Regulation of glutathione metabolism in Ehrlich ascites tumour cells.
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ABSTRACT:
SUBMITTER: Estrela JM
PROVIDER: S-EPMC1133048 | biostudies-other | 1992 Aug
REPOSITORIES: biostudies-other
Similar Datasets
Project description:Ehrlich ascites-tumour cells were investigated with regard to their stability to transport L-lactate by measuring either the distribution of [14C]lactate or concomitant H+ ion movements. The movement of lactate was dependent on the pH difference across the cell membrane and was electroneutral, as evidenced by an observed 1:1 antiport for OH- ions or 1:1 symport with H+ ions. 2. Kinetic experiments showed that lactate transport was saturable, with an apparent Km of approx. 4.68 mM and a Vmax. as high as 680 nmol/min per mg of protein at pH 6.2 and 37 degrees C. 3. Lactate transport exhibited a high temperature dependence (activation energy = 139 kJ/mol). 4. Lactate transport was inhibited competitively by (a) a variety of other substituted monocarboxylic acids (e.g. pyruvate, Ki = 6.3 mM), which were themselves transported, (b) the non-transportable analogues alpha-cyano-4-hydroxycinnamate (Ki = 0.5 mM), alpha-cyano-3-hydroxycinnamate (Ki = 2mM) and DL-p-hydroxyphenyl-lactate (Ki = 3.6 mM) and (c) the thiol-group reagent mersalyl (Ki = 125 muM). 5. Transport of simple monocarboxylic acids, including acetate and propionate, was insensitive to these inhibitors; they presumably cross the membrane by means of a different mechanism. 6. Experiments using saturating amounts of mersalyl as an "inhibitor stop" allowed measurements of the initial rates of net influx and of net efflux of [14C]lactate. Influx and efflux of lactate were judged to be symmetrical reactions in that they exhibited similar concentration dependence. 7. It is concluded that lactate transport in Ehrlich ascites-tumour cells is mediated by a carrier capable of transporting a number of other substituted monocarboxylic acids, but not unsubstituted short-chain aliphatic acids.
| S-EPMC1172721 | biostudies-other
Project description:Ehrlich ascites-tumour cells accumulate Ca2+ when incubated aerobically with succinate, phosphate and rotenone, as revealed by isotopic and atomic-absorption measurements. Ca2+ does not stimulate oxygen consumption by carefully prepared Ehrlich cells, but des so when the cells are placed in a hypo-osmotic medium. Neither glutamate nor malate support Ca2+ uptake in 'intact' Ehrlich cells, nor does the endogenous NAD-linked respiration. Ca2+ uptake is completely dependent on mitochondrial energy-coupling mechansims. It was an unexpected finding that maximal Ca2+ uptake supported by succinate requires rotenone, which blocks oxidation of enogenous NAD-linked substrates. Phosphate functions as co-anion for entry of Ca2+. Ca2+ uptake is also supported by extra-cellular ATP; no other nucleoside 5'-di- or tri-phosphate was active. The accumulation of Ca2+ apparently takes place in the mitochondria, since oligomycin and atractyloside inhibit ATP-supported Ca2+ uptake. Glycolysis does not support Ca2+ uptake. Neither free mitochondria released from disrupted cells nor permeability-damaged cells capable of absorbing Trypan Blue were responsible for any large fraction of the total observed energy-coupled Ca2+ uptake. The observations reported also indicate that electron flow through energy-conserving site 1 promotes Ca2+ release from Ehrlich cells and that extra-cellular ATP increase permeability of the cell membrane, allowing both ATP and Ca2+ to enter the cells more readily.
| S-EPMC1163986 | biostudies-other
Project description:Zinc redistribution between plasma and liver has been examined in mice injected with Ehrlich-ascites-tumour cells. Within 24 h of injection plasma Zn levels decrease and Zn appears in newly synthesized liver metallothionein. This response is dependent upon the number of tumour cells injected into the host. Uptake of Zn into liver and its specific accumulation in a Zn-binding protein, identified as metallothionein, continues for a number of days and reaches a plateau as tumour growth ceases. Over this time period, plasma copper rises. This redistribution also occurs in mice pretreated with cadmium in their drinking water for 1 month at levels of 20, 50, and 100 micrograms/ml. However, in each case there is a lag of 3 days before Zn increases in the livers of these animals which already contain substantial amounts of Cd/Zn-metallothionein. When Ehrlich cells are injected into mice previously placed on a Zn-deficient diet for several days, plasma Zn is already low and no net uptake of Zn into liver metallothionein is apparent. Finally, it is shown that ascites fluid can itself stimulate a transient shift of host of Zn into liver. Heat-inactivated fluid loses this property. It is suggested that, in the peritoneum, tumour cells initiate a stress response mediated by an ascites-fluid factor.
| S-EPMC1152990 | biostudies-other
Project description:1. Adenosine kinase was measured in dialysed extracts from Ehrlich ascites-tumour cells by a chromatographic procedure. 2. In the absence of added Mg(2+) the K(m) values for ATP and adenosine were 0.22mm and 2.8mum respectively. 3. The maximum velocity of adenosine kinase with free ATP was about three times that with the Mg(2+)-ATP complex. Free Mg(2+) was a non-competitive inhibitor of the reaction. A small amount of added Mg(2+), Mn(2+) or Ca(2+) was required for maximum adenosine kinase activity after cation bound to the enzyme had been released by treatment with p-chloromercuribenzoate and then removed by dialysis. 4. GTP, ITP, deoxy-ATP, deoxy-GTP, CTP, xanthosine triphosphate, UTP and thymidine triphosphate could partially or completely replace ATP as a phosphate donor. 5. The reaction of ATP with adenosine kinase was competitively inhibited by AMP, GMP, IMP, ADP, deoxy-ADP and IDP (K(i) 0.2, 1.1, 5.9, 1.2, 0.5 and 0.78mm respectively). Enzymic activity was markedly affected by the relative concentrations of AMP, ADP and ATP in assay mixtures. 6. The results are discussed in terms of possible mechanisms regulating the rate of adenosine kinase in vivo.
| S-EPMC1198536 | biostudies-other
Project description:Ehrlich ascites-tumour cells incubated in a mineral medium supplemented with glucose and glutamine intensely incorporated labelled uridine into free nucleotides and RNA. Detailed kinetic studies of the labelling of total acid-soluble pools of uridine and cytidine nucleotides and of RNA under standard conditions and after chase with non-radioactive uridine were carried out. The relative distribution of the radioactivity between the individual uridine phosphates as well as their total cellular contents were also determined under standard and chase conditions. The labelling kinetics of the mononucleotides and RNA by radioactive uridine and orotate were compared, and some turnover parameters were estimated by mathematical analysis of a steady-state model. The following conclusions were drawn. (1) The observed chase effect of addition of non-radioactive uridine is due to a rapid expansion of the acid-soluble uridine nucleotide pool and consequently a severalfold lowering of its specific radioactivity. (2) The free uridine nucleotides are partly separated into a large and a small compartment, labelled preferentially by exogeneous orotate and uridine respectively. (3) The half-life of the mononucleotides, at least those located in the smaller compartment, is a few minutes only, owing to a rapid exchange with the uridylic acid residues of unstable RNA species. (4) This process of reutilization of RNA-degradation products accounts for 85% (a minimum estimate) of the overall turnover of the nucleotide pool in resting cells.
| S-EPMC1162539 | biostudies-other
Project description:The properties of Ehrlich ascites tumour cells exposed in vivo to cadmium were investigated as a function of the zinc status of the host animals. Tumour-cell growth was inhibited by cadmium in both zinc-sufficient and zinc-deficient animals. However, cells in zinc-sufficient tumours accumulate much less cadmium than those in deficient tumours. The subcellular distributions of cadmium and zinc do not depend on zinc status. Cadmium and zinc are bound to a low-molecular-weight protein with properties similar to metallothionein. Without exposure to cadmium, a zinc- and copper-binding protein is still present that behaves like a metallothionein. This protein can rapidly bind cadmium added to Ehrlich cells in vitro. It is shown that the zinc- and copper-binding protein contains free thiol groups. Ehrlich cells isolated from cadmium-treated animals are viable and show normal incorporation of uridine into RNA, but the cellular uptake of thymidine and its incorporation into DNA are inhibited.
| S-EPMC1161921 | biostudies-other
Project description:The transport and oxidation of succinate by functionally intact Ehrlich ascites-tumour cells was investigated. On the basis of pH dependence and inhibitor sensitivity it was concluded that succinate may be transported across the cell membrane by the organic anion carrier system. Thus the ability of isolated Ehrlich cells to oxidize succinate is real, and is not necessarily a result of damage to cell integrity.
| S-EPMC1164208 | biostudies-other
Project description:1. Aerobic or anaerobic incubation at 37 degrees of Ehrlich ascites-carcinoma cells in Krebs-Ringer bicarbonate medium containing glucose and labelled thiamine results in accumulation in the cell of labelled thiamine, so that the concentration of total labelled thiamine in the cells greatly exceeds (by a factor 7) that in the medium. This concentration ratio is approximately constant for small initial external concentrations of labelled thiamine but diminishes when the latter exceed 0.4mum. 2. All the labelled thiamine in the tumour cells is present as thiamine phosphates. 3. The uptake of labelled thiamine is markedly diminished by decrease of temperature. At 9 degrees concentration ratio (cells/medium) 0.5 is observed whereas at 37 degrees the concentration ratio is 8.6. 4. The extent of phosphorylation of labelled thiamine depends on the period of incubation. 5. The influx of labelled thiamine is diminished by the presence of its analogues, pyrithiamine and Amprol, and also by the presence of thiamine monophosphate and thiamine diphosphate, which are potent inhibitors of thiamine phosphorylation in Ehrlich ascites cells. 6. Labelled thiamine phosphates leak from the cell into the medium, so that eventually all the labelled thiamine, both in the cell and medium, is converted into thiamine phosphates. However, in the presence of 2,4-dinitrophenol (0.1mm) and iodoacetate (1mm) thiamine phosphorylation is diminished, the concentration ratio for labelled thiamine (cells/medium) falls to half its normal value and little or no labelled thiamine phosphates leaks into the medium. 7. In the presence of thiamine phosphates, free labelled thiamine accumulates in Ehrlich ascites cells against a concentration gradient, concentration ratios (cells/medium) greater than unity being evident. 8. The evidence supports the conclusion that thiamine is transferred into the Ehrlich ascites cell by a carrier-mediated energy-assisted process.
| S-EPMC1265068 | biostudies-other
Project description:1. The formation of adenosine 5'-phosphate, guanosine 5'-phosphate and inosine 5'-phosphate from [8-(14)C]adenine, [8-(14)C]guanine and [8-(14)C]hypoxanthine respectively in the presence of 5-phosphoribosyl pyrophosphate and an extract from Ehrlich ascites-tumour cells was assayed by a method involving liquid-scintillation counting of the radioactive nucleotides on diethylaminoethylcellulose paper. The results obtained with guanine were confirmed by a spectrophotometric assay which was also used to assay the conversion of 6-mercaptopurine and 5-phosphoribosyl pyrophosphate into 6-thioinosine 5'-phosphate in the presence of 6-mercaptopurine phosphoribosyltransferase from these cells. 2. At pH 7.8 and 25 degrees the Michaelis constants for adenine, guanine and hypoxanthine were 0.9 mum, 2.9 mum and 11.0 mum in the assay with radioactive purines; the Michaelis constant for guanine in the spectrophotometric assay was 2.6 mum. At pH 7.9 the Michaelis constant for 6-mercaptopurine was 10.9 mum. 3. 25 mum-6-Mercaptopurine did not inhibit adenine phosphoribosyltransferase. 6-Mercaptopurine is a competitive inhibitor of guanine phosphoribosyltransferase (K(i) 4.7 mum) and hypoxanthine phosphoribosyltransferase (K(i) 8.3 mum). Hypoxanthine is a competitive inhibitor of guanine phosphoribosyltransferase (K(i) 3.4 mum). 4. Differences in kinetic parameters and in the distribution of phosphoribosyltransferase activities after electrophoresis in starch gel indicate that different enzymes are involved in the conversion of adenine, guanine and hypoxanthine into their nucleotides. 5. From the low values of K(i) for 6-mercaptopurine, and from published evidence that ascites-tumour cells require supplies of purines from the host tissues, it is likely that inhibition of hypoxanthine and guanine phosphoribosyltransferases by free 6-mercaptopurine is involved in the biological activity of this drug.
| S-EPMC1206406 | biostudies-other
Project description:1. The purine bases adenine, hypoxanthine and guanine were rapidly incorporated into the nucleotide fraction of Ehrlich ascites-tumour cells in vivo. 2. The reaction of 5'-phosphoribosyl pyrophosphate with adenine phosphoribosyltransferase from ascites-tumour cells (K(m) 6.5-11.9mum) was competitively inhibited by AMP, ADP, ATP and GMP (K(i) 7.5, 21.9, 395 and 118mum respectively). Similarly the reactions of 5'-phosphoribosyl pyrophosphate with both hypoxanthine phosphoribosyltransferase and guanine phosphoribosyltransferase (K(m) 18.4-31 and 37.6-44.2mum respectively) were competitively inhibited by IMP (K(i) 52 and 63.5mum) and by GMP (K(i) 36.5 and 5.9mum). 3. The nucleotides tested as inhibitors did not appreciably compete with the purine bases in the phosphoribosyltransferase reactions. 4. It was postulated that the purine phosphoribosyltransferases of Ehrlich ascites-tumour cells may be effectively separated from the adenine nucleotide pool of these cells.
| S-EPMC1265200 | biostudies-other