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)

The genetic type and molecular structure of the precursor forms of collagen synthesized by matrix-free tendon cells isolated from 17-day old chick embryos were examined by chromatographic and electrophoretic techniques. The [14C]proline-labeled collagenous proteins secreted by the cells resolved on diethylaminoethylcellulose into two peaks, A and B. Both peaks contained type I collagenous proteins since on chromatography on carboxymethylcellulose, after limited pepsin proteolysis, both peaks contained alpha1 and alpha2 chains of collagen in a 2:1 ratio, and cyanogen bromide peptide maps of the 14C-labeled protein in both peaks were similar to cyanogen bromide peptide maps derived from authentic type I collagen. Enzymatic digestion with purified mammalian collagenase demonstrated that the collagen precursor in peak B contained noncollagenous peptide extensions at both the amino- and carboxy-terminal ends of the molecule, while peak A had only carboxy-terminal extension peptides. Although both the amino- and carboxy-terminal extensions incorporated radioactive cystine, only the carboxy-terminal extensions contained interchain disulfide bonds. The carboxy-terminal extensions were also shown to incorporate radioactive tryptophan. Since most of the precursor forms of collagen recovered in the incubation medium chromatographed in peak B, it is concluded that matrix-free tendon cells secrete only type I procollagen with extension peptides at both the amino- and carboxy-terminal ends of the molecule.
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PMID:Characterization of procollagen synthesized by matrix-free cells isolated from chick embryo tendons. 18 99

Native type III collagen and procollagen were prepared from fetal bovine skin. Examination of the cleavage products produced by digestion with tadpole collagenase demonstrated that the three palpha1(III) chains of type III procollagen were linked together by disulfide bonds occurring at both the amino-terminal and carboxy-terminal portions of the molecule. Type III collagen contained interchain disulfide bonds only in the carboxy-terminal region of the molecule. After digestion of procollagen with bacterial collagenase an amino-terminal, triple-stranded peptide fragment was isolated. The reduced and alkylated chain constituents of this fragment had molecular weights of about 21 000. After digestion of procollagen with cyanogen bromide a related triple-stranded fragment was isolated. The chains of the cyanogen bromide fragment had a molecular weight of about 27 000. When the collagenase-derived peptide was fully reduced and alkylated, it became susceptible to further digestion with bacterial collagenase. This treatment released a fragment of about 97 amino acid residues which contained 12 cystein residues and had an amino acid composition typical for globular proteins. A second, non-helical fragment of about 48 amino acid residues contained three cysteines. This latter fragment is formed from sequences that overlap the amino-terminal region in the collagen alpha1(III) chain by 20 amino acids and possesses an antigenic determinant specific for the alpha1(III) chain. The collagenase-sensitive region exposed by reduction comprised about 33 amino acid residues. It was recovered as a mixture of small peptides. These results indicate that the amino-terminal region of type III procollagen has the same type of structure as the homologous region of type I procollagen. It consists of a globular, a collagen-like and a non-helical domain. Interchain disulfide bonding and the occurrence of cysteines in the non-helical domain are, however, unique for type III procollagen.
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PMID:Characterization of the amino-terminal segment in type III procollagen. 18 41

Embryonic-chick tendon poly(A)-containing RNA was translated in the wheat-germ and mRNA-dependent rabbit reticulocyte-lysate systems. The ability of each system to synthesize polypeptides similar to pro-alpha chains of collagen was tested on the bases of electrophoretic mobility and susceptibility to highly purified bacterial collagenase. Very small amounts of polypeptides in the size range of pro-alpha chains were synthesized in the wheat-germ system, whereas efficient synthesis of two polypeptides similar to pro-alpha1 and pro-alpha2 chains was achieved in the reticulocyte lysate. The collagenous nature of the major high-molecular-weight products synthesized was demonstrated by their susceptibility to collagenase and ability to act as a substrate for purified collagen proline hydroxylase. Determinations of the relative amounts of these translation products suggest that the 2:1 ratio of pro-alpha1 and pro-alpha2 chains found in type I procollagen is reflected in proportional amounts of translatable mRNA for pro-alpha1 and pro-alpha2 chains. Comparisons of the electrophoretic mobilities of hydroxylated and unhydroxylated reticulocyte-lysate translation products were made with appropriate standards of hydroxylated and unhydroxylated procollagen polypeptides. The results suggest that, in common with a number of secreted proteins, procollagen is synthesized as pre-pro molecules consistent with the ;Signal Hypothesis'.
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PMID:Translation of embryonic-chick tendon procollagen messenger ribonucleic acid in two cell-free protein-synthesizing systems. 22 70

The non-collagenous N-terminal segment of type I procollagen from dermatosparactic sheep skin was isolated in the form of the peptide Col 1 from a collagenase digest of the protein. The peptide has a blocked N-terminus, which was identified as pyrrolid-2-one-5-carboxylic acid. Appropriate overlapping fragments were prepared from reduced and alkylated peptide Col 1 by cleavage with trypsin at lysine, arginine and S-aminoethyl-cysteine residues and by cleavage with staphylococcal proteinase at glutamate residues. Amino acid sequence analysis of these fragments by Edman degradation and mass spectrometry established the whole sequence of peptide Col 1 except for a peptide junction (7--8) and a single Asx residue (44), and demonstrated that peptide Col 1 consists of 98 amino acid residues. The N-terminal portion of peptide Col 1 (86 residues) shows an irregular distribution of glycine, whereas the C-terminal portion (12 residues) possesses the triplet structure Gly-Xy and is apparently derived from the precursor-specific collagenous domain of procollagen. The central region of the peptide contains ten cysteine residues located between positions 18 and 73 and shows alternating polar and hydrophobic sequence elements. The regions adjacent to the cysteine-rich portion have a hydrophilic nature and are abundant in glutamic acid. The data are consistent with previous physicochemical and immunological evidence that distinct regions at the N- and C-termini of the non-collagenous domain possess a less rigid conformation than does the central portion of the molecule.
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PMID:Amino acid sequence of the N-terminal non-collagenous segment of dermatosparactic sheep procollagen type I. 47 72

The presumptive messenger RNAs for type I procollagen were isolated from chick embryo calvaria at various stages of development. Poly(A)-containing RNA fractions from denaturing sucrose gradients directed protein synthesis in a cell-free system derived from wheat germ. Procollagen mRNA activity was detected in a region of about 26 S. Approx. 80% of the labeled proline incorporated into cell-free product was susceptible to digestion by purified bacterial collagenase. The synthesis of procollagen mRNAs was followed during development. Comparison of the in vitro labeled mRNAs from calvaria of day 12--16 embryos indicated that the 26 S component was most pronounced at day 13 and decreased progressively towards day 16. In addition, incubation of calvaria with tritiated nucleosides for 1.5--25 h revealed that 26 S mRNA was significantly labeled only after prolonged periods. The results suggest that procollagen mRNA is a relatively stable species with a prolonged half-life compared to the majority of mRNAs in this tissue.
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PMID:The synthesis of presumptive procollagen messenger ribonucleic acid in the calvaria of the developing chick embryo. 62 42

To investigate the role of transforming growth factor-beta 1 (TGF beta) in bone metabolism, the effects of this agent on the differentiation characteristics of human bone cells were studied in vitro. Human bone cells were isolated from femoral head samples by collagenase digestion. Differentiation characteristics included alkaline phosphatase activity, osteocalcin production, and mRNA levels for alkaline phosphatase, type I alpha 2-procollagen, and osteocalcin. The effect of TGF beta on alkaline phosphatase was not constant, but varied with the incubation conditions. At high cell density and in the presence of serum, TGF beta decreased alkaline phosphatase activity. However, at low cell density and under serum-free conditions, TGF beta stimulated alkaline phosphatase activity. The addition of 1,25(OH)2 vitamin D3 also stimulated alkaline phosphatase. The combination of the two agents gave a greater increase in activity than the sum of the activities when the two agents were given alone. The percentage of cells that stain positively for alkaline phosphatase changed in parallel with the change in specific activity. The percentage of positive cells increased from 17% to 64%, while the specific activity increased from 22 to 169 mU/mg protein. To investigate the mechanism of this stimulation, mRNA levels were measured at 24 hours. Individually, TGF beta and 1,25(OH)2D3 increased message levels for alkaline phosphatase and type I procollagen, but the greatest effect was produced by the combination of the two factors. 1,25(OH)2D3 increased osteocalcin mRNA levels, but TGF beta markedly inhibited this stimulation. TGF beta also inhibited production of osteocalcin by the human bone cells. TGF beta appears to modulate differentiation of human bone cells in combination with 1,25(OH)2D3 and other factors.
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PMID:Differentiation of normal human bone cells by transforming growth factor-beta and 1,25(OH)2 vitamin D3. 153 45

Monoclonal antibodies were prepared that were specific for chicken type I and type III collagens. The specificity of these antibodies was determined by ELISA, inhibition ELISA, and immunoblot assays. The results showed that the monoclonal antibodies were specific for their respective antigens without significant cross reactivity to other types of collagen. An analysis of the location of the epitopes by rotary shadowing that a monoclonal antibody for type I collagen (called DD4) recognized type I procollagen close to the large globular domain at the carboxyl terminus of the molecule. A monoclonal antibody for type III collagen (called 3B2) recognized both the intact type III molecule and also the TCA fragment of type III collagen after mammalian collagenase digestion. The epitope was located approximately one-fifth of the distance from the amino-terminus of the intact molecule. The monoclonal antibodies were used for immunolocalization of type I and type III collagens in cryosections of heart, aorta, kidney, liver, thymus, skin, gizzard and myotendinous junction. In heart, aorta, kidney, liver, thymus and skin, type I and III collagens were colocalized in the connective tissue of each organ. In contrast, gizzard and myotendinous junction showed distinctly different staining patterns for the distribution of type I and type III collagen. The two monoclonal antibodies reported here are potentially useful reagents to study fibril formation involving type I and type III collagens.
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PMID:Monoclonal antibodies that distinguish avian type I and type III collagens: isolation, characterization and immunolocalization in various tissues. 156 Jul 90

Avian skin fibroblasts were isolated, cultured and incubated with [3H]proline for 24 h. The cells exported radiolabeled collagenase-digestible (CDP) and non-collagenase-digestible (NCDP) proteins into the medium. Human, bovine and avian growth hormone (GH) as well as insulin-like growth factor I (IGF-I) attenuated the appearance of [3H]CDP in the medium without affecting [3H]NCDP. The appearance of [3H]CDP was not affected by prolactin. The effects of GH and IGF-I were enhanced by increasing concentrations of fetal calf serum (FCS). A synergism was observed between GH and IGF-I in their effect on CDP. Each peptide, at an ineffective concentration, increased the sensitivity of the cells to the other peptide. Collagenase activity in the medium was enhanced by IGF-I, but not modified by GH, FCS, or by their interaction with IGF-I. GH and IGF-I inhibition of type I procollagen gene expression was demonstrated with the aid of probes containing sequences corresponding to the mRNAs for avian alpha I and alpha II chains. The results suggest that GH and IGF-I cooperate in regulating collagen synthesis, but collagen degradation is affected by IGF-I and not by GH.
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PMID:Growth hormone and insulin-like growth factor I regulate collagen gene expression and extracellular collagen in cultures of avian skin fibroblasts. 165 42

Two substitutions for glycine in the triple-helical domain were found in type I procollagen synthesized by skin fibroblasts from two probands with lethal osteogenesis imperfecta. One was a substitution of valine for glycine alpha 1-637, and the other was a substitution of arginine for glycine alpha 2-694. The effects of the mutations on the zipper-like folding of the collagen triple helix were similar, since there was post-translational overmodification of the collagenase A fragments (amino acids 1-775) but not of more COOH-terminal fragments of the protein. The mutations differed markedly, however, on their effects on thermal unfolding of the triple helix. The collagenase A fragment from the collagen containing the arginine alpha 2-694 substitution was cleaved at about amino acid 700 when incubated with trypsin at 30-35 degrees C. Therefore, there was micro-unfolding of the triple helix at a site close to the glycine substitution. Surprisingly, however, the collagenase A fragment with the valine alpha 1-637 substitution was also cleaved at about amino acid 700 under the same conditions. The results, therefore, demonstrated that although most glycine substitutions delay folding of the triple helix in regions that are NH2-terminal to the site of the substitution, the effects on unfolding can be transmitted to regions that are COOH-terminal to the site of the glycine substitution.
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PMID:Substitutions for glycine alpha 1-637 and glycine alpha 2-694 of type I procollagen in lethal osteogenesis imperfecta. The conformational strain on the triple helix introduced by a glycine substitution can be transmitted along the helix. 187 19

Skin fibroblasts from a proband with a lethal variant of osteogenesis imperfecta synthesized both apparently normal type I procollagen and a type I procollagen that had slow electrophoretic mobility because of posttranslational overmodifications. The thermal unfolding of the collagen molecules as assayed by protease digestion was about 2 degrees C lower than normal. It is surprising, however, that collagenase A and B fragments showed an essentially normal melting profile. Assay of cDNA heteroduplexes with a new technique involving carbodiimide modification indicated a mutation at about the codon for amino acid 550 of the alpha 1(I) chain. Subsequent amplification of the cDNA by the PCR and nucleotide sequencing revealed a single-base mutation that substituted an aspartate codon for glycine at position alpha 1-541 in the COL1A1 gene. The results here confirm previous indications that the effects of glycine substitutions in type I procollagen are highly position specific. They also demonstrate that a recently described technique for detecting single-base differences by carbodiimide modification of DNA heteroduplexes can be effectively employed to locate mutations in large genes.
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PMID:A single base mutation in type I procollagen (COL1A1) that converts glycine alpha 1-541 to aspartate in a lethal variant of osteogenesis imperfecta: detection of the mutation with a carbodiimide reaction of DNA heteroduplexes and direct sequencing of products of the PCR. 203 36


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