Antagonistic effects of insulin and cortisol on coordinate control of metabolism and growth in cultured fibroblasts

A variety of metabolic and biosynthetic pathways in chick embryo fibroblasts are stimulated coordinately by many unrelated exogenous agents. Three of the best characterized components of this coordinate response are the uptake of 2-deoxy-D-glucose (2-dGlc) and of uridine and the incorporation of thymidine into DNA. Insulin stimulates and cortisol inhibits the coordinate response. In cortisol-treated cultures, as little as 10^?3 units/ml of insulin may stimulate thymidine incorporation 4-fold and 10^?1 units/ml may stimulate as much as 40-fold. The higher concentrations of insulin completely override the inhibitory effect of cortisol. They also cause about a 5-fold stimulation of the uptake of 2-dGlc and of uridine and a 2-fold stimulation of proline incorporation into protein. The uptake rates of 2-dGlc and uridine double within 30 minutes after addition of insulin to cortisol-inhibited cultures, but the incorporation of thymidine only begins to increase markedly after a 4-hour delay. When cortisol is added to cultures in the absence of insulin, the rates of uptake of 2-dGlc and uridine begin to decrease within two hours, but the incorporation of thymidine remains constant for two hours before beginning to decrease. Deprivation of Mg²⁺ inhibits the accelerated coordinate response maintained by insulin, but does not further the inhibition induced by cortisol. Results with metabolic inhibitors indicate that the stimulation of 2-dGlc and uridine uptake by insulin do not require RNA synthesis, and also suggest that they do not require protein synthesis. These and other findings can be explained by a model for coordinate control in which insulin increases and cortisol decreases the availability of Mg²⁺ for a wide spectrum of regulatory reactions in different metabolic pathways. In this model both hormones affect only the rates of ongoing reactions and do not instruct the cell to carry out specific new reactions unless the cell was predetermined to do so.     

Coordinate control of Balb/c3T3 cell survival and multiplication by serum or calcium pyrophosphate complexes.

Balb/c3T3 cells in crowded cultures detach from the dish when deprived of serum, and the survivors incorporate 3H-thymidine at a reduced rate. The detachment becomes pronounced two hours after removal of serum, and reaches its maximum rate between two and four hours. Cells in sparse culture are not detached by serum removal, and their rate of 3H-thymidine incorporation is only slightly reduced. As the sparse cultures grow into more crowded cultures, and the serum is depleted, increasing numbers detach. The detached cells are incapable of reattaching when placed in a new dish with ample fresh serum. The cells are leaky to cellular constituents and appear to be dead. Detachment is a consequence rather than the cause of cell death, and can be produced by agents which inhibit cellular energy metabolism. The cells on the dish which survive serum deprivation are fully viable and grow rapidly when serum is added. When they become crowded they are as sensitive to serum deprivation as was the original population. They are therefore not selected for a low serum requirement but apparently survive because they spread into the space vacated by the detaching cells and then behave as sparse cultures in response to serum variations. Insoluble complexes of Ca2+ and pyrophosphate (Ca2+-PPi) show the same concentration dependence in promoting cell survival as in stimulating 3H-thymidine incorporation, showing that a single substance can be responsible for both activities. It is concluded that survival and growth are part of the coordinate response of 3T3 cells to single external effectors. The results are discussed in terms of a simple model in which the coordinate response is regulated by the availability of Mg2+ for transphosphorylation reactions within the cell, and the availability depends on the binding affinity of cellular membranes for Mg2+. The difference between survival and multiplication is postulated to be in the intensity and duration rather than the kind of stimulus.     

Uridine uptake inhibition in Novikoff hepatoma cells by perturbants of intracellular Ca2+ and K+ levels

The rate of uptake of uridine into the acid-soluble fraction of Novikoff hepatoma cells is inhibited by low concentrations of the ionophores A23187 and gramicidin and other perturbants of intracellular cation levels. Inhibition of uridine uptake by A23187 is dependent on Ca2+ and is reduced by serum and high levels of Mg2+. The effectiveness of A23187 is dependent on the Ca2+/Mg2+ ratio rather than the absolute concentration of either ion. Inhibition of uridine uptake by gramicidin is not significantly affected by serum or divalent cations. Other effectors of monovalent cation flux such as ouabain and valinomycin also inhibit uridine uptake. These results indicate that net uptake of uridine may be influenced by intracellular levels of certain monovalent and divalent inorganic cations.     

The logic of the Membrane, Magnesium, Mitosis (MMM) model for the regulation of animal cell proliferation

The addition of animal serum or specific protein growth factors to quiescent, serum-deprived vertebrate cells in culture activates a wide variety of biochemical responses within minutes, which are followed in 5-10h by the initiation of DNA synthesis and then by mitosis. The quintessential early and continuing activation step for the increase in DNA synthesis is the increased initiation rate of protein synthesis, which must be continuously maintained throughout the G1 phase for advancement into S. The aggregate of biochemical reactions to growth factors is called the coordinate response, to indicate that many related and unrelated processes are orchestrated to repetitively reproduce cells. It is, however, crucial to recognize that the coordinate response can be induced for one or more rounds of replication by a variety of non-specific and quasi-specific membrane effectors. The logic of considering this framework of events in growth control implied that a single multi-target second messenger plays a central role in coordinating the events of the overall response. The same reasoning suggested that free Mg(2+) is the unifying regulatory element in that response which includes protein kinase pathways, and that the cytoplasmic activity of Mg(2+) increases with the binding of growth factors to their receptors in the cell membrane, or of less specific perturbations of the membrane. Experimental support of this conclusion is presented here and is represented in the MMM model of cell proliferation control.     

Loss of the post-translational control of nutrient transport in vitro and in vivo virus-transformed chicken cells.

The removal of serum from the medium of uninfected fibroblasts decreased the rate of uptake of uridine, 2-deoxyglucose, alpha-aminoisobutyrate and thymidine. Its subsequent addition rapidly and reversibly stimulated the uptake of all the nutrients but thymidine and this response was not inhibited by treatment of the cells with cycloheximide. The cycloheximide insensitive, rapid increase in the rate of transport has been designated post-translational control. The nutrient transport systems in chick embryo fibroblasts transformed in vitro with avian sarcoma viruses and virus-induced cultured chicken tumor cells do not respond to serum removal or addition. Two possible levels for the control of nutrient transport, i.e., mitogen receptor occupancy and mitrogen-induced activation of the transport system, are presented to explain these observations.     

Specificity of the requirements for magnesium and calcium in the growth and metabolism of chick embryo fibroblasts

The rate of entry of chick embryo fibroblasts (CEF) into the S-period of the cell cycle is reduced by lowering the external supply of Mg²⁺ below 0.2 mM. This slowdown, which is measured by the rate of incorporation of ³H thymidine into DNA, can be largely reversed by doubling or tripling the concentration of Ca²⁺ in the medium, normally 1.7 mM. The Ca²⁺-induced stimulation is shown not to depend on contaminating traces of Mg²⁺ in the added Ca²⁺. The increase in cell number in the Ca²⁺-stimulated cultures is delayed, possibly due to cell detachment. The effect of Ca²⁺ on thymidine incorporation can be simulated almost quantitatively by Sr²⁺. Ba²⁺ does not produce the effect, nor do any of the other cations tested. As little as 0.2 mM Mg²⁺ produces a full stimulation of thymidine incorporation in the absence of added Ca²⁺, and no substitute was found that is effective in the same concentration range. In short term experiments, i.e., 16 hours, even 5.0 mM Ca²⁺ cannot stimulate thymidine incorporation to the extent achieved with 0.2 mM Mg²⁺. Large amounts of Ca²⁺ or Sr²⁺ can accelerate the uptake of 2-dGlc in Mg²⁺-deprived cultures, but they are much less efficient than Mg²⁺ in this regard also. It is suggested that Mg²⁺ is the direct intracellular effector in controlling the diverse reactions of the coordinate response, and that Ca²⁺ and Sr²⁺ act indirectly by making Mg²⁺ available to participate in these reactions.     

Uridine enhances the cytotoxic effect of D-glucosamine in rat C6 glioma cells

This paper studies the influence of uridine on the effects exerted by D-glucosamine in rat C6 glioma cells. 2 mM uridine increased markedly both the cytotoxic effect of the aminosugar and the inhibition of thymidine incorporation into acid-insoluble fraction. Furthermore the complete resumption of the capacity to incorporate either 3H-thymidine or 3H-mannose which was observed after the removal of the aminosugar, was impeded when the cells were treated contemporaneously with D-glucosamine and uridine. An exposure for 4 hr to 20 mM glucosamine alone enhanced about 15-fold the cellular pool of UDP-N-acetylhexosamines; the addition of 2 mM uridine intensified the expansion of this pool, which became about 35-fold the control value. The findings suggest a connection between the accumulation of UDP-N-acetylhexosamines in the cells and the appearance of D-glucosamine cytotoxicity.     

Early cellular responses to diverse growth stimuli independent of protein and RNA synthesis.

Serum, elevated pH, excess Zn++, 9,10 dimethyl-1,2 dibenzanthracene (DMBA) and insulin accelerate the progress of growth-inhibited chick embryo cells into the S-period of DNA synthesis. A comparative study was made of their capacity to elicit other cellular responses within two hours after their application. All the agents studied stimulated the uptake of the glucose analogue 2-deoxy-D-glucose (2-dGlc). Elevated pH elicited a more striking increase than the other agents in the uptake of the amino acid analogue alpha-amino isobutyric acid (AIB). The application of subtoxic concentrations of Zn++ or DMBA did not stimulate the uptake of uridine by cells nor its incorporation into RNA when tested at 2 hours. However, it was found that the stimulation of uridine utilization did occur but was delayed several hours. Similarly, the accelerated onset of DNA synthesis was also delayed for several hours by these agents. Insulin acted like serum in stimulating the utilization of 2-dGlc, AIB and uridine. Serum and DMBA were particularly effective in stimulating the utilization of choline. It was concluded that the utilization of 2-dGlc, uridine and thymidine are affected similarly by all the agents, but that there may be differential effects in the utilization of AIB and choline. The inhibition of RNA synthesis by actinomycin D did not prevent the relative stimulation of 2-dGlc, AIB and choline utilization by serum and pH treatment. The inhibition of protein synthesis by cycloheximide did not prevent the relative stimulation of 2-dGlc and choline utilization by serum and pH treatment. It partially blocked the increased uptake of AIB and had erratic effects on the utilization of uridine. It was concluded that neither RNA nor protein synthesis is required for some, if not all, the early responses to growth stimuli measured here. The inhibited cell appears to be a poised system which carries out a programmed array of reactions characteristic of the cell type following perturbation by a variety of unrelated agents. In vivo specificity is provided by the physiological reagents available (i.e., hormones) and their capacity to interact with different cell types.     

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.     

Thymidine and Thymine Incorporation into Deoxyribonucleic Acid: Inhibition and Repression by Uridine of Thymidine Phosphorylase of Escherichia coli

Thymidine is poorly incorporated into deoxyribonucleic acid (DNA) of Escherichia coli. Its incorporation is greatly increased by uridine, which acts in two ways. Primarily, uridine competitively inhibits thymidine phosphorylase (E.C.2.4.4), and thereby prevents the degradation of thymidine to thymine which is not incorporated into normally growing E. coli. Uridine also inhibits induction of the enzyme by thymidine. It prevents the actual inducer, probably a deoxyribose phosphate, from being formed rather than competing for a site on the repressor. The inhibition of thymidine phosphorylase by uridine also accounts for inhibition by uracil compounds of thymine incorporation into thymine-requiring mutants. Deoxyadenosine also increases the incorporation of thymidine, by competitively inhibiting thymidine phosphorylase. Deoxyadenosine induces the enzyme, in contrast to uridine. But this is offset by a transfer of deoxyribose from deoxyadenosine to thymine. Thus, deoxyadenosine permits incorporation of thymine into DNA, even in cells induced for thymidine phosphorylase. This incorporation of thymine in the presence of deoxyadenosine did not occur in a thymidine phosphorylase-negative mutant; thus, the utilization of thymine seems to proceed by way of thymidine phosphorylase, followed by thymidine kinase. These results are consistent with the data of others in suggesting that wild-type E. coli cells fail to utilize thymine because they lack a pool of deoxyribose phosphates, the latter being necessary for conversion of thymine to thymidine by thymidine phosphorylase.     

A suppressor cell which interferes with antiallotype stimulated DNA synthesis of rabbit B cells

Responsiveness of rabbit spleen cells to anti-allotype antibody was measured in terms of increased thymidine incorporation. Incorporation was enhanced after removal of cells which had ingested or had adhered to magnetic particles. B lymphocytes, prepared from spleen cells by the removal of adherent cells and of RTLA bearing T cells, were more responsive to anti-allotype antibody than were the original spleen suspensions. This increase could not be explained by enrichment in B cells. It was concluded that an adherent cell suppressed B cell transformation. The addition of 2-mercaptoethanol to the cell cultures stimulated with mitogen augmented the incorporation of thymidine. Adherent cells interfered with 2-mercaptoethanol potentiation in the response to anti-allotype antibody but not in the response to Con A. Fractionation of spleen cells, over glass bead columns, yielded nonadherent and adherent cell populations. The responsiveness of nonadherent cells to anti-allotype induced thymidine incorporation was two to six times that of unfractionated cells. The responsiveness of nonadherent cells to stimulation by anti-allotype antibody was reduced after addition of adherent cells. Findings were discussed in terms of the inhibitory role played by adherent cells on anti-allotype antibody induced responsiveness of rabbit B cells and of the possible participation of a third cell type which functions as a promotor of mitogenic T cell stimulation.     

Response of human lymphocytes to mitogen: at what stage is there a requirement for Ca2+?

In order for significant DNA synthesis to be observed in the case of human lymphocytes stimulated for 36 h in presence of phytohemagglutinin (PHA), Ca2+ must be present in the medium continuously for at least 20 h; access to Ca2+ for 10-h periods during the first 30 h was not sufficient to permit DNA synthesis to occur. Addition of the chelator EGTA from 0 to 60 h after stimulation caused severe inhibition of incorporation of labelled thymidine when this was measured after 36 to 144 h of culture. Equimolar calcium reversed the inhibition caused by EGTA. Incorporation of labelled uridine and leucine showed a temporal pattern of dependence on the presence of Ca2+ in the medium similar to that of thymidine. Ca2+ appears not to be required in the medium during the last half (i.e., 20-36 h) of the presynthetic G1 phase nor during S phase since removal of Ca2+ from the medium after 20 h did not prevent a subpopulation of lymphocytes from entering S phase 16 h later.     

Incorporation of thymidine into the DNA of actinomycetes. I. Incorporation of exogenous thymidine into the DNA of Thermoactinomyces vulgaris]

The incorporation of exogenous thymidine and thymine into acid-insoluble material of Thermoactinomyces vulgaris has been studied during germination and subsequent growth. Thymine is not incorporated. The incorporation of thymidine stops after a short time due to the rapid breakdown of thymidine to thymine and deoxyribose-1-phosphate by the inducible thymidine phosphorylase. Deoxyadenosine enhances the incorporation of thymidine as well as of thymine and prolongs the tine of uptake. Uridine stimulates only the incorporation of thymidine but not of thymine. These effects can be explained by the function of these substances within the salvage pathway. Deoxyadenosine acts as donor of deoxyribosyl groups being necessary for the conversion of thymine to thymidine by thymidine phosphorylase and uridine inhibits thymidine phosphorylase, and thereby it prevents the degradation of thymidine to thymine. Thymidine is incorporated into alkali-, RNase-and protease-stable, hot TCA-soluble and DNase-sensitive material. That means that the cellular DNA of T. vulgaris can be specifically labelled by radioactive thymidine in the presence of deoxyadenosine and uridine, respectively.     

Effect of estradiol and relaxin on growth of fibroblastic cells isolated from guinea pig mammary glands

Fibroblasts were isolated from the mammary glands of guinea pigs and grown in 96-well culture plates. They were treated with a factorial arrangement of porcine relaxin (0.0, 0.5, 1.0 or 1.5 micrograms/ml) and estradiol-17 beta (0, 200, 400 or 600 pg/ml). Tritiated thymidine or uridine was added to a final activity of 25 nCi per well and the cells incubated at 37 degrees C for 48 h. Cells were then harvested onto filter paper and counted for tritium. Controls (0.0 micrograms/ml relaxin and 0 pg/ml estradiol) incorporated 3.7 nCi of tritiated thymidine and 4.8 nCi tritiated uridine. Both relaxin and estradiol altered the incorporation of thymidine and uridine. There was also an interaction between the two hormones. Thymidine incorporation with no estradiol and 1.5 micrograms/ml relaxin was 129% of controls. The optimum incorporation of thymidine occurred with 0.5 micrograms/ml relaxin and 400 pg/ml estradiol. This combination of hormones gave a response of 145% of controls. Uridine incorporation followed a different pattern. Relaxin alone at a concentration of 1.5 micrograms/ml gave a near-optimum response of 141% of control. The optimum combination of relaxin and estradiol for uridine incorporation was 1.5 micrograms/ml relaxin and 400 pg/ml estradiol, which gave a response of 156% of controls. These data indicated that relaxin and estradiol alter DNA and RNA synthesis in mammary fibroblasts and thus may be important in controlling the growth of the mammary gland stroma.     

Vasopressin accelerates protein synthesis in neonatal rat cardiomyocytes

Arginine vasopressin (AVP) has been shown to promote vascular smooth muscle cell hypertrophy and hyperplasia of fibroblasts. The present study examines the effect of AVP and endothelin-1 (ET-1) on protein, DNA, and RNA synthesis in primary cultures of serum deprived neonatal rat cardiomyocytes (RC) as assessed by changes in [3H] phenylalanine, [3H] thymidine, and [14C] uridine incorporation respectively. Both AVP and ET-1 evoked significant increases in protein synthesis in RC of 36 ± 12% (p < 0.05) and 53 ± 22% (p < 0.01) respectively. The stimulating action of AVP on [3H] phenylalanine incorporation was abolished by pretreatment with 2-nitro-4carboxyphenyl-N, N-diphenylcarbamate (NCDC), a phospholipase C (PLC) inhibitor. [14C] uridine incorporation was significantly higher in cells incubated with ET-1 (95 ± 12%) but not AVP (9 ± 11%). Neither AVP nor ET-1 significantly affected cell number or [3H] thymidine incorporation, suggesting a lack of a hyperplastic effect. AVP evoked an increase in [Ca2+]i levels (162 ± 12 nmol/L from a basal value of 77 ± 6 nmol/L) which was completely abolished by pretreatment with either NCDC or cyclopiazonic acid (sarcoplasmic reticulum (SR) Ca2+ pump inhibitor) but unaffected by ryanodine (ryanodine sensitive SR Ca2+ store depletor). Taken together, these data suggest that AVP, in a PLC dependent manner, stimulates both protein synthesis and augments [Ca2+]i release in RC from ryanodine insensitive (IP3 sensitive) Ca2+ stores. Thus, AVP may promote cardiac hypertrophy via direct effects on cardiomyocyte protein synthesis secondary to IP3 mediated [Ca2+]i release.     

ZINC REQUIREMENT FOR DNA REPLICATION IN STIMULATED HUMAN LYMPHOCYTES

The requirement for Zn⁺⁺ in DNA replication by phytohemagglutinin-stimulated human lymphocytes was studied. When 6 µM o-phenanthroline, a chelator with a high affinity for Zn⁺⁺, is added to cultures of stimulated lymphocytes a nearly complete inhibition of thymidine incorporation results within a few hours. In contrast, the incorporation of uridine is only slightly reduced and the incorporation of leucine is unaffected. m-Phenanthroline, a nonchelating analogue, does not alter the rate of thymidine incorporation even when present in 10-fold greater amounts than o-phenanthroline. The inhibition of thymidine incorporation by o-phenanthroline could be entirely reversed by the addition of Zn⁺⁺ to the cultures, or could be prevented by the prior addition of either Zn⁺⁺ or Ni⁺⁺. All other divalent cations tested were incapable of reversing the o-phenanthroline inhibition of thymidine incorporation.     

Thymidine transport in phytohaemagglutinin-stimulated pig lymphocytes.

Thymidine and uridine transporters in peripheral pig lymphocytes have structural features in common, but are not identical. Accelerated entry of [3H]thymidine begins 12h after the addition of phytohaemagglutinin. The increased thymidine uptake into the cells is characterized by an increase in Vmax. Without alteration of the apparent Km(0.6+/-0.08muM). Thymidine kinase activity is increased 12h after stimulation. Both the increased thymidine uptake and the increased thymidine kinase activity are inhibited in cultures incubated with puromycin: rates of degradation of the two systems are unchanged after phytohaemagglutinin addition, and indicate similar half-lives of about 2h. Thymidine kinase is rate-limiting for thymidine entry up to 18h after phytohaemagglutinin addition; increase in its synthesis is detectable about 6h before net incorporation of thymidine into DNA is significantly promoted.     

Effects of prostaglandins E1, E2, and F2alpha on the growth of leukaemia cells in culture.

Prostaglandins E1, E2, and F2alpha (PGE1, PGE2, and PGF2alpha) were shown to inhibit the growth of mouse leukaemia lymphoblasts L5178Y in culture. The effects of PGE1 and PGE2 were greater than that of PGF2alpha. PGE1 and PGE2, at the concentration of 100 mug per ml showed significant inhibitory effects on the rates of incorporation of tritiated thymidine, uridine and leucine. At concentrations of 50 and 25 mug per ml, there was significant inhibition of thymidine and uridine incorporation, but not of leucine, PGF2alpha showed significant inhibition of thymidine and uridine incorporation but not leucine incorporation, in all 3 concentrations studied (100, 50, and 25 mug/ml). The ability of the cells to form colonies in soft agar was significantly inhibited by PGE1 and PGE2 at concentrations as low as 1-8 mug/ml. For F2alpha, however, a concentration as high as 56mug/ml was required to show inhibitory effect, but at 1-8 mug/ml it was found to be stimulatory.     

Citric acid arrest and stabilization of nucleoside incorporation into cultured cells

Either citric acid or ascorbic acid (0.23 m final concentration) quickly arrests incorporation of tritiated thymidine or uridine upon addition to cultures of animal cells. The incorporated radioactivity is totally preserved for a day at 37°C without further manipulations. In contrast, radioactivity is extensively lost from cultured cells at 37°C after they are arrested by the conventional method of trichloroacetic acid precipitation following removal of medium and rinsing. The cells arrested with citric or ascorbic acid preserve their morphology and are suitable for autoradiography. The new method has considerable advantages of convenience and accuracy over treatment with trichloroacetic acid.     

Production of 2-Methylisocitric Acid from n-Paraffins by Mutants of Candida lipolytica

Vitamin B₁₂-dependent ribonucleotide reductase purified from Rhizobium meliloti catalyzes the reduction of 5′-diphosphates of guanosine, adenosine, cytidine and uridine (GDP, ADP, CDP and UDP). The enzyme activities were regulated by Mg²⁺ and deoxyribonucleoside triphosphate effectors as follows: in the presence of Mg²⁺, allosteric effector deoxyguanosine triphosphate (dGTP) had the most stimulatory effect on reduction of ADP and UDP; deoxyadenosine triphosphate (dATP) on reduction of CDP; and thymidine triphosphate (dTTP) on reduction of GDP. These stimulatory effectors were active at a low concentration of 10 μm. Other deoxyribonucleotides may be negative or weakly positive effectors. Without effectors, the rate profile of ADP and GDP reduction showed a sigmoidal curve. In the absence of Mg²⁺, the activities of the reductase showed nearly maximal levels, and the addition of effectors rather decreased the activities, except in the case of UDP reduction which was most strongly stimulated by dGTP. The effect of Mg²⁺ can be replaced by Ca²⁺. Monovalent cations such as Na⁺ and K⁺ had a negligible effect on the activities of ribonucleotide reductase.