Transport of calcium ions by Ehrlich ascites-tumour cells.

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.     

L-lactate transport in Ehrlich ascites-tumour cells.

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.     

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

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.     

Studies on the interaction of metal ions with puruvate kinase from Ehrlich ascites-tumour cells and from rabbit muscle

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 NH₄⁺; Na⁺ and Cs⁺ were weak activators but Li⁺ was inactive. 3. Ca²⁺ exhibited `non-competitive' and `apparent competitive' effects in relation to the K⁺ activation. 4. The bivalent metal ion requirements were satisfied by Mg²⁺, Mn²⁺ or Co²⁺; Ba²⁺, Sr²⁺, Ca²⁺, Ni²⁺, Be²⁺ and Cu²⁺ were inactive. Mn²⁺ and Co²⁺ were better activators than Mg²⁺. 5. The bivalent metal ion requirements of purified pyruvate kinase from rabbit muscle were satisfied by Mg²⁺, Mn²⁺, Co²⁺ and to a smaller extent by Ni²⁺. Mn²⁺ and Co²⁺ were better activators than Mg²⁺. 6. Ca²⁺ competitively inhibited the activation by Mg²⁺, Mn²⁺ and Co²⁺ 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.     

Some properties of adenosine kinase from Ehrlich ascites-tumour cells

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.     

The pH-dependence of sugar-transport and glycolysis in cultured Ehrlich ascites-tumour cells.

1. pH-dependence of glycolysis has generally been ascribed to the effects of pH on the activities of glycolytic enzymes. The present study shows that sugar transport is pH-dependent in cultured Ehrlich ascites-tumour cells. 2. The rates of glucose consumption, of 3-O-methylglucose transport, and of 2-deoxyglucose transport and phosphorylation increased as linear functions of pH, as the pH of the cell culture medium was increased from 6.1 to 8.5. Transport of glucose, as measured in ATP-depleted cells, was pH-dependent to the same extent as transport of the non-metabolizable sugars. 3. Glucose consumption rates were about 8-fold higher at pH 8.5 than at pH 6.4. About 65-85% of glucose was converted into lactate. Sugar transport rates were 2.5-fold higher at pH 8.5 than at pH 6.3. 4. pH affected both simple diffusion and facilitated diffusion. pH effect was mainly on the Vmax. of 2-deoxyglucose uptake, and on the rapid-uptake phase of 3-O-methylglucose transport. 5. It was estimated that about 70% of the pH effect on the rates of glucose consumption may be due to the effect on sugar transport and the remainder to the effect on the activities of glycolytic enzymes.     

The time-course of the synthesis of adenosine triphosphate in Ehrlich ascites-tumour cells, and the effect of 2,4-dinitrophenol

1. In Ehrlich ascites-tumour cells kept in nitrogen at 20 degrees for 20-30min., the ATP concentration falls from about 15mumoles/g. dry wt. to 2-3mumoles/g. dry wt. 2. If oxygen is admitted to such cells, the ATP concentration rises again in 1-2min. to about 15mumoles/g. dry wt. 3. If glucose is added, in nitrogen, there is a slower increase in ATP concentration to about 6mumoles/g. dry wt., followed by a fall and then by a still slower rise. 4. With glucose and oxygen, the ATP concentration rises rapidly in 1min. to about 8mumoles/g. dry wt., then falls, and finally increases slowly to reach 15mumoles/g. dry wt. in 2hr. 5. 2,4-Dinitrophenol (0.3mm) has little effect on these processes. 6. At 1.0mm, 2,4-dinitrophenol completely inhibits the ATP synthesis dependent on the endogenous respiration, while leaving that in the presence of glucose only a little impaired in rate, and considerably greater in magnitude. 7. ATP synthesis in the presence of glucose and 1.0mm-2,4-dinitrophenol is about three times as fast in oxygen as in nitrogen.     

Mobilization of intracellular calcium by extracellular ATP and by calcium ionophores in the Ehrlich ascites-tumour cell

We have studied the changes of the intracellular free calcium concentration ([Ca2+]i) effected by external ATP, which induces formation of inositol trisphosphate, and by the divalent cation ionophores ionomycin and A23187. Both, ATP (40 microM) and ionophores (1-80 mumol/l cells ionomycin; 20-400 mumol/l cells A23187), produced a transient rise of [Ca2+]i which reached its maximum within 15-30 s and declined near resting values (about 200 nM) within 1-3 min. When the [Ca2+]i peak surpassed 500 nM a transient cell shrinkage due to simultaneous activation of Ca2+-dependent K+ and Cl- channels was also observed. The cell response was similar in medium containing 1 mM Ca2+ and in Ca2+-free medium, suggesting that the Ca mobilized to the cytosol comes preferently from the intracellular stores. Treatment with low doses of ionophore (1 mumol/l cells for ionomycin; 20 mumol/l cells for A23187) depressed the response to a subsequent treatment, either with ionophore or with ATP. Treatment with ATP did also inhibit the subsequent response to ionophore, but in this case the inhibition was dependent on time, the stronger the shorter the interval between both treatments. This result suggests that the permeabilization of Ca stores by ATP is transient and that Ca can be taken up again by the intracellular stores. Refill was most efficient when Ca2+ was present in the incubation medium. Addition of either ATP or ionomycin (1-25 mumol/l cells) to cells incubated in medium containing 1 mM Ca2+ decreased drastically the total cell Ca content during the following 3 min of incubation. In the case of ATP the total cell levels of Ca returned to the initial values after 7-15 min, whereas in the case of the ionophore they remained decreased during the whole incubation period. These results indicate that Ca released from the intracellular stores by either ATP or ionophores is quickly extruded by active mechanisms located at the plasma membrane. They also suggest that, under the conditions studied here, with 1 mM Ca2+ outside, the Ca-mobilizing effect of ionophores is stronger in endomembranes than in the plasma membrane.     

Interferon alters the pattern of secreted proteins from Ehrlich ascites-tumour cells.

Treatment of Ehrlich ascites-tumour (EAT) cells with interferon (IFN) abolished their ability to secrete a 32 kDa protein that was secreted by growing EAT cells. These IFN-treated cells secreted two proteins (molecular masses 100 and 89 kDa as estimated by SDS/polyacrylamide-gel electrophoresis) that were not detected in two-dimensional gel electrophoresis of the culture fluid of untreated EAT cells. The sequence of 20 amino acids from the N-terminal end of the 32 kDa protein was very similar to portions of sequences of mouse proviral gag proteins.     

Inhibition of purine phosphoribosyltransferases from Ehrlich ascites-tumour cells by purine nucleotides

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.     

Further observations on the inhibitory effect of extracellular potassium ions on glycine uptake by mouse ascites-tumour cells

1. The initial rate of uptake of glycine by the tumour cells was measured as a function of the Na(+) and K(+) concentrations in the solution in which the cells were suspended. When [Gly] was 1mm or 12mm, the rate in the absence of Na(+) was independent of [K(+)] and about 3% or 10% respectively of the rate when [Na(+)] was 150m-equiv./l. 2. The Na(+)-dependent glycine entry rate, v, at a given value of [Na(+)] was successively lowered when [K(+)] was increased from 8 to 47 to 96m-equiv./l. A kinetic analysis indicated that K(+) competitively inhibited the action of Na(+). The results were in fair agreement with previous determinations of the kinetic parameters. 3. The presence of 2mm-sodium cyanide and 10mm-2-deoxyglucose lowered the cellular ATP content to less than 3% of the value in the respiring cells. Although v was then about 50% smaller, the relative effects of K(+) and Na(+) on the system were similar to those observed during respiration. 4. A theoretical analysis indicated that the variation of v with [K(+)] is not a reliable guide to the extent to which the K(+) gradient between the cells and their environment may contribute to the net transport of glycine.     

Inhibition of purine phosphoribosyltransferases of Ehrlich ascites-tumour cells by 6-mercaptopurine

1. The formation of adenosine 5′-phosphate, guanosine 5′-phosphate and inosine 5′-phosphate from [8-¹⁴C]adenine, [8-¹⁴C]guanine and [8-¹⁴C]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° the Michaelis constants for adenine, guanine and hypoxanthine were 0·9 μm, 2·9 μm and 11·0 μm in the assay with radioactive purines; the Michaelis constant for guanine in the spectrophotometric assay was 2·6 μm. At pH 7·9 the Michaelis constant for 6-mercaptopurine was 10·9 μm. 3. 25 μm-6-Mercaptopurine did not inhibit adenine phosphoribosyltransferase. 6-Mercaptopurine is a competitive inhibitor of guanine phosphoribosyltransferase (K_i 4·7 μm) and hypoxanthine phosphoribosyltransferase (K_i 8·3 μm). Hypoxanthine is a competitive inhibitor of guanine phosphoribosyltransferase (K_i 3·4 μm). 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.     

The influence of calcium ions on the adsorption of glycolytic enzymes to cellular structure

In order to provide information on the influence of Ca2+ ions on the adsorption of glycolytic enzymes to cellular structure, the release of these enzymes from digitonized cells has been studied. Increases in the calcium ion concentration were found to cause corresponding decreases in the extent of release of all the glycolytic enzymes, as well as a parallel increase in the extent of polymerization of actin. These observations have been discussed in relation to the effect of physiological concentrations of these ions on the association between glycolytic enzymes and the cytoskeleton.