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)

Studies were undertaken to define the molecular size of the elastin primary gene product. Translation of chick aortic messenger ribonucleic acid (mRNA) in an mRNA-dependent reticulocyte lysate resulted in the synthesis of two major proteins of 70 000 and 73 000 molecular weights. Both proteins were shown to be soluble forms of elastin by isotope incorporation, immunoprecipitation, collagenase and cyanogen bromide sensitivity, and two-dimensional gel electrophoresis. The 70 000-dalton protein behaves similarly to authentic tropoeleastin in sodium dodecyl sulfate gel electrophoresis. There was no evidence for a high molecular weight form of soluble elastin, although procollagen chains were indirectly identified among the aortic mRNA-directed translation products. The same molecular size proteins were also seen in organ cultures of chick embryonic aortas labeled with [3H]valine. However, the 73 000-dalton protein was not extractable in a neutral salt buffer but was found only if the aortas were extracted with urea in the presence of reducing and alkylating reagents. The results from these studies suggest that elastin is first synthesized as two distinct polypeptide chains which differ slightly in size and overall charge. The possibility that these two proteins may associate posttranslationally to form a dimer prior to secretion is postulated to explain the existence of a putative proelastin molecule seen in other systems.
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PMID:Translation of chick aortic elastin messenger ribonucleic acid. Comparison to elastin synthesis in chick aorta organ culture. 735 64

The metacestodes of Taenia pisiformis have been shown to contain a protease inhibitor capable of inactivating the esterolysis of N-alpha-benzoyl-L-arginine ethyl ester (BAEE) and N-benzoyl-L-tyrosine ethyl ester (BTEE) by trypsin and chymotrypsin, respectively, of bovine, dog and rabbit origin, but not affecting the hydrolytic activity of subtilisin, elastase, collagenase, pepsin, rennin and papain. This inhibitor has been demonstrated in whole worm extracts and in the incubation medium of in vitro-maintained, intact living metacestodes. The protease inhibitor which was purified by trichloroacetic acid precipitation, Sephadex G-100 chromatography and affinity chromatography on CNBr-activated Sepharose 4B-bovine, chymotrypsin conjugate was soluble in 5% trichloroacetic acid, withstood heat up to 80 degrees C, tolerated the pH range 1.5 to 9.0, was unaffected by 8 M urea or 0.2 M 2-mercaptoethanol and had a molecular weight of about 7000 to 7200, as calculated from its gel chromatographic behaviour. Complex formation between the inhibitor and the enzymes required 3--4 min for completion. The enzyme-inhibitor complex was not dissociated by 4 M KCl. Activity determinations on bovine TPCK-trypsin and bovine chymotrypsin with BAEE and BTEE assays revealed that the inhibitory actions toward both enzymes are functions of the same or closely adjacent sites of the inhibitor molecule. The supposed function of the inhibitor is discussed.
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PMID:A trypsin and chymotrypsin inhibitor from the metacestodes of Taenia pisiformis. 739 18

The effect of ethanol on the secretion of proteins was studied in hepatocytes isolated from 24-h fasted rats and from fed rats. Hepatocytes were isolated after collagenase disruption of the liver and incubated in a standard medium containing amino acids, bovine albumin, glucose, penicillin and streptomycin in HEPES buffer. Cell viability was determined by urea production and trypan blue exclusion. When studying protein export, a model had to be chosen in which the labeling is accomplished before the addition of the test agents. Cells were incubated with [3H]valine for 2.5 and 7.5 min followed by a 15-mM valine chase and the incubates were adjusted to final concentrations of ethanol of 50 mM, 100 mM, colchicine 5-50 microM or cycloheximide 18 microM. Cells and media were harvested at various times, and counts incorporated into medium and cell protein were determined. Cycloheximide inhibited protein synthesis by 99%, decreased protein secretion by 10-20%, but did not further inihibit protein labeling when given after the chase confirming the chase's effectiveness. Colchicine inhibited protein release by 27-54% depending on the dose. With control cells labeled protein and specifically albumin appeared in the medium 20 min from the start of the pulse and this release of protein was not inhibited by 50 mM or 100 mM ethanol incubated with cells from the same animal whether the donor has been fed or fasted. The values for the ethanol-treated cells ranged from 94.0 to 113% of the control values from 30 to 120 min after the addition of the pulse. Lactate levels were markedly elevated, and urea synthesis decreased in the presence of either 50 mM EtOH or 100 mM EtOH. Thus using a method that can distinguish the effect of ethanol on synthesis from secretion, it is concluded that acute exposure to EtOH does not interfere with protein secretion.
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PMID:Protein secretion in suspensions of isolated rat hepatocytes: no influence of acute ethanol administration. 745 Apr 1

We reported that specific biological activities are confined to three domains of the fibronectin (Fn) molecule [Fukai et al. (1991) J. Biol. Chem. 266, 8807; Fukai et al. (1993) Biochemistry 32, 5746]: the potent ability to stimulate the adipocyte differentiation of ST-13 cells is in the amino-terminal fibrin-binding (Fib 1) domain (referred to as Fib 1 domain activity); the RGD-dependent activities that stimulate NIH-L13 cell migration and inhibit adipocyte differentiation are in the central cell-binding (Cell) domain (Cell domain activity); and the activity that stimulates cell migration in a RGD-independent manner is in the carboxyl-terminal fibrin-binding (Fib 2) domain (Fib 2 domain activity). Human plasma Fn which was purified without exposure to a denaturant, such as urea, exhibited no Fib 1, Fib 2, or Cell domain activity. By exposure to urea or surface adsorption, Fn showed Cell domain activity but not those of the Fib 1 and Fib 2 domains. Whether the cryptic domain activities are disclosed or not depended on whether or not the responsible domains were irreversibly exposed from confined environments of Fn structure as confirmed by light-scattering measurement and enzyme immunoassay using domain-specific monoclonal antibodies. We then investigated the action of matrix metalloproteinases (MMPs) in liberating the Fib 1, Cell, and Fib 2 domain activities. Matrilysin released only the Cell domain activity. In contrast, stromelysin, collagenase, and especially gelatinase A additionally liberated the Fib 1 and Fib 2 domain activities. The Fib 1, Fib 2, and Cell domains acquired much higher activities when they were freed from linkage with adjacent domains.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Release of biological activities from quiescent fibronectin by a conformational change and limited proteolysis by matrix metalloproteinases. 754 73

The authors report a study in which they evaluate the efficacy of some laboratory parameters for monitoring intrasplenic hepatocyte xenotransplantation (mouse to rat) as an alternative to 99Tc-HIDA dynamic scan and histologic exam. Swiss mouse and wistar rat hepatocytes were obtained with collagenase digestion. Wistar male rats were used as recipient and were allocated into three groups: A) omotransplanted rats; B) xenotransplanted rats; C) xenotransplanted and immunosuppressed (Cyclosporin A: 20 mg/kg/daily orally) rats. All rats underwent > 70% hepatectomy. Blood samples were obtained daily from a femoral vein and AST, ALT, ALP, bilirubin, albumin and urea were measured. No statistical differences were observed among groups and the laboratory parameters tested can't be considered a valid technique to xenotransplant rejection monitoring.
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PMID:[Monitoring of hepatocyte xenotransplantation. Usefulness of various laboratory parameters]. 761 63

Type VI collagen beaded microfibrils were extracted from bovine cornea or pig cartilage by limited collagenase digestion. Depolymerization of the microfibril, without strong denaturing reagents linke guanidinium hydrochloride or urea under mild acidic conditions, led to single tetramers and multiples of two to three. However, hyaluronidase digestion in accordance with a published method (Kielty et al. J. Cell Biol. 118:979-990, 1992) was unsuccessful in depolymerizing type VI collagen microfibrils. Also, repolymerization into microfibrils by incubation with hyaluronan was not observed. We further found no binding of native type VI collagen microfibrils to a hyaluronan-Sepharose column. Although a recombinant fragment comprising alpha 3(VI) domains N9-N2 showed apparent binding to the column, electron microscopy did not give any indication of binding of either type VI collagen or fragment N9-N2 to hyaluronan. The present findings suggest that the role of hyaluronan in polymerization of type VI collagen has been overestimated in previous work.
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PMID:Type VI collagen beaded microfibrils from bovine cornea depolymerize at acidic pH, and depolymerization and polymerization are not influenced by hyaluronan. 779 88

In addition to the known 94-kd gelatinase (matrix metalloproteinase 9, MMP-9), HL-60 leukemia cells release a hither-to undescribed 45-kd metalloproteinase into the culture medium. This enzyme cleaves the synthetic substrate Pro-Gln-Gly-Ile-Ala-Gly-Gln-Arg, which represents the cleavage site for collagenases in collagen type I not between isoleucine and alanine--the typical cleavage site for collagenases--but between alanine and glycine. The enzymatic activity was purified through a combination of zinc-chelate-Sepharose column chromatography, precipitation with Fractogel TSK-AF Red and gelatin-Sepharose, and subsequent sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Microsequence analysis of the NH2-terminus of the purified 45-kd proteinase revealed the sequence Asp-Ile-Ser-Lys-Tyr-Thr-Thr-Thr-, which could not be found in other proteins when searched in several protein data bases. Incubation of the enzyme immobilized on nitrocellulose membranes with polyclonal antibodies to collagenase and stromelysin or gelatinases revealed no cross-reactivity. The proteolytic activity was not increased by treatment with trypsin, 8M urea, acid, or organomercurials. The proteinase, which was inhibited by chemical inhibitors of metalloproteinases, such as phenanthrolene or EDTA, is able to degrade several matrix constituents, such as collagen type IV, fibronectin, gelatin, and proteoglycans. In contrast to all known MMPs, the proteolytic activity of the 45-kd enzyme was not abolished upon incubation with recombinant tissue inhibitors of matrix metalloproteinases (TIMP) 1 or 2. Thus, the novel enzyme may influence extracellular matrix (ECM) turnover in vivo because its activity is not influenced by specific inhibitors of MMPs.
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PMID:Leukemic cells (HL-60) produce a novel extracellular matrix-degrading proteinase that is not inhibited by tissue inhibitors of matrix metalloproteinases (TIMPs). 782 72

The cDNA coding for human big endothelin 1 (bigET-1), preceded by an optimized collagenase recognition sequence and followed by a stop codon, was fused in frame to the C-terminal region of alkaline phosphatase (AP). The fusion protein (AP-bigET), expressed in Escherichia coli K12 upon the lowering of organic phosphate concentrations, consisted of alkaline phosphatase (1-447), the collagenase cleavage site (Gly-Pro-Ala)4, and glycylprolyl-bigET-1. AP-bigET accumulated intracellularly in the form of inclusion bodies that were extensively washed and finally extracted by 8 M urea to yield highly enriched AP-bigET. Upon digestion of the fusion protein with collagenase, two disulfide conformeres of glycylprolyl-bigET-1 (bigET-1A and bigET-1B) could be purified by reverse-phase FPLC. Upon treatment with dipeptidylpeptidase IV to remove the N-terminal glycylprolyl-dipeptide, the later-eluting form of bigET-1 (bigET-1B) coeluted with authentic human bigET-1 on reverse-phase HPLC. BigET-1A and bigET-1B were formed at a ratio of 1:3. After reduction and S-pyridylethylation, both conformers coeluted with authentic but reduced bigET-1. Their amino acid sequences were identical. Both forms were converted by digestion with pepsin to the respective ET-1 conformeres (ET-1A and ET-1B) that were purified. In vasoconstriction assays, ET-1B but not ET-1A, at 10(-8) M, evoked a maximal response indistinguishable from that of authentic ET-1.
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PMID:Purification of human big endothelin 1 derived through cleavage with collagenase and dipeptidylpeptidase IV from a fusion protein expressed in Escherichia coli. 790 63

Bone proteins in alveolar bone of mandibles from young adult rabbits (3-month-old) were extracted with 4.0 M guanidine hydrochloride (GuHCl), followed by 0.5 M ethylenediaminetetraacetate, and again with 4.0 M GuHCl (G2-ext). The proteins in the G2-ext were fractionated on a gel-filtration column, followed by an anion-exchange column in the presence of 7.0 M urea. A 28-kDa protein was isolated from the G2-ext. The purified 28-kDa protein showed intense staining with silver on SDS-PAGE slab-gel under reducing conditions. This protein was digested with bacterial collagenase, and a 19-kDa fragment appeared on the gel. However, the protein was not susceptible to reduction with cyanogen bromide. The protein did not bind to hydroxyapatite crystals in the presence of 7.0 M urea, and also did not bind to some lectins. On SDS-PAGE under non-reducing conditions, the protein migrated as two bands; a new band appeared at approximately the 85-kDa region in addition to the original 28-kDa band. The amino acid compositions of the protein were similar to those of the alpha 1-pN-propeptide of type I procollagen obtained from other tissues.
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PMID:Characteristics of a 28-kDa collagenous protein extracted with guanidine from EDTA-demineralized rabbit alveolar bone. 815 87

To use cultured human hepatocytes as a hybrid artificial liver, effective methods for isolating and culturing the hepatocytes from resected surgical specimens were investigated. Two different procedures for isolating hepatocytes, perfusion and agitation with a collagenase solution (Method 1) and perfusion with a mixed solution of collagenase and dispase (Method 2), were examined. The yield of isolated hepatocytes obtained by Method 2 (13.31 x 10(6) cells/g of liver) was significantly higher than that by Method 1 (0.94 x 10(6)). The warm ischemia time (0-90 min) of the liver fragments obtained did not disturb the viability and yield of the isolated hepatocytes. The gluconeogenesis and urea synthesis of the cultured human hepatocytes were well preserved for 10 days. These results show that for prolonged human hepatocyte culture (10 days), isolation from resected human liver tissues by a combination of the proteolytic enzymes collagenase and dispase was effective and warm ischemia was tolerated for up to 90 min, which indicates the possibility of using cultured human hepatocytes as a hybrid artificial liver.
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PMID:Isolation and culture of human hepatocytes from resected liver tissue as a bioreactor for a hybrid artificial liver. 833 42


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