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The effect of calcium ions on the glycolytic activity of Ehrlich ascites-tumour cells.

ABSTRACT: 1. Added Ca(2+) inhibited lactate formation from sugar phosphates by intact Ehrlich ascites-tumour cells. Lactate formation from glucose by these cells was unaffected by added Ca(2+). 2. The Ca(2+) inhibition of lactate formation by intact cells occurred in the extracellular medium. 3. Intact ascites-tumour cells did not take up Ca(2+)in vitro. 4. Glycolysis of sugar phosphates by cell extracts as well as pyruvate formation from 3-phosphoglycerate and phosphoenolpyruvate was inhibited by Ca(2+). 5. It was concluded that Ca(2+) inhibited the pyruvate-kinase (EC 2.7.1.40) reaction. Further, Ca(2+) inhibition of pyruvate kinase could be correlated with the overall inhibition of glycolysis. 6. Concentrations of Ca(2+) usually present in Krebs-Ringer buffers, inhibited glycolysis and pyruvate-kinase activity by approx. 50%. 7. The inhibition of glycolysis by added Ca(2+) could be partially reversed by K(+) and completely reversed by Mg(2+) or by stoicheiometric amounts of EDTA. 8. The hypothesis is advanced that the inability of tumour cells to take up Ca(2+) is a factor contributing towards their high rate of glycolysis.

SUBMITTER: Bygrave FL

PROVIDER: S-EPMC1270131 | biostudies-other | 1966 Nov

REPOSITORIES: biostudies-other

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Transport of calcium ions by Ehrlich ascites-tumour cells.

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

Calcium-binding protein from mouse Ehrlich ascites-tumour cells is homologous to human calcyclin.

Project description:A Ca2+-binding protein was purified from mouse Ehrlich ascites-tumour cells. The protein forms monomers and disulphide-linked dimers, which can be separated by reverse-phase h.p.l.c. A partial amino acid sequence analysis demonstrated that the protein has an EF-hand structure. A striking homology was found to rat and human calcyclin (a member of the S-100 protein family), which is possibly involved in cell-cycle regulation.

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L-lactate transport in Ehrlich ascites-tumour cells.

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.

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Calcium-ion transport by intact Ehrlich ascites-tumour cells. Role of respiratory substrates, Pi and temperature.

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Some properties of adenosine kinase from Ehrlich ascites-tumour cells.

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Project description:1. Pyruvate kinase (ATP-pyruvate phosphotransferase, EC 2.7.1.40) from Ehrlich ascites-tumour cells was purified approximately fivefold by chromatography on DEAE-cellulose. The enzyme was shown to have an absolute requirement for one univalent and for one bivalent metal ion. 2. The univalent metal ion requirements were satisfied by K(+), Rb(+) or NH(4) (+); Na(+) and Cs(+) were weak activators but Li(+) was inactive. 3. Ca(2+) exhibited ;non-competitive' and ;apparent competitive' effects in relation to the K(+) activation. 4. The bivalent metal ion requirements were satisfied by Mg(2+), Mn(2+) or Co(2+); Ba(2+), Sr(2+), Ca(2+), Ni(2+), Be(2+) and Cu(2+) were inactive. Mn(2+) and Co(2+) were better activators than Mg(2+). 5. The bivalent metal ion requirements of purified pyruvate kinase from rabbit muscle were satisfied by Mg(2+), Mn(2+), Co(2+) and to a smaller extent by Ni(2+). Mn(2+) and Co(2+) were better activators than Mg(2+). 6. Ca(2+) competitively inhibited the activation by Mg(2+), Mn(2+) and Co(2+) for both the tumour and rabbit enzymes. 7. It is concluded that there are no significant differences in metal ion specificity between the tumour and rabbit enzymes. 8. The possible role of metal ions in regulating enzymic and metabolic activities is considered further.

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The transport and oxidation of succinate by Ehrlich ascites-tumour cells.

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.

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INHIBITION OF PRUINE PHOSPHORIBOSYLTRANSFERASES OF EHRLICH ASCITES-TUMOUR CELLS BY 6-MERCAPTOPURINE.

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

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