Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts:   Target Concepts:
Query: EC:3.4.24.3 (collagenase)
 18,340 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Cells were isolated from the major arteries of 17-day chick embryos by digestion of the tissue with collagenase and trypsin. The cells, when examined immediately after isolation, exhibited a high degree of viability and they were shown to synthesize and secrete procollagen at a high and constant rate for several hours when incubated in suspension in modified Krebs medium. Continuous labelling of the cells with [(14)C]proline demonstrated a lag of about 30min between the time at which the synthesis of non-diffusible peptide-bound hydroxy[(14)C]proline became linear and the time at which its secretion into the medium became linear. This lag time compares with that of 18min observed for freshly isolated matrix-free cells from embryonic-chick tendon, which synthesize and secrete the same type of collagen. Gel-filtration chromatography and polyacrylamide-gel electrophoresis indicated that the collagenous polypeptides secreted into the medium were in the precursor form, known as procollagen, and that the constituent pro-alpha-chains were linked by interchain disulphide bonds and were also in a triple-helical conformation. Characterization of the secreted procollagen by gel-filtration chromatography, polyacrylamide-gel electrophoresis, DEAE-agarose chromatography, and polyacrylamide-gel electrophoresis of peptides obtained by CNBr cleavage, indicated that the predominant form was type-I procollagen. This work extends the range of freshly isolated matrix-free cell systems, which have been characterized for use in studies on the biosynthesis and secretion of procollagen, and it indicates differences in the rates of secretion of procollagen in different cell types secreting the same type of procollagen.
 ...
PMID:Synthesis of procollagen by matrix-free cells from embryonic-chick arteries. 59 39

Type I procollagen secreted by matrix-free chick embryo tendon cells was labeled with L-[3,3'-3H] cystine and purified by DEAE-cellulose chromatography. After bacterial collagenase digestion, the NH2- and COOH-terminal propeptides were partially characterized by ion exchange chromatography and gel filtration. Similar experiments were then conducted after labeling with either D-[6-3H] glucosamine, D-[2-3H] mannose, or D-[U-14C] glucose. On the basis of these studies and subsequent carbohydrate analysis, it was concluded that the COOH-terminal peptide contained greater than 90% of the radioactive carbohydrate which consisted predominantly of glucosamine and mannose with traces of galactosamine and galactose. Only radioactive glucosamine could be detected in the NH2-terminal propeptide. Under conditions which inhibit hydroxylation of lysine and glycosylation of hydroxylysine, unhydroxylated procollagen (protocollagen) could still be labeled with [3H] glucosamine and [3H] mannose. This suggested that glycosylation of the propeptides is at least initiated at the level of the rough endoplasmic reticulum.
 ...
PMID:Localization and partial composition of the oligosaccharide units on the propeptide extensions of type I procollagen. 61 65

Collagen synthesis was studied in monolayer cultures of rabbit corneal endothelial cells by following [14C]proline and [3H]glucosamine or [3H]fucose incorporation into a fraction enriched for collagen and its precursor molecules. Sodium dodecyl sulfate gel electrophoresis of this fraction showed that it consisted of a high-molecular-weight (greater than 300,000 daltons) polypeptide. This component was collagenase sensitive and, in the presence of dithiothreitol, gave rise to two polypeptides of the apparent molecular weights of 200,000 and 160,000 daltons. Pepsin digestion of this material destroyed all the high-molecular-weight material and gave rise to a single collagenase-sensitive component of an apparent molecular weight of 115,000 daltons. This 115,000 dalton material is similar to previously observed basement membrane collagens, and the 160,000 and 200,000 dalton components are probably precursor chains of basement membrane collagen. The very-high-molecular-weight material (greater than 300,000 daltons) may represent a disulfide-linked complex of these precursor chains. DEAE-cellulose column chromatography confirmed the presence of a single procollagen species distinct from the collagen fraction. Amino acid analysis of collagen and procollagen fractions showed a decreased hydroxyproline value as compared with previously reported basement membrane collagens or collagen precursors.
 ...
PMID:Biochemical characterization of procollagen-collagen synthesized by rabbit corneal endothelial cells in culture. 75 87

Type I procollagen secreted by matrix-free cells from chick embryo tendons was purified by DEAE-cellulose chromatography. Electron microscopy of segment-long-spacing aggregates of the procollagen demonstrated the presence of both NH2-terminal and COOH-terminal extensions not found in collagen. The procollagen was digested with bacterial collagenase and the COOH-terminal fragments were isolated by gel filtration and polyacrylamide gel electrophoresis in sodium dodecyl sulfate. Analysis of tryptic peptides demonstrated that the COOH-terminal extensions on the pro alpha 1 and pro alpha 2 chains had different primary structures.
 ...
PMID:Segment-long-spacing aggregates and isolation of COOH-terminal peptides from type I procollagen. 106 85

In this report we describe the purification of bovine interstitial collagenase and provide information on its substrate specificity, kinetic parameters of catalytic activity, and amino terminal protein sequence. In addition, we present a simplified protocol for the purification of bovine tissue inhibitor of metalloproteinases (TIMP). Collagenase was purified by sequential chromatography through heparin-Sepharose, DEAE-Sepharose, and green-agarose, resulting in a product that was greater than 95% pure as judged by polyacrylamide electrophoresis. Typical of other interstitial collagenases, the isolated bovine protein was activated by protease and organomercurial treatment. It also demonstrated a kinetics and substrate specificity similar to those of human collagenase. TIMP was purified by sequential chromatography through heparin-Sepharose and DEAE-Sepharose followed by reverse-phase HPLC. The purified protein had a size, N-terminal sequence, and inhibitor activity similar to those of other mammalian TIMPs. Partial peptide sequences suggested that bovine collagenase and TIMP have strong sequence homology to their human homologues.
 ...
PMID:Purification and characterization of bovine interstitial collagenase and tissue inhibitor of metalloproteinases. 131 Nov 65

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

Activation of procollagenase constitutes a crucial event in collagenolytic activity regulation. In this study we have purified by DEAE-cellulose, Ultrogel AcA-44, and zinc chelate sepharose chromatographies, a procollagenase-activator from the culture medium of the guinea pig carrageenin granuloma model. On SDS-PAGE, the activator migrates as a principal band of Mr approximately 44,000. The molecule activates procollagenase from human lung fibroblasts in a concentration dependent manner and an enhancement of collagenase activity of trypsin-treated crude culture medium was observed. A loss of about 50% of its activity occurs after heating. In addition, this activator degrades gelatin and casein. All these data suggest that this procollagenase-activator might be stromelysin. The activator was found in both phases of the granuloma, at 7 days when collagen is actively deposited and an important proportion of the collagenolytic activity remains in latent form; and at 14 days, when this enzymatic activity is fully expressed.
 ...
PMID:Purification of a procollagenase-activator present in medium of cultured guinea pig carrageenin granuloma. 166 Aug 1

To obtain an adequate amount of human prolactin (hPRL) for elucidation of the structure-function relationship, we have expressed the hPRL cDNA in Escherichia coli (E. coli) by using a high-expression vector. The vector contained a chimeric gene encoding a fusion of protein A, a peptide sensitive to collagenase digestion and hPRL, which was inserted downstream of the right direction promotor of lambda phage. The resulting protein fusion was purified through three column chromatographies of immunoglobulin G-linked Sepharose 4B, DEAE-5PW, and phenyl-5PW. In a typical experiment, a final sample with a purity of more than 80% was obtained with a recovery of more than 40% judged by enzyme-linked immunosorbent assay (ELISA). The fusion thus obtained was digested with collagenase, and protein reactive to anti-hPRL antibody was purified through phenyl-5PW column chromatography. The hPRL sample was found to be identical to authentic hPRL with respect to the amino acid composition and an N-terminal sequence of 20 residues, except that it contained an additional four amino acids at the N-terminal end. This peptide was presumed to be derived from the collagenase-target sequence. The hPRL thus obtained was found to be as active as the authentic hormone either immunologically judged by ELISA or biologically judged by the growth stimulatory effect on rat Nb2 lymphoma cells.
 ...
PMID:Efficient production of biologically active human prolactin in Escherichia coli. 166 26

Cementum forms the interface through which soft connective tissue of the periodontium is attached to the root surface. The interactions between cementum and connective tissue are not completely understood and whether cementum influences periodontal connective tissue formation and regeneration is not clear. We have examined the effect of cementum components on the attachment of gingival fibroblasts. Cementum was harvested from healthy human and bovine teeth and extracted sequentially in 0.5 M CH3COOH, 4 M guanidine and bacterial collagenase. Fibroblast attachment was measured using 51Cr-labelled human gingival fibroblasts on tissue culture plates previously incubated with cementum components. Results showed that all three extracts mediated fibroblast attachment and attachment was dependent on concentration and incubation time. The attachment activity was not destroyed by digestion with bacterial collagenase or by antibodies to fibronectin and laminin. However, it was inhibited by a peptide containing the amino acid sequence RGD. By gel filtration or HPLC using a DEAE-cellulose column several proteins with attachment activity were fractionated. SDS-polyacrylamide gel electrophoresis revealed that HPLC fraction eluted by 0.2-0.3 M NaCl contained a protein with molecular weight 55 kDa as a major component. This protein was isolated and shown to promote fibroblast attachment, and optimal attachment occurred at a concentration of 2 micrograms/ml. We conclude that cementum contains substances capable of mediating fibroblast attachment and that these substances play an important role in periodontal connective tissue formation and regeneration by facilitating fibroblast attachment to root surfaces.
 ...
PMID:Isolation of a fibroblast attachment protein from cementum. 213 24

Constituents of the bone marrow microenvironment have the capacity to influence both normal and malignant hematopoietic cell behavior. For example, HL-60 human promyelocytic leukemia cells in vitro display a more mature phenotype when grown on a bone marrow stroma-derived matrix. To elucidate which component(s) of the stromal matrix is capable of modulating HL-60 cell phenotype, matrices were treated with a variety of chemicals and enzymes prior to being used in the differentiation assay. Treatment of matrices with collagenase, pronase, chondroitinase, or chloroform:methanol:ether could not abolish the differentiation-promoting activity of bone marrow stroma. In contrast, the activity was destroyed by alkali treatment (0.5 M NaOH for 18 h) or heparinase/heparitinase enzymes. Heparin added to cultures increased maturation of HL-60 cells as determined by esterase production, Fc rosette formation, and morphological appearance. Other stromal components such as laminin, fibronectin, collagen I, collagen IV, or chondroitin sulfate did not alter the HL-60 leukemia cell phenotype. Stroma-derived matrix material which labeled with [35S]sulfate and eluted on a DEAE ion-exchange column as a high ionic fraction in 1.5 M LiCl and 7.5% sodium dodecyl sulfate contained the active fraction. A heparan sulfate proteoglycan component isolated by polyacrylamide-agarose gel electrophoresis induced a more mature HL-60 phenotype, and digestion with heparinase/heparitinase in the presence of protease inhibitors abrogated the effects on HL-60 phenotype. We conclude that a heparan sulfate-associated fraction of the bone marrow matrix plays a key role in the regulation of leukemic cell maturation.
 ...
PMID:A heparan sulfate-containing fraction of bone marrow stroma induces maturation of HL-60 cells in vitro. 214 Feb 91


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