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)

A quantitative evaluation of lectin binding to adult rat hepatocyte cell surfaces was done using cells isolated by two different collagenase perfusion methodologies and cultured as monolayers with two different tissue culture media formulations (protocol I vs. protocol II). The presence of alpha-D-mannosyl and alpha-D-glucosyl groups was detected by the binding of Concanavalin A (Con A), Lens culinaris agglutinin (LCA), and Pisum sativum agglutinin (PSA) to freshly isolated cells. Furthermore, beta-D-galactose [Ricinus communis agglutinin (RCA)] and sialic acid residues [wheat germ (WGA)] were also found. Protocols I and II served as models for evaluation of: a) the stripping effect of collagenase separation procedures, b) the restoration in culture of collagenase-stripped sugar residues, c) the effect of the culture environment on cell viability [as measured by lactic acid dehydrogenase (LDH) leakage] and the protein content of hepatocytes, and d) the presence of cell surface sugar residues as a function of culture duration. The ultrastructural morphology of freshly isolated and cultured hepatocytes was also evaluated. These studies indicated that a decline in lectin binding invariably occurred earlier than a massive leakage of LDH and a decrease in the protein content of the cells in culture. Ultrastructurally, autophagocytosis was an early phenomenon in cells isolated and cultured by protocol I, which was also inferior to protocol II regarding the preservation of hepatocyte glycocalyces. Sugar residues lost due to the collagenase-stripping effect were restored, as shown by lectin binding, within the first 24 h of culture. This stripping effect was confirmed by quantitative evaluations of lectin binding to hepatocytes in culture after an incubation with collagenase. This study shows that the binding of peroxidase-labeled lectins is a useful tool for quantitative evaluation of the sugar composition of hepatocyte cultures.
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PMID:A quantitative analysis of lectin binding to adult rat hepatocyte cell surfaces. 283 55

The zonal distribution of GSH metabolism was investigated by comparing hepatocytes obtained from the periportal (zone 1) or perivenous (zone 3) region by digitonin/collagenase perfusion. Freshly isolated periportal and perivenous cells had similar viability (dye exclusion, lactate dehydrogenase leakage and ATP content) and GSH content (2.4 and 2.7 mumol/g respectively). During incubation, periportal cells slowly accumulated GSH (0.35 mumol/h per g), whereas in perivenous cells a decrease occurred (-0.14 mumol/h per g). Also, in the presence of either L-methionine or L-cysteine (0.5 mM) periportal hepatocytes accumulated GSH much faster (3.5 mumol/h per g) than did perivenous cells (1.9 mumol/h per g). These periportal-perivenous differences were also found in cells from fasted rats. Efflux of GSH was faster from perivenous cells than from periportal cells, but this difference only explained 10-20% of the periportal-perivenous difference in accumulation. Furthermore, periportal cells accumulated GSH to a plateau 26-40% higher than in perivenous cells. There was no significant difference in gamma-glutamylcysteine synthetase or glutathione synthetase activity between the periportal and perivenous cell preparations. The periportal-perivenous difference in GSH accumulation was unaffected by inhibition of gamma-glutamyl transpeptidase or by 5 mM-glutamate or -glutamine, but was slightly diminished by 2 mM-L-methionine. This suggests differences between periportal and perivenous cells in their metabolism and/or transport of (sulphur) amino acids. Our results suggest that a lower GSH replenishment capacity of the hepatocytes from the perivenous region may contribute to the greater vulnerability of this region to xenobiotic damage.
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PMID:Glutathione replenishment capacity is lower in isolated perivenous than in periportal hepatocytes. 290 50

Intact rat liver cells from the perivenous region were isolated by collagenase perfusion after first destroying the periportal region by a brief portal infusion of digitonin. Periportal cells were isolated after retrograde digitonin infusion. Significantly higher alanine aminotransferase, gamma-glutamyltransferase and lactate dehydrogenase activities and lower glutamate dehydrogenase and pyruvate kinase activities in periportal than in perivenous cells demonstrate marked separation. The high yield allows further characterization in vitro of the cell populations.
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PMID:Digitonin-collagenase perfusion for efficient separation of periportal or perivenous hepatocytes. 299 54

A specific and potent synthetic inhibitor of mammalian tissue collagenase and related metallo-proteinases inhibits the collagen matrix resorption induced by parathyroid hormone (PTH) in cultured embryonic mouse calvaria. The inhibition is reversible, dose-dependent and virtually complete at 50 microM inhibitor concentration whereas that due to a less potent stereoisomer is much weaker. The PTH-enhanced secretion of calvarial lysosomal enzymes and the small spontaneous leakage of lactate dehydrogenase are not affected by the inhibitor. These results suggest that collagenase plays a critical role in bone resorption. Its role is discussed in relation to that of cysteine-proteinases that have also been implicated in this process.
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PMID:A new synthetic inhibitor of mammalian tissue collagenase inhibits bone resorption in culture. 300 46

Intact periportal (pp) or perivenous (pv) hepatocytes were prepared by digitonin-collagenase liver perfusion. The degree of separation was indicated by significant differences between the pp and pv cells in their activity of the pp markers, alanine aminotransferase (pp/pv = 2.1), gamma-glutamyltranspeptidase (3.4) and lactate dehydrogenase (1.3), and of the pv markers, glutamate dehydrogenase (0.73) and pyruvate kinase (0.81). This pattern was not altered by a 3-day pretreatment with phenobarbital (PB). The hepatocytes isolated from the pv area contained higher activities of microsomal NADPH-cytochrome c reductase, 7-ethoxycoumarin O-deethylase, 7-ethoxyresorufin O-deethylase and benzo(a)pyrene hydroxylase, and of cytosolic glutathione transferase. Cytochrome P-450 and UDP-glucuronosyltransferase were slightly higher in pv cells. Treatment with PB induced NADPH-cytochrome c reductase, glutathione transferase, cytochrome P-450 and UDP-glucuronosyltransferase but the degree of induction was found to be at least as strong in pp cells as in pv cells. The induction of 7-ethoxyresorufin O-deethylase and 7-ethoxycoumarin O-deethylase was clearly more prominent in pp cells. On the other hand, PB reduced the activities of benzo(a)pyrene hydroxylase and alcohol dehydrogenase in both cell types. These results demonstrate by direct enzyme assay of separated cells the dominance of the pv-region for metabolizing drugs in the normal liver. Contrary to several other studies, however, our data indicate that induction by PB occurs panacinarily, i.e., relatively more in the pp region, thus diminishing rather than exaggerating the original pv dominance.
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PMID:Effect of phenobarbital on the distribution of drug metabolizing enzymes between periportal and perivenous rat hepatocytes prepared by digitonin-collagenase liver perfusion. 302 20

We modified and improved enzyme digestion and density gradient separation procedures to obtain fractions of proximal and distal renal tubules with high yield and viability. Kidneys from two anesthetized adult Wistar rats were flushed with Krebs-Henseleit buffer (KHB) and then perfused in situ with recirculated KHB containing collagenase and hyaluronidase at 125 mmHg. Cortices were excised, minced, and incubated in KHB containing enzymes for 35 min at 37 degrees C. Dissociated tubules were removed at 10-min intervals, rinsed, and placed in KHB containing 10% calf serum, vitamins, and amino acids at 4 degrees C. Separation was achieved by suspending the tissue in 45% isosmotic Percoll layered over an undiluted Percoll cushion and centrifuging. Proximal tubules sedimented near the cushion. Distal segments were isolated in the uppermost bands of a second 35% Percoll separation. Viability was greater than 95% as measured by lactate dehydrogenase leakage and quantitated by oxygen consumption and ATP content. Basal oxygen consumption was greater than 33 nmol O2 X min-1 X mg protein-1 in all fractions and was stimulated by succinate and inhibited by amiloride and ouabain. Basal ATP content averaged 9.7 nmol/mg ATP. An average 3.3-fold separation for the proximal fraction and 24.5-fold separation for the distal fraction was assessed by the enrichment of six specific enzyme markers, with several of the markers indicating separations up to 32-fold. Isolated tubules also displayed functional responses to parathyroid hormone and vasopressin. Distal, but not proximal, segments demonstrated significantly increased adenosine 3',5'-cyclic monophosphate formation with vasopressin.
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PMID:Improved separation method for rat proximal and distal renal tubules. 303 59

Benzyl chloride (BCl) is used in the manufacture of basic and acidic dyes, pharmaceutical products, resins, and synthetic tannins. BCl is known to have caused liver malfunctions in some workers exposed to 2 ppm BCl vapors. This study was conducted to investigate the effect of BCl on isolated male rat hepatocytes using several toxicity parameters. The hepatocytes were isolated by a collagenase perfusion technique and were incubated in airtight tubes with 1.8 and 3.6 mM BCl in a shaking water bath at 37 degrees C for 10, 30, 60, and 120 min. Throughout the incubation period the cell viability was determined by trypan blue exclusion and leakage of cytosolic enzymes such as lactate dehydrogenase (LDH), aspartate transaminase (AST), and alanine transaminase (ALT). Exposure to BCl resulted in a significant decrease in cell viability as assessed by trypan blue and significant increase in leakage of these enzymes compared to the controls.
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PMID:Effect of benzyl chloride on rat hepatocytes. 319 58

Fat-storing cells and other non-parenchymal cells (endothelial and Kupffer cells) were isolated from rat liver by a combined pronase-collagenase procedure and subsequent Visotrast-370 density gradient centrifugation. The lactate dehydrogenase isoenzyme pattern of fat-storing cells was found different from that of other non-parenchymal liver cells. Fat-storing cells contain LDH-4 as the main isoenzyme and do not contain LDH-1, whereas the other non-parenchymal cells have all five lactate dehydrogenase isoenzymes, among which LDH-5 is dominating. All non-parenchymal liver cell populations contain the M-type pyruvate kinase. The alkaline phosphatase of fat-storing cells has the same electrophoretic mobility as that of the other non-parenchymal cells.
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PMID:Isoenzyme patterns of pyruvate kinase, lactate dehydrogenase, and alkaline phosphatase in isolated fat-storing cells of rat liver. 320 45

Parenchymal cells were prepared from the livers of male Sprague-Dawley rats by collagenase perfusion and purified by a self-generating Percoll gradient. The method consisted of mixing 31% Percoll and 5 x 10(6) cells/ml, followed by centrifugation at 10,000 x g for 10 min. A self-generated gradient provided a rapid and efficient recovery of highly viable parenchymal cells. The parenchymal cells were determined to be very stable during incubation at 37 degrees C for at least 2 h. Cell integrity was evaluated by trypan blue dye exclusion, lactate dehydrogenase leakage, and membrane peroxidation. In addition, drug metabolism and conjugation were evaluated as markers of intracellular integrity. With increasing p-nitroanisole (pNA) concentration, the formation of p-nitrophenol (pNP) increased. The rate of sulfation was maximal at a pNA concentration of 0.25 mM and decreased greatly above 1.0 mM. Glucuronidation increased from 0.25 mM to a maximum rate of 2.0 mM pNA. Above 1.0 mM pNA, nonconjugated pNP increased proportionately to the decrease in sulfation. These results indicate that the cell integrity was maintained, and that these cells can be used as a model for studying drug metabolism.
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PMID:A rapid method of preparing hepatic parenchymal cells for studying drug metabolism. 337 62

The dynamics of the release of human placental lactogen (hPL) under basal conditions and response to various secretogogues has been studied in perifused enriched hPL-producing cells from term placentae prepared by the isopycnic centrifugation of collagenase/hyaluronidase-dispersed placental cells on Percoll gradients. Under basal conditions, the perifused cells released hPL at a relatively constant rate for up to 24 h in culture. The mean rates of hPL release from cells (5 x 10(6) cells) from 18 normal full-term placentae varied from 1.8 to 20.2 ng/5 min (mean 7.7 ng/5 min). The cells from term placentae, however, did not release detectable amounts of chorionic gonadotrophin or the cytosolic enzymes lactic dehydrogenase and alkaline phosphatase. The amounts of hPL released by the perifused cells were inversely related to cell density with mean rates of hPL release by 2, 5, and 10 x 10(6) cells of 15.8, 8.6, and 5.7 ng/10(6) cells/0.5 h. The perifused cells responded to provocative stimuli (high-density lipoproteins (HDL), apolipoproteins AI, AII, and CI, partially purified hPL-releasing factor, phorbol esters, sn-1,2-diacylglycerol, and cAMP) in a manner qualitatively similar to enriched trophoblast cells and placental explants in static culture. Release of hPL in response to HDL, apoproteins AI, AII, and CI, and partially purified hPL-releasing factor was dose-dependent and occurred within 5 min of exposure. Basal and stimulated hPL release by perifused trophoblast cells that had been previously frozen at -70 degrees C for four weeks was identical to that of freshly dispersed cells from the same placenta. These experiments indicate that perifused trophoblast cells may be used as a model system to examine the dynamics of hPL release under basal conditions and in response to provocative stimuli.
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PMID:Characterization of placental lactogen release from perifused human trophoblast cells. 339 89


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