Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.4.24.11 (CD10)
 9,792 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The capillary endothelial cells of the median eminence represent a potential site for the degradation/modification of both circulating and hypothalamic peptides passing through the hypophysial portal system toward the pituitary. This study examines endothelial cell peptidase expression in vitro by monitoring the metabolism of gonadotropin-releasing hormone (GnRH) by cultured endothelial cells from sheep median eminence. Cleavage of GnRH by median eminence endothelial cell membranes generated GnRH1-5 as the primary stable product, which was then degraded to GnRH1-3 and free amino acids. Degradation of GnRH was completely inhibited by TPCK, ZnCl2 and N-ethylmaleimide, and partially inhibited by EDTA and by a specific inhibitor of the metalloendopeptidase EC 3.4.24.15, CFP-AAY-pAB. Interestingly, an increase in GnRH1-9 production was seen with the latter inhibitors, suggesting a two-step mechanism of GnRH degradation involving a primary cleavage at the Pro9-Gly10-NH2 bond, inhibitable by TPCK, ZnCl2, and NEM, followed by cleavage by EC 3.4.24.15 to generate GnRH1-5. Phosphoramidon and angiotensin converting enzyme inhibitors (as well as other non-specific inhibitors) were without effect, indicating that endopeptidase EC 3.4.24.11 and angiotensin converting enzyme are not involved. Neither bovine aortic endothelial cell nor AtT-20 cell membranes exhibited this pattern of peptidase activity. Degradation of GnRH by intact median eminence endothelial cells in culture was also observed, suggesting an extracellular orientation for these enzymes; the potential role of such peptidases in the fine regulation of both pituitary function and local blood flow is currently under investigation.
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PMID:Characterization of membrane-associated peptidase activities expressed by endothelial cells of the ovine median eminence. 804 22

Inactivation of Streptomyces griseus metallo-endopeptidase II (SGMPII) by ClCH2CO-DL-(N-OH)Leu-OCH3 and by ClCH2CO-DL-(N-OH)Leu-Ala-Gly-NH2 was studied kinetically. These reagents cause irreversible inhibition of the enzyme in a pseudo-first order reaction, and the inhibition reaction exhibits saturation kinetics. The second-order rate constants for inactivation of SGMPII by ClCH2CO-DL-(N-OH)Leu-OCH3 and by ClCH2CO-DL-(N-OH)Leu-Ala-Gly-NH2 were measured to be 0.12 and 8.9 M-1.s-1, respectively. The order of affinities of metallo-endopeptidases towards these irreversible inhibitors is thermolysin > SGMPII > Pseudomonas aeruginosa elastase. A competitive inhibitor of SGMPII, L-Val-L-Trp, protects the enzyme against inactivation by ClCH2CO-DL-(N-OH)Leu-Ala-Gly-NH2 in a competitive manner. Furthermore, the pH profile of the inactivation closely resembles that for the hydrolysis of synthetic peptide substrates by the enzyme. These findings suggest that these reagents bind reversibly and react irreversibly at the active site of the enzyme.
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PMID:Inhibition of Streptomyces griseus metallo-endopeptidase II (SGMPII) by active-site-directed inhibitors. 805 68

To assess clearance mechanisms of atrial and brain natriuretic peptides in the circulation, we examined the effects of a neutral endopeptidase (NEP) inhibitor and a clearance receptor ligand on plasma concentrations of the peptides in normal rats. Plasma concentrations of endogenous alpha-rat atrial natriuretic peptide (alpha-rANP) were not significantly elevated by intravenous infusion of a NEP inhibitor, phosphoramidon, but were elevated threefold by intravenous infusion of a clearance receptor ligand, des(Gln18-Gly22)-rANP(4-23)-NH2 [C-ANF(4-23)]. On the other hand, the clearance of alpha-rANP given intravenously at the pharmacological dose, 600 pmol/min/kg for 2 min, was decreased to one-third by the administration of phosphoramidon, although the administration of C-ANF(4-23) did not significantly decrease the clearance. The clearance of rat brain natriuretic peptide (rBNP) given at 600 pmol/min/kg for 2 min was approximately 38% lower than that of alpha-rANP. The effect of phosphoramidon on the clearance of rBNP was not significant and was similar to that of C-ANF(4-23). These results suggest that clearance receptor is involved in the clearance of the physiological levels of alpha-rANP and that NEP plays a major role in the clearance of a pharmacological dose of alpha-rANP, at which clearance receptors are thought to be saturated, and also indicate a pharmacokinetic difference between alpha-rANP and rBNP.
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PMID:Clearance mechanisms of atrial and brain natriuretic peptides in rats. 814 57

The complete primary structure of the subfragment-2 (S-2) from adult chicken cardiac ventricular muscle myosin has been determined by analysis of peptides derived from digests of S-2 with cyanogen bromide, lysyl endopeptidase, arginyl endopeptidase, and from hydrolysates of CNBr fragments with formic acid. This region composed of 520 amino-acid residues which span the connecting segment between subfragment-1 (S-1) and S-2 to the NH2-terminal portion of light meromyosin (LMM). Comparing this sequence with the partial sequence of the rod from the same chicken ventricular muscle myosin deduced from its nucleotides of cDNA which lacks 64 NH2-terminal amino-acid residues, 14 amino-acid differences and 3 deletion/insertions were recognized. Furthermore, the sequence of S-2 from adult chicken ventricular myosin was compared with corresponding sequences of rat alpha and beta cardiac myosin heavy chains (MHC) and human alpha and beta cardiac MHCs. The results show 83.7%, 82.1%, 83.1% and 82.1% sequence identities, respectively with almost similar degrees of similarities to both alpha- and beta-MHCs. However, sequences of isoform-specific regions in this S-2 from adult chicken ventricular myosin showed clearly a higher homology to those of alpha-MHCs than to beta-MHCs of mammalian cardiac myosins.
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PMID:Primary structure of subfragment-2 from adult chicken cardiac ventricular muscle myosin. 821 95

A heptapeptide was isolated from brain-corpora cardiaca-corpora allata-suboesophageal ganglion extracts of the locust, Locusta migratoria. Biological activity was monitored during HPLC purification by observing the myotropic effect of column fractions on the isolated hindgut of Leucophaea maderae. The primary structure of this myotropic peptide was established as: Phe-Thr-Pro-Asn-Trp-Gly-Thr-NH2. The chromatographic and biological properties of the synthetic peptide were the same as those of the native peptide, thus confirming structural analysis. This heptapeptide is identical to the carboxyterminal heptamer of AKH-I and therefore designated as AKH-I4-10. AKH-I4-10 has no adipokinetic activity. AKH-I4-10 is most likely a breakdown product of Lom-AKH-I, suggesting that an endopeptidase which cleaves between Asn and Phe is present in the brain complex of L. migratoria. Such an endopeptidase has recently been characterized in in synaptic membranes of the nervous system of Schistocerca gregaria.
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PMID:Isolation, identification, and synthesis of AKH-I4-10 from Locusta migratoria. 822 63

Kidney plasma membranes of Aplysia californica were shown to contain an endopeptidase activity which cleaved [Leu]enkephalin (Tyr-Gly-Gly-Phe-Leu) and [Leu]enkephalinamide (Tyr-Gly-Gly-Phe-Leu-NH2) at the Gly3-Phe4 bond, as determined by reverse-phase h.p.l.c. analysis of metabolites. The optimal pH was shown to be 6.5. The bivalent cation chelating agent, 1,10-phenanthroline protected [Leu]enkephalin from degradation, suggesting that this enzyme is a metallopeptidase. The degradation of [Leu]enkephalin was also abolished by the neutral endopeptidase-24.11 inhibitors RB104 (2-[(3-iodo-4-hydroxyl)-phenylmethyl]-4-N-[3-(hydroxyamino-3-oxo-1- phenylmethyl)-propyl]amino-4-oxobutanoic acid), HABCO-Gly [(3-hydroxy-aminocarbonyl-2-benzyl-1-oxypropyl)glycine], phosphoramidon and thiorphan, with IC50 values of 1 nM, 1 microM, 20 microM and 30 microM respectively. By contrast, the angiotensin-converting enzyme inhibitor captopril and the serine proteinase inhibitor phenylmethanesulphonyl fluoride were without effect. Phase separation experiments using Triton X-114 showed that about 64% of the neutral endopeptidase activity in the Aplysia kidney membrane corresponds to an integral membrane protein. A specific radioiodinated inhibitor ([125I]RB104) was shown to bind the Aplysia endopeptidase with high affinity; the KD and Bmax. values were 21 +/- 5 pM and 20.3 +/- 5 fmol/mg of proteins respectively. This inhibitor was used to determine the molecular form of the enzyme, after separation of solubilized membrane proteins on SDS/PAGE and transfer on to nitrocellulose membranes. A single protein band with an apparent molecular mass of 140 kDa was observed. The labelling was abolished by specific neutral endopeptidase inhibitors. This study provides the first biochemical characterization of an endopeptidase with catalytic properties similar to those of neutral endopeptidase-24.11 in the mollusc Aplysia californica.
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PMID:Identification and characterization of a neutral endopeptidase activity in Aplysia californica. 825 38

gamma-Glutamyl hydrolase has been partially purified and characterized from conditioned culture medium of H35 hepatoma cells. Evidence for heterogeneity of the enzyme is derived from its elution as three distinct peaks of enzymatic activity when the enzyme is purified by TSK-butyl-Sepharose column chromatography. These three enzyme fractions appear to have identical catalytic properties but, as yet, the basis for their resolution is not understood. A rapid, sensitive and simple assay based on reverse-phase HPLC fluorescent detection with pre-column derivatization using o-phthalaldehyde (OPA) was developed to separate OPA-derivatives of poly-gamma-glutamates and glutamic acid. Using this assay and the standard HPLC assay for pteroylpolyglutamates, the enzyme appears to be an endopeptidase with respect to pteroylpenta-gamma-glutamate (PteGlu5), methotrexate penta-gamma-glutamate (4-NH2-10-CH3PteGlu5) and p-aminobenzoyl-penta-gamma-glutamate (pABAGlu5). The initial products are PteGlu1 (or 4-NH2-10-CH3PteGlu1 or pABAGlu1) and intact tetra-gamma-glutamate, which is subsequently degraded to glutamic acid. When penta-gamma-glutamate is the substrate, the cleavage of the gamma-bonds by the enzyme is less ordered, with the early appearance of mono-, di-, tri- and tetraglutamate. Poly-alpha-glutamate is not a substrate nor are pABA-gamma-Glu5 or penta-gamma-glutamate covalently linked to albumin. 4-NH2-10-CH3PteGlu2 or Glu5 bound to dihydrofolate reductase is not a substrate for the enzyme, offering further evidence that protein-associated poly-gamma-glutamates are poor substrates for gamma-glutamyl hydrolase from H35 hepatoma cells.
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PMID:The properties of the secreted gamma-glutamyl hydrolases from H35 hepatoma cells. 834 22

A lysed preparation of isolated insulin secretory granules efficiently cleaved murine proopiomelanocortin (mPOMC) at physiologically important Lys-Arg processing sites. This processing was mostly attributed to an activity that co-eluted with the proinsulin processing type-II endopeptidase from anion exchange chromatography (Lys-Arg-directed; Davidson, H. W., Rhodes, C. J., and Hutton, J. C. (1988) Nature 333, 93-96). The principal peptide hormone products generated by the insulin secretory granule lysate were identified by specific radioimmunoassay and NH2-terminal microsequencing analysis of high performance liquid chromatography-separated products as alpha-melanocyte-stimulating hormone, corticotropin-like intermediate, gamma-lipotropin, beta-endorphin-(1-31), 18-kDa NH2-terminal fragment and, to a lesser extent, adrenocorticotrophin and beta-lipotropin. This processing had an acidic pH optimum (pH 5-5.5) and was Ca(2+)-dependent (K0.5 activation = 5-80 microM). With increasing Ca2+ concentrations there was an increase in the extent to which mPOMC was processed. The in vitro processing of mPOMC by the insulin secretory granule endopeptidase activity reported here is in excellent agreement with the in vivo processing of this prohormone by a combination of PC2 and PC3, candidates of prohormone endpeptidase, in gene transfer studies with cells that express the regulated secretory pathway (Thomas, L., Leduc, R., Thorne, B. A., Smeekens, S. S., Steiner, D. F., and Thomas, G. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 5297-5301).
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PMID:Processing of proopiomelanocortin by insulin secretory granule proinsulin processing endopeptidases. 838 98

Rabbit neutral endopeptidase-24.11 is a type II transmembrane protein with a 27-amino acid residue positively charged NH2-terminal cytoplasmic domain, a 23-amino acid residue hydrophobic signal peptide/membrane anchor domain, and a large catalytic COOH-terminal domain exposed on the exoplasmic side of the membrane. In order to study the mechanism of membrane anchoring of neutral endopeptidase-24.11, we created mutants in which the cytoplasmic tail was deleted. Expression of these mutants in COS-1 cells resulted in the secretion of approximately 10-20% of the protein into the culture medium, due possibly to the cleavage of part or all of the signal peptide/membrane anchor domain by the rough endoplasmic reticulum signal peptidase. In a second set of mutants, a hydrophilic sequence (GSQNS) was inserted midway in the signal peptide/membrane anchor domain of neutral endopeptidase-24.11. When this hydrophilic sequence was introduced into the full-length neutral endopeptidase-24.11, approximately 20% of the enzyme activity was recovered in the culture medium. This proportion increased to 93% when the cytosolic tail was deleted. Sequencing of the [3H]tyrosine- or [3H]isoleucine-labeled secreted protein indicated that proteolysis, possibly by signal peptidase, occurred on the COOH-terminal side of the signal peptide/membrane anchor domain. We conclude that the efficient cleavage of the signal peptide/membrane anchor domain and secretion of the protein require both the deletion of the cytosolic domain and the presence of a hydrophilic sequence.
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PMID:Transformation of the signal peptide/membrane anchor domain of a type II transmembrane protein into a cleavable signal peptide. 842 44

Asparaginyl endopeptidase was highly purified from mature seeds of the jack bean (Canavalia ensiformis). The final enzyme preparation showed a single peak in high-performance liquid chromatography on a reversed-phase column, and the material in the peak gave the following NH2-terminal amino acid sequence on Edman degradation for 25 cycles: H-Glu-Val-Gly-Thr-Arg-Trp-Ala-Val-Leu-Val-Ala-Gly-Ser-Asn-Gly-Tyr-Gly-Asn-Tyr- Arg-His-Gln-Ala-Asp-Val-. Behavior of the enzyme toward various protease inhibitors suggested that it belongs to a family of cysteine proteases. Strict substrate specificity of this enzyme was verified by the use of 14 polypeptide substrates including those derived from proteins. Almost all the peptide bonds on the carboxyl side of Asn residues were susceptible to the enzyme. The exceptions were cases where the residue was at the NH2 terminus or the second position from the NH2 terminus of substrates and where it was N-glycosylated Asn. Peptide bonds on the carboxyl side of any other amino acid residues were not cleaved. These properties promise the high utility of this novel endopeptidase in protein sequence analysis. Identity of jack bean asparaginyl endopeptidase with a processing enzyme responsible for maturation of concanavalin A from its precursor is also discussed.
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PMID:Asparaginyl endopeptidase of jack bean seeds. Purification, characterization, and high utility in protein sequence analysis. 842 28


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