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)

Chemotactic factor-enriched butanol extracts from Escherichia coli culture filtrates were fractionated and purified by high pressure liquid chromatography. The yield from individual fractions of biological activity (lysosomal enzyme secretion) and antigenic activity (competition with [3H]fMet-Leu-Phe for binding to rabbit anti-fMet-Leu-Phe) revealed an average 50% recovery of original material. Five peaks of biological activity were separated as demonstrated by enzyme-releasing activity. Three of these peaks coincided exactly with peaks of antigenic activity, suggesting that at least 3 and as many as 5 distinct formyl-methionyl peptides had been separated. The majority of recovered activity appeared in peak 3 and represented 70% of the total biological and antigenic activities recovered. The five peak fractions were subsequently analyzed by dipeptidyl carboxypeptidase gas chromatography-mass spectrometry (DCP/GC-MS) to determine amino acid sequences. After digestion, the formyl-Met peptide was demonstrated in only one of the five peak fractions (peak 3). Furthermore, both the GC retention times and mass spectra indicated that peak 3 contained formyl-methionyl-leucyl-phenylalanine. The DCP/GC and MS data were confirmed with tests made on authentic fMet-Leu-Phe. Butanol extracts from E. coli filtrates to which were added synthetic fMet-Leu-Phe resulted in increased biological and antigenic activity in the precise high pressure liquid chromatography fractions of peak 3 where the fMet-Leu-Phe produced by E. coli was found. Finally, the analysis of recovered biological and antigenic activities indicated that the formyl peptides were found in nanomolar concentrations in culture filtrates. These results demonstrate that the NH2-terminal formyl peptides produced by E. coli, of which formyl-methionyl-leucyl-phenylalanine appears to be the major component, are the peptide mediators responsible for leukocyte chemotactic activity in the bacterial culture extracts.
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PMID:Purification and identification of formyl-methionyl-leucyl-phenylalanine as the major peptide neutrophil chemotactic factor produced by Escherichia coli. 637 Oct 5

Rabbit testicular dipeptidyl carboxypeptidase activity was purified by a procedure exploiting its affinity for N-alpha-[1-(S)-carboxy-3-phenylpropyl]-L-lysyl-L-proline. The molecular, catalytic, and immunological properties of the testicular enzyme are presented and compared with the corresponding properties of pulmonary angiotensin-converting enzyme. Although catalytically similar and immunologically related to pulmonary dipeptidyl carboxypeptidase, the testicular enzyme has a molecular weight (100,000) which is lower by a factor of about one-third and differs in its NH2 and COOH termini. Furthermore, we present evidence that the testicular enzyme is not a post-translation product of the pulmonary type enzyme. These data suggest that testicular and pulmonary dipeptidyl carboxypeptidase are two distinct proteins which are catalytically similar and immunologically closely related.
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PMID:Molecular and catalytic properties of rabbit testicular dipeptidyl carboxypeptidase. 675 23

Angiotensin I-converting enzyme (ACE; kininase II) contains two very similar domains (the NH2- and COOH-terminal domains (N and C domains, respectively)), each bearing an active site. These active sites hydrolyze the same peptides, but do not have the same catalytic properties and substrate specificities. In an attempt to develop domain-specific immunological probes, two series of monoclonal antibodies (mAbs), 19 clones in all, were produced and tested against human ACE. These mAbs recognized at least nine different epitopes within three antigenic regions of the ACE molecule. Testing on wild-type recombinant ACE and several mutants with only one intact domain showed that these epitopes were all located in the N domain. None of the mAbs recognized the C domain. This particular specificity and analysis of results obtained with several polyclonal antibodies to human ACE suggest that ACE immunogenicity is determined mainly by the N domain. Two mAbs (3A5 and i2H5) recognizing epitopes from different antigenic regions of ACE inhibited the enzymatic activity of the N (but not of the C) domain. mAb 3A5 had the same inhibitory potency toward hippuryl-His-Leu, benzyloxycarbonyl-Phe-His-Leu, and angiotensin I hydrolysis, with 50% inhibition achieved at a mAb/ACE molar ratio of 6. mAb i2H5 was roughly three times more effective than mAb 3A5 inhibiting the hydrolysis of benzyloxycarbonyl-Phe-His-Leu and the natural substrates angiotensin I and bradykinin (50% inhibition at a molar ratio of 1-2), but was less effective in inhibiting hippuryl-His-Leu cleavage (50% inhibition at a molar ratio of 22-25), indicating that this substrate interacts with a specific subsite. mAb i2H5 almost completely inhibited the hydrolysis of the luteinizing hormone-releasing hormone by the isolated N domain. Both the primary carboxyl- and amino-terminal cleavages of this peptide were suppressed. This antibody suppressed the primary amino-terminal cleavage of the luteinizing hormone-releasing hormone by wild-type ACE by > 90%, indicating that this particular ACE function is mediated mainly by the N domain active site. These data provide evidence for structural differences between the two homologous domains of ACE despite their high degree of sequence homology and show that monoclonal antibodies are able to distinguish between the two active sites in ACE.
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PMID:Structure-function analysis of angiotensin I-converting enzyme using monoclonal antibodies. Selective inhibition of the amino-terminal active site. 752 12

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

The detergent extract of rabbit liver microsomes contains an endopeptidase (MEP) with substrate specificity for peptides containing Arg residues at the P1 and P4 positions in the cleavage site (Kawabata, S., and Davie, E. W. (1992) J. Biol. Chem. 267, 10331-10336). These sequences occur in many proproteins such as the vitamin K-dependent proproteins and prohormones. A cDNA coding for MEP has been obtained from three overlapping clones isolated from two rabbit liver lambda gt10 cDNA libraries. The longest open reading frame of the 3507-base pair cDNA codes for a protein of 704 amino acids, of which 406 residues were confirmed by amino acid sequence analysis. MEP contains a putative active site of -His-Glu-X-X-His-, which is typical of mammalian zinc metallopeptidases. Based on a hydropathy plot, MEP is a hydrophilic protein with no transmembrane domain and no NH2-terminal signal sequence. Amino acid sequence analysis identified Asn at the three potential N-glycosylation sites in the enzyme, indicating that MEP contains no N-linked sugar. MEP is homologous with rat testes metalloendopeptidase 24.15 (60% identity), rat mitochondrial intermediate peptidase (24% identity), Escherichia coli dipeptidyl carboxypeptidase (25% identity), and the open reading frame YCL57w present in yeast chromosome III (35% identity).
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PMID:Rabbit liver microsomal endopeptidase with substrate specificity for processing proproteins is structurally related to rat testes metalloendopeptidase 24.15. 850 89

The reaction of human erythrocyte acetylcholinesterase (AChE) with a set of structurally related phosphoramidates was studied in order to investigate the properties of phosphorylated enzyme and the effects of 4 oximes PAM-2, TMB-4, HI-6 and BDB-106 on the reactivation of inhibited AChE. Second-order rate constant of the phosphorylation reaction of the compounds towards the active site of AChE range between 5.0 x 10(2) and 4.9 x 10(6) M-1min-1 and their inhibitory power (I50) was from 7.3 x 10(-5) to 5.7 x 10(-9) M for 20 min incubation at 37 degrees C. The oximes used were weak reactivators of inhibited AChE except for (C4H9O)(NH2)P(O)DCP (DCP, -O-2,5-dichlorphenyl group) and (C6H13O)(NH2)P(O)SCH3 where we have obtained good reactivation. Imidazole oxime BDB-106 proved to be a potent reactivator of tabun-inhibited AChE.
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PMID:Oxime-induced reactivation of acetylcholinesterase inhibited by phosphoramidates. 861 58

For facilitating crystallization and structural studies of the testicular isozyme of angiotensin-converting enzyme (ACE,), we attempted the production of enzymatically active ACET proteins which are unglycosylated or underglycosylated. Expression in Escherichia coli of the rabbit ACET cDNA resulted in the synthesis of an unglycosylated but inactive protein. Similarly, unglycosylated ACET synthesized in HeLa cells, by using a cDNA in which all five potential N-glycosylation sites had been mutated, was inactive and rapidly degraded. Several ACET variants carrying mutations in one or more of the potential N-glycosylation sites were used to examine the role of glycosylation at specific sites on ACET synthesis, transport to the cell surface, cleavage processing, and enzyme activity. These experiments demonstrated that allowing glycosylation only at the first or the second site, as counted from the NH2 terminus, was sufficient for normal synthesis and processing of active ACET. In contrast, ACETg3, which had only the third glycosylation site available, was unglycosylated, enzymatically inactive and rapidly degraded. N-Glycosylated ACET could also be produced in yeast. Surprisingly, the mutant ACETg3 was synthesized, N-glycosylated, and properly transported in yeast. Wild type and mutant ACE proteins were cleavage-secreted from yeast and enzymatically active.
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PMID:Different glycosylation requirements for the synthesis of enzymatically active angiotensin-converting enzyme in mammalian cells and yeast. 862 43

The intestinal oligopeptide transporter (OPT) mediates the absorption of di-/tripeptides, beta-lactam antibiotics, angiotensin converting enzyme (ACE) inhibitors and renin inhibitors. This suggests that the targeting of molecules to this transporter could result in orally-absorbed drugs. Results from a recent study with renal brush border membrane vesicles (BBMV) suggested that an alpha-NH2 group is required for interaction with the renal OPT. In general, structural requirements for interaction with the renal and intestinal OPT are similar. However, these recent findings do not agree with earlier studies, which showed that an alpha-NH2 group is not essential for interaction with the intestinal OPT. Thus, it appears that the renal and intestinal OPT may differ in their recognition of compounds containing an alpha-NH2 group. In this study, molecular modeling was used to determine the tridimensional structures of various cephalosporins for which Ki values had been determined using renal BBMV. All cephalosporins which interact with the OPT have two, energetically equivalent, conformations. Most compounds which do not interact with the OPT cannot adopt the two conformations. A key factor which influences the conformation seems to be the substituent group at the alpha position; an electron drawing group at that position alters the common conformations. For the OPT substrates, the distances between the -NH2 and -COOH groups are comparable to those of the tripeptide, GlyGlyGly; and the distances between -NH2 and carbonyl group in the beta-lactam ring are close to the distance between N-terminal and C-terminal in the dipeptide, GlyGly. The corresponding distances in cephamycin C (in which a -NH2 group is located in a different position) and the tetrapeptide, GlyGlyGlyGly, are longer than those in alpha-NH2 cephalosporins and GlyGlyGly. Cephamycin C and the tetrapeptide have low affinity for the renal OPT, suggesting that the distances between functional groups are critical for affinity. The alpha-NH2 group had no effect on the conformations of the molecules. We concluded that the alpha-NH2 group may interact directly with the renal oligopeptide transporter. Whether this is unique to the renal transporter or could be applied to the intestinal transporter will require further investigation.
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PMID:Molecular modeling study of structural requirements for the oligopeptide transporter. 879 74

In this study, we demonstrated that NH2-terminal Ser and Ile residues of pRL1b (SI-pRL1a) (SIIPGLPLSL) are not involved in the recognition by RLmale 1-specific cytotoxic T lymphocyte. The sensitization activity observed with pRL1b (SI-pRL1a) was not greater than that of peptides substituted with irrelevant amino acids at these positions. In serum-free medium, pRLla retained sensitization activity, but pRL1b (SI-pRL1a) did not. Furthermore, addition of bestatin to serum-containing medium blocked sensitization by pRL1b (SI-pRL1a). On the other hand, the addition of captopril enhanced it, probably by inhibiting the degradation of pRL1a by ACE. pRL1a-D peptide with D-Ile in place of the L-Ile residue of pRL1a (IPGLPLSL) showed sensitization, but SI-pRLla-2,3D peptide, which has D-Iles in place of the L-Ile residues of pRLlb (SI-pRL1a), and which was not cleaved between the two D-Iles, did not. The findings suggest that pRL1a is the antigenic peptide bound to L(d) molecules and pRL1b (SI-pRL1a) peptide is its natural precursor, which generates pRL1a via proteolysis.
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PMID:Production of murine leukemia RLmale1 rejection antigen peptide pRL1a by proteolysis of natural precursor pRL1b. 904 46

Degradation of yolk protein is essential for the early development of the avian embryo. In Japanese quail (Coturnix coturnix japonica), proteolysis in the surrounding tissue of the yolk, the yolk-sac membrane, can be inhibited by class-specific inhibitors of cysteine proteinases as well as of aspartic proteinases. Purification of the enzymes leads to one cysteine proteinase and one aspartic proteinase with an apparent molecular mass of 29 kD and 44 kD, respectively. Both enzymes were purified in a two-chain form, although a single-chain form is also present in the homogenate of yolk-sac membrane. The cysteine proteinase was identified by NH2-terminal sequence analysis as well as by kinetic studies as a new cathepsin B from quail. Like mammalian cathepsin B, this avian cathepsin B exhibits two different kinds of proteolytic activity, an endopeptidase activity and a dipeptidyl carboxypeptidase activity. Chicken egg white cystatin, a protein-aceous cysteine proteinase inhibitor, inhibits quail cathepsin B with an equilibrium dissociation constant (Ki) of 3.3 nM. Likewise the aspartic proteinase was identified as a new cathepsin D from quail. This avian cathepsin D has a different processing site to all known mammalian cathepsins D. In quail cathepsin D one NH2-termini is homologous to amino acids 211-230 in mammalian cathepsin D. This is more than 100 amino acids downstream of the mammalian processing site. Comparison of the enzymatic properties of quail and bovine cathepsin D indicate that the different processing site has no influence on the enzymatic properties.
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PMID:Proteolytic enzymes in yolk-sac membrane of quail egg. Purification and enzymatic characterisation. 941 5


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