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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)

We have developed a preparation of monolayer cultures of bovine parathyroid cells in order to elucidate the control mechanism of the biosynthesis and secretion of parathyroid hormone (PTH) at cellular level. Dispersion of parathyroid cells was performed by stirring minced bovine parathyroid tissues in Hanks' BSS containing 0.3 yields to 0.5 percent collagenase at 37 degrees C for 60 min. Dispersed cells were cultured at 37 degrees C in MEM-Hanks' BSS containing 10 percent fetal calf serum and 15 mM HEPES. On the 5th day of the culture, the medium was replaced with 1 percent BSA-MEM-Hanks-HEPES buffer, and the cells were incubated with 3H-leucine or in the media containing various concentrations of calcium, magnesium, PGE1, PGE2 or DBcAMP. At the end of incubation, the cells were detouched and homogenized in 8M urea, 0.2 N HCL and 0.01 M cysteine solution. The isolation of proparathyroid hormone (ProPTH) and PTH was performed through the preparation of TCA-powder followed by CMC column chromatography. PTH in the incubation medium was determined by radioimmunoassay. It was demonstrated that the monolayer cultures of bovine parathyroid cells were synthesizing ProPTH and converting it to PTH. The cultures exhibited linear secretion rates of PTH into the medium. The secretion of PTH was markedly increased by PGE1, PGE2 or DBcAMP in the range of 10(-7) yields to 10(-5)M in the former and 10(-5) yields to 10(-3)M in the latter, while calcium or magnesium changed secretion rate in the range of 0.3 yields to 4.4 mM.
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PMID:[Studies on the biosynthesis and secretion of parathyroid hormone in monolayer cultures of bovine parathyroid cells (I) (author's transl)]. 20 10

1. Gap (communicating) junctions are plasma-membrane specializations of characteristic morphology that form transmembrane channels allowing direct communication between cells. Their preparation is described starting from mouse liver plasma membranes and the constituent polypeptides are deduced. 2. Gap junctions co-purify with collagen fibres when the plasma-membrane residues insoluble in N-dodecyl sarcosinate are fractionated on sucrose gradients. Sucrose-density perturbation by relipidation of isolated gap junctions or the use of urea to remove non-junctional membranes both failed to diminish the collagen content of fractions. 3. Removal of collagen by treatment with purified collagenase preparations yielded morphologically satisfactory gap-junction fractions. Analysis by polyacrylamide-gel electrophoresis of the polypeptides present in gap junctions prepared by procedures omitting or using collagenases indicated two non-glycosylated polypeptides, a major component of apparent mol.wt. 38000 and a minor 40000-mol.wt. component. These two polypeptides were also present in plasma membranes and the intermediate fractions. 4. Proteolysis of the gap-junction polypeptides yielding components of mol.wt. 34000, 25000 and below 20000 occurred when iodinated gap junctions were subject to prolonged collagenase treatment, thus explaining the variable polypeptide composition of gap junctions reported by others. 5. The morphological properties of the isolated gap junctions prepared by the various procedures are described.
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PMID:Preparation of hepatic gap (communicating) junctions. Identification of the constituent polypeptide subunits. 20 88

Solubilization of the normal glomerular basement membrane with various solvents revealed that the material is held together by hydrogen and disulfide linkages as well as ionic salt bridges which ionize at around pH 10.0. Pronase digestion indicated that differences in susceptibility to enzyme digestion exist between normal and nephritic membrane. Titration of a urea-insoluble material indicated that some alteration must have taken place in the association between various components of the nephritic basement membrane. Chemical analysis of alkali-solubilized fractions suggested that greater alkali susceptibility of the nephritic material may be present. A collagen-like material resembling both tendon and dog basement membrane collagen in its amino acid composition was isolated. It contained 10% hexose, but in addition to glucose and galactose, mannose was also detected. A glycopeptide fraction obtained by pronase and collagenase digestion has a carbohydrate composition similar to the collagen-like material above. These substances probably represent incompletely digested fragments of the basement membrane.
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PMID:Studies of normal and nephritic rat glomerular basement membrane. 23 74

Elastin was extracted from human aortic plaque and adjacent grossly normal intima by the following methods: (1) 0.1 N NaOH at 100 degrees C, (2) hot NaOH and 0.2 M EDTA, (3) 5 M guanidine--HCl and collagenase, (4) guanidine--collagenase and dithioerythritol--urea--sodium dodecyl sulfate, (5) guanidine--collagenase and EDTA, (6) 10% NaCl and collagenase, and (7) NaCl--collagenase and EDTA. All elastin samples contained small amounts of carbohydrate and hydroxyproline. The lipid content of non-plaque intimal elastin samples was small (2--3%), whereas it increased to 4--6% in plaque intima. The lipid composition of elastin preparations varied significantly with the extraction procedure. Elastin from plaque intima contained significantly more cholesterol (50--60%) and less triglyceride and phospholipid than elastin of non-plaque intima (30--50% cholesterol). The contents of free and esterified cholesterol were comparable in all preparations. The main phospholipid in all samples was sphingomyelin, which comprised between 50 and 80% of the total phospholipid. Compared with NaOH-purified elastin, the other elastin samples were characterized by an increased phosphatidyl--choline content, while they all contained an almost equal amount of phosphatidylethanolamine. In elastin samples from plaque intima, the polar amino acids were increased, whereas cross-linking amino acids were decreased. The polarity and hydroxyproline content of elastin samples were slightly decreased after treatment with EDTA or dithioerythritol--urea--sodium dodecyl sulfate.
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PMID:Elastin--lipid interaction action in the arterial wall. Part 1. Extraction of elastin from human aortic intima. 46 28

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

Viable pancreatic islets were isolated from the pancreas of humans using modifications of the collagenase digestion and Ficoll gradient techniques. Gel filtration of tissue extracts following islet incubation in the presence of 3H- tryptophan indicated that radioactivity becomes incorporated into at least two islet proteins. The larger of the two (LGI) has the approximate molecular size of proinsuiln and the smaller coelutes with glucagon as determined by column standardization. Radioimmunoassay of the gel filtration eluate for glucagon revealed that both molecules have glucagon immunoreactivity. Gel filtration in the presence of 8M urea did not alter the elution pattern of the LGI molecule. Polyacrylamide gel electrophoresis was performed on these glucagon immunoreactive molecules. The 3H radioactivity and the glucagon immunoreactivity of the smaller molecule were found to co-migrate electrophoretically with crystalline porcine glucagon and monodesamidoglucagon. With electrophoresis at high pH the electrophoretic mobility of the LGI molecule proved to be lower than that of glucagon. Partial tryptic degradation of the human LGI molecule yields 3H-tryptophan labeled products having charge and immunologic characteristics indistinguishable from porcine glucagon and monodesamidoglucagon. Although further investigation is indicatend may thus serve as a precursor or an intermediate in human glucagon biosynthesis.
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PMID:Glucagon biosynthesis in human pancreatic islets: preliminary evidence for a biosynthetic intermediate. 109 15

The insoluble component of stratum corneum of rat epidermis yields two major bands after extraction with 8 M urea-mercaptoethanol-dithiothreitol. The ratio of these two bands is about 1:1 in terms of protein stain intensity and S-[14C]carboxymethyl label. Both polypeptides were purified to homogeneity by DE-52-cellulose, sodium dodecyl sulfate hydroxylapatite C column chromatography, and preparative DodSO4-polyacrylamide gel electrophoresis. The heavier polypeptide contains 30% alpha helix and the lighter contains 27% alpha helix as determined by circular dichroism studies. Both are sensitive to Pronase and resistant to trypsin, collagenase, and elastase. The lighter chain is stable to pepsin but the heavier can be partially degraded to a smaller polypeptide with a molecular weight similar to that of light chain. Amino acid analysis shows that the light chain contains 12 more tyrosine residues than does the heavy chain, suggesting that the light chain is not generated from the heavy chain. However, the two chains may have a common peptide region. Antiserum prepared against the heavier polypeptide can be completely absorbed by purified lighter polypeptide and vice versa indicating that both chains have some common antigenic determinants. Antibody against either chain can cross-react with the stratum corneum and keratohyalin granules in the epidermis of newborn rat as indicated by fluorescent microscopic observation. Similarly, this antibody also cross-reacts with the cell surface or the contents of spinous and granular cells, and very weakly with basal cells, indicating that the two proteins may be present in the lower strata as well as the stratum corneum.
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PMID:Two polypeptide chain constituents of the major protein of the cornified layer of newborn rat epidermis. 116 97

The distribution spaces at equilibrium for 3H2O, [14C]urea and s-O-[14C]-methylglucose were measured in white fat cells using centrifugation through silicone oil at 2500 X g; no significant differences were observed. L-[14C]Glucose added immediately before the centrifugation was used as a marker for the extracellular water space. The calculated intracellular water content of the cells after the centrifugation through oil (e.g. 3H2O space minus L-[14C]glucose space) is an unbiased measure of the water content of the fat cells in suspension as judged by the following criteria: (1) The intracellular distribution space for 3-O-[14C]'methylglucose at equilibrium (methylglucose space minus L-glucose space) was not different from that calculated from a methylglucose wash-out curve. (2) The intracellular content of L-[14C]glucose (half time of efflux about 60 min) in cells preloaded during incubation of the tissue with collagenase was not different in cells recovered by (a) centrifugation through oil at 2500 X g, (b) centrifugation through oil at 600 X g, (c) centrifugation at 600 X g in the absence of oil and (d) filtration on Millipore filters. The intracellular content of water determined on cells from single rats weighing 120-150 g was 2.75 +/- 0.55 mul/100 mul fat cells (+/- S.D., n = 30). The intracellular content of potassium, determined on cells from the same rats, was 252 +/- 62 nmols/100 mul fat cells (+/- S.D., n = 30). The concentration of potassium in the intracellular water was calculated as 104 +/- 15 mM (+/- S.D., n = 30).
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PMID:The content of water and potassium in fat cells. 126 18

A new molecule, type XIV collagen, with domains homologous to type IX and XII collagens has been recently discovered in pepsin extracts of fetal bovine tissues (Dublet, B., and van der Rest, M. (1991) J. Biol. Chem. 266, 6853-6858). In the present study, we describe the purification and the characterization of the intact native form of this newly discovered collagen. By using only two chromatographic steps we were able to obtain pure type XIV collagen. Furthermore, minor modifications of the protocol allowed us to perform the simultaneous large scale purification of type XII and type XIV collagens from the same tissue. Intact type XIV collagen migrates on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) as two bands of 220 and 290 kDa (reducing conditions). After collagenase treatment, a single band of 190 kDa is observed, which represents the large non-collagenous domain of the molecule (NC3). Rotary shadowing electron micrographs of intact type XIV collagen show a cross-shaped structure formed by a thin tail attached through a central globule to three identical "fingers." These properties are similar to those previously described for intact chicken type XII collagen (Dublet, B., Oh, S., Sugrue, S. P., Gordon, M. K., Gerecke, D. R., Olsen, B. R., and van der Rest, M. (1989) J. Biol. Chem. 264, 13150-13156), but the two molecules are different gene products and have charge and glycosylation differences. Finally, we show that the three chains of purified type XIV collagen have an apparent molecular mass of approximately 220 kDa and are not cross-linked to each other by bonds other than disulfide bridges. The same observation was made for type XII collagen. In both cases, the 290-kDa migrating band in SDS-PAGE is due to incomplete denaturation in electrophoresis sample buffer in the absence of urea.
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PMID:Purification and characterization of native type XIV collagen. 132 5

Polyhydroxyethylmethacrylate (PHEMA) based microcarriers with different bulk structures were prepared by a phase inversion polymerization technique. PHEMA surfaces were further modified chemically by glow-discharge treatment, and biologically by covalent attachment of fibrinogen and collagen. Hepatocytes were isolated from young male Wistar rats using an in situ portal vein collagenase perfusion technique. Freshly isolated hepatocytes were seeded at 6 x 10(5) cells/mL and microcarrier concentration was 10 g/L. Stationary microcarrier cultures were carried out in standard (nontissue culture) polystyrene petri dishes in a humidified 5% CO2 incubator at 37 +/- 0.5 degrees C. Cell attachment was followed by light microscopy by taking samples from the culture medium every 30 min. Urea and protein syntheses by microcarrier-attached hepatocytes were determined by standard techniques. Nonswellable (highly cross-linked) hydrophilic PHEMA microcarriers did not support cell attachment and viability. However, swellable (low cross-linked) PHEMA microcarriers (pretreated in FBS) allowed high attachment and cell spreading. PHEMA microcarriers treated in dimethylaminoethylmethacrylate (DMAEMA) glow-discharge plasma also improved the cell attachment characteristics of the PHEMA microcarriers. The highest attachment efficiencies (immobilization yields) were observed with the biologically modified PHEMA microcarriers, especially modified with fibronectin. Metabolic activity, as estimated by urea and protein syntheses, was also higher in these microcarriers.
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PMID:Hepatocyte immobilization on PHEMA microcarriers and its biologically modified forms. 134 12


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