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)

Rabbit antisera were produced against purified calf dermatosparatic procollagen and against the purified procollagens obtained from the culture medium of calf dermatosparatic cells. These antisera and their derived gamma-globulins were characterized by immunoprecipitation, double immunodiffusion and immunoelectrophoresis. Antiserum directed against dermatosparatic procollagen cross-reacted with the two different forms of procollagen obtained from the culture medium of dermatosparatic calf cells. Antiserum directed against onw of these procollagens, namely (pro alpha1)2 pro alpha2, cross reacted with dermatosparatic procollagen and also cross-reacted with the other procollagen,(pro alpha1)3. Antiserum directed against procollagen (pro alpha1)3 cross-reacted with dermatosparatic procollagen and with the procollagen (pro alpha1)2 pro alpha2. None of the antisera reacted with authentic calf skin collagen, or with the collagen extracted from the cell layer of the dermatosparatic calf cells in culture. Reduction andalkylation of the procollagens abolished the antigen-antibody reactions, while prior digestion of the antigens with bacterial collagenase did not eliminate the immunological reaction. Antigenic determinants in the cell culture procollagens were found at the COOH-terminal non-collagen peptide as well as at the NH2-terminal non-collagen peptide.
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PMID:Immunological properties of procollagens obtained from the culture medium of dermatosparactic cells. 5 Feb 85

An enzyme which catalyzes the deamidation of thyroliberin (TRF; less than Glu-His-Pro-NH2) has been purified 110-fold from extracts of bovine anterior pituitary by ammonium sulfate fractionation, ion exchange chromatography on DEAE-cellulose, and gel filtration. This enzyme of 76,000 molecular weight (as estimated by gel filtration) exhibits maximal activity at neutral pH (optimum pH 7.4 to 7.6) in buffers of high ionic strength supplemented with thiol-protecting agents. As indicated by the strong inhibition of the enzymatic activity by N-ethylmaleimide and Hg2+, as well as by the extreme sensitivity toward diisopropyl fluorophosphate, -SH, and -OH residues apparently represent essential functional groups of the enzyme. The stereospecific deamidation of TRF (Km = 4.1 . 10(-4) M) is inhibited competitively by TRF analogues which contain proline or by the proline containing biologically active peptides luliberin (LH-RF), oxytocin, vasopressin, angiotensin II, and Substance P. TRF analogues without proline or peptide amides without proline are ineffective. This enzyme cleaves the appropriate Pro-X bonds in luliberin, angiotensin II, pyroGlu-His-Pro-Gly-NH2, and the collagenase substrate Z-Gly-Pro-Leu-Gly-Pro. Thus, it may be characterized as a post-proline-cleaving enzyme.
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PMID:Characterization of "thyroliberin-deamidating enzyme" as a post-proline-cleaving enzyme. Partial purification and enzyme-chemical analysis of the enzyme from anterior pituitary tissue. 11 64

Tadpole collagenase (EC 3.4.24.3) cleaved chick cranial bone procollagen into two triple-stranded fragments, PCA and PCB. Only PCB, with an estimated molecular weight of about 60,000 for each component chain after reduction, was found to contain interchain disulfide bonds. The analogous cleavage of collagen is known to produce a large NH2-terminal fragment with a molecular weight of 70,000 for each chain and a small COOH-terminal fragment containing chains of about 25,000 molecular weight. Since PCB was too small to represent the product NH2-terminal to the site of collagenase cleavage, localization of interchain disulfide bonds to a COOH-terminal domain in procollagen was indicated. This assignment was conformed by Dintzis-type short-term labeling experiments. Procollagen obtained by acid extraction of bone lacked the COOH-terminal disulfide-bonded domain. The findings support a model for procollagen consisting of three proalpha chains each containing nonhelical NH2-terminal extensions of 20,000 molecular weight and COOH-terminal extensions of about 35,000 molecular weight, the latter linked by interchani disulfide bonds.
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PMID:Interchain disulfide bonds in procollagen are located in a large nontriple-helical COOH-terminal domain. 17 50

Collagenase cleavage of human Type II and III collagens has been studied using a highly purified preparation of rabbit tumor collagenase. Progress of the reactions in solution was followed by viscometry and the results indicated that under the conditions employed Type III collagen molecules were cleaved at approximately five times the rate of Type II molecules. Cleavage products of the reactions were isolated in denatured form by agarose molecular sieve chromatography. The molecular weights and amino acid compositions of the products demonstrated that Type II and III molecules had been cleaved at the characteristic three-quarter, one-quarter locus, giving rise to a large fragment derived from the NH2-terminal portion of the molecule and a smaller fragment representing the COOH-terminal region. The amino acid sequence at the NH2-terminal portion of the smaller fragment derived from Type II collagen was determined to be Ile-Ala-Gly-Gln-Arg, and the corresponding region from Type III collagen was found to have the sequence Leu-Ala Gly-Leu-Arg. These sequences for alpha1(II) and alpha1(III) chains adjacent to the site of collagenase cleavage along with previous data for alpha1(I) and alpha2 chains indicate that the minimum specific sequence required for collagenase cleavage is Gly-Ile-Ala or Gly-Leu-Ala. Inspection of the available sequence data for collagen alpha chains indicates that the latter sequences are found in at least three additional locations at which collagenase cleavage does not occur. Each of the sequences which are apparently not substrates for collagenase, however, are followed by a Gly-X-Hyp sequence. We suggest, then, that a minimum of five residues in collagen alpha chains COOH-terminal to the cleavage site comprise the substrate recognition site.
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PMID:Cleavage of Type II and III collagens with mammalian collagenase: site of cleavage and primary structure at the NH2-terminal portion of the smaller fragment released from both collagens. 17 19

98% of the collagen in mature connective tissue is in the form of insoluble collagen fibers, consisting of bundles of polymeric collagen (PC) fibrils. The enzymes concerned in connective tissue remodeling degrade PC rather than tropocollagen (TC). TC is the most usual substrate for collagenase assays, and we believe it is essential to employ PC in any study of the activity of collagenolytic enzymes. In order to facilitate the study of enzymic degradation of PC we have labelled PC with fluorescein iso-thiocyanate to produce F-PC fibrils, containing 5 fluorescein labelled epilson-NH2 groups of lysine per TC molecule within the PC. The fluorescent F-PC is degraded at the same rate as PC with the release of hydroxyprolyl peptides but has the great advantage that the solubilised F-peptides can be quantitated by their fluorescent emission. The technique is described in detail employing bacterial collagenase and mammalian collagenase preparations to illustrate the methodology. The advantages of the fluorescent technique over the collagenolytic assay methods currently in use are outlined.
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PMID:Indoluble collagen II. The use of fluorescein labelled polymeric collagen fibrils in a very sensitive assay procedure for enzymes degrading insoluble collagen. 17 6

Hepatocytes isolated by the collagenase digestive method were transplanted into the spleens of syngeneic rats. Morphology and function of the hepatocytes in the spleen were investigated for 12 to 17 months after transplantation. The transplanted hepatocytes proliferated and reconfigured in the spleen without direct perfusion of portal venous blood and with the presence of an intact host liver. Fourteen to 17 months after transplantation, the hepatocytes which had formed a demarcated nodule occupied approximately 40% of the area of the splenic parenchyma without undifferentiation on microscopic examination. However, the weight of the hepatized spleen did not increase beyond the weight of a normal spleen and the weight of the host liver that had normal morphology also did not differ from a normal liver. Light and electron microscopic studies demonstrated differentiated cord structure and normal architecture for each heptocyte. Furthermore, the hepatized spleen synthesized albumin and glycogen as demonstrated by immunofluorescence and histochemical studies. Ammonia tolerance and indocyanine green clearance tests revealed functioning hepatocytes in the spleen proper. These results indicate that our experimental model lends itself well to investigations in cell growth mechanism and that hepatocellular transplantation has potential clinical application to compensate for impaired hepatic function.
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PMID:Morphology and function of isolated hepatocytes transplanted into rat spleen. 51 33

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.
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PMID:Localization and partial composition of the oligosaccharide units on the propeptide extensions of type I procollagen. 61 65

Using hepatocytes isolated by collagenase perfusion, we studied the kinetic characteristics of the uptake process for procaine amide ethobromide (PAEB). Determination of initial uptake velocities (Vo) at substrate concentrations from 30 to 400 micrometer demonstrated a saturable process with a Km of 54 +/- 10 micrometer and a Vmax of 0.13 +/- 0.01 nmol/min/mg of protein. Pretreatment of cells with metabolic inhibitors and reduction of the incubation temperature significantly reduced the Vo of 100 micrometer PAEB. Replacement of sodium ions with lithium had no effect, while replacement with choline decreased Vo by 75%. The intracellular concentration of PAEB was 18 times the medium concentration after 90 min, but 33% of that was in the acetylated form. Uptake of N4-acetyl PAEB occurred at a much lower rate and reached a cell/medium ratio of only 6 after 90 min. Only one of seven quaternary amines tested inhibited PAEB uptake at an inhibitor/substrate ratio (I/S) of 7.5, while four out of five tertiary amines significantly decreased Vo at an I/S of 0.75 and all five decreased it at a ratio of 7.5. Some organic acids and steroidal compounds also significantly decreased PAEB Vo at an I/S of 0.75, while others from each group had no effect at an I/S of 7.5. Because uptake is saturable, requires metabolic energy, and occurs against an electrochemical gradient, it is suggested that the hepatic accumulation of PAEB occurs via an active, carrier-medicated transport process.
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PMID:Carrier-mediated transport of the organic cation procaine amide ethobromide by isolated rat liver parenchymal cells. 70 24

1. Isolated parenchymal cells were prepared by collagenase perfusion of livers from fed rats that had been previously injected with [(3)H]leucine to label liver proteins. When these cells were incubated in a salts medium containing glucose, gelatin and EDTA, cellular integrity was maintained over a period of 6h. 2. Cells incubated in the presence of 2mm-leucine to minimize radioactive isotope reincorporation released [(3)H]leucine into the medium at a rate accounting for the degradation of 4.5% of the labelled cell protein per h. 3. Degradation of [(3)H]protein in these cells was inhibited by insulin and by certain amino acids, of which tryptophan and phenylalanine were the most effective. 4. Protein degradation was decreased by several proteinase inhibitors, particularly those that are known to inhibit lysosomal cathepsin B, and by inhibitors of cell-energy production. 5. Ammonia inhibited degradation, but only at concentrations above 1.8mm. Aliphatic analogues of ammonia were effective at lower concentrations than was ammonia. 6. High concentrations of ammonia inhibited degradation by 50%. The extent of this inhibition could not be increased further by the addition of the cathepsin B inhibitor leupeptin, which by itself inhibited degradation by approx. 30%. 7. The sensitivity of proteolysis in isolated hepatocytes to these various inhibitory agents is discussed in relation to their possible modes of action.
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PMID:Inhibition of protein degradation in isolated rat hepatocytes. 88 Feb 45

A modified form of procollagen was extracted with 10 M urea from the skin of lambs with dermatosparaxis, a disease which is produced by a genetic defect in the conversion of procollagen to collagen. The extracts contained little if any alpha1 and alpha2 chains of normal type I collagen, and instead they contained the larger polypeptides palpha1 and palpha2 together with high polymers. palpha1 was purified by ion-exchange chromatography and gel filtration. The polypeptide was shown to be related to alpha1 by its chromatographic behavior, its amino acid composition, and the peptides obtained after cleavage with cyanogen bromide. The molecular weight of palpha1 by gel filtration was 112 300 +/- 6300. After digestion of palpha1 with bacterial collagenase, a fragment of about 100 amino acid residues was obtained which was similar in amino acid composition and antigenic activity to a comparable fragment previously obtained from the NH2-terminal region of palpha1 chains from dermatosparaxic cattle. However, after cleavage of palpha1 with cyanogen bromide, a larger NH2-terminal fragment of about 160 amino acid residues was obtained. The larger cyanogen bromide fragment contained 8 residues of hydroxyproline, 12 residues of proline, and 19 residues of glycine not found in the NH2-terminal fragment isolated after digestion with bacterial collagenase. The results indicated that, in addition to containing amino acid sequences similar to those found in globular proteins, the peptide extensions on the NH2-terminal end of the palpha1 chain of procollagen also contain amino acid sequences similar to those found in the triple-helical region of the collagen molecule. The molecular weight of palpha2 by gel filtration was 102 400 +/- 6800. No additional peptide fragment was recovered after digestion of palpha2 with bacterial collagenase.
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PMID:NH2-terminal extensions on skin collagen from sheep with a genetic defect in conversion of procollagen into collagen. 94 80


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