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)

A fluorescent peptide substrate to explore the protease specificity for the amino acid regions C- and N-terminal to the cleavage site has been designed. Intramolecular quenching of indole fluorescence by an N-terminal dansyl group separated by six amino acid residues forms the basis of this assay. For a particular enzyme, specificity can be designed into the peptide sequence by means of the number of residues that separate the two chromophores. In the present instance, the heptapeptide Dns-Gly-Lys-Tyr-Ala-Pro-Trp-Val is used to assay angiotensin converting enzyme (ACE), Astacus protease, carboxypeptidase A, alpha-chymotrypsin, and trypsin, all of which cleave the peptide in accord with their known specificity: Trypsin and Astacus protease hydrolyze only the Lys-Tyr and Tyr-Ala bonds, respectively. alpha-Chymotrypsin primarily cleaves the Tyr-Ala bond while ACE makes three successive dipeptidyl cleavages from the C-terminus. Carboxypeptidase rapidly hydrolyzes first the Trp-Val and then the Pro-Trp bond. For all of the enzymes, catalytic activity (kcat/Km) is in the range from 10(5) to 10(6) M-1 s-1. Hydrolysis causes a fluorescence increase in the 310 to 410 nm region of 8.6- to 13.6-fold depending on the enzyme that is assayed. Assays can be designed based on the increase in tryptophan fluorescence or by individual product analyses using thin-layer or high-performance liquid chromatography. The specificity and sensitivity of such internally quenched fluorescent oligopeptides would seem to be ideal for the assay of specific endoproteases.
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PMID:A fluorescent oligopeptide energy transfer assay with broad applications for neutral proteases. 255 28

Endopeptidase (EP) 24.15 cleaves the Tyr5-Gly6 bond of luteinizing hormone-releasing hormone (LHRH) (pGlu-His-Trp-Ser-Tyr-Gly-Leu-Arg-Pro-Gly-NH2), and is the primary LHRH degrading enzyme in pituitary and hypothalamic membrane preparations. Potent and specific inhibitors were used to identify the enzymes involved in the in vivo degradation of LHRH. After i.c.v. administration of LHRH, only about 1% of the peptide was recovered from brain after 1 hr. Concurrent administration of LHRH and N-[1-(RS)-carboxy-3-phenylpropyl]-Ala-Ala-Phe-p-aminobenzoate (cFP-AAF-pAB), a specific inhibitor of EP 24.15, led to a more than 10-fold increase in LHRH recovery. Administration of N-[1-(RS)-carboxy-3-phenylpropyl]-Phe-pAB (cFP-F-pAB) or captopril, inhibitors of "enkephalinase" (EP 24.11) and angiotensin converting enzyme, respectively, did not significantly increase LHRH recovery. Intravenous administration of LHRH and either cFP-F-pAB or cFP-AAF-pAB but not captopril, led to an increase in the half-life of LHRH from 10 min to 15 and 20 min, respectively. Concurrent administration of both inhibitors resulted in a dramatic 8-fold increase in the half-life of LHRH, similar to values reported for "superactive" analogs of LHRH which are rendered resistant to enzymatic degradation by introduction of a D-amino acid in position 6. Concentrations of plasma LHRH 65 to 80 min after administration of inhibitors were 100- to 200-fold higher than those in controls. The potentiating effect of cFP-F-pAB resulted from inhibition of the in vivo degradation of cFP-AAF-pAB by EP 24.11.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Inhibition of endopeptidase 24.15 slows the in vivo degradation of luteinizing hormone-releasing hormone. 268 86

The role of rat intestinal angiotensin-converting enzyme (ACE; E.C 3.4.15.1) in the digestion and absorption of dietary protein was investigated. Enzyme activity was associated with the brush-border membrane fraction, with the highest activity in the proximal to midregion of the small intestine. Preliminary enzyme characterization studies were carried out using purified brush-border membrane preparations. When a variety of N-blocked synthetic peptides were used as potential substrates for ACE, activity was highest with those containing proline at the carboxy terminal position. The hydrolytic rates observed with these prolyl peptides were comparable to those observed when major digestive peptidases of the brush-border membrane such as aminopeptidase N and dipeptidyl aminopeptidase IV were assayed. When isolated rat jejunum was perfused in vivo with solutions of Bz-Gly-Ala-Pro, the dipeptide Ala-Pro was the main hydrolytic product detected in the perfusates. Absorption rates of the constituent amino acids, alanine and proline, depended on the concentration of peptide perfused. Captopril, an active site specific ACE inhibitor, significantly inhibited hydrolysis and absorption of constituent amino acids from Bz-Gly-Ala-Pro. These results show that intestinal brush-border membrane ACE functions as a digestive peptidase in addition to its role as a regulator of biologically active peptides in other tissues.
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PMID:Role of rat intestinal brush-border membrane angiotensin-converting enzyme in dietary protein digestion. 282 4

The authors present a simple and clinically applicable method for the serial monitoring of pulmonary microvascular enzyme function in vivo. This method requires the intravenous injection of trace amounts of a radiolabelled substrate and the collection of a single arterial blood sample. Simultaneous measurement of pulmonary blood flow, (e.g., by dye- or thermo-dilution) and the determination of blood hematocrit are also needed for the calculations. This method was compared to the multiple blood sample indicator dilution method in normal anesthesized rabbits. Both methods gave identical results for the metabolism of the synthetic, hemodynamically inactive tripeptide, 3H-benzoyl-Phe-Ala-Pro (3H-BPAP), by pulmonary microvascular endothelial angiotensin converting enzyme. The parameters measured were: 1) substrate utilization, expressed linearly and logarithmically, and 2) the apparent first order reaction constant. The new method was also used for the simultaneous measurement of single pass, transpulmonary metabolism of 3H-BPAP by angiotensin converting enzyme and of 5'-adenosine monophosphate by 5'-nucleotidase in rabbits in vivo. The authors propose that similar enzyme kinetic measurements could be used in clinical studies to test their usefulness as an aid in the early diagnosis of incipient pulmonary endothelial dysfunction, e.g., adult respiratory distress syndrome.
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PMID:Monitoring of pulmonary endothelial enzyme function: an animal model for a simplified clinically applicable procedure. 282 40

The synthesis of a new class of potential angiotensin converting enzyme (ACE) inhibitors, analogs of enalapril, is reported. In these molecules the C-terminal amino acidic sequence Ala-Pro of enalapril was replaced by the sequence Pro-Pro. The compounds were tested both in vitro and in vivo. They showed no in vivo activity but only a week in vitro inhibitory activity.
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PMID:Prolyl derivatives of enalapril as potential angiotensin converting enzyme inhibitors. 285 88

A glutamic acid residue at the active site of bovine lung angiotensin I-converting enzyme, a zinc-metallo peptidyl dipeptidase, was esterified with p-[N,N-bis(chloroethyl)amino]phenylbutyryl-L-[U-14C]proline (chlorambucyl-L-[U-14C]-L-proline), an affinity label for this enzyme (Harris, R.B., and Wilson, I.B. (1983) J. Biol. Chem. 258, 1357-1362). The radiolabeled enzyme was digested with BrCN and only 1 of the 30 cleavage peptides resolved by reverse-phase high performance liquid chromatography (HPLC) contained the bound radiolabel. This active-site peptide (Mr = 16,000) was digested with trypsin and the labeled peptide formed (T-2) was further degraded with thermolysin. The thermolytic peptides were resolved by reverse-phase HPLC. Only 1 of the 5 peptides obtained (Th-1, Mr = 1290) contained the bound radiolabel. Th-1 (12 residues) was subjected to manual Edman degradation and the following partial sequence was determined: H2N-Phe-Thr-Glu-Leu-Ala-Asp-Ser-Glu... The radiolabel was released at cycle 3 and the amount recovered was equivalent to the amount of phenylthiohydantoin-Glu detected on HPLC. Thus, glutamic acid is esterified with chlorambucyl-L-[U-14C]proline in confirmation of our earlier findings. The sequence determined is homologous in 5 residues with the corresponding sequences of bovine carboxypeptidase A and B, two other mammalian zinc proteases. There is little sequence homology with thermolysin, a bacterial zinc protease that also contains an essential active-site glutamic acid residue.
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PMID:Sequencing of an active-site peptide of angiotensin I-converting enzyme containing an essential glutamic acid residue. 285 12

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

The mRNA encoding angiotensin I-converting enzyme, a zinc-metallo dipeptidyl carboxyhydrolase, has been identified in extracts prepared from bovine lung tissue. Bovine lung poly(A) + mRNAs were subjected to electrophoresis and northern blot hybridization analysis using a radiolabeled synthetic 24-deoxyoligonucleotide probe complementary to eight codons for amino acids at the active-site of the enzyme (Harris, R.B. & Wilson, I.B., J. Biol. Chem. 260, 2208-2211, 1985). This amino acid sequence contains the catalytic glutamic acid residue. A single RNA species (approximately equal to 4 kb) was detected which is 1 kb larger than predicted from the molecular weight of the enzyme. The excess nucleic acid composition may be due to leader and/or trailer sequences or the RNA may encode a high molecular weight precursor form of the enzyme. We have cloned an EcoR1-HindIII digest fragment (1400 bp) of the duplex cDNA derived from the bovine lung converting enzyme poly(A) + mRNA and also Bal31 deletion fragments generated from the 1400 bp clone. Several of the Bal31 clones contain the active-site sequence codons of the enzyme and the complete cDNA sequence of one of these (72 bp) has been determined. We found the amino acid sequence at the active site to be -Phe-Thr-Glu-Leu-Ala-Asn-Ser-, containing the catalytic Glu residue. This sequence is identical with the sequence that we previously determined by manual Edman degradation analysis of the appropriate active-site peptide except that we now find Asn instead of Asp. We have sequenced 670 bp of the 1400 bp clone but have not yet overlapped the active-site sequence.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Hybridization and partial cDNA sequence analyses of bovine lung angiotensin I-converting enzyme. 288 36

Protamine given to neutralize heparin after extracorporeal circulation can trigger a catastrophic reaction in some patients. While searching for a biochemical basis for this reaction, protamine was tested as an inhibitor of human plasma carboxypeptidase N (CPN) or kininase I, the inactivator of anaphylatoxins and kinins. Human plasma and CPN purified from human plasma, (Mr = 280 K) or its isolated active subunit (Mr = 48 K) were the sources of enzyme. The hydrolysis of furylacryloyl (FA)-Ala-Lys was measured in a UV spectrophotometer and that of bradykinin and the synthetic C-terminal octapeptide of anaphylatoxin C3a (C3a8) by high performance liquid chromatography. Protamine inhibited the hydrolysis of FA-Ala-Lys by CPN, (IC50 = 3.2 X 10(-7) M); added human serum albumin (30 mg/ml) increased the IC50 to 7 X 10(-6) M. When plasma was the source of CPN, the IC50 was 2 X 10(-6) M. Protamine more effectively inhibited the hydrolysis of bradykinin and C3a8. The IC50 for protamine was 5 X 10(-8) M with CPN and bradykinin, 7 X 10(-8) M with CPN and C3a8 and with the 48 K subunit and bradykinin it was 7 X 10(-8) M of protamine. Heparin competes with CPN for protamine, because in high concentration (18 U/ml) it reverses the inhibition by protamine. Protamine did not inhibit angiotensin I converting enzyme (kininase II) or the endopeptidase 24.11 (enkephalinase). Kinetic studies showed the mechanism of protamine inhibition to be partially competitive; about 10-20% of the hydrolysis of bradykinin by CPN was not inhibited by protamine. Thus, by blocking the inactivation of mediators released in shock, protamine inhibition of CPN may be partially responsible for the catastrophic reaction observed to occur in some patients.
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PMID:Protamine inhibits plasma carboxypeptidase N, the inactivator of anaphylatoxins and kinins. 291 61

A new amino alcohol modification designed to mimic the putative transition-state of amide bond cleavage by proteolytic enzymes has been incorporated into the scissile bond position of N-benzoyl-Phe-Ala-Pro, a known substrate of angiotensin converting enzyme (ACE). The resulting modified tripeptides (i.e. 4) are shown to be a new class of potent inhibitors of converting enzyme.
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PMID:Design of peptide derived amino alcohols as transition-state analog inhibitors of angiotensin converting enzyme. 298 76


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