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)

Human bone cell cultures were established by maintaining collagenase-treated bone fragments in low Ca++ medium. The resulting cell cultures exhibited a high level of alkaline phosphatase activity and produced a significant increase in intracellular cAMP when exposed to the 1-34 fragment of human parathyroid hormone. With continued culture, the cells formed a thick, extracellular matrix that mineralized when cultures were provided daily with normal levels of calcium, fresh ascorbic acid (50 micrograms/ml) and 10 mM beta-glycerol phosphate. Biosynthetically, these cells produced type I collagen (without any type III collagen), and the bone-specific protein, osteonectin. In addition, the cells produced sulfated macromolecules electrophoretically identical to those positively identified as the bone proteoglycan in parallel cultures of fetal bovine bone cells. This technique provides a useful system for the study of osteoblast metabolism in vitro.
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PMID:Human bone cells in vitro. 299 72

The antihyperglycemic agent, metformin (dimethylbiguanide), inhibits hepatic gluconeogenesis. To investigate the mechanism involved, glucose production from collagenase-isolated hepatocytes of starved rats was determined after 1 hr incubations with different substrates. In the absence of insulin, glucose production from 10(-2) M lactate-10(-3) M pyruvate, 10(-2)M M alanine, 10(-2) M glutamine and 5 x 10(-3) M glycerol was decreased (35-78%) by high concentrations (10(-2) and 10(-3) M) of metformin. Lower concentrations of metformin were not effective in the absence of insulin, but a therapeutic concentration (10(-5) M) of metformin acted synergistically with insulin (10(-8) M) to suppress gluconeogenesis from each of the substrates by an additional 10-14% compared with insulin (10(-8) M) alone. The synergistic antigluconeogenic effect of metformin (10(-5) M) with insulin (10(-8) M) was achieved without alteration of the contents of NADH and NAD+ in digitonin-separated cytosolic and mitochondrial-rich hepatocyte fractions. Mitochondrial ATP was also unaltered by the metformin (10(-5) M)-insulin (10(-8) M) combination. However, the antigluconeogenic effect of 10(-2) M metformin alone was associated with an increased (by 109%) mitochondrial NADH:NAD+ ratio. Thus reduced gluconeogenesis by high concentrations of metformin (e.g. 10(-2) M) may involve changes of redox state. However, therapeutic concentrations of metformin (e.g. 10(-5) M) potentiate the antigluconeogenic effect of insulin to a similar extent from a range of substrates, without altering energy status or redox state.
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PMID:Inhibition of hepatic gluconeogenesis by metformin. Synergism with insulin. 305 29

Confluent monolayers of normal human hepatocytes obtained by collagenase perfusion of liver fragments were incubated in a serum-free medium. Intracellular apolipoproteins apo AI, apo C, apo B, and apo E were detected between Day 1 and Day 6 of the culture by immunoenzymatic staining using polyclonal antibodies directed against these apoproteins and monoclonal antibodies directed against both forms of apo B (B100 and B48). Translation of mRNA isolated from these hepatocytes in an acellular system revealed that apo AI and apo E were synthesized as the precursor forms of mature plasma apo AI and apo E. Three lipoprotein fractions corresponding to the density of very low density lipoprotein (VLDL), low density lipoprotein (LDL), and high density lipoprotein (HDL) were isolated from the medium at Day 5 of culture and examined by electron microscopy after negative staining. VLDL and LDL particles are similar in size and shape to plasma lipoproteins; spherical HDL are larger than normal plasma particles isolated at the same density. Their protein represented 44, 19.5, and 36.5% respectively, of the total lipoprotein protein. The secretion rate of VLDL protein corresponded to that measured in primary cultures of rat hepatocytes. After incorporation of [3H]glycerol, more than 92% of the [3H]triglyceride secreted into the medium was recovered in the VLDL fraction. These results demonstrate that primary cultures of normal human hepatocytes are able to synthesize and secrete lipoproteins and thus could be a useful model to study lipoprotein metabolism in human liver.
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PMID:Synthesis and secretion of lipoproteins by human hepatocytes in culture. 312 44

We studied PGE2-release from isolated human gastric mucosal cells. Mucosa was obtained at surgery and cells were dispersed by collagenase and pronase. Centrifugation with Percoll yielded a fraction of light density cells (70-75% parietal cells; 2-4% mast cells) revealing maximal rates of PGE2-release. A radioimmunoassay was used to measure PGE2-release into the incubation medium. Calcium ionophore A23187 which aids calcium transport across membranes caused a 3.5-fold increase of PGE2-release; this effect was abolished in calcium-free incubation medium. PGE2-release was also stimulated by phospholipase C (100 mU/ml) which is known to induce phosphoinositol breakdown, as well as by 1-oleyl-2-acetyl-sn-glycerol (OAG; 10 microM) and by 12-O-tetradecanoyl-13-acetate (TPA; 10 microM) which cause direct activation of protein kinase C without preceding induction of phosphoinositol breakdown. The response to TPA was potentiated by A23187. The calmodulin antagonist naphthalene sulfonamide W 7 reduced PGE2-release in response to A23187 and TPA (IC50: 1 microM). Our data indicate that PGE2-release of human gastric mucosal cells is stimulated by calcium influx as well as by indirect (phospholipase C) and direct (OAG, TPA) activation of protein kinase C. Stimulation of PGE2-release involves calmodulin-mediated mechanisms.
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PMID:[Calcium, phospholipase C and protein kinase C stimulate prostaglandin secretion of isolated gastric mucosa cells of the human]. 347 5

The sensitivity to lipolytic agents is altered in diabetic vs. control animals. Because of its role as a diabetogenic hormone and its ability to elicit lipolysis, GH was studied in isolated fat cells (IFC) from control and streptozotocin-diabetic (STZ-DM) rats. IFCs from the epididymal fat of 150 to 200-g normal and STZ-DM Holtzman rats were prepared by collagenase digestion. Lipolysis was measured by glycerol release after either incubation or perifusion with the following concentrations: epinephrine (EPI), 0.01-0.1 microM; theophylline, 0.01-1.0 mg/ml; adenosine deaminase (ADA), and bovine GH (bGH), 0.01-1.0 microgram/ml. Rats, rendered diabetic by STZ (65 mg/kg), were used on day 3. In a dose-response study comparing glycerol release from control and STZ-DM IFC, IFC were preincubated with 1.0 microgram/ml bGH and then incubated with varying concentrations of EPI or bGH. In STZ-DM, we noted increased lipolytic sensitivity to low concentrations of EPI or bGH. Furthermore, in perifusion, STZ-DM IFC did not require obligatory preincubation with bGH for optimal glycerol release. The addition of ADA increased glycerol release from incubated IFC (STZ-DM and controls). In both systems an enhanced lipolytic response to theophylline was seen in the presence of bGH in control and STZ-DM. It was thus concluded that IFC from normal animals do not respond to GH without preincubation. IFC from STZ-DM rats show a lipolytic response to GH without preincubation. Preincubation with GH increases the lipolytic response of IFC from STZ-DM to all lipolytic agents compared to control responses. In addition, ADA greatly enhanced lipolysis in IFC from STZ-DM compared to that in controls. Together these data demonstrate enhanced sensitivity to both lipolytic stimuli and adenosine suppression of lipolysis in IFC from STZ-DM.
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PMID:Lipolysis in diabetic adipocytes: differences in response to growth hormone and adenosine. 362 74

Endurance training helps muscle tissue oxidize lipids and therefore helps conserve glycogen. It was thought interesting to find out if, in addition to this preferential use of fatty acids by muscle tissue, there is an increase in the capacity of adipose tissue to mobilize lipids. So the response to epinephrine of collagenase-isolated fat cells obtained after biopsies of fat performed in the periumbilical region of 10 trained marathon runners (T) and 10 sedentary subjects (S), all males, was studied in vitro. Glycerol release, chosen as adipocyte lipolysis indicator, was measured by bioluminescence. Lipolysis was studied with increased epinephrine concentration. This caused a significant increase in lipolysis only in the T subjects. The dose-response curves were significantly different for T and S subjects at 10(-6) M and above (P less than 0.05). To determine the modification mechanisms observed, lipolysis with isoproterenol and epinephrine plus propranolol were studied. Isoproterenol significantly increased lipolysis in both groups. The dose-response curves were significantly different at 10(-7) M (P less than 0.01) and above. In both groups, epinephrine plus propranolol significantly decreased lipolysis without distinction between T and S. It is concluded that in male subjects endurance training increases the sensitivity of subcutaneous abdominal adipose tissue to the lipolytic action of epinephrine; this effect seems to be related to an increased response of the beta-adrenergic pathways.
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PMID:Effect of physical training in humans on the response of isolated fat cells to epinephrine. 373 12

The activity of chymase was markedly inhibited by phosphoglycerides such as phosphatidic acid, phosphatidylserine, and phosphatidylinositol, but was not affected by acylglycerides, phosphoglyceroserine, serine, inositol, or glycerol. These results suggest that both the nonpolar hydrophobic hydrocarbon tails and the polar hydrophilic head are essential for the inhibitory effects of phosphoglycerides. Binding of a primary amine to an anionic polar head of phosphatidic acid, such as in phosphatidylserine and phosphatidylethanolamine, slightly decreased the inhibitory effect of phosphatidic acid and, conversely, binding of a strong cation to the head, such as in phosphatidylcholine, resulted in its activation of chymase. Phosphatidic acid containing an unsaturated fatty acid, such as dioleoyl phosphatidic acid, caused the same extent of inhibition as natural phosphatidic acid from bovine brain, but was 20 times more inhibitory than phosphatidic acid containing a saturated fatty acid, such as distearoyl phosphatidic acid. The inhibition by phosphatidylserine was noncompetitive and pseudoirreversible, and the Ki value was 0.54 microM. The inhibition of chymase by phosphatidylserine was pH dependent, being strong at pH 8.5 to 9.5 but weak below pH 7.5. Phosphatidylserine specifically inhibited chymase and elastase; it did not inhibit the other chymotrypsin-type serine endopeptidases tested, trypsin, papain, collagenase, carboxypeptidase A, or cathepsin D.
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PMID:Inhibition of chymase activity by phosphoglycerides. 388 53

When adipose tissue cells were incubated with collagenase for different periods of time, cell populations with different mean cell sizes were obtained from the same tissue sample. Lipid synthesis from glucose was studied as a function of adipose cell size and number. The incubations were performed in Parker medium 199, which is suitable for tissue culture of human adipose tissue. The results show that the larger cells of a specimen have a greater rate of lipid synthesis than the smaller cells of the same specimen. This is mainly due to an increase in the synthesis of glyceride-glycerol. Addition of insulin stimulated lipid synthesis. However, the larger adipose cells were less sensitive to the stimulating effect of insulin than the smaller cells.
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PMID:Effect of cell size on lipid synthesis by human adipose tissue in vitro. 432 18

1. A method is described for extracting separately mitochondrial and extramitochondrial enzymes from fat-cells prepared by collagenase digestion from rat epididymal fat-pads. The following distribution of enzymes has been observed (with the total activities of the enzymes as units/mg of fat-cell DNA at 25 degrees C given in parenthesis). Exclusively mitochondrial enzymes: glutamate dehydrogenase (1.8), NAD-isocitrate dehydrogenase (0.5), citrate synthase (5.2), pyruvate carboxylase (3.0); exclusively extramitochondrial enzymes: glucose 6-phosphate dehydrogenase (5.8), 6-phosphogluconate dehydrogenase (5.2), NADP-malate dehydrogenase (11.0), ATP-citrate lyase (5.1); enzymes present in both mitochondrial and extramitochondrial compartments: NADP-isocitrate dehydrogenase (3.7), NAD-malate dehydrogenase (330), aconitate hydratase (1.1), carnitine acetyltransferase (0.4), acetyl-CoA synthetase (1.0), aspartate aminotransferase (1.7), alanine aminotransferase (6.1). The mean DNA content of eight preparations of fat-cells was 109mug/g dry weight of cells. 2. Mitochondria showing respiratory control ratios of 3-6 with pyruvate, about 3 with succinate and P/O ratios of approaching 3 and 2 respectively have been isolated from fat-cells. From studies of rates of oxygen uptake and of swelling in iso-osmotic solutions of ammonium salts, it is concluded that fat-cell mitochondria are permeable to the monocarboxylic acids, pyruvate and acetate; that in the presence of phosphate they are permeable to malate and succinate and to a lesser extent oxaloacetate but not fumarate; and that in the presence of both malate and phosphate they are permeable to citrate, isocitrate and 2-oxoglutarate. In addition, isolated fat-cell mitochondria have been found to oxidize acetyl l-carnitine and, slowly, l-glycerol 3-phosphate. 3. It is concluded that the major means of transport of acetyl units into the cytoplasm for fatty acid synthesis is as citrate. Extensive transport as glutamate, 2-oxoglutarate and isocitrate, as acetate and as acetyl l-carnitine appears to be ruled out by the low activities of mitochondrial aconitate hydratase, mitochondrial acetyl-CoA hydrolyase and carnitine acetyltransferase respectively. Pathways whereby oxaloacetate generated in the cytoplasm during fatty acid synthesis by ATP-citrate lyase may be returned to mitochondria for further citrate synthesis are discussed. 4. It is also concluded that fat-cells contain pathways that will allow the excess of reducing power formed in the cytoplasm when adipose tissue is incubated in glucose and insulin to be transferred to mitochondria as l-glycerol 3-phosphate or malate. When adipose tissue is incubated in pyruvate alone, reducing power for fatty acid, l-glycerol 3-phosphate and lactate formation may be transferred to the cytoplasm as citrate and malate.
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PMID:The intracellular localization of enzymes in white-adipose-tissue fat-cells and permeability properties of fat-cell mitochondria. Transfer of acetyl units and reducing power between mitochondria and cytoplasm. 439 82

Fat cells were isolated by collagenase digestion of adipose tissue from male dogs. The cells were shown to be responsive to both histamine and norepinephrine in producing glycerol, a marker for lipolysis. The histamine-stimulated response was shown to be mediated by H2-receptors because it was inhibited by the H2-receptor antagonists metiamide and cimetidine, but not by H1 blockers such as tripelennamine, or by other agents such as propranolol. Conversely, norepinephrine-stimulated lipolysis was inhibited by propranolol but not by metiamide. The KD estimated for cimetidine and metiamide was approximately 10(-6) M, which compares favorably with that observed in other H2-receptor systems. The relative potency of various histamine H2-receptor agonists in this system was comparable to that seen in other H2-receptor systems. This assay has the advantage of utilizing a homogeneous cell preparation and is not complicated by tachyphylaxis. The dogs which are the source of the adipose tissue can potentially be used for other studies after a suitable recovery period. This system appears useful in screening novel compounds for H2-receptor antagonist or agonist activity.
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PMID:Development of an assay for H2-receptor antagonists using isolated fat cells. 610 50


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