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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)

1. Tubule fragments were isolated after treatment of rat kidney cortex with collagenase. The formation of glucose and lactate on incubation with 5mM-pyruvate was then measured under various conditions. 2. When tubule fragments were isolated from fed rats in the absence of Ca2+ and then incubated with various Ca2+ concentrations, an incubation period of 15--30 min was necessary to establish a metabolic steady state. Under these conditions glucose formation was increased by Ca2+, adrenaline or 3':5'-cyclic AMP to a greater extent than was lactate formation. Data show that appreciable lactate formation could not have resulted from glycolytic metabolism of glucose formed by gluconeogenesis during incubation. 3. When tubule fragments were isolated from fed rats in the presence of 1.27 mM-Ca2+ and adjustments made to the Ca2+ concentration at the commencement of incubation, metabolic steady state was rapidly established. Under these conditions lactate formation was almost insensitive to Ca2+ concentration (0.16--4.5 mM), whereas glucose formation varied with Ca2+ concentration in a sigmoidal manner. 3':5'-Cyclic AMP decreased this sigmoidicity. 4. Ca2+ depletion of the tissue before incubation appeared to change permanently the relationship between extracellular Ca2+ concentration and the measured rates of metabolic processes. 5. Under conditions of metabolic steady state, glucose formation by tubule fragments from fed rats was less sensitive than lactate formation to inhibition by 3-mercaptopicolinate or 2-n-butylmalonate. Lactate formation by tubule fragments prepared from 48 h-starved rats was more sensitive to these inhibitors. 6. Estimates were made of the rate of futile cycling of C3 species through pyruvate kinase. This was greater in the starved than in the fed state, was decreased by 3':5'-cyclic AMP in both the fed and the starved state, but was unaffected by Ca2+. 7. These results suggested that formation of lactate and glucose is less tightly linked in kidney cortex than in liver. A considerable amount of the supply of reducing equivalents for lactate formation did not appear to be associated with an energy-dependent translocation from mitochondria to cytosol involving a pyruvate leads to oxaloacetate leads to phosphoenolpyruvate leads to pyruvate cycle.
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PMID:A study of regulation of gluconeogenesis and the supply of cytosolic reducing equivalents for lactate formation in rat kidney-cortical-tubule fragments incubated with pyruvate. 21 19

The use of pancreatic lobule preparations is one of the well-established approaches to study stimulus-secretion coupling in the exocrine pancreas in vitro. We have developed a kinetic system for the perfusion and intermittent incubation of rat pancreatic lobules. This model allows repeated hormone stimulation for up to 3 h while permitting rapid changes of the cellular environment with no accumulation of secretory or metabolic products. Tissue viability could be demonstrated over 6 h by in vivo toluidine blue exclusion, histology and electron microscopy. Lactate dehydrogenase leakage from cells over 6 h was only 2.4% of total content. No activated trypsin was detected in the perfusion medium. A biphasic dose response was established for cholecystokinin stimulation with a maximal response at 10(-8) M. We conclude that kinetic perfusion and incubation are technically feasible with rat pancreatic lobules. This in vitro model appears particularly suited for the investigation of pharmacologic and metabolic effects on the pancreatic acinar cell when rapid changes of the cellular environment are required and when the accumulation of secretory and metabolic products must be avoided. The technique described requires neither protease inhibition in the medium nor collagenase treatment of the cells.
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PMID:In vitro perfusion and incubation of rat pancreatic lobules in a kinetic system. 172 99

The dentin was removed from bovine tooth germs, followed by the separation of the extracellular matrix vesicle fraction after collagenase treatment. Lactate dehydrogenase (LDH)-containing vesicles with a density different from that of matrix vesicles were detected in the matrix vesicle fraction. LDH in these vesicles did not result from cell lysis and vesicle capture during the preparation of the matrix vesicle fraction. The isoenzyme pattern of LDH in LDH-containing vesicles was similar to that of cytosolic LDH of odontoblasts. Other cytosolic enzymes were not detected in LDH-containing vesicles, suggesting the presence of a mechanism for specific uptake of cytosolic LDH during the in vivo formation of the vesicles.
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PMID:Presence of vesicles containing lactate dehydrogenase in the dentin of bovine tooth germs. 196 Feb 56

Lactate transport was studied in giant (median diameter 6.3 microns) sarcolemmal vesicles obtained by collagenase treatment of rat skeletal muscle. The lactate transport displayed stereospecificity, had a high temperature coefficient, and could be inhibited up to 90% with known transport inhibitors (PCMBS and cinnamate). In equilibrium exchange experiments, the L-lactate flux demonstrated saturation kinetics with Km = 23.7 mM and Vmax = 108 pmol cm-2 s-1. With lactate present on only one side of the membrane, (zero trans conditions), Vmax was reduced to 48 pmol cm-2 s-1. The flux rate displayed transacceleration. The lactate flux was coupled to a parallel H+ flux. Under equilibrium exchange conditions, the carrier-mediated lactate flux was not pH-dependent. In the zero trans experiments, H+ on the trans side acted as an inhibitor. The loaded form of the carrier reorients faster than the unloaded form, and the protonated form with no lactate bound reorients slowly or is immobile. When compared to intact muscles, the giant sarcolemmal vesicles retain their transport characteristics both qualitatively and quantitatively.
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PMID:Muscle lactate transport studied in sarcolemmal giant vesicles. 204 48

Two cell populations were isolated from calvaria of chick embryos: PF cells were liberated by collagenase treatment from the periosteum, OB cells from the periosteum-free calvarium. Both populations were cultured in plastic culture dishes. After 6 d of culture, monolayers of each cell type either were scraped off the culture dishes, transplanted on the chorio-allantoic membrane of 7-d-old quail eggs, and cultured there for 6 d, or were used for biochemical experiments. OB transplants proved capable of producing calcified bone matrix, whereas PF transplants formed only fibrous tissue. Biochemically, OB cells showed high cAMP production in the presence of parathyroid hormone (PTH), whereas cAMP production was not stimulated in PF cultures. Lactate production was stimulated by PTH in both populations although somewhat differently. Citrate decarboxylation was high in OB cells and was inhibited by PTH but was low in PF cells, where it was stimulated by the same hormone. The differences in hormonal response between the two cell types made it possible to conclude that PF cultures are relatively free of OB cells. The PF contamination in OB cultures was more difficult to assess. The experiments described in this report show that the OB population contains osteoblasts or osteoblastlike cells which are, under favorable circumstances, capable of bone formation.
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PMID:Bone formation and calcification by isolated osteoblastlike cells. 617 44

Synthetic hydroxyapatite (HA) and calcium pyrophosphate dihydrate (CPPD) microcrystals are phagocytosed by rabbit articular-cartilage chondrocytes in primary culture. The ingestion of crystals greatly stimulated the release of collagenase, neutral protease, and prostaglandins E2 and F2 alpha into the ambient medium. Lactate dehydrogenase was not released by either crystal despite electron microscopic evidence of cell damage by HA crystals (partial loss of phagolysosomal membrane and increased myelin figures). HA, but not CPPD crystals, stimulated release of beta-glucuronidase. HA crystal concentrations from 50 to 200 micrograms ml-1 induced a dose-dependent release of collagenase and of extracellular protein. Both phagocytosis and collagenase release were greatly attenuated when HA crystals were added to the chondrocyte monolayers in the absence of serum. As HA and CPPD crystals have been identified in human articular cartilage in association with degenerative changes, it is possible that the cell-crystal interaction described here may be pathogenetically important.
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PMID:Phagocytosis of hydroxyapatite or calcium pyrophosphate dihydrate crystals by rabbit articular chondrocytes stimulates release of collagenase, neutral protease, and prostaglandins E2 and F2 alpha. 630 68

Cells with electron-microscopic characteristics of myofibroblasts were isolated from baboon liver biopsy specimens by collagenase digestion and Percoll density gradient centrifugation and then cultured. The cultures consisted of only one cell type. By immunofluorescence, these cells synthesized collagen types I, III, and IV and laminin. Typical features of myofibroblasts were maintained throughout many passages in the culture. To study the effects of ethanol (and its oxidation product acetaldehyde and associated metabolite lactate) on myofibroblast collagen synthesis, the cell cultures were incubated for 24 h in a medium containing either 50 mM ethanol, 200 microM acetaldehyde, or 5 mM lactate. The cells did not contain significant alcohol dehydrogenase activity. Acetaldehyde stimulated significantly (p less than 0.05) myofibroblast collagen synthesis without changing noncollagen protein synthesis or proline pools. Lactate caused a significant (p less than 0.02) increase in intracellular proline pool and collagen synthesis. Ethanol itself did not have any effect on collagen synthesis of myofibroblasts. The stimulation of collagen synthesis of hepatic myofibroblasts by acetaldehyde and lactate may contribute to the development of alcoholic liver fibrosis, as alcohol intake is known to elevate acetaldehyde and lactate in tissues and blood.
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PMID:Acetaldehyde and lactate stimulate collagen synthesis of cultured baboon liver myofibroblasts. 638 Dec 14

The effect of ethanol on the secretion of proteins was studied in hepatocytes isolated from 24-h fasted rats and from fed rats. Hepatocytes were isolated after collagenase disruption of the liver and incubated in a standard medium containing amino acids, bovine albumin, glucose, penicillin and streptomycin in HEPES buffer. Cell viability was determined by urea production and trypan blue exclusion. When studying protein export, a model had to be chosen in which the labeling is accomplished before the addition of the test agents. Cells were incubated with [3H]valine for 2.5 and 7.5 min followed by a 15-mM valine chase and the incubates were adjusted to final concentrations of ethanol of 50 mM, 100 mM, colchicine 5-50 microM or cycloheximide 18 microM. Cells and media were harvested at various times, and counts incorporated into medium and cell protein were determined. Cycloheximide inhibited protein synthesis by 99%, decreased protein secretion by 10-20%, but did not further inihibit protein labeling when given after the chase confirming the chase's effectiveness. Colchicine inhibited protein release by 27-54% depending on the dose. With control cells labeled protein and specifically albumin appeared in the medium 20 min from the start of the pulse and this release of protein was not inhibited by 50 mM or 100 mM ethanol incubated with cells from the same animal whether the donor has been fed or fasted. The values for the ethanol-treated cells ranged from 94.0 to 113% of the control values from 30 to 120 min after the addition of the pulse. Lactate levels were markedly elevated, and urea synthesis decreased in the presence of either 50 mM EtOH or 100 mM EtOH. Thus using a method that can distinguish the effect of ethanol on synthesis from secretion, it is concluded that acute exposure to EtOH does not interfere with protein secretion.
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PMID:Protein secretion in suspensions of isolated rat hepatocytes: no influence of acute ethanol administration. 745 Apr 1

Brain edema has been shown to increase brain lactate, but the effect on pH is unclear. We used in vivo nuclear magnetic resonance (NMR) spectroscopy to measure lactate and pH in a region of brain edema. Ninety-five anesthetized rats underwent proton and 31P-NMR spectroscopy with a 7-T 89-mm vertical bore spectrometer, using a surface coil over the edematous regions and distant from a hemorrhage produced by the injection of bacterial collagenase. Brain water content was measured from multiple regions after the NMR measurements in all rats. Lactate was significantly increased 4 h after the hemorrhage and remained elevated for 48 h, but brain pH was unaffected. The increase in lactate correlated (P < 0.01) with the increase in water content in the measured region. We conclude that lactate and pH are dissociated in a region of primarily vasogenic edema.
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PMID:Brain lactate and pH dissociation in edema: 1H- and 31P-NMR in collagenase-induced hemorrhage in rats. 821 65

Lactate dehydrogenase-containing vesicles have been isolated from the extracellular matrix of the calvaria of new-born mice. The calvariae, intramembranous ossification tissue, were removed from 2-day-old mice, followed by the separation of the extracellular matrix vesicle fraction after collagenase digestion. Lactate dehydrogenase-containing vesicles with a density different from that of matrix vesicles were detected in the matrix vesicle fraction. Lactate dehydrogenase in these vesicles did not result from cell lysis and vesicle capture during the preparation of the matrix vesicle fraction. The isoenzyme pattern of lactate dehydrogenase in lactate dehydrogenase-containing vesicles was nearly identical to that of cytosolic lactate dehydrogenase of the cell fraction. Other cytosolic enzymes were not detected in lactate dehydrogenase-containing vesicles, suggesting the presence of a mechanism for specific uptake of cytosolic lactate dehydrogenase during the in vivo formation of the vesicles. This is the first report on the presence of lactate dehydrogenase-containing vesicles in the intramembranous ossification site.
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PMID:Presence of lactate dehydrogenase-containing vesicles in an intramembranous ossifying tissue: new-born mouse calvaria. 834 86


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