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)

Flavobacterium meningosepticum, Elder strain (ATCC 33958), secretes into the medium a neutral zinc endoprotease as a major component of the extracellular proteins. The enzyme was purified to homogeneity in a simple two-step procedure involving ammonium sulfate precipitation and hydrophobic interaction chromatography. The molecular weight of this metalloprotease was determined to be about 27,000 (P27) by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. P27 was comparable to thermolysin in the relative rates of elastin-orcein, azocasein, and azoalbumin hydrolysis. P27 and thermolysin hydrolyzed equally well 2,4-dinitrophenyl-Pro-Gln-Gly-Ile-Ala-Gly-Gln-D-Arg and 2,4-dinitrophenyl-Pro-Leu-Gly-Leu-Trp-Ala-D-Arg-NH2 at the same primary sites that are susceptible to cleavage by vertebrate collagenases, Gly-Ile, and Gly-Leu. P27 was also capable of partially hydrolyzing Type I acid-soluble calf skin collagen and slowly hydrolyzing N-[3-(2-furyl)acryloyl]-Leu-Gly-Pro-Ala, a bacterial collagenase substrate not cleaved by thermolysin. P27 was further differentiated from thermolysin from the inability of the former to hydrolyze N-[3-(2-furyl)acryloyl]-Gly-Leu-NH2. In addition, a vertebrate elastase substrate succinyl-Ala-Ala-Ala-p-nitroanilide was hydrolyzed by P27 but not by thermolysin. P27 is a newly described and unique enzyme from the standpoint of substrate specificity and from the fact that it is resistant to inhibition by phosphoramidon, an inhibitor of a number of zinc endopeptidases, including thermolysin.
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PMID:Purification and characterization of a neutral zinc endopeptidase secreted by Flavobacterium meningosepticum. 818 8

The protein fractions precipitated by ammonium sulfate from the bovine, human and Greenland's seal blood sera enhanced the pain sensitivity of mice, rats and rabbits. The proteins fraction of the seal blood serum was divided in six subfractions by ion-exchange chromatography. One of these subfractions clearly showed hyperalgesic properties, while the others had an opposite effect. The collagenase hydrolysate of the same protein fraction had an analgetic activity. The results of this and previous studies suggest the occurrence of one more nociception-regulating protein-peptide system in mammals.
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PMID:[The hyper- and hypoalgesic effects of human and animal blood serum proteins]. 822 80

For the collagenases PMNL-CL and FIB-CL, the presence of the N-terminal Phe79 correlates with an increase in proteolytic activity. We have determined the X-ray crystal structure of the recombinant Phe79-Gly242 catalytic domain of human neutrophil collagenase (PMNL-CL, MMP-8) using the recently solved model of the Met80-Gly242 form for phasing and subsequently refined it to a final crystallographic R-factor of 18.0% at 2.5 A resolution. The PMNL-CL catalytic domain is a spherical molecule with a flat active site cleft separating a smaller C-terminal subdomain from a bigger N-terminal domain, that harbours two zinc ions, namely a 'structural' and a 'catalytic' zinc, and two calcium ions. The N-terminal segment prior to Pro86, which is disordered in the Met80-Gly242 form, packs against a concave hydrophobic surface made by the C-terminal helix. The N-terminal Phe79 ammonium group makes a salt link with the side chain carboxylate group of the strictly conserved Asp232. Stabilization of the catalytic site might be conferred via strong hydrogen bonds made by the adjacent, likewise strictly conserved Asp233 with the characteristic 'Met-turn', which forms the base of the active site residues.
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PMID:Structural implications for the role of the N terminus in the 'superactivation' of collagenases. A crystallographic study. 830 85

We have developed a new assay method for collagenase activity, in the presence of two different types of collagenase inhibitors, in culture medium of periodontal ligament (PDL) cells. Human PDL cells were cultured with serum-free alpha-MEM for 5 days after reaching confluency, and the culture medium was then harvested. Collagenase and collagenase inhibitors in the medium were concentrated by ammonium sulfate precipitation. The precipitate was dissolved, and the suspension was applied to an Ultrogel AcA 54 column for partial purification. Collagenase inhibitor activity was detected as two peaks; a small inhibitor molecule of about 32 kDa sensitive only to dithiothreitol (DTT), and a large one of about 70 kDa considerably more sensitive to 4-aminophenylmercuric acetate (APMA) than to DTT. Therefore, pretreatment of the sample preparation with both DTT and APMA was necessary in order to inactivate the inhibitors prior to assay of latent collagenase. This observation indicates that PDL cells secrete collagenase and two collagenase inhibitors without any stimulation in vitro.
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PMID:Collagenase and collagenase inhibitors secreted by cultured human periodontal ligament cells. 841 Feb 4

The effects of various anions on decreasing the solubility of acidic Hypoderma lineatum collagenase at pH 7.2 and 18 degrees C were qualitatively defined by replacing the crystallizing agent of known crystallization conditions by various ammonium salts. The solubility curves measured in the presence of the sulfate, phosphate, citrate, and chloride ammonium salts gave the following ranking of anions: HPO4(2-)/H2PO4- > SO4(2-) > citrate 3-/citrate2- >> Cl-. This order is in agreement with the Hofmeister series. In a previous study on the solubility at pH 4.5 of lysozyme, a basic protein, the effectiveness of anions in decreasing the solubility was found to be in the reverse order. This suggests that the effectiveness of anions in the crystallization of proteins is dependent on the net charge of the protein, i.e., depending on whether a basic protein is crystallized at acidic pH or an acidic protein at basic pH.
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PMID:Relative effectiveness of various anions on the solubility of acidic Hypoderma lineatum collagenase at pH 7.2. 853 49

The colH gene encoding 116-kDa collagenase of Clostridium histolyticum (cColH) was cloned into an Escherichia coli-Bacillus subtilis shuttle vector to develop a method for purification of recombinant collagenase (rColH). When plasmid pJCM310 containing the colH gene was introduced into B. subtilis DB104 and the transformant was grown in LB broth at 37 C, stability of the plasmid was not maintained. However, stability was partly improved by growing the transformant in a modified LB broth containing 0.5 M sodium succinate with gentle shaking at 35 C. When the transformant was grown to an optical density of 0.4 at 600 nm in this medium, pJCM310 was stable and rColH was produced in sufficient amounts. rColH was purified to homogeneity by ammonium sulfate precipitation, gel filtration and ion-exchange chromatography. The yield of rColH from an 800-ml culture was 0.53 mg and its specific activity was estimated to be 1,210 U per mg of protein. The purified rColH was capable of degrading native type-I collagen fibril from bovine achilles tendon, as was demonstrated by zymography. A comparison of the N-terminal amino acid sequence between cColH and rColH revealed that rColH has 10 extra N-terminal amino acid residues. However, the peptide mapping of rColH with V8 protease was virtually identical to that of cColH. Furthermore, the molecular mass of rColH was estimated to be 112,999 Da by mass spectrometry, coinciding with the value of 112,977 Da, which was predicted from the nucleotide sequence of the colH gene. Therefore, the recombinant B. subtilis culture is capable of serving as a useful source for enzyme purification.
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PMID:Expression of the colH gene encoding Clostridium histolyticum collagenase in Bacillus subtilis and its application to enzyme purification. 901 90

Our previous studies have shown that the degree of damage to a liposome corresponds to the variability of the animal species from which the serum comes, and that a complement activating factor (CAF) plays an important role in inducing the activation of the complement system, ultimately leading to the lysis of the liposomes. In this study, our attention focused on the characterization of the bovine serum factor (bCAF) that is involved in complement-mediated immune lysis of the liposome. The active fraction containing CAF partially purified with PEG and ammonium sulfate results in marked activation of the complement system via the alternative pathway when interacted with CAF-depleted serum, whereas the active fraction or CAF-depleted serum alone does not activate the complement. The interaction between lipopolysaccharide (LPS), heparin, zymosan or their mixture in place of CAF and CAF-depleted serum does not result in any significant activation of the complement system. Results from pretreatment with rabbit anti-bovine IgM IgG and rabbit anti-bovine IgG IgG indicate that activation of the complement system is not attributable to the antibody which is generally involved in activation of complement via the classical pathway. The results have further been proven by pretreatment with Concanavalin A (Con A) sepharose and protein G sepharose ruling out the possibility of antibody-mediated activation of complement. Our studies on collagenase and trypsin digestion demonstrate that the relative activity of CAF does not diminish with increase in collagenase concentration, and decreases with increase in trypsin concentration, strongly indicating that CAF does not have a collagen-like domain in its structure. The relative activity of CAF is dramatically inhibited after reduction with 2-mercaptoethanol (2-ME), clearly demonstrating that CAF is sensitive to reduction with 2-ME and confirming a sulhydryl-dependent protein. The optimal activity of CAF is observed in the range of 35-45 degrees C and its half-life at 37 degrees C is about 105 h. Furthermore, the relative activity of CAF increases and gradually approaches a plateau level with the increase of Mg2+ concentration. Obviously, complement activation induced by CAF depends on adequate Mg2+ concentration, confirming that this dependence is characteristic of the alternative pathway.
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PMID:Characterization of bovine serum factor triggering the lysis of liposomes via complement activation. 958 79

The pancreatic regenerating gene (reg I) is expressed in the exocrine pancreas and is involved in islet regeneration. Reg I protein has been shown to be mitogenic to beta- and ductal cell lines, but not mature islets. In this study, we tested the effect of two isolates of reg I on primary cultures of ductal cells. Rat pancreatic ductal cells were isolated by collagenase digestion and isolated colonies were maintained in culture. The cells were proven to be ductal in origin by their morphology and by immunofluorescent staining with epithelial markers. Reg I was isolated from human pancreatic extracts or from the rat acinar cell line AR42J by sequential ammonium sulfate precipitation and acid precipitation. Cells were cultured with doses of reg I for 72 h, pulsed with 10 microM bromodeoxyuridine (BrdU) for 2 h. After fixation, nuclei were double-stained with propidium iodide and BrdU monoclonal antibody. The percentages of nuclei positive for BrdU were calculated from at least five colonies per group. A 10-nM concentration of human reg I increased BrdU incorporation by 2.3-fold over controls, rat reg I increased it by 1.4-fold (p < 0.05). When compared to their effects on the ductal cell line ARIP, both human and rat reg I were 100 times more potent on the primary cultures of ductal cells. We conclude that human and rat reg I proteins are mitogenic to primary cultures of ductal cells. Although principally a product of the acinar cell, reg I appears to be a stimulus of ductal cell growth and, in this fashion, may modulate the expansion of the pancreatic ductal population during islet regeneration.
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PMID:Effect of reg protein on rat pancreatic ductal cells. 978 38

A cellular suspension from rat submandibular glands was prepared with collagenase. The intracellular pH (pHi) was estimated with 2',7'-bis-(2-carboxy-ethyl)-5(6)-carboxyfluorescein (BCECF). After exposure to NH4Cl, the pHi transiently increased (diffusion of NH3) and then dropped (influx of NH4+). Isoproterenol increased 2.5-fold the rate of NH4+ influx; bumetanide, an inhibitor of the Na+-K+-2Cl(-)-cotransporter blocked the response to isoproterenol, confirming that the beta-adrenergic agonist stimulated the cotransporter. Forskolin (1 micromol/L) mimicked the response to isoproterenol. VIP (1 nmol/L(-1) micromol/L) also increased the activity of the cotransporter. Cyclic AMP rather than calcium was the mediator of this activation since 1) carbachol which increased the [Ca2+]i fivefold increased the uptake of NH4+ by only 50%; 2) only high concentrations of VIP significantly increased the [Ca2+]i; 3) incubation in the presence of EGTA had no effect on the response to VIP; 4) low concentrations (nmol/L) of the neuropeptide increased the intracellular level of cAMP; and 5) the stimulation of the cotransporter by VIP, forskolin, and isoproterenol was inhibited by H8, an inhibitor of cAMP-dependent protein kinase. It is concluded that the Na+-K+-2Cl(-)-cotransporter of rat submandibular glands is activated by isoproterenol, forskolin, and neuropeptides of the VIP family by a mechanism involving cAMP-dependent processes. The activation of the cotransporter by VIP could partly explain the potentiating effect of VIP on the response to sialagogues like substance P or muscarinic agonists.
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PMID:Activation of the Na+-K+(NH4+)-2Cl(-)- cotransporter from rat submandibular glands in response to VIP. 988 83

Calbindin D28k has been reported to be involved in the transcellular calcium transport along the rat distal tubule. It has also been shown that chronic metabolic acidosis (CMA) induces significant hypercalciuria. The present study investigated whether CMA affects the mRNA and the protein expression of calbindin D28k along isolated distal tubule (DT) of rats. The animals were made acidotic by adding 0.28 mol/L NH4Cl to the drinking water for 7 d. This maneuver was associated with an increase in plasma ionized calcium. Inulin clearance experiments demonstrated that metabolic acidosis did not affect GFR, but it significantly increased both total and fractional urinary calcium excretion. To define the role of calbindin D28k, total RNA was extracted from DT, identified, and microdissected from collagenase-treated kidneys. cDNA was synthesized from RNA using reverse transcriptase and oligo(dT)(12-18) primers. Calbindin D28k mRNA abundance was semiquantified by a competitive reverse transcription-PCR, using an internal standard of cDNA that differed from the wild-type calbindin D28k by a deletion of 86 bp. The reverse transcription-PCR was performed starting from the same amount of total RNA. For each set of experiments, control and acidotic rats were studied in parallel. The identity of the DT was further verified by the presence of the thiazide-sensitive NaCl cotransporter (rTSC1) mRNA. Calbindin D28k mRNA abundance was 0.89 +/- 0.21 amol/ng total RNA in DT of CMA rats (n = 5) compared with 0.30 +/- 0.12 amol/ng total RNA of control rats (n = 5) (P < 0.05). Using specific rabbit polyclonal anti-calbindin D28k antibody, Western blotting was performed starting from thin slices of outer cortex. Densitometric analysis revealed that in acidotic rats (n = 7) there was a 17 +/- 5% (P < 0.05) increase in calbindin D28k protein abundance compared with controls (n = 7). These results indicate that in the rat, ammonium chloride loading induces an increase in filtered ionized calcium load that is associated with a significant upregulation of calbindin D28k both at the mRNA and protein level. These last effects will help to reduce the concomitant hypercalciuria, thus mitigating the consequence of CMA on calcium metabolism.
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PMID:Effect of chronic metabolic acidosis on calbindin expression along the rat distal tubule. 1066 27


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