Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.4.15.1 (ACE)
18,300 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Neurotensin (NT) endopeptidase (EC 3.4.24.16) has been purified about 800-fold from pig brain by four sequential chromatographic steps depending on ion-exchange and hydrophobic interactions. Two types of preparation were studied: one from a Triton X-100-solubilized membrane fraction, and the other from the soluble fraction containing 90% or more of the total activity in the homogenate. NT endopeptidase activity was monitored by high-precision liquid chromatography of the two peptide products, characterized as NT-(1-10) and NT-(1-8), resulting from cleavage of the Pro10-Tyr11 and Arg8-Arg9 bonds respectively. As purification proceeded, from both membranes and cytosol, the yield of the two products achieved a constant ratio of 5:1 and this ratio was reproduced in repeated purifications. However, a distinct peptidase which hydrolysed exclusively at the Arg8-Arg9 bond was partially resolved from NT endopeptidase by chromatography on hydroxyapatite, and this activity was further purified and assigned to endopeptidase-24.15 (EC 3.4.24.15). SDS/PAGE of both preparations of neurotensin endopeptidase revealed a major band of apparent Mr 75000, and treatment of the membrane-associated form with N-Glycanase gave no evidence that the enzyme was a glycoprotein. The membrane-associated and cytosol forms of NT endopeptidase activities, monitored for both NT-(1-10) and NT-(1-8) products, were compared in their responses to 1,10-phenanthroline, EDTA, dithiothreitol (DTT) and some synthetic site-directed inhibitors of endopeptidase-24.15 or peptidyl dipeptidase A. The effects revealed no significant differences between the two preparations, nor did the reagents discriminate between the activities generating the two NT fragments. The partially purified form of endopeptidase-24.15 was also included in this comparison: while some responses were similar, this peptidase was distinguishable in its activation by DTT and its relative resistance to inhibition by EDTA. Both forms of NT endopeptidase were found to hydrolyse other substrates, including Boc-Phe-Ala-Ala-Phe-4-aminobenzoate, bradykinin and substance P (these at faster rates than neurotensin), as well as dynorphin A-(1-8) and luliberin. The bonds hydrolysed in these neuropeptides, as well as in angiotensins I and II and alpha-neoendorphin, were defined. These studies confirm that NT endopeptidase is distinct from endopeptidase-24.15. They further show that the former is a soluble enzyme, not an integral membrane protein, that it is not peptide-specific and that it might be more appropriately named. enzyme, not an integral membrane protein, that it is not peptide-specific and
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PMID:Purification and properties of a neurotensin-degrading endopeptidase from pig brain. 190 21

Conversion of the octapeptide dynorphin (Dyn) A-(1-8) to Leu5-enkephalin (LE) by endopeptidase EC 3.4.24.15 (EP-24.15) in vivo was examined using the technique of ventriculocisternal perfusion. Peptides were administered intracerebroventricularly in the presence or absence of the EP-24.15 inhibitor N-[1-(R,S)-carboxy-3-phenylpropyl]-Ala-Ala-Phe-p-aminobenzoate (cFPAAF-pAB) via cannulae placed into the lateral ventricle of urethane-anesthetized rats. The concentration of Dyn-like peptides and LE within the CSF was monitored by radioimmunoassay in samples of CSF taken from a second cannula placed in the cisterna magna. In the absence of inhibitor, less than 5% of the Dyn A-(1-8) administered was recovered in CSF. Immunoreactive LE, which is normally not found in CSF, increased rapidly in content following Dyn A-(1-8) infusion, an observation suggesting that the larger peptide is converted to LE. When the inhibitor cFPAAF-pAB was coadministered with Dyn A-(1-8), the concentration of immunoreactive Dyn A-(1-8) after 5 min was 40 times higher than that found in the absence of inhibitor. The angiotensin converting enzyme inhibitor captopril reduced the degradation of Dyn A-(1-8) to a much lesser degree. The inhibitor of EP-24.15 also afforded some protection of other Dyn-like peptides. No EP-24.15 activity was found in rat CSF, whereas high activity was found in the choroid plexus. Taken together, these data clearly indicate that an ectoenzyme form of EP-24.15 rapidly converts intracerebroventricularly administered Dyn-like peptides to LE.
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PMID:An inhibitor of endopeptidase-24.15 blocks the degradation of intraventricularly administered dynorphins. 197 55

Opioid peptides are present in human cerebrospinal fluid (CSF), and their levels are reported to change in some pathologic conditions. However, less is known about their degradation in CSF. In the present study, human CSF was found to contain aminopeptidase activity which hydrolyzed alanyl-, leucyl- and arginyl-naphthylamides in a ratio of 100:28:27. Twelve CSF samples hydrolyzed alanyl-2-naphthylamide and degraded Met5-enkephalin (N-terminal hydrolysis) at rates of 188 +/- 38 and 420 +/- 79 pmol/min/mL respectively. Further, the distribution of alanyl-naphthylamidase activity in individual samples (39-437 pmol/min/mL) was closely correlated with that of Met5-enkephalin degradation (37-833 pmol/min/mL). Both alanyl-naphthylamidase and enkephalin degradation were optimal at pH 7.0 to 7.5 and were inhibited by aminopeptidase inhibitors amastatin (IC50 = 20 nM), bestatin (4-7 microM) and puromycin (30-35 microM). Conversely, degradation was unaffected by inhibitors of neutral endopeptidase (phosphoramidon), carboxypeptidase N (MERGETPA) or angiotensin converting enzyme (captopril). The Km of Met5-enkephalin for the CSF aminopeptidase activity was 201 +/- 19 microM (N = 4). Rates of hydrolysis of the Tyr1-Gly2 bond of larger opioid peptides decreased with increasing peptide length. Pooled, concentrated CSF hydrolyzed Leu5-enkephalin, dynorphin A fragments [1-7], [1-10] and [1-13] and dynorphin A at rates of 2.05 +/- 0.27, 1.27 +/- 0.18, 0.94 +/- 0.06, 0.55 +/- 0.14 and 0.16 +/- 0.03 nmol/min/mL respectively. When analyzed by rocket-immunoelectrophoresis against antisera to aminopeptidase M (EC 3.4.11.2), the concentrated CSF formed an immunoprecipitate which could be stained histochemically for alanyl-naphthylamidase activity. These data are consistent with a significant role for aminopeptidase M activity in the degradation of low molecular weight opioid peptides in human CSF.
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PMID:N-terminal degradation of low molecular weight opioid peptides in human cerebrospinal fluid. 197 24

Since both aminopeptidases and angiotensin I-converting enzyme are reported to degrade circulating enkephalins, we have examined the degradation of low-molecular-weight opioid peptides by a vascular plasma membrane-enriched fraction previously shown to contain both angiotensin I-converting enzyme (EC 3.4.15.1) and aminopeptidase M (EC 3.4.11.2). Except for an enkephalin analog resistant to amino-terminal hydrolysis, [D-Ala2]enkephalin, the purified vascular plasma membrane preferentially degraded low-molecular-weight opioids by hydrolysis of the N-terminal Tyr-1--Gly-2 bond. Enkephalin degradation was optimal at pH 7.0 and was inhibited by the aminopeptidase inhibitors amastatin (I50 = 0.08 microM), bestatin (9.0 microM) and puromycin (80 microM). Maximal rates of hydrolysis, calculated per mg plasma membrane protein, were highest for the shorter peptides (18.3, 15.6 and 16.6 nmol/min per mg for Met5-enkephalin, Leu5-enkephalin and Leu5-enkephalin-Arg6, respectively) and decreased with increasing peptide length (0.7 nmol/min per mg for dynorphin (1-13)). No significant hydrolysis of beta- and gamma-endorphin was detected. Km values decreased significantly with increasing peptide length (Km = 72.9 +/- 2.7, 43.6 +/- 4.7 and 21.4 +/- 0.9 microM for Met5-enkephalin, Leu5-enkephalin-Arg6 and Met5-enkephalin-Arg6-Phe7, respectively). However, no further decreases were seen with even larger sequences, i.e., dynorphin(1-13). Other peptides hydrolyzed by the plasma membrane aminopeptidase (angiotensin III, kallidin and hepta(5-11)-substance P) inhibited enkephalin degradation in a competitive manner. Thus, localization, specificity and kinetic data are consistent with identification of aminopeptidase M as a vascular enzyme with the capacity to differentially metabolize low-molecular-weight opioid peptides within the microenvironment of vascular cell surface receptors. Such differential metabolism may play a role in modulating the vascular effects of peripheral opioids.
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PMID:Degradation of low-molecular-weight opioid peptides by vascular plasma membrane aminopeptidase M. 287 42

Brain contains a membrane-bound form of endopeptidase-24.15, a metalloendopeptidase predominantly associated with the soluble protein fraction of brain homogenates. Subcellular fractionation of the enzyme in rat brain showed that 20-25% of the total activity is associated with membrane fractions including synaptosomes. Solubilization of the enzyme from synaptosomal membranes required the use of detergents or treatment with trypsin. The specific activity of the enzyme in synaptosomal membranes measured with tertiary-butoxycarbonyl-Phe-Ala-Ala-Phe-p-aminobenzoate as substrate was higher than that of endopeptidase-24.11 ("enkephalinase"), a membrane-bound zinc-metalloendopeptidase believed to function in brain neuropeptide metabolism. Purified synaptosomal membranes converted efficiently dynorphin1-8, alpha- and beta-neoendorphin into leucine enkephalin and methionine-enkephalin-Arg6-Gly7-Leu8 into methionine enkephalin in the presence of captopril, bestatin, and N-[1-(R,S)-carboxy-2-phenylethyl]-Phe-p-aminobenzoate, inhibitors of angiotensin converting enzyme (EC 3.4.15.1), aminopeptidase (EC 3.4.11.2), and membrane-bound metalloendopeptidase (EC 3.4.24.11), respectively. The conversion of enkephalin-containing peptides into enkephalins was virtually completely inhibited by N-[1-(R,S)-carboxy-2-phenylethyl]-Ala-Ala-Phe-p-aminobenzoate, a specific active-site-directed inhibitor of endopeptidase-24.15, indicating that this enzyme was responsible for the observed interconversions. The data indicate that synaptosomal membranes contain enzymes that can potentially generate and degrade both leucine- and methionine-enkephalin.
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PMID:Synaptosomal membrane-bound form of endopeptidase-24.15 generates Leu-enkephalin from dynorphin1-8, alpha- and beta-neoendorphin, and Met-enkephalin from Met-enkephalin-Arg6-Gly7-Leu8. 287 74

In this article is given a survey on physiology and pharmacology of the opioid system. Opioid peptides are naturally occurring morphine-like acting metabolites of glycoprotein precursors. Of the opioid peptides proved hitherto in the organism met-, leu-enkephalin, dynorphin and beta-endorphin are characterized more in detail. Opioids react directly with opioid receptors. The opioid receptors are not a homogeneous system. Their distribution is depending on organs and species. Opioid peptides and receptors were proved within as well as outside the central nervous system. The main quantity of the endogenous beta-endorphin is stored in the pituitary gland. High concentrations of met-, leu-enkephalin and dynorphin are present in the gastrointestinal tract and in the adrenal glands. Opioid peptides are inactivated by enzymatic hydrolysis, in which case the splitting of the N-terminal tyrosine is decisive. The inactivation may be performed by amino peptidases, peptidyl dipeptide hydrolases or the angiotensin converting enzyme. The effect of the opioid peptides can be inhibited by opioid antagonists (naloxone, naloxazone). Up to now there are contradictory findings as to the presence of an endogenous opioid antagonist. In general, the presence of different opioid peptides and their different receptor preference indicate multiple functions in the organism. However, their physiological function is up to now only little clarified.
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PMID:[Stress and the endogenous opioid system. I. Physiology and pharmacology of opioid peptides]. 298 51

Cathepsin B from brain exhibited both endopeptidase and dipeptidyl carboxypeptidase activity. Recently the factors, contributing to dipeptidyl carboxypeptidase properties of brain cathepsin B, were identified: I. occupation of the enzyme S3 subsite, 2. free C-terminal group of the substrate, 3. specific interaction between the split off dipeptide and the enzyme active site. The identification was carried out using angiotensin I, its C-end tripeptide and chromophore oligopeptides containing p-nitrophenylalanine residue. C-terminal dipeptide was split off in the proopioid peptides dynorphins 1-7 and 1-8, Met-enkephalin-Arg6-Phe7, Met-enkephalin-Arg6-Gly7-Leu8; the enzyme hydrolyzed also the C-terminal dipeptide bond in Leu- and Met-enkephalins without the subsequent hydrolysis of the remaining tripeptide. D-Ala2, D-Leu5-enkephalin were not hydrolyzed; the bond Arg9-Pro10 was resistant to proteolysis in dynorphin 1-11. Cathepsin B split off the C-terminal dipeptide in synthetic substrates Leu-Trp-Met-Arg-Phe-Ala and Trp-Met-Arg-Phe-Ala but not in Met-Arg-Phe-Ala. These results 06.08 M-15 demonstrated the essential role of branched-chain amino acid residue at the position of P2 and/or P3 of substrates for the enzyme dipeptidyl carboxypeptidase activity. The data obtained suggest that Arg residue at the position P2 (dynorphin 1-7) slowed down, D-amino acid at the position P2 (D-Ala2, D-Leu5-enkephalin) and Pro-Lys bond at the position P1-P2 (dynorphin 1-11) inhibited the cathepsin B dipeptidyl carboxypeptidase activity.
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PMID:[Brain cathepsin as dipeptidylcarboxypeptidase transforming provasopressor, pro-opioid and model peptides]. 331 15

In order to identify which peptidases are involved in the catabolism of neurotensin in the CNS, [3H-Tyr3,11]-neurotensin was superfused over rat hypothalamic slices in the presence and absence of peptidase inhibitors. The degree of degradation of the peptide was determined by reverse phase HPLC separation of 3H-labelled neurotensin from 3H-labelled products. Very little degrading activity was released from the slice into the medium during the superfusion. In the absence of inhibitors, 20 to 50% of 3H-neurotensin was degraded giving mainly 3H-Tyr along with other unidentified 3H-labelled products. Inhibitors of endopeptidase 24.11 (phosphoramidon) and proline endopeptidase (antibody) had no effect on the degradation. Captopril, an inhibitor of angiotensin converting enzyme, had a small inhibitory effect. In contrast, dynorphin(1-13), an inhibitor of a soluble, thiol dependent metallopeptidase which hydrolyses neurotensin at Arg8-Arg9, gave greater than 80% inhibition of 3H-neurotensin degradation in the slice preparation. 1,10-Phenanthroline, an inhibitor of metallopeptidases, was also an effective inhibitor. The dynorphin sequence responsible for the inhibition contains the Arg6-Arg7 bond. Other peptides (bradykinin and angiotensin) which are substrates of the soluble metallopeptidase also inhibited neurotensin breakdown by the slice. This evidence suggests that this thiol dependent metalloendopeptidase is the major neurotensin catabolizing enzyme in hypothalamic slices.
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PMID:Peptidases involved in the catabolism of neurotensin: inhibitor studies using superfused rat hypothalamic slices. 352 99

Synaptosomal membrane (SPM) bound exo- and endopeptidases cleave the dynorphins and Met-enkephalin-Arg-Gly-Leu at several sites to produce shorter fragments; among these are dynorphin 1-8 from 1-17, and Met-enkephalin from Met-enkephalin-Arg-Gly-Leu. The most vulnerable site is the Tyr-Gly bond cleaved by membrane-bound aminopeptidase(s), with the shorter peptides degraded more rapidly than the longer ones. A purified metalloendopeptidase sensitive to phosphoramidon inactivates the shorter peptide sequences at the Gly3-Phe4 bond, and the 1-13 and 1-17 sequences also at the Arg7-Ile8 bond. The kcat/Km ratios for purified metalloendopeptidase were 20-30 times higher for Leu-enkephalin and the proenkephalin octapeptide than for dynorphins 1-8, 1-13, and 1-17. Dynorphins 1-13 and 1-17 may serve as precursors for the widely distributed CNS neuropeptide dynorphin 1-8 since they were cleaved by a separate SPM endopeptidase insensitive to phosphoramidon. SPM monocarboxypeptidase converted dynorphin 1-13 to 1-12 (release of Lys) and dipeptidyl carboxypeptidase converted dynorphin 1-8 to 1-6; enkephalin octapeptide served as a precursor of Met-enkephalin by sequential action (release of Leu and Arg-Gly) of both carboxypeptidases.
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PMID:Membrane-bound enzymes and their role in processing of the dynorphins and of the proenkephalin octapeptide Metenkephalin-Arg-Gly-Leu. 614 75

Cathepsin B was purified about 11,000-fold from monkey skeletal muscle by ammonium sulfate fractionation and sequential column chromatographies monitored by assaying of Z-Phe-Arg-MCA hydrolase activity. The purified enzyme gave a single protein band on SDS-polyacrylamide gel electrophoresis, and its molecular weight was estimated to be 24,000 by gel filtration. It had a pH optimum of 6.5, required a thiol reducing agent for activation, and was inhibited by various thiol protease inhibitors. These properties were similar to those reported for cathepsins B from other sources. Although the enzyme scarcely hydrolyzed ordinary proteins, such as casein, hemoglobin, and bovine serum albumin, it degraded myosin and actin among various myofibrillar proteins. These results strongly suggested that skeletal muscle cathepsin B may participate in the degradation of muscle proteins in vivo. In addition, cathepsin B was shown to hydrolyze various neuropeptides such as Leu-enkephalin, beta-neoendorphin, alpha-neoendorphin, dynorphin(1-13), and substance P. It appeared to act on these peptides mainly as a dipeptidyl carboxypeptidase, although not so rigorously, presumably due to its endopeptidase activity.
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PMID:Purification and characterization of cathepsin B from monkey skeletal muscle. 672 39


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