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 isolation of tubules and cells from human kidney cortex was realized by an enzymatic method. Tubules and cells were released from slices of kidney cortex by collagenase. The yield amounted to 80 % of the wet weight of incubated cortex slices. Thus numerous experiments with isolated tubules from one organ could be performed. Glucose production from different substrates was measured in order to test the biochemical integrity of the isolated cells. The highest rates of glucose formation were obtained with fructose as precursor. Glucose production was higher from lactate than from pyruvate. With proline and glutamine as substrates only small amounts of glucose were produced. Glucose formation from 10 mmol/1 pyruvate was linear with time up to 80 minutes. Ado-3':5'-P stimulated glucose formation at 10 mumolar concentration and inhibited gluconeogenesis at 1 mmolar, 0.1 mmolar and 1 mumolar concentrations.
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PMID:[An enzymatic method for the isolation of tubules and cells from human kidney cortex]. 16 42

Renal gluconeogenesis was studied in suspended tubule fragments isolated by collagenase treatment of rat kidney cortices. Angiotensin II increased glucose formation from pyruvate, lactate, and to a lesser extent from oxoglutarate and glutamine, but not from other substrates such as malate, succinate, dihydroxyacetone or fructose. Stimulation was significant with peptide concentration exceeding 1 . 10(-8) M and was also shown with an 8-Sar derivative. Other peptides such as 4-Ala-8-Ile-angiotensin II, hexapeptide and bradykinin had no effect. The stimulatory action of angiotensin II was additive to that of L-lysine, and 3',5'-adenosine cyclic monophosphate, suggesting a different mechanism of action. In the presence of maximally stimulatory concentrations of oleate, phenylephrine and 3',5'-guanosine cyclic monophosphate, however, the stimulatory effect of angiotensin II was absent. Cyclic GMP levels, however, did not increase in tubules after angiotensin II and phenylephrine addition, making a messenger function of this nucleotide unlikely. Omission of Ca2+ from the medium markedly reduced basal gluconeogenesis but did not result in a complete loss of angiotensin II effect. Reduction of medium potassium to 2 mM, however, increased basal gluconeogenesis and blunted the peptide effect. 1 mM ouabain was also able to inhibit the stimulatory effect of angiotensin II. Therefore changes in intracellular potassium levels are discussed as a possible mechanism of angiontensin action, whereas calcium seems not to be specifically linked to this metabolic action of angiotensin on the proximal tubule.
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PMID:Stimulation of renal gluconeogenesis by angiotensin II. 45 78

Kidney-cortex tubule suspensions were prepared by collagenase treatment of kidney cortex from fed and starved rats. This preparation, consisting mainly of proximal convoluted tubules was incubated with three major renal substrates, L-lactate, glutamine and oleate to study the dose dependence of substrate uptake rates from medium substrate combinations. All three substances, when added at near physiological concentrations, modified the uptake rate and fate of the other substrates. In accordance with previous observations, oleate inhibited lactate uptake, and lactate decreased glutamine metabolism. Glutamine on the other hand led to a marked increase in lactate uptake. Both, glutamine and lactate increased oleate metabolism. Glucose was the main product of lactate and glutamine metabolism, lactate being preferentially taken up for this process. Oleate led to a net synthesis of triglycerides in the tubules, which was stimulated by the addition of lactate and glutamine. More than 75% of the oleate taken up was recovered as triglycerides. In the absence of fatty acids, triglyceride content of tubules decreased. The results indicate that oleate is taken up in preference to lactate and glutamine when all three substrates are offered to the tubule. Glucose and triglycerides are the main metabolic products of tubular substrate metabolism. Whereas glucose is released into the medium, triglycerides are stored in the tubule cell.
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PMID:Metabolism of isolated kidney tubules. Interactions between lactate, glutamine and oleate metabolism. 49 17

Rat kidneys extract citrulline derived from the intestinal metabolism of glutamine and convert it stoichiometrically into arginine. This pathway constitutes the major endogenous source of arginine. We investigated the localization of enzymes of arginine synthesis, argininosuccinate synthase and lyase, and of breakdown, arginase and ornithine aminotransferase, in five regions of rat kidney, in cortical tubule fractions and in subcellular fractions of cortex. Argininosuccinate synthase and lyase were found almost exclusively in cortex. Arginase and ornithine aminotransferase were found in inner cortex and outer medulla. Since cortical tissue primarily consists of proximal convoluted and straight tubules, distal tubules and glomeruli, we prepared cortical tubule fragments by collagenase digestion of cortices and fractionated them on a Percoll gradient. Argininosuccinate synthase and lyase were found to be markedly enriched in proximal convoluted tubules, whereas less than 10% of arginase and ornithine aminotransferase, were recovered in this fraction. Arginine production from citrulline was also enriched in proximal convoluted tubules. Subcellular fractionation of kidney cortex revealed that argininosuccinate synthase and lyase are cytosolic. We therefore conclude that arginine synthesis occurs in the cytoplasm of the cells of the proximal convoluted tubule.
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PMID:Cellular and subcellular localization of enzymes of arginine metabolism in rat kidney. 131 26

Equine articular chondrocytes were isolated from explant cartilage cultures by digestion in a 0.075% collagenase solution for 15 to 19 hours. Cartilage from late-term fetal and neonatal foals resulted in mean chondrocyte yield of 51.99 x 10(6) cells/g of cartilage (wet weight), compared with a yield of 17.83 x 10(6) cells/g for foals 3 to 12 months old. Propagation of chondrocytes in monolayer and 3-dimensional culture was accomplished, using Ham's F-12 as the basal medium, with supplements of fetal bovine serum (10%), ascorbic acid, alpha-ketoglutarate, and L-glutamine. The medium was buffered with HEPES, and penicillin and streptomycin were added for microorganism control. In primary monolayer cultures of freshly isolated chondrocytes, the population doubling time was approximately 6 days. Dedifferentiation of chondrocytes toward a more fibroblastic-appearing cell was observed after the fifth passage (subculture), but was hastened by lower cell-plating density. Chondrocytes were frozen for periods of up to 9 months, using 10% dimethyl sulfoxide as the cryoprotectant. Cell viability of late-term fetal and neonatal foal chondrocytes after storage at -196 C decreased from 86% at 3 weeks to 31% at 12 weeks. Viability of cells derived from older foals and young adult horses was considerably better than that of cells from neonatal foals. Frozen chondrocytes can be stored for extended periods and thawed for immediate implantation or can be sustained in vitro in monolayer or 3-dimensional culture. Such cultures would be suitable for cartilage resurfacing experiments or in vitro assessment of various pharmaceuticals.
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PMID:Isolation, propagation, and cryopreservation of equine articular chondrocytes. 147 23

A genetic approach to define the role of collagenase in physiological and pathological bone remodeling is to identify spontaneous mutations in the collagenase gene which alter enzymatic activity. Alternatively it is possible, though site-directed mutagenesis, to alter genes encoding critical amino acid sequences in the collagen substrate, in a manner analogous to the successful development of animal models for osteogenesis imperfecta. We have thus utilized this approach to alter the Col1a1 gene to encode amino acid substitutions in sequences around the known collagenase cleavage site (glycine-isoleucine at positions 775-776) in type I collagen, and transfect these genes into homozygous Mov-13 fibroblasts, in which the endogenous Col1a1 gene is inactive. Nonconservative substitutions of proline for isoleucine at the P1' site and double substitutions of proline for glutamine (P2) and alanine (P2') resulted in type I collagen resistant to hydrolysis by collagenase. Furthermore, in normal fibroblasts transfected with a mutant Col1a1 gene encoding collagenase resistance in which an additional methionine substitution at position 776 provided a marker for the mutant protein, mutant and wild type triple helical molecules were synthesized and secreted as heterotrimers. A single mutant alpha 1(I) chain did not prevent cleavage of the wild type alpha 1(I) chain but it is likely that the uncleaved alpha 1(I) chain would prevent dissociation of the triple helical fragments containing the other cleaved chains. Introduction of these genes into transgenic mice should result in abnormal phenotypes characterized by altered connective tissue remodeling.
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PMID:Site-directed mutagenesis of type I collagen: effect on susceptibility to collagenase. 148 89

We investigated the steroid biosynthetic capabilities of ovarian granulosa and thecal elements of the viviparous dogfish, Squalus acanthias. In this report we present evidence that granulosa cells secrete quantitatively important amounts of progesterone (P), testosterone (T), and estradiol-17 beta (E), while theca has a more limited capacity to synthesize T and E. Ovarian granulosa cells were obtained from animals at each stage of gestation. After collagenase dispersion, an aliquot of 250,000 cells was incubated at 18 degrees C in basal medium, containing Eagle's salts, glutamine, penicillin, streptomycin and adjusted with 136 mM sodium chloride and 350 mM urea. After a 4 hour incubation, the content of P, T, and E in medium was determined by radioimmunoassay. P was not detectable at any time, while E was present throughout the cycle, being maximal when gestation is three quarters complete (Stage C). T gradually increased from Stage B toward late pregnancy. In Stage C granulosa cells, E production increased in the presence of graded doses of T substrate. Also, a homologous pituitary extract (1/25 equivalents) and the calcium ionophore A23187 stimulated production of all 3 steroids. Using radioisotopes, granulosa cells showed a wide range of synthetic capacities. In Stage C thecal tissue, E production also increased in the presence of graded doses of T substrate, while pituitary extract only increased T. When granulosa and theca were recombined, in the presence of pituitary extract, P levels decreased with a corresponding increase in T, when compared to granulosa alone. These data suggest a possible interaction between granulosa and theca for steroid biosynthesis.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Regulation of ovarian steroidogenesis in vitro in the viviparous shark, Squalus acanthias. 172 88

As a first step in attempting to isolate the Na(+)-dependent System N transporter from rat liver we have investigated the use of prophase-arrested oocytes from Xenopus laevis for the functional expression of rat liver glutamine transporters. Individual oocytes, defolliculated by collagenase treatment, were injected with 50 nl of a 1 mg.ml-1 solution of poly(A)+ RNA (mRNA) isolated from rat liver. 50 microM L-[3H]glutamine uptake was measured 1-5 days post-injection: after 48 h, poly(A)+ RNA-injected oocytes showed a 60 +/- 12% increase in Na(+)-dependent glutamine uptake compared to controls. This increased uptake showed characteristic features of hepatic System N: that is, it tolerated Li(+)-for-Na+ substitution and was inhibited by the System N substrate L-histidine (5 mM) in Li medium, unlike endogenous Na(+)-dependent glutamine transport. In subsequent experiments rat liver poly(A)+ RNA, size-fractionated by density gradient fractionation, was injected into oocytes. Injection of poly(A)+ RNA of 1.9-2.8 kilobases (kb) in size resulted in a significant stimulation of Na(+)-dependent glutamine transport to 0.362 +/- 0.080 pmol.min-1/oocyte from 0.178 +/- 0.060 pmol.min-1/oocyte in vehicle-injected oocytes (p less than 0.01). A lighter fraction, with poly(A)+ RNA of less than 1.9 kilobases size resulted in a similar increase in Na(+)-dependent glutamine uptake which was largely Li(+)-tolerant: Li(+)-stimulated glutamine uptake in oocytes injected with this fraction increased to 0.230 +/- 0.070 pmol.min-1/oocyte from 0.098 +/- 0.029 pmol.min-1/oocyte in controls (p less than 0.05). This enhanced rate of Li(+)-stimulated glutamine uptake was inhibited 28 and 70%, respectively, by 1 and 5 mM L-histidine. Na(+)-independent uptake of glutamine rose by 72 +/- 12% in oocytes injected with poly(A)+ RNA of 2.8-3.6 kb (p less than 0.001). These results demonstrate that glutamine transporters, with characteristics associated with hepatic Systems N, L, and A (or ASC), can be expressed in X. laevis oocytes injected with specific size fractions of rat liver mRNA.
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PMID:Expression of rat liver glutamine transporters in Xenopus laevis oocytes. 174 Apr 35

This study assessed and compared the rate of glucose utilization, activity of the hexose-monophosphate shunt (HMS), and the oxidation of glutamine, lactate, and palmitate in Kupffer (KC), endothelial (EC), and parenchymal liver cells (PC). Cells were isolated by collagenase and pronase digestion followed by centrifugal elutriation. The freshly isolated cells were incubated in the presence of 5 mM glucose, 0.5 mM glutamine, 1 mM lactate, and 0.4 mM palmitate, and the oxidation rate of individual substrates was determined by the measurement of 14CO2 production. Glucose utilization was assessed by detritiation of [2-3H]glucose. Glucose flux through HMS was 2.6, 1.6, and 0.72 nmol.h-1.mg protein-1 in KC, EC and PC, respectively. The oxidation rate of palmitate in PC (3.5 nmol.h-1.mg protein-1) was about twofold greater than in nonparenchymal cells. Glutamine oxidation was 6.1, 4.2, and 2.1 nmol.h-1.mg protein-1 in KC, EC, and PC, respectively. In contrast, oxidation of exogenous lactate by PC (32.1 nmol.h-1.mg protein-1) was about seven- to eightfold greater than by KC or EC. Presence of prevailing lactate concentrations did not inhibit glucose oxidation in these cells, while it attenuated glucose utilization by PC. Our data show that in the presence of a physiological substrate mixture, less than 20% of the ATP generated from exogenous substrates is derived from glycolysis in KC or EC. Oxidation of glutamine and palmitate are the main sources for energy in these cells. In PC, however, lactate and palmitate oxidation is responsible for approximately 90% for the ATP production derived form the oxidation of exogenous substrates.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Glutamine and fatty acid oxidation are the main sources of energy for Kupffer and endothelial cells. 187 92

Previous investigators have reported a protective effect of some prostaglandins and of the prostaglandin E2 analogue enprostil on carbon tetrachloride (CCl4)-induced injury of liver cells. In the present study liver cells were isolated from the rat liver by collagenase perfusion and suspended in F-10 medium, containing 20% foetal bovine serum, 1% gentamicin, and 1% glutamine. In the first study cells were cultured in T-flasks with 3 ml suspension of 6 x 10(6) cells/ml, and in the second study (extended dose response) cells were cultured in tissue culture wells with 0.5 ml cell suspension. Misoprostol was added to groups of cultures 15 min before CCl4, 2 microliters/ml, and the number of living cells was counted 45 min after the first addition. The number of living cells was compared with those of other groups with CCl4 only and control groups. In the first experiment misoprostol was given in doses of 200, 400, and 800 ng/ml medium and in the second experiment in 0.1, 1, 10, 100, and 1000 ng/ml medium. CCl4 is an agent well known to be toxic to liver cells, and in cultures to which only CCl4 was added, the number of living cells was significantly reduced compared with controls. When 0.1 ng misoprostol was added before CCl4, no significant difference in the number of living cells was shown compared with cultures with CCl4 only. On the other hand, misoprostol given in doses from 1 ng to 1000 ng before CCl4 resulted in a higher number of living cells, indicating a protective effect.
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PMID:Effect of the prostaglandin E1 analogue misoprostol on the carbon tetrachloride-induced injury of rat liver cells in culture. 194 73


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