkephalin-Arg6 to Leu5-enkephalin. Carboxypeptidase activity was significantly higher in cultured endothelial (1.47 +/- 0.4 units/mg) than in cultured smooth muscle cells (0.16 +/- 0.4 units/mg). Both the vascular and endothelial activities had neutral pH optima and were activated 4- to 5-fold by 0.1 mM CoCl2. The carboxypeptidase N inhibitor MERGETPA (D-L-mercaptoethanol-3-guanidino-ethylthiopropanoic acid) inhibited the plasma membrane bound carboxypeptidase with an I50 of 0.3 microM. Conversion was also inhibited by o-phenanthroline and EDTA, whereas inhibitors of aminopeptidases (bestatin, puromycin), endopeptidases (phosphoramidon), "enkephalinase" (ZINCOV) or enkephalin convertase (PCMS) were without effect. The affinity of the endothelial plasma membrane carboxypeptidase for bradykinin (Km = 56.8 +/- 4.7 microM) was higher than that for Leu5-enkephalin-Arg6 (Km = 92.7 +/- 10.1 microM), whereas the maximal rates of conversion (calculated per mg of endothelial plasma membrane protein) were similar (17.1 and 21.3 nmoles/min/mg respectively). These results demonstrate that a carboxypeptidase is present on the cell surface of vascular endothelium which can convert kinins and enkephalins in the micro-environments of vascular cell surface receptors.

Topics: 3-Mercaptopropionic Acid; Animals; Carboxypeptidases; Cell Membrane; Chromatography, High Pressure Liquid; Chromatography, Thin Layer; Edetic Acid; Endothelium; Enkephalins; Glycopeptides; Hydrogen-Ion Concentration; Kinetics; Kinins; Leucine; Lysine Carboxypeptidase; Microscopy, Phase-Contrast; Muscle, Smooth, Vascular; Phenanthrolines; Puromycin; Swine

We previously reported that the cell fusion that occurs during muscle development, when mononucleated myoblasts fuse to form multinucleated myotubes, requires endogenous metalloendoprotease activity at the time of fusion. We report here that myoblasts contain both soluble and membrane-associated metalloendoproteases, and that these proteases have different inhibitor specificities. Several inhibitors, previously shown to block myoblast fusion, inhibit only soluble and not membrane-associated metalloendoprotease activity in myoblasts. Another metalloendoprotease inhibitor, phosphoramidon, which had no effect on fusion, inhibits only the membrane-associated metalloendoprotease. These observations implicate a soluble metalloendoprotease in myoblast fusion. Two soluble metalloendoproteases can be demonstrated by column chromatofocusing, with pI values at pH 5.9 and 4.8. The soluble metalloendoprotease eluted at pH 5.9 is not inhibited by an inhibitor which blocks fusion, while the soluble metalloendoprotease eluted at pH 4.8 is inhibited. Of the three metalloendoprotease activities identified in myoblasts, the metalloendoprotease required in myoblast fusion appears to be the soluble metalloendoprotease with a pI of 4.8.

Topics: Animals; Anti-Bacterial Agents; Cell Fusion; Cell Line; Cell Membrane; Clone Cells; Cytosol; Glycopeptides; Kinetics; Metalloendopeptidases; Mice; Muscles; Phenanthrolines; Protease Inhibitors

A membrane fraction from the electric organ of Torpedo marmorata hydrolyses the Gly3-Phe4 bond of [D-Ala2, Leu5]enkephalin as well as the Gly-His bond of benzoyl-Gly-His-Leu. The hydrolysis of benzoyl-Gly-His-Leu is completely inhibitable by Captopril (I50 = 19nM), consistent with peptidyl dipeptidase activity, but enkephalin hydrolysis is inhibited to a maximum of only 70%. The residual activity hydrolysing enkephalin is inhibited by phosphoramidon (I50 = 15nM) and therefore resembles endopeptidase-24.11, a mammalian plasma-membrane enzyme implicated in the metabolism of neuropeptides. Both enkephalin-hydrolysing activities in Torpedo electric organ are inhibited by 1,10-phenanthroline, like their mammalian counterparts. The peptidases may function in the hydrolysis of endogenous peptides or in neurotransmitter exocytosis in the electric organ.

Topics: Animals; Captopril; Cell Membrane; Electric Organ; Endopeptidases; Enkephalin, Leucine; Enkephalin, Leucine-2-Alanine; Glycopeptides; Hydrolysis; In Vitro Techniques; Metalloendopeptidases; Nerve Tissue Proteins; Oligopeptides; Phenanthrolines; Torpedo