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 mechanism of congenital osteopetrosis in microphthalmic (mi) mice has been examined in bone organ cultures. Resorption was measured by the release of previously incorporated 45Ca in fetal long bones and newborn calvaria from mi mice and heterozygous or homozygous normal litter mates. Bones from mi mice showed a generalized resorption defect with decreased spontaneous or control resorption and failure to respond to parathyroid hormone (PTH), prostaglandin E2, 1,25 dihydroxy vitamin D3, vitamin A, or osteoclast activating factor (OAF) from human peripheral leukocytes or mouse spleen cells. Bones from heterozygotes showed a smaller response to PTH than bones from homozygous normals. Mutant bones failed to show an increase in lysosomal enzyme release in response to PTH or vitamin A, agents which increased release from bones of homozygous normals. Proline incorporation into collagenase-digestible protein was similar in cultures of normal and mutant bone and was inhibited by PTH and OAF. These results indicate that congenital osteopetrosis in mi mice is due to a generalized defect in the function and hormonal response of osteoclasts and suggests that this cell line is separate from the osteoblast cell line which shows no impairment of hormonal response.
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PMID:Studies on congenital osteopetrosis in microphthalmic mice using organ cultures: impairment of bone resorption in response to physiologic stimulators. 87 Jun 7

We investigated the effects of insulin-like growth factor I/somatomedin C (IGF-I/SM-C), and the interaction of IGF-I and 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) on mouse clonal osteoblasts, MC3T3-E1. IGF-I stimulated [3H]thymidine incorporation into the DNA of the cells at concentrations of 1.3-130 X 10(-9) M. The alkaline phosphatase (ALP) activity in cultures was also raised by the hormone at the same concentrations. The optimal dose of IGF-I was 13 X 10(-9) M. Co-addition of IGF-I (1.3-130 X 10(-9) M) and 1,25(OH)2D3 (10(-11) to 10(-10) M) to the culture of MC3T3-E1 cells caused a synergistic increase in ALP activity. 25(OH)D3 and 24,25(OH)2D3 showed a similar effect with IGF-I at 1000-2000 times higher concentrations than 1,25(OH)2D3. [3H]Proline incorporation into collagenase digestible protein (CDP) in media was stimulated dose-dependently by IGF-I up to 2.2-fold over the control levels at 130 X 10(-9) M. Addition of 1,25(OH)2D3 (5 X 10(-11) M) and IGF-I further elevated the proline incorporation into CDP. However, the increment in CDP synthesis, induced by the two hormones was less than the increment in ALP activity. Thus, we conclude (1) that IGF-I stimulates both cell replication and differentiated functions in cultured murine osteoblasts and (2) that IGF-I and 1,25(OH)2D3 have the synergistic effect on ALP activity and the additive effect on collagen synthesis in MC3T3-E1 cells.
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PMID:Cooperation of synthetic insulin-like growth factor I/somatomedin C and 1,25-dihydroxyvitamin D3 on regulation of function in clonal osteoblastic cells. 272 Feb

We previously constructed a bifunctionally active membrane-bound fusion protein, in which Escherichia coli proline carrier (the product of the putP gene) was linked with beta-galactosidase (the product of the lacZ gene) through a collagen linker (Hanada, K., Yamato, I., and Anraku, Y. (1987) J. Biol. Chem. 262, 14100-14104). The proline carrier was purified from this site specifically cleavable fusion protein. Cytoplasmic membranes overproducing the fusion protein were solubilized with dodecylmaltoside, and the solubilized fraction was subjected to anti-beta-galactosidase IgG-Sepharose chromatography. The fusion protein was specifically adsorbed to the immunoaffinity resin and then treated with collagenase for splitting the proline carrier moiety of the fusion protein from the beta-galactosidase moiety. The collagenase used for the collagenolysis was then removed by anti-collagenase IgG-Sepharose chromatography. In this way, the proline carrier was purified to more than 95% homogeneity of the protein. Proline transport in proteoliposomes reconstituted with the purified carrier was dependent on the membrane potential and the chemical gradient of Na+ across the membrane with apparent Michaelis constants for proline and for Na+ stimulation of 3.6 microM and 31 microM, respectively. These results indicated that the proline carrier mediates electrogenic Na+/proline symport.
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PMID:Purification and reconstitution of Escherichia coli proline carrier using a site specifically cleavable fusion protein. 313 Mar 79

A new method for the simultaneous determination of newly synthesized collagen and noncollagen proteins has been developed. Because tryptophan is not found in collagen noncollagen proteins were specifically labeled with [3H]tryptophan. [14C]Proline was used to label both groups of proteins. To calculate the 14C-labeled noncollagen protein the 3H radioactivity of the protein mixture was divided by the ratio of 3H:14C in noncollagen protein of a representative sample. This value was obtained by collagenase digestion. The remaining 14C radioactivity in the protein mixture was attributed to [14C]collagen. There was a very good correlation between the dual label method and the widely used collagenase digestion method for the measurement of collagen and noncollagen protein production and for the calculation of the relative rate of collagen synthesis. This new method provides a simple and accurate analysis of collagen production, and it is suitable for rapid processing of a large number of biological samples.
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PMID:[3H]tryptophan-[14C]proline dual label method for the simultaneous determination of collagen and noncollagen protein production. 652 75

Cultured pulmonary artery smooth muscle cells derived from the medial vessel layer of weanling rabbits were grown in the presence or absence of sodium ascorbate. The connective tissue elements insoluble elastin and collagen were identified and quantified. Formation and accumulation of alpha-aminoadipic acid gamma-semialdehyde (allysine) and the intermolecular cross-links desmosine (Des), isodesmosine (Ides), and aldol condensation product (Aldol) were evaluated from [14C]lysine pulse-chase experiments. [14C]Des, [14C]Ides, peptide-bound [14C]lysine, [14C]allysine, and [14C]Aldol were determined from amino acid analysis. The latter two components were determined after reduction with NaBH4. [14C]Proline conversion to hydroxy[14C]proline and collagenase susceptibility were used to identify and quantify collagen synthesis. Ascorbate dramatically affects insoluble elastin synthesis, accumulation, and cross-link formation. Cells grown in the presence of ascorbate synthesize and accumulate significantly less insoluble elastin than non-ascorbate cultures. Those elastin molecules which do become incorporated into the extracellular matrix in the presence of ascorbate contain a slightly elevated content of hydroxyproline and lysine and, most importantly, are turned over more rapidly.
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PMID:Effects of ascorbate on insoluble elastin accumulation and cross-link formation in rabbit pulmonary artery smooth muscle cultures. 681 21

We have developed monoclonal antibody 5109 against a unique highly acidic sequence in type II collagen. When paired with previously reported monoclonal antibody 9A4, 5109 can be used as the capture antibody in an ELISA assay for the neoepitope generated by collagenase-cleavage of type II collagen. The assay detects the sequence ZGlyGluX(759)GlyAspAspGlyProSerGlyAlaGluGlyProX(771)GlyProGlnGly(775) where Z is a variable length polypeptide, X is proline or hydroxyproline, and Gly(775) corresponds to C-terminal amino acid of the 3/4 piece after collagenase cleavage. Antibody 5109 detects the first and 9A4 the second underlined sequence. Antibody 5109 recognizes its epitope with a K=1.2x10(-8) M independently of hydroxylation of X(759). When X(771) is proline, the sequence is 90x more sensitively detected by this ELISA than when it is hydroxyproline. Type II collagen of human articular cartilage was fragmented by cyanogen bromide (CNBr) and trypsin. The immunoreactive fragment was captured with 5109 and sequenced. Proline(771) averaged 81% hydroxylated. Other 3rd position prolines were >97% hydroxylated. In urine of control individuals of 50-70 years of age, we failed to detect the presence of the collagen fragment in a majority (8/10) of specimens. The two controls with measurable levels averaged 123 pM. In a similar age cohort of osteoarthritic patients, the majority (9/10) showed measurable values of urinary collagen fragments averaging 312 pM. This assay can be used for monitoring type II collagen metabolism in patients with osteoarthritis.
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PMID:Analysis of collagenase-cleavage of type II collagen using a neoepitope ELISA. 1115 May 34

AIM:To study the mechanism of Fuzhenghuayu (FZHY) decoction on anti-liver fibrosis.METHODS:FZHY 10% decoction sera was incubated with rat normal subcultured hepatic stellate cells (HSC) and fibrotic primarily cultured HSC, normal and fibrotic hepatocytes and subcultured skin fibroblasts seperately. Cell intracellular and extracellular collagen synthesis rates were measured by the method of (3)H Proline impulse and collagenase digestion.RESULTS:For primarily cultured HSC and hepatocytes, both of intracellular and extracellular collagen synthesis rates decreased in the drug sera group. For the normal subcultured HSC and primarily cultured hepatocytes, the extracellular collagen secretion was decreased obviously by the drug sera, and intracellular collagen synthesis rates were inhibited to some extents.For fibroblasts, both intracellular and extracellular collagen synthesis rates were inhibited somewhat, but no significant differences were found.CONCLUSION:The mechanism of FZHY decoction on anti-liver fibrosis may be associated with inhibition of liver collagen production.
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PMID:Effects of Fuzhenghuayu decoction on collagen synthesis of cultured hepatic stellate cells, hepatocytes and fibroblasts in rats. 1181 68

Collagen gel sandwich and immobilization cultures of rat hepatocytes are two recently developed organotypical culture models. Basic information with respect to the maintenance of xenobiotic biotransformation pathways and the expression of key enzyme activities, however, is lacking, making their use in pharmaco-toxicological studies rather speculative. The expression of the glutathione S-tranferase (GST; EC 2.5.1.18) activity, a key phase II enzyme, has been chosen to study the various problems that may arise in expressing the results of cytosolic enzyme activities when rat hepatocytes are cultured using both new culture models. Collagen gel matrix easily entraps culture medium proteins. These interfere with the cytosolic protein content, a parameter versus which cytosolic enzyme activities, including GSTs, are usually expressed. The following solutions are proposed: expression of the cytosolic enzyme activity results versus either (i) microsomal proteins, these are not contaminated by medium proteins, or versus (ii) cytosolic proteins after a complete collagenase digestion (0.05% collagenase type I of Sigma, 45 min, 37 degrees C) of the collagen matrix. Expression of enzyme activities versus cellular DNA appears to be unacceptable since unreliable results were obtained due to entrapped DNA in the collagen matrix. Once it was known how to express cytosolic enzyme activity, the maintenance of GST activities was investigated in both culture models using 1-chloro-2,4-dinitrobenzene (CDNB) and 1,2-dichloro-4-nitrobenzene (DCNB) as substrates for total and Mu class GSTs, respectively. Two culture media were compared, control medium (DMEM) with and without supplementation of l-proline (final concentration 60 mug/ml). In both culture models, after an initial decrease, total GST activities increased significantly up to values higher than those observed for freshly isolated cells. The Mu class GST activities were maintained constant for 7 days and increased thereafter. l-Proline supplementation of the culture medium prevented the initial decline in total and Mu class GST activities in both culture configurations but did not seem to be of crucial importance in the maintenance of GST activities in both culture models.
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PMID:Collagen gel sandwich and immobilization cultures of rat hepatocytes: Problems encountered in expressing glutathione S-transferase activities. 2065 79