Calcyclin-like protein from Ehrlich ascites tumour cells. Ca2+ and Zn2+ binding, distribution and target protein

The calcyclin-like protein from Ehrlich ascites tumour cells is a 10.5 kDa heat stable protein, which binds two Ca2+ ions each with different affinity. Upon Ca2+ binding, the protein changes its conformation exposing hydrophobic regions. In this conformation it is able to interact with fluphenazine and with a 36 kDa protein immunologically similar to mammalian calpactin. Calcyclin-like protein binds Zn2+ and forms dimers like other members of the S-100 protein family. The calcyclin-like protein is present in several mouse tissues such as stomach, skeletal muscle, heart, spleen, lung and kidney, but seems to be absent from brain, intestine and liver as well as from some tumorigenic cells lines.     

Mechanism of action of calcium ionophores on intact cells: ionophore-resistant cells

Calcium ionophores are generally assumed to directly facilitate the transport of Ca2+ across the plasma membrane. The ability of Ca2+ ionophores ionomycin and A23187 to increase Ca2+ concentration in the cytosol ([Ca2+]i) in different cells was analyzed in detail using fluorescent Ca2+ probes. In fura-2-loaded cells, the dependence of the level of [Ca2+]i on ionomycin and A23187 concentrations had a complex character and could not be explained by ionophoric properties only. The Ca2+ signal induced by the Ca2+ ionophores consisted of three components. The first component was due to the activation of Ca2+ influx through native Ca2+ channels and was sensitive to drugs which inhibited the receptor-operated Ca2+ influx. The second component originated from phospholipase C-dependent mobilization of Ca2+ from intracellular stores. An additional influx of Ca2+ into the cells was activated in this case by a store-regulated mechanism. The third ionophoric component was very small at low concentrations of the ionophores. The effect of the ionophores on Ca2+ influx and Ca2+ mobilization was demonstrated on different cells such as Ehrlich ascites tumour cells, murine peritoneal neutrophils, macrophages, and T-lymphocytes. Thymocytes, neutrophils, and Ehrlich ascites tumour cells were more sensitive to the Ca2+ ionophores. Memory T-cells and brown preadipocytes were ionophore-resistant. The insensitivity to Ca2+ ionophores correlated with the absence of Ca2+ in the intracellular Ca2+ stores and the low activity of plasma membrane store-regulated Ca2+ channels.     

The role of mitochondria in modifying the cellular ionic environment. Calcium-induced respiratory activities in mitochondria isolated from various tumour cells

Cyclic stimulation by Ca(2+) of respiration in mitochondria isolated from Ehrlich ascites-tumour cells occurs only when low phosphate concentrations (approx. 0.5mm) are also included in the incubation system. Under these circumstances the extra oxygen consumed is related stoicheiometrically to the amount of Ca(2+) taken up by the mitochondria; the values are similar to those obtained with mitochondria from rat liver in the absence of added phosphate. In contrast with liver mitochondria, up to 280nmol of Ca(2+)/mg of protein can be added to ascites mitochondria without causing any deleterious effect. Respiration in mitochondria isolated from the Yoshida ascites hepatoma (HA 130) and from the Morris hepatomas 5123C and 9618A is also stimulated by Ca(2+) in a cyclic manner. However, that in mitochondria from regenerating rat liver responds to Ca(2+) in the same way as those from normal rat liver. ADP-stimulated respiration in mitochondria from Ehrlich ascites tumour cells, but not from rat liver, is inhibited by low amounts of Ca(2+).     

The role of extracellular calcium ions in HVJ (Sendai virus)-induced cell fusion

The biochemical and biophysical roles of extracellular calcium ions in HVJ (Sendai virus)-induced cell fusion were studied. (1) Various kinds of cell, such as Ehrlich ascites tumor cells, mouse melanoma cells (B16-CW1 cells) and human epidermoid carcinoma cells (KB cells), could fuse in Ca2+-free medium containing a cheletor, glycoletherdiaminetetraacetic acid, in the same way as in Ca2+-containing medium. (2) The ATP content in Ehrlich ascites tumor cells decreased rapidly when the cells were treated with the virus in Ca2+-free medium but not in Ca2+-containing medium. (3) Intracellular adenine nucleotides leaked out into the reaction medium when the cells were treated with the virus in Ca2+-free medium but not in Ca2+-containing medium. (4) On addition of the virus, O2 consumption of Ehrlich ascites tumor cells decreased in Ca2+-free medium, but not in Ca2+-containing medium. (5) HVJ (Sendai virus) did not affect production of lactate by Ehrlich ascites tumor cells in both Ca2+-free medium and Ca2+-containing medium. These observations suggest that the role of extracellular Ca2+ in virus-induced cell fusion is to maintain the ATP and other intracellular metabolite contents at normal levels instead of triggering the fusion reaction itself.     

Calcyclin is a calcium and zinc binding protein

Calcyclin, a cell cycle regulated protein, was recently purified from Ehrlich ascites tumour (EAT) cells and shown to be a calcium binding protein. Here we show that calcyclin monomer and dimer also bind zinc ions. Zinc binding sites seem to be different from calcium binding sites since: preincubation with Ca2+ lacks effect on the binding of Zn2+, and Ca2+ (but not Zn2+) increases tyrosine fluorescence intensity. Binding of Zn2+ reduces the extent of the conformational changes induced by Ca2+, and seems to affect Ca2(+)-binding. The data suggest that Ca2+ and Zn2+ might trigger the biological activity of calcyclin.     

Purification and characterization of the Ca2+-ATPase of plasma membranes from Ehrlich ascites mammary carcinoma cells

Ca2+-ATPase was isolated from plasma membranes of Ehrlich ascites mammary carcinoma cells by means of calmodulin affinity chromatography. The purification procedure included removal of endogenous calmodulin from a Triton X-100 solubilizate of the membranes by DEAE ion-exchange chromatography as an essential step. With respect to its molecular mass, activation by calmodulin, Ca2+-dependent phosphorylation and highly sensitive inhibition by orthovanadate, the purified enzyme resembles the Ca2+-ATPase of erythrocyte membranes. In contrast to the strong calmodulin dependence of the isolated enzyme the Ca2+-ATPase in native Ehrlich ascites carcinoma cell membranes cannot be remarkably stimulated by added calmodulin. It is suggested that the membrane-bound Ca2+-ATPase in the presence of Ca2+ is activated by interaction with endogenously bound calmodulin.     

Intracellular signalling involved in activation of the volume-sensitive K+ current in Ehrlich ascites tumour cells

The cell swelling-activated K+ channel in Ehrlich ascites tumour cells has a conductance of 5 pS estimated from noise analysis of the volume-sensitive whole-cell K+ current (I(K,vol)). I(K,vol) exhibits Goldman-Hodgkin-Katz type behaviour and is insensitive to clotrimazole, apamin and charybdotoxin (ChTX), but inhibited by clofilium. Its small conductance, lack of intrinsic voltage-dependence and peculiar pharmacological profile are similar to properties described for the two-pore domain background K+ TASK channels. Neither Ca2+ nor ATP work as initiators in the activation of I(K,vol). In contrast, several investigations in Ehrlich cells suggest an important role for leukotriene D4 (LTD4) in the activation of I(K,vol). Under isotonic conditions, LTD4 activates Ca2+-dependent, ChTX-sensitive K+ channels as well as Ca2+-independent. ChTX-insensitive K+ channels. The LTD4-activated, ChTX-insensitive K+ current exhibits a current-voltage relation, pharmacological profile and single channel conductance similar to that of I(K,vol), indicating that LTD4 is the signalling molecule responsible for activation of the volume-sensitive K+ channels in Ehrlich cells. Hypotonic swelling of Ehrlich cells results in translocation of the 85-kDa cytosolic (c) PLA2alpha to the nucleus where it is activated. This activation leads to an increase in arachidonic acid release followed by an increased release of leukotrienes, and is essential in cell swelling-induced activation of I(K,vol) and of the organic osmolyte channels.     

Uncoupler-stimulated release of Ca2+ from Ehrlich ascites tumor cell mitochondria

Ruthenium red-insensitive, uncoupler-stimulated release of Ca2+ from Ehrlich ascites tumor cell mitochondria is much slower than from rat liver mitochondria under comparable conditions. In the presence of Pi and at moderate or high Ca2+ loads, ruthenium red-insensitive Ca2+ efflux elicited with uncoupler is approximately 20 times more rapid for rat liver than Ehrlich cell mitochondria. This is attributed to resistance of tumor mitochondria to damage by Ca2+ due to a high level of endogenous Mg2+ that also attenuates Ca2+ efflux. Calcium release from rat liver and tumor mitochondria is inhibited by exogenous Mg2+. This applies to ruthenium red-insensitive spontaneous Ca2+ efflux associated with Ca2+ uptake and uncoupling, and (b) ruthenium red-insensitive Ca2+ release stimulated by uncoupling agent. The endogenous Mg2+ level of Ehrlich tumor mitochondria is approximately three times that of rat liver mitochondria. Endogenous Ca2+ is also much greater (six fold) in Ehrlich tumor mitochondria compared to rat liver. Despite the quantitative difference in endogenous Mg2+, the properties of internal Mg2+ are much the same for rat liver and Ehrlich cell mitochondria. Ehrlich ascites tumor mitochondria exhibit slow, metabolically dependent Mg2+ release and rapid limited release of Mg2+ during Ca2+ uptake. Both have been observed with rat liver and other types of mitochondria. The proportions of apparently "bound" and "free" Mg2+ (inferred from release by the ionophore, A23187) do not differ significantly between tumor and liver mitochondria. Thus, the endogenous Mg2+ of tumor mitochondria has no unusual features but is simply elevated substantially. Ruthenium red-insensitive Ca2+ efflux, when expressed as a function of the intramitochondrial Ca2+/Mg2+ ratio, is quite similar for tumor and rat liver. It is proposed, therefore, that endogenous Mg2+ is a major regulatory factor responsible for differences in the sensitivity to damage by Ca2+ and Ca2+ release by Ehrlich ascites tumor mitochondria compared to mitochondria from normal tissues.     

Inhibition of oxidative phosphorylation in ascites tumor mitochondria and cells by intramitochondrial Ca2+

Accumulation of Ca2+ (+ phosphate) by respiring mitochondria from Ehrlich ascites or AS30-D hepatoma tumor cells inhibits subsequent phosphorylating respiration in response to ADP. The respiratory chain is still functional since a proton-conducting uncoupler produces a normal stimulation of electron transport. The inhibition of phosphorylating respiration is caused by intramitochondrial Ca2+ (+ phosphate). ATP + Mg2+ together, but not singly, prevents the inhibitory action of Ca2+. Neither AMP, GTP, GDP, nor any other nucleoside 5'-triphosphate or 5'-diphosphate could replace ATP in this effect. Phosphorylating respiration on NAD(NADP)-linked substrates was much more susceptible to the inhibitory effect of intramitochondrial Ca2+ than succinate-linked respiration. Significant inhibition of oxidative phosphorylation is given by the endogenous Ca2+ present in freshly isolated tumor mitochondria. The phosphorylating respiration of permeabilized Ehrlich ascites tumor cells is also inhibited by Ca2+ accumulated by the mitochondria in situ. Possible causes of the Ca2+-induced inhibition of oxidative phosphorylation are considered.     

Identification of Ca2+-pump-related phosphoprotein in plasma membrane vesicles of Ehrlich ascites carcinoma cells

Plasma membrane vesicles of Ehrlich ascites carcinoma cells have been isolated to a high degree of purity. In the presence of Mg2+, the plasma membrane preparation exhibits a Ca2+-dependent ATPase activity of 2 mumol Pi per h per mg protein. It is suggested that this (Ca2+ + Mg2+)-ATPase activity is related to the measured Ca2+ transport which was characterized by Km values for ATP and Ca2+ of 44 +/- 9 microM and 0.25 +/- 0.10 microM, respectively. Phosphorylation of plasma membranes with [gamma-32P]ATP and analysis of the radioactive species by polyacrylamide gel electrophoresis revealed a Ca2+-dependent hydroxylamine-sensitive phosphoprotein with a molecular mass of 135 kDa. Molecular mass and other data differentiate this phosphoprotein from the catalytic subunit of (Na+ + K+)-ATPase and from the catalytic subunit of (Ca2+ + Mg2+)-ATPase of endoplasmic reticulum. It is suggested that the 135 kDa phosphoprotein represents the phosphorylated catalytic subunit of the (Ca2+ + Mg2+)-ATPase of the plasma membrane of Ehrlich ascites carcinoma cells. This finding is discussed in relation to previous attempts to identify a Ca2+-pump in plasma membranes isolated from nucleated cells.     

Fractionation of the detergent-resistant filamentous network of Ehrlich ascites tumour cells

Previous investigations of the filamentous network in eukaryotic cells have been based on observations by electron and fluorescence microscopy. In order to examined, in more detail, the interconnection of the various components of th filamentous network, we have treated Ehrlich ascites tumour cells with Triton X-100 in the presence of Mg++, disassembled the detergent-resistant, residual cell structure with Tris-EDTA and subjected the postnuclear supernatant to sucrose density gradient equilibrium centrifugation. Using this technique we are able to demonstrate 1) the association of the major part of intermediate-sized filament protein (vimentin) with unfolded ribosomal subunits, 2) the nearly identical sedimentation behavior of the boundary lamina and actin, and a minor part of the intermediate-sized filament protein respectively, and 3) the association of a Ca++-dependent protease specific for vimentin intermediate-sized filament protein with the Triton X-100 resistant, residual cell structure. Furthermore, we are able to confirm, by labelling intact Ehrlich ascites tumour cells with [3H] concanavalin A and recovering radioactivity in the lighter sucrose gradient fractions, that the detergent-resistant boundary lamina is derived from the plasma membrane. The presence of coated vesicles in Triton X-100-treated cells as well as of coated pits in the derived membrane point at the same origin of the boundary lamina. The results of the fractionation study are correlated with structures observed by electron microscopy of ultrathin sections of the intact filamentous network.     

Purification of myosin from Ehrlich ascites tumour cells (phosphorylation of its light chain and heavy chain)

1. The myosin molecule from Ehrlich ascites tumour cells consists of heavy chains of about 200 kDa and three species of light chains of 20, 19 and 15 kDa. 2. The heavy chain can be phosphorylated in vitro either by endogenous Ca2+-independent kinase or by casein kinase II. 3. The 20 and 19 kDa light chains can be phosphorylated either by an endogenous kinase or by myosin light chain kinase from chicken gizzard. 4. The Ca2+-ATPase activity of the purified myosin was 0.3 mumol/min mg protein. The Mg2+-ATPase activity was activated 14-fold by actin upon the light chain phosphorylation.     

Increased activity of the eEF-2 specific, Ca2+ and calmodulin dependent protein kinase III during the S-phase in Ehrlich ascites cells.

The activity of the eukaryotic elongation factor (eEF-2) specific, Ca2+ and calmodulin dependent protein kinase III (CaM PK III) was studied in homogenated Ehrlich ascites tumour cells. The eEF-2 kinase activity was determined in the presence of an excess of the substrate, i.e., non-limiting concentrations of eEF-2. The homogenates showed both kinase and phosphatase activity. The latter activity was inhibited by the protein phosphatase 2A inhibitor okadaic acid. Analysis of the kinase using cells from defined stages of the cell cycle showed that the highest activity was found in cells from the early S-phase, whereas the phosphatase activity was most pronounced during the G2+M phase.     

The proton stoichiometry of electron transport in Ehrlich ascites tumor mitochondria.

Initial rate measurements of the stoichiometric relationships between H+ ejection, K+ and Ca2+ uptake, and electron transport were carried out on mitochondria from Ehrlich ascites tumor cells grown in mice. With succinate as substrate and N-ethylmaleimide to prevent interfering H+ reuptake via the phosphate carrier, close to 8 H+ were ejected per oxygen atom reduced (H+/O ejection ratio = 8.0); with the NAD-linked substrates pyruvate or pyruvate + malate, the H+/O ejection ratio was close to 12. The average H+/site ratio (H+ ejected/2e-/energy-conserving site) was thus close to 4. The simultaneous uptake of charge-compensating cations, either K+ (in the presence of valinomycin) or Ca2+, was also measured, yielding average K+/site uptake ratios of very close to 4 and Ca2+/site ratios close to 2. It was also demonstrated that each calcium ion enters the respiring tumor mitochondria carrying two positive electric charges. These stoichiometric data observed in mitochondria from Ehrlich ascites tumor cells thus are in complete agreement with similar data on normal rat liver and rat heart mitochondria and suggest that the H+/site ratio of mitochondrial electron transport may be 4 generally. It was also observed that the rate of deltaH+ back-decay in anaerobic tumor mitochondria following oxygen pulses is some 6- to 8-fold greater than in rat liver mitochondria tested at equal amounts of mitochondrial protein.     

K+ conductance activated during regulatory volume decrease. The channels in Ehrlich cells and their possible molecular counterpart

K+ currents activated by hypotonic cell swelling have been studied in Ehrlich ascites tumour cells by the whole-cell recording mode of the patch-clamp technique. K+ together with Cl- currents developed in the absence of added intracellular Ca2+ and with strong buffering of internal Ca2+ in experiments conducted at 37 degrees C. Manipulation of the extracellular medium with other cations suggests a selectivity sequence of K+ > Rb+ > NH4+ > or = Na+ approximately equals Li+ approximately equals Cs+. The current-voltage relationship of the volume-sensitive K+ current was well fitted with the Goldman-Hodgkin-Katz current equation between -130 and 20 mV at both physiological and high K+ extracellular solutions. The class III antiarrhytmic drug clofilium blocked the volume-sensitive K+ current in a voltage-independent manner. Clofilium was also found to be a strong inhibitor of the regulatory volume decrease (RVD) response of Ehrlich cells. The leukotriene D4 (LTD4) can activate the same current in isotonicity, consistent with a role for this compound in the signalling process of volume regulation. It is suggested that K+ channels activated by cell swelling belong to the so-called background K+ channel group. These are voltage-independent channels which underlie the resting potential of many cells and have recently been identified as belonging to a family of K+ channels with two pore domains in tandem (2P-4TM). Preliminary experiments show the presence of the TASK-2 channel, a member of the 2P-4TM family inhibited by acid extracellular pH, in Ehrlich cells and suggest that it might underlie the swelling-induced K+ current.     

Antioxidant effect of zinc and zinc-metallothionein in the acute cytotoxicity of hydrogen peroxide in Ehrlich ascites tumour cells

This study was concerned with the role of zinc (Zn) and zinc-metallothionein (Zn-MT) in oxidative stress. Hydrogen peroxide-induced oxidative injury was examined in Ehrlich ascites tumour cells isolated from control host mice, mice pretreated with 10 mg/kg ZnSO4 (i.p.) to increase cellular Zn/Zn-MT levels, and mice exposed to Zn-deficient diet to reduce the cellular Zn/Zn-MT levels. The results of the present study showed that Ehrlich cells with seven-fold differences in Zn-MT concentrations could be obtained by manipulating the Zn status of host mice and that high Zn and Zn-MT levels can make Ehrlich cells more resistant to H2O2-induced oxidative injury (cell viability, lipid peroxidation, [Ca2+]i) while cells with reduced Zn/Zn-MT levels were more susceptible to this treatment. H2O2 treatment resulted in oxidation of MT thiolate groups and loss of its metal binding capacity with translocation of Zn released from oxidized MT to other cellular sites. Preincubation of Ehrlich cells with ZnSO4 in vitro also conferred some degree of resistance to H2O2 toxicity, suggesting the inherent antioxidative property of Zn ions. These data suggested that Zn-MT can be considered as an antioxidant by virtue of its thiolate groups and its Zn ions that are released in the presence of oxidative stress.     

Membrane-bound Ca2+ in the mitochondria of Ehrlich ascitic tumor cells

Ca2+ transport in mitochondria of Ehrlich ascite tumour cells and in liver mitochondria has been compared. It has been shown that in tumour cell mitochondria unlike liver ones even small amounts of Ca2+ caused marked increase in membrane-bound Ca2+ level. Therefore, a decrease in the electro-neutral Ca2+ efflux, stabilization of mitochondria membranes and inhibition of phosphorylated respiration were observed. It has been proposed that high content of membrane-bound Ca2+ is predetermined by a higher affinity of membrane phospholipids to Ca2+.     

The inhibitory effect of various fatty acids on aerobic glycolysis in Ehrlich ascites tumour cells

Inhibition of glycolysis in Ehrlich ascites tumour cells by saturated fatty acids, added either in form of potassium salts or incorporated into phosphatidylcholine liposomes, increases with the increasing carbon atom chain length and is independent of the concentration within the range of 0.1 to 1.0 mM. In contrast, the inhibition of glycolysis in the cytosolic fraction from Ehrlich ascites cells depends on the concentration of fatty acids. The content of ATP in Ehrlich ascites cells incubated with fatty acids increases with increasing carbon atom chain length, which leads to a crossing-over in the concentrations of pyruvate and 2-phosphoenolpyruvate. Lowering of the sum of both these metabolites by palmitate and stearate points to the inhibition not only of pyruvate kinase but also of other enzymes of early steps of glycolysis. Fatty acids in intact Ehrlich ascites cells inhibit all three key glycolytic enzymes but added to the cytosolic fraction affect mainly the activity of phosphofructokinase. The inhibition of pyruvate kinase by fatty acids is smaller in the cytosolic fraction from tumour cells than from liver and muscles.     

Ehrlich tumour cells: Ca(2+)-uptake modification by aluminium lactate.

Aluminium lactate provokes the same modification of 45Ca(2+)-uptake by Ehrlich ascites tumour cells as does ferric lactate. The increase of uptake is metal-complex concentration-dependent, and in contrast to ferric lactate, is only partially inhibited by albumin. Under the same experimental conditions, chromic lactate provoked no modification of that uptake. A plasma membrane rigidity increase provoked by coordination binding of aluminium to phospholipids is likely to be the main cause of Ca(2+)-uptake modification by aluminium lactate.