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

A recombinant 19-kDa human fibroblast collagenase catalytic fragment modeled on a naturally occurring proteolytic product was purified from E. coli inclusion bodies. Following renaturation in the presence of zinc and calcium, the fragment demonstrated catalytic activity with the same primary sequence specificity against small synthetic substrates as the full-length collagenase. Unlike the parent enzyme, it rapidly cleaved casein and gelatin but not native type I collagen. Intrinsic fluorescence of the three tryptophan residues was used to monitor the conformational state of the enzyme, which underwent a 24-nm red shift in emission upon denaturation accompanied by quenching of the fluorescence and loss of catalytic activity. Low concentrations of denaturant unfolded the fragment while the full-length enzyme displayed a shallow extended denaturation curve. Calcium remarkably stabilized the 19-kDa fragment, zinc less so, while together they were synergistically stabilizing. Among divalent cations, calcium was the most effective stabilizer, EC50 approximately 60 microM, and similar amounts were required for substrate hydrolysis. Catalytic activity was more sensitive to denaturation than was tryptophan fluorescence. Least sensitive was the polypeptide backbone secondary structure assessed by CD. These observations suggest that the folding of the 19-kDa collagenase fragment is a multistep process stabilized by calcium.
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PMID:Metal ion stabilization of the conformation of a recombinant 19-kDa catalytic fragment of human fibroblast collagenase. 131 76

Canine jejunal epithelial cells were isolated and maintained in short-term culture to study cholecystokinin (CCK) release. Sequential digestion of jejunal mucosa with collagenase and ethylenediaminetetraacetic acid was followed by counterflow elutriation to enrich CCK-containing cells. After 40 hours in culture on collagen-coated plates, 8.4% of the initially seeded cells were attached; 8.7% of them stained positive with a C-terminal CCK/gastrin antibody and 2.5% stained positive with a gastrin-specific antibody. Basal release of CCK into the culture medium amounted to 1.3% of total cell content over 105 minutes. Receptor-independent stimulation of protein kinase C by the phorbol ester beta-phorbol-12-myristate-13-acetate caused significant CCK release. The inactive form, 4 alpha-phorbol-12-myristate-13-acetate, had no effect. Activation of adenylate cyclase by 10(-5) mol/L forskolin evoked a 2.5-fold increase in CCK concentrations, which was completely abolished by 10(-8) mol/L somatostatin. L-phenylalanine stimulated CCK release at 20 and 50 mmol/L, whereas D-phenylalanine caused significant hormone output only at 50 mmol/L. L-tryptophan had no effect. Cholecystokinin release stimulated by L-phenylalanine was not influenced by the addition of either somatostatin or somatostatin antibody. In conclusion, a system of isolated canine jejunal epithelial cells was developed in short-term culture. This preparation proved suitable for the study of CCK release on a cellular basis.
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PMID:Cholecystokinin release from isolated canine epithelial cells in short-term culture. 172 60

The sequence specificities of human fibroblast and neutrophil collagenases have been investigated by measuring the rate of hydrolysis of 60 synthetic oligopeptides covering the P4 through P'5 subsites of the substrate. The choice of peptides was patterned after both known cleavage sites in noncollagenous proteins and potential cleavage sites (those containing Gly-Ile-Ala, Gly-Leu-Ala, or Gly-Ile-Leu sequences) found in types I, II, III, and IV collagens. The initial rate of hydrolysis of the P1-P'1 bond of each peptide has been measured under first-order conditions ([SO] much less than KM), and kcat/KM values have been calculated from the initial rates. The amino acids in subsites P4 through P'4 all influence the hydrolysis rates for both collagenases. However, the effects of substitutions at each site are distinctive and are consistent with the view that human fibroblast and neutrophil collagenases are homologous but nonidentical enzymes. For peptides with unblocked NH2 and COOH termini, occupancy of subsites P3 through P'3 is necessary for rapid hydrolysis. Compared with the alpha 1(I) cleavage sequence, none of the substitutions investigated at subsites P3, P2, and P'4 produces markedly improved substrates. In contrast, many substitutions at subsites P1, P'1, and P'2 improve specificity. The preferences of both collagenases for alanine in subsite P1 and tryptophan or phenylalanine in subsite P'2, is noteworthy. Human neutrophil collagenase accommodates aromatic residues in subsite P'1 much better than human fibroblast collagenase. The subsite preferences observed for human fibroblast collagenase in these studies agree well with the residues found at cleavage sites in noncollagenous substrates. However, the sequence specificities of these collagenases cannot explain the failure of these enzymes to hydrolyze many potentially cleavable but apparently protected sites in intact collagens. This represents additional support for the notion that the local structure of collagen is important in determining the location of collagenase cleavage sites.
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PMID:Sequence specificities of human fibroblast and neutrophil collagenases. 184 91

The poor growth associated with protein-calorie malnutrition occurs despite circulating growth hormone levels that are normal or elevated and is thought to be mediated partly by blunted generation of insulinlike growth factor I (IGF-I) in the liver. To explore underlying mechanisms, we asked whether altered availability of amino acids could regulate hepatic IGF-I release independent of the contributions of regulatory hormones. Normal rat hepatocytes were isolated by collagenase digestion and maintained in serum-free medium with fixed concentrations of insulin and dexamethasone. Levels of immunoassayable albumin and IGF-I accumulation in daily changes of medium were sustained for 3-5 days, and all studies were performed within this period. Cellular viability and content of DNA were unaffected by deprivation of the essential amino acids lysine or tryptophan and the nonessential amino acids cysteine and/or cystine. However, deletion of tryptophan or lysine from the culture medium led to 63 and 76% declines in IGF-I release, respectively (both P less than 0.001 vs. complete medium), although omission of cysteine or cysteine plus cystine produced no significant change. Over 5 days of culture, release of albumin was maintained in complete medium, but omission of tryptophan depressed albumin release over days 2-5 (P less than 0.001). In complete medium, IGF-I release rose for 3 days and then declined. In tryptophan-deficient medium, IGF-I levels were comparable to control values after 24 h but did not rise at 48 h and then fell rapidly after 72 h in culture, with values significantly below levels in complete medium (all P less than 0.005).(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Nutrition and somatomedin. XXIII. Molecular regulation of IGF-I by amino acid availability in cultured hepatocytes. 190 9

An extensive series of N-(monoethylphosphoryl)peptides was synthesized and their inhibition of purified human skin fibroblast collagenase examined. At the cleavage site S1 all reported compounds have the (EtO)(OK)P(O) group and the peptide side chain extended toward the C-terminal end (up to P5') of the substrate sequence. These phosphoramidates with a tetrahedrally hybridized phosphorus atom are thought to be transition state analogue inhibitors. They exhibited fair inhibitory potency against this vertebrate collagenase having Ki values in the micromolar range. The most potent of these, (EtO)(OK)P(O)-Ile-TrpNHCH3 (68), inhibits with a Ki value of 1.5 microM and is nearly 100 times stronger than (EtO)(OK)P(O)-Ile-Ala-GlyOK (51) (Ki of 140 microM), which has the sequence matching that of the alpha 1 (I) chain of collagen in P1', P2', P3' after the cleavage site. Several compounds were prepared in an attempt to identify the nature of the S2', S3', and S4' binding sites. Alanine at the P2' position was replaced by leucine, phenylalanine, tryptophan, or tyrosine derivatives, resulting in Ki values in a significantly lower range, 1.0-40 microM, compared to 51. No upper size limitation or specificity has been found at this position, yet similar replacements at the P3' position, which is occupied naturally by a glycine residue, gave weaker inhibitors: (EtO)(OK)P(O)-Ile-Tyr(OBzl)-PheOK (57) had a Ki of 120 microM. Hexapeptide derivatives had weaker activities in the 270 microM-2 mM range. All inhibitors were evaluated by using the synthetic thio peptolide spectrophotometric assay.
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PMID:Phosphoramidate peptide inhibitors of human skin fibroblast collagenase. 215 7

Isolated adrenocortical cells from White Leghorn chickens (Gallus domesticus) were compared to those from rats (Rattus norvegicus). Cells were prepared from collagenase-dispersed adrenal glands of sexually mature male animals. Corticosterone was measured by radioimmunoassay after incubation for 2 h with steroidogenic agents. Of the four ACTH analogues used, three were 6-17 times more potent with rat cells than with fowl cells (potencies were indicated by half-maximal steroidogenic concentrations). However, 9-tryptophan (O-nitrophenylsulfenyl) ACTH was 8 times more potent with fowl cells than with rat cells, thus suggesting that ACTH receptor differences exist between the two cell types. In addition, cAMP analogues were 10 times more potent with rat cells than with fowl cells suggesting that fowl corticosteroidogenesis is less dependent on cAMP than is rat corticosteroidogenesis. At equal cell concentrations, rat cells secreted 20-40 times more corticosterone than did chicken cells when they were maximally stimulated. Although rat cells converted 8 times more pregnenolone to corticosterone than did fowl cells, the half-maximal steroidogenic concentration for pregnenolone-supported corticosterone synthesis was the same for both cell types (about 5 microM). This suggests that fowl cells have lower steroidogenic enzyme content rather than lower steroidogenic enzyme activity. An unusual feature seen in the isolated fowl adrenocortical cells was an abundance of intracellular filaments.
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PMID:Isolated adrenocortical cells of the domestic fowl (Gallus domesticus): steroidogenic and ultrastructural properties. 298 70

To determine the nature of the pancreatic islet cell cholecystokinin (CCK) receptor, we studied CCK receptor binding and biologic activity in isolated rat pancreatic islets. Binding of 70 pM 125I-CCK to collagenase-prepared isolated rat pancreatic islets at 24 degrees C was one-half maximal after 5 min and maximal at 60 min. At 60 min, specific binding was 12% of total radioactivity per 100 micrograms islet protein; nonspecific binding (in the presence of 1 microM CCK 8) was less than 2% of total radioactivity. Unlabeled CCK 33 inhibited labeled hormone binding one-half maximally at 2 nM; Scatchard analysis showed one binding site (Kd, 2.3 +/- 0.4 nM; Bmax, 8.1 pmol/mg protein). The agonist selectivity of this binding site was: CCK 8 = CCK 33 greater than desulfated-CCK 8 greater than CCK 4. Two CCK antagonists were studied; N-carbobenzoxy-L-tryptophan was more potent than dibutyryl-cGMP. When the effect of CCK on insulin release from the islets was studied, the order of potency of CCK agonists and antagonists on insulin secretion was the same as the order of their ability to inhibit 125I-CCK binding. The effect of CCK on insulin secretion was dependent on the glucose concentration in the media. CCK had no effect at 5.6 mM glucose and was fully effective at 11.0 mM glucose. These data, therefore, indicate that: specific binding sites for CCK are present in rat pancreatic beta cells; and CCK acts in concert with glucose to stimulate insulin secretion.
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PMID:Evidence that cholecystokinin interacts with specific receptors and regulates insulin release in isolated rat islets of Langerhans. 300 Aug 56

We examined whether the generation of reactive oxygen metabolites (as quantified by measuring luminol-amplified chemiluminescence) by isolated rat glomeruli could be triggered enzymatically. No response was observed with thrombin (1 or 10 U/ml), collagenase (100, 200, or 400 U/ml), or plasmin (0.1 or 1 U/ml). In contrast, chymotrypsin and trypsin caused a dose-dependent (10-200 micrograms/ml) increase in chemiluminescence from glomeruli. The peak response with chymotrypsin (100 micrograms/ml) and trypsin (50 micrograms/ml) was as follows: resting, 16 +/- 2 X 10(3) cpm/mg protein, n = 17; chymotrypsin, 233 +/- 58 X 10(3) cpm/mg protein, n = 17; and trypsin, 221 +/- 38 X 10(3) cpm/mg protein, n = 10. Tubules had only a minor response. Soybean trypsin inhibitor and aprotinin caused marked inhibition, indicating the dependency of the chemiluminescence response on the protease enzyme activity. The chemiluminescence response was by glomeruli rather than by "contaminating" leukocytes, since a similar marked response (n = 6) was observed in glomeruli isolated from cyclophosphamide-treated leukopenic (leukocyte less than 1,000/mm3) rats. Superoxide dismutase, a scavenger of superoxide, and free-radical scavengers benzoate and tryptophan inhibited the glomerular chemiluminescence response to trypsin and chymotrypsin. Neutral proteases from infiltrating leukocytes and/or renal tissue have been shown to be released in glomerular diseases; our results, which show the generation of chemiluminescence in response to neutral proteases, suggest a potential mechanism for the production of reactive oxygen metabolites in glomerular diseases.
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PMID:Trypsin- and chymotrypsin-induced chemiluminescence by isolated rat glomeruli. 359 31

A collagenase secreted by tadpole (Rana catesbiana) back-skin explants in culture has been purified to electrophoretic homogeneity by successive chromatography on sulfopropyl Sephadex, Sephacryl S-200, collagen Sepharose, and heparin Sepharose. The purified enzyme has a molecular weight of approximately 49,000 and an isoelectric pH of 5.0. The enzyme is more active versus soluble collagen than reconstituted fibrils and exhibits very low activity against gelatin (specific activities: Type I collagen, 7660 units/mg; Type I gelatin, 66 units/mg). The collagenase obeys simple Michaelis-Menten kinetics using soluble type I collagen (Km), 0.35 microM; Vm, 1380 units/mg, at 25 degrees C and pH 7.4) and is inhibitable by chelating agents specific for transition metals. Methylene blue catalyzes the photoinactivation of this collagenase, suggesting the presence of essential histidine, tryptophan, tyrosine, or methionine residues.
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PMID:Purification and characterization of tadpole back-skin collagenase with low gelatinase activity. 609 65

A study of the influence of chemical modifications on the activity of Achromobacter iophagus collagenase (EC 3.4.24.8) has led to the following conclusions: a modification of 4 out of 80 COOH groups with carbodiimide led to 90% loss of enzymic activity. A 70% inactivation was found after modification of two tyrosines out of 30 with tetranitromethane. The modification of four to six tryptophans out of 16 with 2-hydroxy-5-nitrobenzyl bromide decreased enzyme activity to 36%. This inactivation is accelerated in the presence of collagen. An increase of reagent/enzyme molar ratio led to a modification of 16 tryptophan residues and denaturation of Acahromobacter collagenase. A modification of two arginines out of 18 with 1,2-cyclohexanedione and eight NH2 groups out of 24 with 2,3-dimethyl maleic anhydride does not change the collagenolytic activity. All NH2 groups become available for 2,3-dimethyl maleic anhydride after dissociation of the dimer. A possible analogy of hydrolytic site of collagenase with that of two other known bacterial metalloproteinases (thermolysin and Bacillus subtilis neutral proteinase (EC 3.4.24.4)) is discussed.
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PMID:Chemical modifications of Achromobacter collagenase and their influence on the enzymic activity. 625 92


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