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 binding of Ca2+ to a salivary phosphoprotein, protein C, was studied by equilibrium dialysis. In 5mM-Tris/HCl buffer, pH 7.5, protein C bound 190 nmol of Ca2+/mg of protein. The apparent dissociation constant, K, was determined to be 1.9 x 10(-4)M and the binding of Ca2+ to the protein was non-co-operative. The binding of Ca2+ to protein C apparently depends on groups which ionize above pH 5.0. Ca2+ binding decreased with increased concentration of the dialysis buffer and on addition of SrCL2, MgCl2 and MnCl2 to the dialysis buffer. Digestion of protein C with trypsin or collagenase or heating of the protein to 60 degrees or 100 degrees C had little or no effect on the Ca2+ binding. Digestion of protein C with alkaline phosphatase caused a decrease in the amount of protein-bound Ca2+. This was also found for another salivary phosphoprotein, protein A. In the absence of Ca2+ the S020,w for protein C was 1.29 S and in the presence of Ca2+ it was 1.46S. Ca2+ may cause a conformational change in the protein or an aggregation of the protein molecules. No conformational changes of protein C in the presence of Ca2+ could be detected by circular dichroism or nuclear magnetic resonance.
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PMID:The binding of calcium to a salivary phosphoprotein, protein C, and comparison with calcium binding to protein A, a related salivary phosphoprotein. 1 96

Two metabolically distinct types of bone cell populations were isolated from mouse calvaria by a repetitive digestive procedure with a mixture of collagenase and trypsin. Cells released early in the digestion showed approximately two-fold increases in cAMP when treated with either parathormone or calcitonin. These populations were denoted CT type. Later eluting cells showed larger parathormone-induced increases in cAMP but did not respond to calcitonin. These populations were denoted PT type. Six metabolic and enzymatic activites were measured in the two types of populations: acid and alkaline phosphatases, hyaluronate synthesis, citrate decarboxylation, prolyl hydroxylase, and general protein synthesis. Although each of these activites was present in both cell types, the basal levels of acid phosphatase and hyaluronate synthesis were higher in the CT cells, whereas alkaline phosphatase, citrate decarboxylation, and prolyl hydroxylase were higher in te PT cells. Parathormone stimulated acid phosphatase and hyaluronate synthesis by 100-200% only in the CT cells; in inhibited alkaline phosphatase, citrate decarboxylation, and prolyl hydroxylase by 75-90% only in the PT cells. Calcitonin alone had no effect on any of these activities other than cAMP production, but in inhibited the action of parathormone in the CT cells. The sensitivities, time courses of development,and magnitudes of these hormonal effects were similar to those observed previously in intact calvaria, indicating that the isolated cell system is a reliable model for the study of bone metabolism. Based on the metabolic responses of the cells, we postulate that the CT type of populations is enriched in osteoclasts and, possibly, osteocytes, and the PT type of population is enriched in osteoblasts.
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PMID:Biochemical characterization with parathormone and calcitonin of isolated bone cells: provisional identification of osteoclasts and osteoblasts. 18 58

We have previously shown that bone cells possess glucocorticoid receptors and that, in addition to being inhibitory to cell growth, glucocorticoid treatment potentiates the ability of parathyroid hormone (PTH) to stimulate cyclic AMP (cAMP) formation. This study extends those observations to specific subpopulations of bone cells and explores the mechanism of the cAMP augmentation. Subpopulations of cultured bone cells derived from 20-d-old fetal rat calvaria were enriched for "osteoblast-like" (OB) and "osteoclast-like" (OC) cells by sequential collagenase digestion. OC cells released during the first 30 min of collagenase digestion were characterized by low alkaline phosphatase activity, a cAMP response to salmon calcitonin (CT), but only a small cAMP response to bovine PTH. In contrast, OB cells released between 30 and 120 min of collagenase digestion, possessed high alkaline phosphatase activity, responded with a large cAMP rise to PTH, but exhibited no response to CT. Glucocorticoid receptors, with similar properties, were demonstrated in both populations (K(d) congruent with 5 nM, N(maximum) congruent with 400 fmol/mg cytosol protein). Dexamethasone equivalently inhibited cell growth and alkaline phosphatase activity in both populations. Dexamethasone potentiation of cAMP generation occurred after PTH but not CT stimulation. A greater enhancement of cAMP generation observed in OB cells appears to result from two glucocorticoid actions: (a) stimulation of adenylate cyclase and (b) inhibition of phosphodiesterase. Only the latter mechanism was found in OC cells. Dexamethasone-treated cells showed an increase in both sensitivity and maximal response of cAMP to PTH. The possible relationship of these actions to the mechanism of glucocorticoid-induced osteopenia is discussed.
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PMID:Glucocorticoid receptors and actions in subpopulations of cultured rat bone cells. Mechanism of dexamethasone potentiation of parathyroid hormone-stimulated cyclic AMP production. 22 Feb 82

When liver cells were dispersed with collagenase, their 5'-nucleotidase activity decreased to half the initial level, but it increased to the original level again on culture of the cells for a few days. The activity of another membrane enzyme, alkaline phosphatase, did not decrease on dispersion of the cells, but it increased about 10-fold on culture of the cells. These inductions did not require any hormone, but the effects were greater at a high cell density. These enzymes are located in both the plasma membranes and the cytoplasm, but the enzymes in these two locations can be distinguished by differences in their pH optima, substrate specificities, and susceptibilities to inhibitors. The increases were found to be due to increases in the activity of only the enzymes in the plasma membranes. The increases in enzyme activities were inhibited by actinomycin D, cycloheximide, and puromycin. The activities of leucine aminopeptidase and aminopeptidase B, other membrane enzymes, remained constant during dispersion and culture of the cells. These results show that enzymes in the cell membranes are affected in different ways by cell dispersion with collagenase and subsequent culture of the cells.
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PMID:Biochemical studies on liver functions in primary cultured hepatocytes of adult rats. III. Changes of enzyme activities on cell membranes during culture. 52 39

1. This paper describes improved methods of obtaining, purifying and studying bulk suspensions of isolated living hepatocytes and other cells of adult rats and urodeles. 2. The cells were isolated largely by dissolving the hepatic ground substance through the extracorporeal portal perfusion and further incubation of the excised liver with 0.05% collagenase and 0.1% hyaluronidase. The different kinds of cells were then separated from one another by counter-current centrifugation. The isolated cells were examined by differential interference, phase-contrast, amplitude-contrast, ultraviolet, fluorescence and electron microscopy. Various cytochemical tests were carried out. Whenever possible, for each method of examination, the isolated cells were compared with cells of the same kind which had not undergone isolation. 3. Dye-exclusion, lysochromy, fluorescence and differential interference microscopical analysis indicated viability rates between 75 and 99%. Succinate dehydrogenase activity was preserved at a high level in nearly all isolated cells. In hepatocytes, the essentially extracellular cells. In hepatocytes, the essentially extracellular 'soluble' alkaline phosphatase activity of bile canaliculi was retained. Living hepatocytes were studied by super-modulating methods of microscopy for the first time, with somewhat unexpected findings. It now seems probable that previous methods of tissue preparation produced gross alterations in hepatocyte mitochondria. The assessment of the viability of isolated cells was re-examined. 4. The methods described may permit a more meaningful correlation between biochemical, cytochemical, ultrastructural and biophysical findings than that obtainable by the use of current methods.
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PMID:Improved isolation, separation and cytochemistry of living cells. 110 11

In a series of four studies, adult female Swiss-Webster mice were used to measure the effects of salmon calcitonin on two biochemical indices of local and systematic bone formation: (1) skeletal alkaline phosphatase activity--in serum and in extracts of calvaria and tibiae, and (2) calvarial collagenase-digestible protein synthesis--measured, acutely, in vitro. Subcutaneous calcitonin doses ranged from 50 to 400 mU/mouse/day (0.95-18.1 U/kg/day), and treatment schedules were continuous (daily) for 2-14 days, acute, or intermittent (2 days/week for 6 weeks). The effects of calcitonin on these bone formation indices (skeletal alkaline phosphatase and collagenase-digestible protein synthesis) were biphasic with respect to dose and treatment time, being increased in response to short-term, low-dose treatment, but not long-term, continuous treatment. The effects of long-term intermittent calcitonin treatment were dose-dependent increases in skeletal alkaline phosphatase in calvaria and serum (r = 0.948, P less than 0.02, and r = 0.960, P less than 0.01, respectively).
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PMID:Two biochemical indices of mouse bone formation are increased, in vivo, in response to calcitonin. 131 Aug 83

Lymphocytes show heterogeneity both in phenotype and in locomotor activity; methods which permit the simultaneous assessment of both are therefore useful. The activation of locomotion may be dependent on interactions between lymphocytes and accessory cells, making it necessary to study mixed cell populations. We describe here two in vitro procedures which do this. Firstly the lymphocyte polarization assay has been adapted by using the alkaline phosphatase-anti-alkaline phosphatase method (APAAP) after fixation with glutaraldehyde. Secondly, we have allowed lymphocytes to invade collagen gels and then digested the gel with collagenase to recover the locomotor population. This can then be phenotyped by immunofluorescence using a fluorescence-activated cell sorter (FACS). Collagenase did not affect the staining pattern with commonly-used markers such as CD3, -4, -8, -19, -29, -45RO and -45RA.
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PMID:Methods for phenotyping polarized and locomotor human lymphocytes. 131 36

Transforming growth factor-beta (TGF beta) produced by osteoblasts is present in high levels in bone and influences bone formation, replication of bone cells, and expression of osteoblast protein products. Interactions between bone active hormones and locally released and activated TGF beta were studied by examining the influence of TGF beta preincubation on PTH, calcitonin (CT), and vitamin D receptors in an osteoblastic cell line (UMR 106-06). Preincubation of UMR 106-06 cells with 1 ng/ml TGF beta for 3 days increased specific binding of [125I]PTH-related protein (PTHrP)(1-84) to 140% of that in control cells, but [125I]salmon CT binding decreased to 50% of controls. Binding isotherms indicated that the changes in binding were due to altered receptor numbers since affinities for 125I-labeled PTH and CT remained unchanged. The effect on receptor levels was time dependent, requiring 24 h preincubation with TGF beta for measurable changes, and dose dependent, with maximal effects seen with 1 ng/ml TGF beta. Binding of [3H]1,25(OH)2 vitamin D3 was increased to 130% of control in cytosolic extracts of UMR 106-06 cells pretreated for 3 days with 1 ng/ml TGF beta. Scatchard plots suggested an increase in receptor number without change in affinity. The adenylate cyclase response to PTH increased to 150% of control cells after 3 days of treatment with 1 ng/ml TGF beta; however, the adenylate cyclase response to CT was little changed. Forskolin- and cholera toxin-stimulated adenylate cyclase responses were increased by TGF beta treatment to 130-160% of control, indicating an increase in the stimulatory subunit of the G protein. Increased abundance of both Gs and Gi proteins were indicated by increased cholera toxin- or pertussis toxin-dependent [32P] NAD ribosylation of 47-kilodalton (kDa) and 42-kDa or 40-kDa proteins, respectively, in TGF beta-treated cells. Our data support a complex regulatory effect of TGF beta on UMR 106-06 cells with increases in PTH receptors, vitamin D receptors, and G proteins, whereas there is an apparent down-regulation of CT receptors. TGF beta might induce a more differentiated osteoblast phenotype of these cells, which already express differentiated features such as high alkaline phosphatase activity, PTH and vitamin D receptors, and collagenase production. Since low doses of PTH stimulate bone formation in vivo, TGF beta released or activated at sites of new bone formation might locally modulate PTH activity be allowing increased PTH receptor and postreceptor effectiveness.
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PMID:Transforming growth factor-beta modulates receptor binding of calciotropic hormones and G protein-mediated adenylate cyclase responses in osteoblast-like cells. 132 61

The effects of tissue maturation on the cellular composition and biochemical characteristics of bone were studied in neonatal, young adult, and aging mice. Osteoblast subclasses were isolated on Percoll density gradients. Neonatal calvariae consisted almost exclusively of cells banding at low and intermediate buoyant density. High buoyant density cells constituted 5-10% of total cells at 10 days of age but increased to 50-60% by 5 weeks of age. These latter cells were released late during collagenase digestion. This indicates that they arise from the deeper layer of bone. For this reason, we consider them putative osteocytes. We established that constitutive secretion of IGF-I and TGF-beta and activities of cellular alkaline phosphatase paralleled those of the tissue of origin in all cell groups and was highest in cells of intermediate buoyant density. These activities declined rapidly after cessation of growth at 5 weeks of age in both bone and isolated cells. Between 5 and 8 weeks of age, the hormonal response to PTH also declined dramatically. The maximum cAMP induced by PTH declined by about 70% in highly responsive cells of intermediate buoyant density and fell to insignificant levels in cells of high buoyant density. We found that a cyclic AMP response to PTH was positively correlated with stimulated secretion of IGF-I by this hormone in cells from animals of all ages. Despite their inability to respond to PTH with increases in cAMP and IGF-I, adult bone cells of high buoyant density continued to respond to PTH with increases in the secretion of TGF-beta.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Maturation-associated changes in the cellular composition of mouse calvariae and in the biochemical characteristics of calvarial cells separated into subclasses on Percoll density gradients. 132 39

Rat calvaria bone cells isolated by collagenase digestion form a bone-like matrix which mineralizes in vitro in the presence of beta-glycerophosphate, in less than 2 weeks. The purpose of this work was to investigate, in this mineralizing rat osteoblastic cell culture, the synthesis of collagen, osteocalcin, and bone alkaline phosphatase (ALP). The results obtained indicate (1) After 15 days in culture, the extracellular-matrix contains collagen type I, V, and to some extent type III. Metabolic labeling at day 14, during the phase of nodules mineralization as well as new nodules formation, shows that collagen types I and type V are synthesized; (2) During the phase of cell growth, no osteocalcin could be detected in the medium, however, at the point of nodule formation, the osteocalcin level reached values of 3.55 +/- 1.39 ng/ml, followed by a 30-fold increase after nodules became mineralized. At day 14, after metabolic labeling, de novo synthesized osteocalcin was chromatographed on an immunoadsorbing column. With urea-SDS PAGE the apparent molecular weight was determined to be 9,000 daltons. (3) Specific activity of ALP was found to be 10 nmol/min/mg of proteins at cell confluence. At day 15, when nodules are mineralized, this activity was increased by 40-fold. The Michaelis constant was 1.58 10(-3) M/L. ALP was inhibited by L-homoarginine and levamisole but not by L-phenylalanine. ALP was shown to be heat sensitive at 56 degrees C with two slopes of inhibition.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Expression of collagen, osteocalcin, and bone alkaline phosphatase in a mineralizing rat osteoblastic cell culture. 137 88


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