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

---

Query: EC:3.4.24.3 (collagenase)
18,340 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Our investigations indicate that a variety of neutral serine proteases exist in highly purified, IL-2-activated rat NK (A-NK) cells. These enzymatic activities are not restricted to only cytolysin-containing granules and are not defined by only the assay of N-alpha-benzyloxycarbonyl-L-lysine thiobenzylesterase activity. These activities, which we term A-NKP 1, A-NKP 2, A-NKP 3, and A-NKP 4, cleave, respectively, the following fluorogenic peptide substrates: Boc-Phe-Ser-Arg-7-amino-4-methylcoumarin (AMC, trypsin-like); Suc-Ala-Ala-Phe AMC (chymotrypsin-like); Suc-Gly-Pro-Leu-Gly-Pro AMC (collagenase-like), and Z-Phe-Arg AMC (another trypsin-like enzyme). The proteases A-NKP 1, A-NKP 2, and A-NKP 3 are not cell surface-associated and appear to be cytosolic as defined by isopycnic sucrose density gradient centrifugation. In contrast, A-NKP 4 appears to be located in lysosomes. Treatment of rat A-NK cells with protease inhibitors that inhibit A-NKP 2 and A-NKP 3 also substantially inhibit A-NK cell-mediated cytotoxicity against both NK-sensitive and -resistant targets (YAC-1 and P815, respectively). These results indicate that A-NKP2 and A-NKP 3 may play a role in IL-2-activated NK cell-mediated cytotoxicity. A variety of proteolytic enzymes, in addition to granzymes, therefore exist in A-NK cells. Our studies indicate that a prerequisite to a thorough understanding of the role of proteases in killer cell function is the investigation of several classes of enzymes in addition to granzymes contained in lytic granules.
...
PMID:Nongranular proteolytic enzymes of rat IL-2-activated natural killer cells. I. Subcellular localization and functional role. 151 70

Antihemorrhagic proteins from Crotalus atrox serum were tested for their ability to inhibit the proteolytic activity of the hemorrhagic toxin-e from Crotalus atrox venom and of several other proteolytic enzymes: trypsin, collagenase and thermolysin. The antihemorrhagic proteins inhibited the proteolytic activity of hemorrhagin-e when tested on gelatin type I and collagen type IV, the proteolytic activity of trypsin on photofilm gelatin and the proteolytic activity of whole venom when tested on azocollagen and photofilm gelatin. The antihemorrhagins failed to inhibit the proteolytic activity of trypsin when tested on the specific synthetic substrate N-acetyl-DL-phenylalanine-beta-naphthyl ester (APNE), the activity of microbial collagenase on N-(3-[2-furyl]acryloyl)-Leu-Gly-Pro-Ala (FALGPA) or on azocollagen and the activity of thermolysin on N-(3-[2-furyl]acryloyl)-Gly-Leu amide (FAGLA). It is tentatively suggested that the antihemorrhagins from snake blood serum are proteinase inhibitors that underwent specialization towards the neutralization of the proteolytic activity of hemorrhagic toxins.
...
PMID:Inhibition of the proteolytic activity of hemorrhagin-e from Crotalus atrox venom by antihemorrhagins from homologous serum. 151 50

(7-methoxycoumarin-4-yl)Acetyl-Pro-Leu-Gly-Leu-(3-[2,4-dinitrophenyl]-L- 2,3-diaminopropionyl)-Ala-Arg-NH2 (Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH2) has been synthesised as a fluorogenic substrate for the matrix metalloproteinases. The highly fluorescent 7-methoxycoumarin group is efficiently quenched by energy transfer to the 2,4-dinitrophenyl group. The punctuated metalloproteinase (PUMP, EC 3.4.24.23) cleaves the substrate at the Gly-Leu bond with a 190-fold increase in fluorescence (lambda cx 328 nm, lambda cm 393 nm). In assays of the human matrix metalloproteinases. Mca-Pro-Leu-Gly-Leu-Dpa-Ala-Arg-NH2 is about 50 to 100 times more sensitive than dinitrophenyl-Pro-Leu-Gly-Leu-Trp-Ala-D-Arg-NH2 and continuous assays can be made at enzyme concentrations comparable to those used with macromolecular substrates. Specificity constants (kcat/Km) are reported for both synthetic substrates with PUMP, collagenase, stromelysin and 72 kDa gelatinase.
...
PMID:A novel coumarin-labelled peptide for sensitive continuous assays of the matrix metalloproteinases. 153

A metal-dependent peptidase was isolated from the homogenate of human uterus by standard chromatographic techniques and purified to apparent homogeneity. The peptidase hydrolysed the synthetic vertebrate collagenase substrate 2,4-dinitrophenyl-Pro-Gln-Gly-Ile-Ala-Gly-Gln-D-Arg (Dnp-peptide), the synthetic bacterial collagenase substrate 4-phenylazobenzyloxycarbonyl-Pro-Leu-Gly-Pro-D-Arg (PZ-peptide) and gelatinolytic peptides of gelatin, but was inactive against collagen type I, gelatin and casein. The cleavage site for the Dnp-peptide was the Gly-Ile bond. The enzyme was not only inhibited by metal chelators, such as EDTA, 1,10-phenantroline and dithiothreitol but also by thiol reagents, such as mersalylic acid and N-ethylmaleimid. However, E-64, an inhibitor for thiolproteinases, and leupeptin, an inhibitor for thiol- and serine proteases, did not exhibit any inhibitory activity. Pepstatin, an inhibitor for aspartate proteinases, and inhibitors for serine proteinases like phenylmethanesulfonyl fluoride and Trasylol were ineffective as well. The purified peptidase displayed a single band in the SDS-PAGE with an apparent molecular mass of 65 kDa. Employing isoelectric focusing an IP of 5.0 could be determined. The enzyme's properties are discussed in relation to the proteinase EC 3.4.24.11 and to proteinases of the collagenase family as well as the possibility to discriminate these three metalloproteinase classes by employing the Dnp-peptide.
...
PMID:Isolation and properties of a metal-dependent endopeptidase from human uterus hydrolysing synthetic collagenase substrates. 165 Feb 34

The interaction of succinyl-Pro-Ala, a competitive inhibitor of Achromobacter iophagus collagenase, with the enzyme was studied by longitudinal proton and tritium relaxation. Specific deuterium and tritium labeling of the succinyl part at vicinal positions allowed the measurement of the cross-relaxation rates of individual proton or tritium spin pairs in the inhibitor-enzyme complex as well as in the free inhibitor. Overall correlation times, internuclear distances, and qualitative information on the internal mobility in Suc1 (as provided by the generalized order parameter S2) could be deduced by the comparison of proton and tritium cross-relaxation of spin pairs at complementary positions in the -CH2- CH2- moiety as analyzed in terms of the model-free approach by Lipari and Szabo. The conformational and motional parameters of the inhibitor in the free and enzyme-bound state were directly compared by this method. The measurement of proton cross-relaxation in the Ala residue provided additional information on the inhibitor binding. The determination of the order parameter in different parts of the inhibitor molecule in the bound state indicates that the succinyl and alanyl residues are primarily involved in the interaction with the enzyme activity site. The succinyl moiety, characterized in solution by the conformational equilibrium among the three staggered rotamers--i.e., trans: 50%; g+: 20%; g-: 30%--adopted in the bound state the unique trans conformation.
...
PMID:Proton and tritium NMR relaxation studies of peptide inhibitor binding to bacterial collagenase: conformation and dynamics. 165 Nov 24

Several N-carboxyalkyl peptides were synthesized and tested as inhibitors of pig synovial collagenase, 72-kDa gelatinase and stromelysin (matrix metalloproteinases MMP-1, MMP-2, and MMP-3). The most potent of the series, CH3CH2CH2(R,S)CH(COOH)-NH-Leu-Phe-Ala-NH2, competitively inhibited cleavage of dinitrophenyl-Pro-Leu-Gly-Leu-Trp-Ala-D-Arg-NH2 at the Gly-Leu bond by MMP-1 and MMP-2 (KI = 30 and 40 microM, respectively). A similar inhibitory potency was found for MMP-1 with soluble Type I collagen and MMP-3 with substance P as substrate. The inhibitor was coupled to EAH-Sepharose 4B through a C-terminal amide. In the presence of 2 M NaCl at pH 7.2, this matrix bound MMP-1, MMP-2, and MMP-3 from concentrated culture medium of pig synovial membranes. The enzymes coeluted at pH 4.1 and subsequently were resolved by chromatography on DEAE-Sephacel and heparin-Sepharose. Purified MMP-1 catalyzed the o-phenanthroline-sensitive cleavage of collagen into TCA and TCB fragments as well as slower hydrolysis of the alpha 2 chain. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of MMP-1 indicated a predominant polypeptide of approximately 44 kDa and minor species of approximately 24 and 21 kDa. The 44-kDa species and one of the smaller polypeptides reacted with an antiserum to residues 195-207 of human fibroblast MMP-1, indicating that porcine MMP-1 contains a similar sequence and that the smaller components were probably derived from MMP-1. Neither MMP-2 nor MMP-3 reacted with this antiserum. Purified porcine MMP-2 degraded gelatin but not collagen and exhibited an apparent Mr of approximately 71 kDa. Additional smaller polypeptides were present, one of which may correspond to tissue inhibitor of metalloproteinases. MMP-3 showed doublets of approximately 47/46 and 26/25 kDa and cleaved substance P at the Gly6-Phe7 bond. This procedure provides a rapid means of obtaining all three MMPs from one source in approximately 15% yield each.
...
PMID:Application of N-carboxyalkyl peptides to the inhibition and affinity purification of the porcine matrix metalloproteinases collagenase, gelatinase, and stromelysin. 165 8

The midgut chymotrypsins (EC 3.4.4.5) of three species of shrimps, Penaeus monodon, Penaeus japonicus and Penaeus penicillatus were purified and studied in detail to clarify previous ambiguity in their identification. In each of the species there are two major forms of chymotrypsin, both single-chained with three disulfide bonds. One has a pI of 3.2 and Mr 27,000 or 28,000, while the other has a pI of 3.0 and Mr 25,000 or 26,000. The N-terminal amino acid sequences of the P. monodon enzymes are homologous to those of the crab (Uca pugilator) collagenase and to the other chymotrypsins. However, the active sites of the shrimp chymotrypsins are different from that of the well studied bovine alpha-chymotrypsin in some respects: (1) in spite of showing the typical specificity of chymotrypsin, the shrimp enzymes are more stringently selective for substrates with extended polypeptide chain; (2) some titration agents of alpha-chymotrypsin, including t-cinnamoylimidazole, 4-nitrophenyl guanidinobenzoate and its fluorescent derivative, do not react with the shrimp enzymes, neither do some of the alpha-chymotrypsin inhibitors: Tosyl-PheCH2Cl, methyl-4-nitrobenzenesulfonate and benzeneboronic acid; (3) the shrimp chymotrypsins are more reactive than the bovine enzyme toward native protein substrates including collagen; (4) the kinetic-salt-effects of the shrimp enzyme toward N-succinyl- and acetyl-Ala-Ala-Pro-Phe-4-nitroanilide mainly reflect electrostatic rather than hydrophobic interactions between the substrates and the enzyme. The shrimp enzymes are acid-labile but resistent to autolysis. Our results suggest that most Crustacea decapods contain chymotrypsins as one of the major digestive endopeptidases.
...
PMID:The midgut chymotrypsins of shrimps (Penaeus monodon, Penaeus japonicus and Penaeus penicillatus). 165 78

To develop a potent and specific collagenase inhibitor, a series of tetrapeptidyl hydroxamic acids were synthesized, based on the previous findings with tripeptidyl derivatives (Chem. Pharm. Bull., 38, 1007-1011, 1990). Among the series of tetrapeptidyl derivatives synthesized, R-Gly-Pro-Leu-Ala-NHOH and R-Gly-Pro-D-Leu-D-Ala-NHOH were found to be highly specific and potent inhibitors against vertebrate collagenase with an IC50 of 10(-6) M order, where R stands for Boc or acyl group. Analysis of their structure-activity relationships showed a characteristic feature of the substrate-binding site of collagenase as follows: 1) the S1 subsite forms a shallow hydrophobic pocket, although glycine residue corresponds to the subsite of the natural collagen substrate: 2) the S2 subsite constitutes a bulky pocket with less requirement for hydrophobicity: 3) the S3 subsite preferentially accommodates Pro residue: and 4) the accommodation of the P4-P1 subsites of peptidyl collagenase inhibitor to the S4-S1 subsites is required to form a tight binding of its hydroxamic acid moiety to the zinc ion at the catalytic site of the enzyme. The introduction of an enantiometric dipeptide unit, D-Leu-D-Ala, to the P2-P1 subsites demonstrated an increased binding capacity to the extended S4-S1 subsites of collagenase, thus providing proteinase-resistant inhibitor.
...
PMID:Vertebrate collagenase inhibitor. II. Tetrapeptidyl hydroxamic acids. 165 22

Mammalian collagenases cleave all three alpha chains of native, triple-helical types I, II, and III collagens after the Gly residue of the partial sequence Gly-[Ile or Leu]-[Ala or Leu] at a single locus approximately three-fourths from the amino terminus. There are an additional 31 sites in the triple-helical regions of types I, II, III, and IV collagens that contain the same partial sequence but are not hydrolyzed. A model has been developed to explain this remarkable specificity. The mammalian collagenase cleavage site in interstitial collagens is distinguished by: (a) a low side-chain molal volume-, high imino acid (greater than 33%)-containing region that is tightly triple-helical, consisting of four Gly-X-Y triplets preceding the cleavage site, (b) a low imino acid-containing (less than 17%), loosely triple-helical region consisting of four Gly-X-Y triplets following the cleavage site, and (c) a maximum of one charged residue for the entire 25 residue cleavage site region, which is always an Arg that follows the cleavage site in subsite P'5 or P'8. In addition, the high imino acid-containing region cannot have an imino acid adjacent to the cleaved Gly-[Ile or Leu] bond (i.e. in subsite P2). Careful scrutiny of the 31 non-cleaved sequences reveals that none of those sites shares all of the characteristics of the cleavage site. The criterion of this model thus explain both cleaved and non-cleaved sequences in the triple-helical regions of types I, II, III, and IV collagen, and are supported by all known experimental and theoretical results on collagen catabolism and structure.
...
PMID:A model for interstitial collagen catabolism by mammalian collagenases. 166 5

When human fibroblast collagenase was incubated with ClCH2CO-(N-OH)Leu-Ala-Gly-NH2 (2-5 mM) in Tris buffer, pH 7.4 at 25 degrees C, a slow, time-dependent inhibition of the enzyme was observed. Dialysis against a buffer to remove free inhibitor did not reactivate the enzyme. A reversible competitive inhibitor, phthaloyl-GlyP-Ile-Trp-NHBzl (50 microM) partially protected the enzyme from inactivation by the compound. From the concentration dependent rates of inactivation Ki = 0.5 +/- 0.1 mM and k3, the rate constant for inactivation = 3.4 +/- 0.3 x 10(-3) min-1 were determined. The inactivation followed the pH optimum (6.5-7.0) for the enzyme activity, suggesting direct involvement of the same active site residue(s). The reaction mode of the inhibitor may be analogous to that of the inactivation of Pseudomonas aeruginosa elastase [Nishino, N. and Powers, J. (1980) J. Biol. Chem., 255, 3482] in which the catalytic glutamate carboxyl was alkylated by the inhibitor after its binding to enzyme through the hydroxamic Zn2+ ligand. All carboxyl groups in the inactivated collagenase were modified with 0.1 M ethyl dimethylaminopropyl carbodiimide/0.5 M glycinamide in 4 M guanidine at pH 5. The inactivator-affected carboxyl group was then regenerated with 1 M imidazole at pH 8.9, 37 degrees C for 12 h and the protein was radiolabeled with 3H-glycine methyl ester and carbodiimide to incorporate 0.9 residue glycine per mol enzyme.
...
PMID:Inactivation of human fibroblast collagenase by chloroacetyl N-hydroxypeptide derivatives. 166 36


<< Previous 1 2 3 4 5 6 7 8 9 10 Next >>

---