Control of normal and transformed cell proliferation by growth factor-nutrient interactions

The proliferation of normal mammalian cells, similar to that of single bacterial and lower eukaryotic cells, is restricted by space and nutrients. Cultured human lung fibroblasts have been used as a model to show that, in the absence of spatial restrictions, the requirement for specific nutrients limits normal cell proliferation. Serum-derived hormonelike growth factors transiently reduce the requirement for Ca2+, K+, Mg2+, phosphate ions, and 2-oxocarboxylic acids for normal cell proliferation. Oncogenic transformation by virus causes a constitutive reduction in the requirement for multiple nutrients for proliferation. A constitutive reduction in the proliferative requirement for Ca2+, K+, and Mg2+ allows transformed cells to escape the restrictions imposed on normal cell growth by suboptimal external concentrations of Ca2+, K+, Mg2+, and hormonelike growth factors. An understanding of the processes that determine the nutrient requirements of different normal cell types and their alteration by hormonelike growth factors and oncogenic agents is needed to understand and suppress the growth advantage possessed by malignant cells.     

Extracellular regulation of fibroblast multiplication: A direct kinetic approach to analysis of role of low molecular weight nutrients and serum growth factors

The principles of enzyme kinetic analysis were applied to quantitate the relationships among serum-derived growth factors, nutrients, and the rate of survival and multiplication of human fibroblasts in culture. The survival or multiplication rate of a population of cells plotted against an increasing concentration of a growth factor or nutrient in the medium exhibited a hyperbolic pattern that is characteristic of a dissociable, saturable interaction between cells and the ligands. Parameters equivalent to the K_m and V_max of enzyme kinetics were assigned to nutrients and growth factors. When all nutrient concentrations were optimized and in steady state, serum factors accelerated the rate of multiplication of a normal cell population. The same set of nutrients that supported a maximal rate of multiplication in the presence of serum factors supported the maintenance of non-proliferating cells in the absence of serum factors. Therefore, under this condition, serum factors are required for cell division and play a purely regulatory iole in multiplication of the cell population. The quantitative requirement for 18 nutrients of 29 that were examined was significantly higher (P < 0.001) for cell multiplication in the presence of serum factors than for cell maintenance in the absence of serum factors. This indicated specific nutrients that may be quantitatively important in cell division processes as well as in cell maintenance. The quantitative requirement for Ca²⁺, Mg²⁺, K⁺, Pi, and 2-oxocarboxylic acid for cell multiplication was modified by serum factors and other purified growth factors. The requirement for over 30 other nutrients could not clearly be related to the level of serum factors in the medium. Serum factors also determined the Ca²⁺, K⁺, and 2-oxocarboxylic acid requirement for maintenance of non-proliferating cells. Therefore, when either Ca²⁺, K⁺, or 2-oxocarboxylic acid concentration was limiting, factors in serum played a role as cell “survival or maintenance” factors in addition to their role in cell division as “growth regulatory” factors. However, with equivalent levels of serum factors in the medium, the requirement for Ca²⁺, K⁺, and 2-oxocarboxylic acids was still much higher for multiplication than for maintenance. Kinetic analysis revealed that the concentrations of individual nutrients modify the quantitative requirement for others for cell multiplication in a specific pattern. Thus, specific quantitative relationships among different nutrients in the medium are important in the control of the multiplication rate of the cell population. When all nutrient concentrations were optimal for multiplication of normal cells, the multiplication response of SV40-virus-transformed cells to serum factors was similar to that of normal cells. When serum factors were held constant, transformed cells required significantly less (P < 0.001) of 12 of the 26 nutrients examined. Therefore, the transformed cells only have a growth advantage when the external concentration of specific nutrients limits the multiplication rate of normal cells. Taken together, the results suggest that the control of cell multiplication is intimately related to external concentrations of nutrients. Specific growth regulatory factors may stimulate cell proliferation by modification of the response of normal cells to nutrients. Transforming agents may confer a selective growth advantage on cells by a constitutive alteration of their response to extracellular nutrients.     

Complexation and ionophoric properties of taxol and colchicine: complex formation and transport of sodium, potassium, magnesium and calcium ions across a liquid membrane

We report the activities of taxol (an anticancer drug) and colchicine, which are inhibitors of microtubule organization, on the complexation and transport of Na+, K+, Mg2+ and Ca2+ ions across a liquid membrane, using a spectrophotometric procedure. Taxol, a diterpenoid compound, that has been demonstrated to possess a potent antitumour activity, is shown to extract Na+, K+, Mg2+ and Ca2+ ions from the aqueous solution to the organic phase with preference for Ca2+ ions. A kinetic study of the transport and complexation of Na+, K+, Mg2+ and Ca2+ ions through a liquid membrane revealed that the K+ ion is more rapidly transported and the Ca2+ ion is more rapidly complexed than other ions. However, colchicine, another alkaloid compound, extracted and transported only the divalent ions tested, Mg2+ and Ca2+. In both complexation and transport, the flux of the ions increases with the concentration of taxol or colchicine. Complexation and ionophoric properties of taxol and colchicine sheds new lights on therapeutic properties of these drugs. The treatment of disease states by the administration of these drugs to alter membrane permeability will prove to be a valuable therapeutic concept.     

Calcium,magnesium, and serum factors in multiplication of normal and transformed human lung fibroblasts

Summary Serum factors determine the extracellular requirement for both Ca²⁺ and Mg²⁺ for multiplication of normal human lung fibroblasts in vitro. Serum factors also affect the extracellular Ca²⁺ requirement for transformed fibroblasts but to a different extent than for normal cells. Transformed cells exhibit a reduced requirement for both Ca²⁺ and Mg²⁺ for multiplication. The apparent reduction in Ca²⁺ requirement of transformed cells is dependent on the level of serum factors in the medium. The reduced Mg²⁺ requirement for transformed cells is more striking than the loss of Ca²⁺ and independent of the level of serum factors in the medium. A sequential effector relationship among serum factors, Ca²⁺ and Mg²⁺, in a proliferative control system for normal cells is proposed. Alteration or bypass of an intracellular Mg²⁺-requiring process is proposed as a major lesion in the transformed cells. This alteration causes an observed loss of requirements for both Ca²⁺ and serum factors for the multiplication of transformed cells. This work was supported by Grant CA-15305 from the National Cancer Institute, Contract 223-74-1156 from the Bureau of Biologics, Food and Drug Administration, HEW Biomedical Research Support Grant S07RR05800, and the W. Alton Jones Foundation.     

Effect of anthelmintics and phenothiazines on adenosine 5'-triphosphatases of filarial parasite Setaria cervi

S. cervi showed particulate bound Ca2+ ATPase and Na+,K(+)-ATPase activities while Mg2+ ATPase was detected in traces. ATPase of S. cervi was also differentiated from the nonspecific p-nitrophenyl phosphatase activity. Female parasite and microfilariae exhibited higher Ca2+ ATPase and Na+,K(+)-ATPase activities than the male adults and the enzyme Na+,K(+)-ATPase was mainly concentrated in the gastrointestinal tract of the filarial parasite. Na+,K(+)-ATPase of the filariid was ouabain-sensitive while Ca2(+)-ATPase activity was regulated by concentration of Ca2+ ions and inhibited by EGTA. Phenothiazines, viz. trifluoperazine, promethazine and chlorpromazine caused significant inhibition of Ca2+ ATPase and Na+,K(+)-ATPase. Diethylcarbamazine was a potent inhibitor of these ATPases. Mebendazole, levamisole and centperazine also caused significant inhibition of the ATPases indicating this enzyme system as a common target for the action of anthelmintic drugs.     

Metal ion requirements for sequence-specific endoribonuclease activity of the Tetrahymena ribozyme

A shortened form of the self-splicing intervening sequence RNA of Tetrahymena thermophila acts as an enzyme, catalyzing sequence-specific cleavage of RNA substrates. We have now examined the metal ion requirements of this reaction. Mg2+ and Mn2+ are the only metal ions that by themselves give RNA enzyme activity. Atomic absorption spectroscopy indicates that Zn, Cu, Co, and Fe are not present in amounts equimolar to the RNA enzyme and when added to reaction mixtures do not facilitate cleavage. Thus, these ions can be eliminated as cofactors for the reaction. While Ca2+ has no activity by itself, it alleviates a portion of the Mg2+ requirement; 1 mM Ca2+ reduces the Mg2+ optimum from 2 to 1 mM. These results, combined with studies of the reactivity of mixtures of metal ions, lead us to postulate that two classes of metal ion binding sites are required for catalysis. Class 1 sites have more activity with Mn2+ than with Mg2+, with the other divalent ions and Na+ and K+ having no activity. It is not known if ions located at class 1 sites have specific structural roles or are directly involved in active-site chemistry. Class 2 sites, which are presumably structural, have an order of preference Mg2+ greater than or equal to Ca2+ greater than Mn2+ and Ca2+ greater than Sr2+ greater than Ba2+, with Zn2+, Cu2+, Co2+, Na+, and K+ giving no detectable activity over the concentration range tested.     

盐胁迫下柚和橘幼苗体内矿质元素变化的比较研究

采用沙培法,对盐胁迫下坪山柚和福橘幼苗体内矿质元素的变化进行了研究。结果表明,随着NaCl浓度的增加,坪山柚和福橘幼苗根部及地上部Na^＋、Cl-含量增加,且相同浓度下,福橘比坪山柚高。40mmol／L NaCI胁迫下,坪山柚和福橘幼苗地上部的K^＋、Fe含量,根部的Ca^2＋、Mg^2＋、Zn含量显著下降,而根部Fe含量及地上部Zn含量显著增加。随NaCl浓度增大,坪山柚根部K^＋含量,地上部Ca^2＋、Mg^2＋含量变化不明显,而福橘根部、地上部上述离子含量在NaCl浓度≥160mmol／L时均显著下降。因此,根部K^＋含量,地上部Ca^2＋、Mg^2＋含量存在品种问差异,或许可作为耐盐性鉴定指标。NaCl胁迫降低坪山柚和福橘幼苗根部及地上部P、Mn含量,而Cu含量在较高浓度NaCl胁迫下显著增加。NaCl胁迫明显降低坪山柚和福橘幼苗地上部K^＋／Na^＋、Ca^2＋／Na^＋和Mg^2＋／Na^＋值,其中K^＋／Na^＋值的变化可考虑作为柑橘耐盐性鉴定的指标。     

Conduction of Monovalent and Divalent Cations in the Slow Vacuolar Channel

The conduction properties of individual physiologically important cations Na+, K+, Mg2+, and Ca2+ were determined in the slowly activating (SV) channel of sugar beet vacuoles. Current-voltage relationships of the open channel were measured on excised tonoplast patches in a continuous manner by applying a +/-140 mV ramp-wave protocol. Applying KCl gradients of either direction across the patch we have determined that the relative Cl- to K+ permeability was < or =1%. Symmetrical increase of the concentration of tested cation caused an increase of the single channel conductance followed by saturation. Fitting of binding isotherms at zero voltage to the Michaelis-Menten equation resulted in values of maximal conductance of 300, 385, 18, and 13 pS, and of apparent dissociation constants of 64, 103, 0.04, and 0.08 mm for Na+, K+, Mg2+, and Ca2+, respectively. Deviations from the single-ion occupancy mechanism are documented, and alternative models of permeation are discussed. The magnitude of currents carried by divalent cations at low concentrations can be explained by an unrealistically wide (approximately 140 A) radius of the pore entrance. We propose instead a fixed negative charge in the pore vestibules, which concentrates the cations in their proximity. The conduction properties of the SV channel are compared with reported characteristics of voltage-dependent Ca2+-permeable channels, and consequences for a possible reduction of postulated multiplicity of Ca2+ pathways across the tonoplast are drawn.     

Effects of depletion of K+, Na+, or Ca2+ on DNA synthesis and cell cation content in chick embryo fibroblasts.

Decreasing the K+ concentration of the medium from 5 mM to 0.59 mM decreased the K+ content of chick embryo fibroblasts to 22% of control values and increased the Na+ content to 820% of control values. The alteration of monovalent cation content occurred within two hours but had no effect on the rate of DNA synthesis, as measured by 3H-thymidine incorporation, for at least 16 hours. By decreasing the Na+ concentration in the medium, a 50% reduction in cellular Na+ could be obtained with no effect on thymidine incorporation. Since these changes in cellular Na+ or K+ are much larger than any known to occur under physiological conditions but have no effect on thymidine incorporation, we conclude that Na+ and K+ do not play a critical role in determining multiplication rate. Addition of 1.8 mEGTA to cells in media containing 1.7 mM Ca2+ and 0.8 mM Mg2+ inhibited thymidine incorporation and sharply decreased cellular K+ and increased cellular Na+ content. However, there was no reduction in total cellular Ca2+ levels. Likewise, decreasing the Ca2+ concentration of the medium below 0.01 mM inhibited thymidine incorporation, decreased cellular K+ and Mg2+, and increased cellular Na+ but did not affect total cellular Ca2+ levels. Inhibition of DNA synthesis, therefore, could not be correlated with changes in cellular Ca2+ levels.     

Interaction of divalent metal ions with human serum albumin

ESR study was carried out of the interaction between Zn2+, Cu2+, Ca2+, Mg2+ ions and human serum albumin (HSA) in the presence of Mn2+ ions which depends on pH. Competitive binding of these ions with "manganese-binding" sites of albumin was shown to depend on pH. An analysis of concentration dependence of binding these ions with human serum albumin confirmed earlier supposition about the nature of the binding sites of Mn2+ ions with HSA.     

Method for determination of sodium, potassium, calcium, magnesium, chloride, and phosphate in the rat choroid plexus by flame atomic absorption and visible spectroscopy

A rapid simple technique for the measurement of Na+, K+, Mg2+, Ca2+, PO4(3-), and Cl- was developed to analyze ion contents in the choroid plexus of the rat. The technique involves digestion in piperidine, precipitation of proteins with HClO4, and analysis of Na+, K+, Ca2+, and Mg2+ by atomic absorption spectroscopy and Cl- and PO4(3-) by visible spectroscopy. The coefficient of variation for the measurement of eight replicates was 1-3% for all ions. Analysis of choroid plexuses from eight rats yielded coefficients of variation of about 6% and the values for Na+, K+, and Cl- compared favorably to previous works. The analytical procedure described in this paper allows the determination of six major physiologic ions in rat choroid plexus (4 mg wet wt).     

Blockade of current through single calcium channels by Cd2+, Mg2+, and Ca2+. Voltage and concentration dependence of calcium entry into the pore

We studied the blocking actions of external Ca2+, Mg2+, Ca2+, and other multivalent ions on single Ca channel currents in cell-attached patch recordings from guinea pig ventricular cells. External Cd or Mg ions chopped long-lasting unitary Ba currents promoted by the Ca agonist Bay K 8644 into bursts of brief openings. The bursts appear to arise from discrete blocking and unblocking transitions. A simple reaction between a blocking ion and an open channel was suggested by the kinetics of the bursts: open and closed times within a burst were exponentially distributed, the blocking rate varied linearly with the concentration of blocking ion, and the unblocking rate was more or less independent of the blocker concentration. Other kinetic features suggested that both Cd2+ and Mg2+ lodge within the pore. The unblocking rate was speeded by membrane hyperpolarization or by raising the Ba concentration, as if blocking ions were swept into the myoplasm by the applied electric field or by repulsive interaction with Ba2+. Ca ions reduced the amplitude of unitary Ba currents (50% inhibition at approximately 10 mM [Ca]o with 50 mM [Ba]o) without detectable flicker, presumably because Ca ions exit the pore very rapidly following Ba entry. However, Ca2+ entry and exit rates could be resolved when micromolar Ca blocked unitary Li+ fluxes through the Ca channel. The blocking rate was essentially voltage independent, but varied linearly with Ca concentration (rate coefficient, 4.5 X 10(8) M-1s-1); evidently, the initial Ca2+-pore interaction is outside the membrane field and much faster than the overall process of Ca ion transfer. The unblocking rate did not vary with [Ca]o, but increased steeply with membrane hyperpolarization, as if blocking Ca ions were driven into the cell. We suggest that Ca is both an effective permeator and a potent blocker because it dehydrates rapidly (unlike Mg2+) and binds to the pore with appropriate affinity (unlike Cd2+). There appears to be no sharp dichotomy between "permeators" and "blockers," only quantitative differences in how quickly ions enter and leave the pore.     

The effect of membrane-bound calcium on the activity of adenosine triphosphatase from erythrocytes and erythrocyte permeability for monovalent cations]

The activities of Ca2+, Mg2+-ATPase and Na+, K+-ATPase and the permeability of reconstituted human erythrocytes for Na and K ions were measured, using Ca2+-EGTA, Ca2+ATP and Ca2+-sodium citrate buffers. It was found that the increase in the Ca2+/chelate ratio caused stimulation of Ca2+, Mg2+- and Na+, K+-Atpases and an increase in the rate constants of ouabain--dependent 42K+ influx and 22Na+ efflux from the erythrocytes. The use of the Ca2+-sodium citrate system as a calcium buffer did not change the parameters of the functional state of erythrocyte membranes. The data obtained are discussed in terms of a possible role of calcium ions, which are bound to the inner surface of the erythrocyte membrane, in the regulation of the systems of active and passive transport of cations.     

A cation channel for K+ and Ca2+ from Tetrahymena cilia in planar lipid bilayers

The single-channel properties for monovalent and divalent cations of a voltage-independent cation channel from Tetrahymena cilia were studied in planar lipid bilayers. The single-channel conductance reached a maximum value as the K+ concentration was increased in symmetrical solutions of K+. The concentration dependence of the conductance was approximated to a simple saturation curve (a single-ion channel model) with an apparent Michaelis constant of 16.3 mM and a maximum conductance of 354 pS. Divalent cations (Ca2+, Ba2+, Sr2+, and Mg2+) also permeated this channel. The sequence of permeability determined by zero current potentials at high ionic concentrations was Ba2+ greater than or equal to K+ greater than or equal to Sr2+ greater than Mg2+ greater than Ca2+. Single-channel conductances for Ca2+ were nearly constant (13.9 pS-20.5 pS) in the concentrations between 0.5 mM and 50 mM Ca-gluconate. In the experiments with mixed solutions of K+ and Ca2+, a maximum conductance of Ca2+ (gamma Camax) and an apparent Michaelis constant of Ca2+ (K Cam) were obtained by assuming a simple competitive relation between the cations. Gamma Camax and K Cam were 14.0 pS and 0.160 mM, respectively. Single-channel conductances in mixed solutions were well-fitted to this competitive model supporting that this cation channel behaves as a single-ion channel. This channel had relatively high-affinity Ca2+-binding sites.     

Mg(Ca)-ATPase of arterial muscle cells]

We could show an ATPase in mitochondrial and microsomal fractions of sheep arteria carotis communis and arteria coronaria of cattle which can be stimulated by Ca2+ of Mg2+, respectively. The enzyme has a higher affinity for Ca2+ than for Mg2+. The maximum activity of the Mg(Ca)-ATPase was found at 2-4 mM Ca2+ or Mg2+, respectively. Higher concentrations of these ions inhibit the enzyme. Mn2+, Sr2+ and Co2+ can substitute Ca2+ in splitting of ATP by the ATPase of both fractions of ateria coronaria of cattle. The ions K+ and Na+, variation of temperature and pH and a variety of pharmacological active compounds has the same effect on the ATPase stimulated by Ca2+ or Mg2+. These findings prove that Ca2+ and Mg2+ act at the same site of the ATPase of the mitochondrial and microsomal fraction of vascular smooth muscle.     

A model for the phosphorylation of the Ca2+ + Mg2+-activated ATPase by phosphate.

We have shown that changes in fluorescence intensity for the Ca2+ + Mg2+-activated ATPase of sarcoplasmic reticulum labelled with fluorescein isothiocyanate following the addition of Ca2+ can give the ratio of the two conformations (E1 and E2) of the ATPase. We show that the fluorescence response to Ca2+ is unaffected by Mg2+, phosphate or K+, implying that these ions bind equally well to the E1 and E2 conformations. A model is presented for phosphorylation of the ATPase by phosphate as a function of pH, Mg2+, K+ and Ca2+.     

Modulation of Na+-K+-ATPase by calcium ions

The modulatory effects of calcium ions on highly active Na+, K(+)-ATPase from calf brain and pig kidney tissues have been studied. The inhibitory action of Ca2+free on this enzyme depends on the level of ATP (but not AcP). The reduction of pH from 7.4 to 6.0 noticeably increases, but the elevation of pH to 8.0, in its turn, decreases the inhibition of ATP-hydrolyzing activity by calcium. With the increase of K+ concentration (in contrast to Na+) the sensibilization of Na+, K(+)-ATPase to Ca ions is observed. In the presence of potassium ions Mg2+free effectively modifies the inhibitory action of Ca2+free on this enzyme. Ca2+free (0.16-0.4 mM) decreases the sensitivity of Na+, K(+)-ATPase to action of the specific inhibitor ouabain in the presence of ATP. In the presence of AcP (phosphatase reaction) such a change of enzyme sensitivity to ouabain isn't observed. The influence of membranous effects of Ca2+ on the interaction of Na+, K(+)-ATPase with the essential ligands and cardiosteroids is discussed.     

Na+/Ca2+ antiport in cultured arterial smooth muscle cells. Inhibition by magnesium and other divalent cations

Cultured smooth muscle cells from rat aorta were loaded with Na+, and Na+/Ca2+ antiport was assayed by measuring the initial rates of 45Ca2+ influx and 22Na+ efflux, which were inhibitable by 2',4'-dimethylbenzamil. The replacement of extracellular Na+ with other monovalent ions (K+, Li+, choline, or N-methyl-D-glucamine) was essential for obtaining significant antiport activity. Mg2+ competitively inhibited 45Ca2+ influx via the antiporter (Ki = 93 +/- 7 microM). External Ca2+ or Sr2+ stimulated 22Na+ efflux as would be expected for antiport activity. Mg2+ did not stimulate 22Na+ efflux, which indicates that Mg2+ is probably not transported by the antiporter under the conditions of these experiments. Mg2+ inhibited Ca2+-stimulated 22Na+ efflux as expected from the 45Ca2+ influx data. The replacement of external N-methyl-D-glucamine with K+, but not other monovalent ions (choline, Li+), decreased the potency of Mg2+ as an inhibitor of Na+/Ca2+ antiport 6.7-fold. Other divalent cations (Co2+, Mn2+, Cd2+, Ba2+) also inhibited Na+/Ca2+ antiport activity, and high external potassium decreased the potency of each by 4.3-8.6-fold. The order of effectiveness of the divalent cations as inhibitors of Na+/Ca2+ antiport (Cd2+ greater than Mn2+ greater than Co2+ greater than Ba2+ greater than Mg2+) correlated with the closeness of the crystal ionic radius to that of Ca2+.     

Cationic specificity of a Ca2+-accumulating system in smooth muscle cell mitochondria

In the experiments conducted with application of an isotopic technique (45Ca2+) on the myometrium cells suspension treated by digitonin solution (0.1 mg/ml) some properties of Ca ions accumulation system in the mitochondria--cationic and substrate specificity as well as effects of Mg2+ and some other bivalent metals ions on the Ca2+ accumulation velocity have been estimated. Ca ions accumulation from the incubation medium containing 3 mM sodium succinate Na, 2 mM Pi (as potassium K(+)-phosphate buffer, pH 7.4 at 37 degrees C), 0.01 mM (40CaCl2 + 45CaCl2) and 100 nM thapsigargin--selective inhibiting agent of endoplasmatic reticulum calcium pump were demonstrated as detected just only in presence of Mg, while not Ni, Co or Cu ions. The increase of Mg2+ concentration from 1 x 10(-6) to 10(-3) M induced the ATP dependent transport activation in the myometrium mitochondria. Under [Mg2+] increase till 40 mM this cation essentially decreased Ca2+ accumulation (by 65% from the maximal value). The optimum for Ca2+ transport in the myometrium cells suspension is Mg2+ 10 mM concentration. Ka activation apparent constant along Mg2+ value (in presence 3 mM ATP and 3 mM sodium succinate) is 4.27 mM. The above listed bivalent metals decreased Mg2+, ATP-dependent accumulation of calcium, values of inhibition apparent constants for ions Co2+, Ni2+ and Cu2+ were--2.9 x 10(-4) M, 5.1 x 10(-5) M and 4.2 x 10(-6) M respectively. For Mg2+, ATP-dependent Ca2+ transport in the uterus myocytes mitocondria a high substrate specificity is a characteristic phenomenon in elation to ATP: GTP, CTP and UTP practically fail to provide for Ca accumulation process.     

The stability of polypurine tetraplexes in the presence of mono- and divalent cations.

As with other guanine-rich sequences, poly[d(GGA)], poly[d(GA)] and poly[d(GAA)] probably form four-stranded or tetraplex structures. Thermal denaturation profiles were measured for these polymers at pH8 in the presence of Na+, NH4+, K+, Cs+, Mg2+, Ca2+ and Ba2+. For poly[d(GA)], Na+, NH4+, K+ stabilize the tetraplex to similar extents and the Tm increases with increasing ionic strength. In contrast the Tms with Mg2+, Ca2+ and Ba2+ are significantly different and reach maxima at about 5mM of cation. The tetraplex from poly [d(GAA)] behaves in a similar manner. Thermal denaturation profiles for poly[d(GGA)] yield transitions whose hyperchromicity depends both on the concentration and nature of the ion. A reversible cooperative transition is not observed at concentrations greater than 0.15M K+, 1mM Ca2+ or 0.3 mM Ba2+ and hysteresis is evident at some concentrations. These results are consistent with the idea that K+ and ions of a similar size can form a coordination complex with the 6-Keto group of eight guanines (G8-DNA). Unlike the tetraplex polymer this G8-DNA does not melt cooperatively.