Fluorescamine-induced membrane permeability in mitochondria.

1. Addition of fluorescamine (75 microM) to mitochondria induced an increase in membrane permeability. 2. The leakiness of the inner mitochondrial membrane is characterized by extensive release of accumulated Ca2+, collapse of the transmembrane potential, mitochondrial swelling and efflux of matrix proteins, among them, malate dehydrogenase. 3. These effects were diminished by supplementing the media with 1 mM phosphate, and partially prevented by Mg2+. 4. These results indicate that the primary amino groups of membrane components contribute, partially, to the maintenance of the permeability barrier in mitochondria.     

The subcellular localization of neutral sphingomyelinase in rat liver.

The subcellular distribution of neutral sphingomyelinase activity has been determined in rat liver. Neutral sphingomyelinase is present in the plasma membrane. This enzyme requires either Mg2+ or Mn2+ for full activity; these cations cannot be replaced by Co2+ or Ca2+. The plasma membrane sphingomyelinase is strongly inhibited by Hg2+. A small amount of neutral spingomyelinase activity appears to be present in microsomes. No neutral sphingomyelinase activity is present in liver mitochondria or bytosol. Lysosomal sphingomyelinase is fully active at pH 4.4--4.8 without added divalent cations. However, between pH 5.0 and 7.5 lysosomal sphingomyelinase activity is stimulated by Mg2+, Mn2+, Co2+, and Ca2+. Below pH 4.8, Mg2+ inhibits the reaction. In contrast to the results obtained with the neutral sphingomyelinase activity of plasma membranes and microsomes, lysosomal sphingomyelinase is unaffected by sulfhydryl inhibitors.     

Thermostable DNA polymerase from Thermus thermophilus B35: influence of divalent metal ions on the interaction with deoxynucleoside triphosphates

The interaction of DNA polymerase from Thermus thermophilus B35 (Tte-pol) with deoxynucleoside triphosphates in the presence of different divalent metal ions has been studied. DNA synthesis and competitive inhibition of the polymerase reaction by non-complementary dNTPs are described with corresponding kinetic schemes. The co-factor properties of some metals (Mg2+, Mn2+, Co2+, Ni2+, Cu2+, Ca2+, Cd2+, and Zn2+) were investigated, and their activating concentration ranges were determined. It was found that kcat values are significantly decreased and Km values slowly decrease when Mn2+ displaces Mg2+. The value of Kd for DNA template-primer is Me2+-independent, whereas Kd values for non-complementary dNTPs decrease in the presence of Mn2+. Tte-pol processivity but not DNA synthesis efficiency is Me2+-type independent.     

Formation of a hexagonal lattice structure by an R-form lipopolysaccharide of Klebsiella: effect of various divalent cations on the lattice formation

The R-form lipopolysaccharide from Klebsiella pneumoniae strain LEN-111 (O3-:K1-), from which cationic material had been removed by electrodialysis, was previously shown to form a hexagonal lattice structure with the lattice constant of 14 to 15 nm when suspended in 50 mM tris(hydroxymethyl)aminomethane buffer at pH 8.5 containing 10 mM Mg2+. Under this experimental condition, effects of other divalent metal cations on the hexagonal assembly of the electrodialyzed LPS were compared with that of Mg2+. The Zn2+, Hg2+, Cu2+, and Ni2+ could produce essentially the same hexagonal lattice structure with the lattice constant of 14.5 to 15.0 nm as that formed with Mg2+. The Cd2+, Co2+, and Fe2+ produced the hexagonal lattice structure with the lattice constant of 15.5 to 16.0 nm, and Ba2+, Sr2+, and Ca2+ produced that with the lattice constant of 18 to 19 nm. In addition, the hexagonal lattice structures formed with the latter three cations were less orderly than those formed with the other cations. When the higher concentrations of Ba2+, Sr2+, and Ca2+ were used, the lattice constants were not shortened. The length of lattice constants of the hexagonal lattice structures formed with the divalent cations did not relate to the quantity of the cations bound to the LPS. Among the divalent cations tested, Hg2+ was bound to the LPS in the smallest amount (its atomic ratio to P, 0.07), and Zn2+ and Fe2+ were bound in very large amounts (their atomic ratios to P, 2.94 and 8.28, respectively).     

The effects of amphiphilic cationic drugs and inorganic cations on the activity of phosphatidate phosphohydrolase.

1. Phosphatidate phosphohydrolase from the particle-free supernatant of rat liver was assayed by using emulsions of phosphatidate as substrate. 2. The inhibition of the phosphohydrolase by chlorpromazine was of a competitive type with respect to phosphatidate. The potency of various amphiphilic cationic drugs as inhibitors of this reaction was related to their partition coefficients into a phosphatidate emulsion. 3. The effect of chlorpromazine on the phosphohydrolase activity was complementary rather than antagonistic towards Mg2+. Chlorpromazine stimulated the phosphohydrolase activity in the absence of added Mg2+ and was able to replace the requirement for Mg2+. However, at optimum concentrations of Mg2+, chlorpromazine inhibited the reaction, as did Ca2+. The phosphohydrolase activity was also stimulated by Co2+ and to a lesser extent by Mn2+, Fe2+, Fe3+, Ca2+, spermine and spermidine when Mg2+ was not added to the assays. 4. It is concluded that the inhibition of phosphatidate phosphohydrolase by amphiphilic cations can largely be explained by the interaction of these compounds with phosphatidate, which changes the physical properties of the lipid, making it less available for conversion into diacylglycerol. 5. The implications of these results to the effects of amphiphilic cations in redirecting glycerolipid synthesis at the level of phosphatidate are discussed.     

Calcium and magnesium regulation of phosphorylation by ATP and ITP in sarcoplasmic reticulum vesicles.

Membrane phosphorylation and nucleoside triphosphatase activity of sarcoplasmic reticulum vesicles isolated from rabbit skeletal muscle were studied using ATP and ITP as substrates. The Ca2+ concentration was varied over a range large enough to saturate either the high affinity Ca2+-binding site or both high and low affinity binding sites. In intact vesicles, which are able to accumulate Ca2+, the steady state level of enzyme phosphorylated by either ATP or ITP is already high in 0.02 mM Ca2+ and does not vary as the Ca2+ concentration is increased to 10 mM. Essentially the same pattern of membrane phosphorylation by ATP is observed when leaky vesicles, which are unable to accumulate Ca2+, are used. However, for leaky vesicles, when ITP is used as substrate, the phosphoenzyme level increases 3- to 4-fold when the Ca2+ concentration is raised from 0.02 to 20 mM. When Mg2+ is omitted from the assay medum, the degree of membrane phosphorylation by ATP varies with Ca2+ in the same way as when ITP is used in the presence of Mg2+. Membrane phosphorylation of leaky vesicles by either ATP or ITP is observed in the absence of added Mg2+. When these vesicles are incubated in media containing ITP and 0.1 mM Ca2+, addition of Mg2+ up to 10 mM simultaneously decreases the steady state level of phosphoenzyme and increases the rate of ITP hydrolysis. When ATP is used, the addition of 10 mM Mg2+ increases both the steady state level of phosphoenzyme and the rate of ATP hydrolysis. When the Ca2+ concentration is raised to 10 or 20 mM, the degree of membrane phosphorylation by either ATP or ITP is maximal even in the absence of added Mg2+ and does not vary with the addition of 10 mM Mg2+. In these conditions the ATPase and ITPase activities are activated by Mg2+, although not to the level observed in 0.1 mM Ca2+. An excess of Mg2+ inhibits both the rate of hydrolysis and membrane phosphorylation by either ATP or ITP.     

Mechanistic studies on E. coli DNA topoisomerase I: divalent ion effects.

E. coli DNA topoisomerase I catalyzes the hydrolysis of short, single stranded oligodeoxynucleotides. It also forms a covalent protein-DNA complex with negatively supercoiled DNA in the absence of Mg2+ but requires Mg2+ for the relaxation of negatively supercoiled DNA. In this paper we investigate the effects of various divalent metals on catalysis. For the relaxation reaction, maximum enzyme activity plateaus after 2.5 mM Mg2+. However, the rate of cleavage of short oligodeoxynucleotide increased linearly between 0 and 15 mM Mg2+. In the oligodeoxynucleotide cleavage reaction, Ca2+, Mn2+, Co2+, and Zn2+ inhibit enzymatic activity. When these metals are coincubated with Mg2+ at equimolar concentrations, the normal effect of Mg2+ is not detectable. Of these metals, only Ca2+ can be substituted for Mg2+ as a metal cofactor in the relaxation reaction. And when Mg2+ is coincubated with Mn2+, Co2+, or Zn2+ at equimolar concentrations, the normal effect of Mg2+ on relaxation is not detectable. We propose that Mg2+ allows the protein-DNA complex to assume a conformation necessary for strand passage and enhance the rate of enzyme turnover.     

Mutants in three genes affecting transport of magnesium in Escherichia coli: genetics and physiology.

Mutants in three genes affecting two Mg2+ transport systems are described. System I, for which Co2+, Mn2+, and Mg2+ are substrates, is inactive in corA mutants corB mutants express system I after growth on high (10 mM) Mg2+ but not low (0.1 mM) Mg2+. Both corA and corB mutants are resistant to Co2+ or Mn2+. corA mutants are sensitive to CA2+. Transport system II is specific for Mg2+ and is repressed by growth on 10 mM Mg2+. mgt mutations inactivate system II. Growth on mgt mutants in normal except on very low (1 muM) concentrations of Mg2+, corA mgt strains exhibit no high-affinity, energy-dependent transport of Mg2+ and require 10 mM Mg2+ for optimal growth. The three genes are not linked. The corA locus is contransducible with ilv at 75 min, corB is cotransducible with pyrB at 85 min, and mgt is cotransducible with malB and mel at 81 min on the genetic map.     

Substitution studies of the second divalent metal cation requirement of protein tyrosine kinase CSK

In addition to a magnesium ion needed to form the ATP-Mg complex, we have previously determined that at least one more free Mg2+ ion is essential for the activation of the protein tyrosine kinase, Csk [Sun, G., and Budde, R. J. A. (1997) Biochemistry 36, 2139-2146]. In this paper, we report that several divalent metal cations, such as Mn2+, Co2+, Ni2+, and Zn2+ bind to the second Mg2+-binding site of Csk with up to 13200-fold higher affinity than Mg2+. This finding enabled us to substitute the free Mg2+ at this site with Mn2+, Co2+, Ni2+, or Zn2+ while keeping ATP saturated with Mg2+ to study the role of the free metal cation in Csk catalysis. Substitution by these divalent metal cations resulted in varied levels of Csk activity, with Mn2+ even more effective than Mg2+. Co2+ and Ni2+ supports reduced levels of Csk activity compared to Mg2+. Zn2+ has the highest affinity for the second Mg2+-binding site of Csk at 0.65 microM, but supports no kinase activity, acting as a dead-end inhibitor. The inhibition by Zn2+ is reversible and competitive against free Mg2+, noncompetitive against ATP-Mg, and mixed against the phosphate accepting substrate, polyE4Y, significantly increasing the affinity for this substrate. Substitution of the free Mg2+ with Mn2+, Co2+, or Ni2+ also results in lower Km values for the peptide substrate. These results suggest that the divalent metal activator is an important element in determining the affinity between Csk and the phosphate-accepting substrate.     

The effect of divalent cations on bovine spermatozoal adenylate cyclase activity.

The effect of divalent cations on bovine sperm adenylate cyclase activity was studied. Mn2+, Co2+, Cd2+, Zn2+, Mg2+ and Ca2+ were found to satisfy the divalent cation requirement for catalysis of the bovine sperm adenylate cyclase. These divalent cations in excess of the amount necessary for the formation of the metal-ATP substrate complex were found to stimulate the enzyme activity to various degrees. The magnitude of stimulation at saturating concentrations of the divalent cations was strikingly greater with M2+ than with either Ca2+, Mg2+, Zn2+, Cd2+ or Co2+. The apparent Km was lowest for Zm2+ (0.1 - 0.2 mM) than for any of the other divalent cations tested (1.2 - 2.3 mM). The enzyme stimulation by Mn2+ was decreased by the simultaneous addition of Co2+, Cd2+, Ni2+ and particularly Zn2+ and Cu2+. The antagonism between Mn2+ and Cu2+ or Zn2+ appeared to have both competitive and non-competitive features. The inhibitory effect of Cu2+ on Mn2+-stimulated adenylate cyclase activity was prevented by 2,3-dimercaptopropanol, but not by dithiothreitol, L-ergothioneine, EDTA, EGTA or D-penicillamine. Ca2+ at concentrations of 1-5 mM was found to act synergistically with Mg2+, Zn2+, Co2+ and Mn2+ in stimulating sperm adenylate cyclase activity. The Ca2+ augmentation of the stimulatory effect of Zn2+, Co2+, Mg2+ and Mn2+ appeared to be specific.     

Properties of Na+, K+-ATPase of liver plasma membranes in the hamster

This study was designed to establish the properties of liver plasma membranes (LPM) Na+,K+-ATPase in the hamster and to determine whether a similar assay may be used to measure enzyme activity in the hamster and in the rat. Maximal Na+,K+-ATPase activity was obtained when the assay medium contained 5 mM Mg APT2- with or without 1 mM free Mg2+, 120 mM Na+, 12,5 mM K+. The incubation must be performed at 37 degrees C, pH 7.4. In the absence of free Mg2+, the saturation curve with respect to the substrate Mg ATP2- resulted in biphasic complex kinetics with a maximal activity at a substrate concentration of 5 mM. In the presence of 1 mM free Mg2+ activation of Na+,K+-ATPase and modification of the kinetics were observed: the biphasic curve tended to disappear and to become of the Michaelis-Menten type. The apparent Km for Mg APT2- was 0.36 mM and the Vmax 34.5 mumol.h-1.mg protein-1. In the presence of 10 mM free Mg2+ a decrease in the Vmax was observed without any effect on the apparent Km for Mg APT2-. It is concluded that the same incubation medium may be used to assay LPM N+,K+-ATPase from hamster and rat and that the addition of 1 mM free Mg2+ to the incubation medium is recommended to obtain Michaelis-Menten kinetics in order to eliminate complex kinetics due to the absence of free Mg2+.     

Calorimetric studies of the interaction of magnesium and phosphate with Na+, K+) ATPase: evidence for a ligand-induced conformational change in the enzyme.

The phosphorylation of (Na+, K+)ATPase from the electric organ of the electric eel is dependent on Mg2+. The amount of phosphoenzyme formed was increased by K+ and decreased by Na+. Kinetic analyses indicate that a ternary complex of ATPase, Pi and Mg2+ is formed prior to phosphorylation of the protein. Calorimetric studies revealed extraordinarily large enthalpy changes associated with the binding of Mg2+ (-49 kcal/mol) and of Pi (-42 kcal/mol), indicating a thermodynamically significant conformational change in the enzyme. The dissociation constant for the binding of Mg2+ and Pi derived from calorimetric measurements is in good agreement with the value obtained from the kinetic studies. These results indicate that ion binding induces a conformational change in the enzyme which is a prerequisite for phosphorylation by Pi.     

Aminoacyl transfer RNA formation. VII. Lack of correlation between aminoacylation and PPi-ATP exchange catalyzed by isoleucyl-tRNA synthetase of Escherichia coli in the presence of various divalent cations.

Isoleucyl-tRNA formation and isoleucine-dependent PPi-ATP exchange catalyzed by purified isoleucyl-tRNA synthetase [EC 6.1.1.5] of Escherichia coli were studied in the presence of various amounts of either Mg2+, Ca2+, Fe2+, Ni2+, or Cu2+. In the presence of Mg2+, isoleucine-dependent PPi-ATP exchange was observed in parallel with isoleucyl-tRNA formation, while in the presence of Ca2+, isoleucyl-tRNA formation was observed without isoleucine-dependent PPi-ATP exchange. Moreover, isoleucine-dependent PPi-ATP exchange was much more in the presence of Fe2+ than in the presence of Mg2+, while little isoleucyl-tRNA was formed in the presence of Fe2+. In the presence of Ni2+ or Cu2+, neither reaction was observed. These data, indicating that formation of an isoleucyl-AMP-enzyme complex is not a necessary step in isoleucyl-tRNA formation, support the existence of a concerted mechanism of isoleucyl-tRNA formation in E. coli.     

Effects of trifluoperazine on calcium binding by calmodulin. Heat capacity and entropy changes

The possible structural changes of the calmodulin-trifluoperazine (TFP) complex caused by Ca2+ binding have been analyzed by microcalorimetric titrations. Titrations of calmodulin with Ca2+ in the presence of 8-fold molar excess TFP have been made both in the absence and presence of Mg2+, at pH 7.0, and at 5, 15, and 25 degrees C. At high concentrations of TFP calmodulin forms a complex with TFP even in the absence of Ca2+. The reaction of the calmodulin-TFP complex with Ca2+ is exothermic, both in the presence and absence of Mg2+. In the presence of Mg2+ the reaction is driven almost entirely by a favorable enthalpy change. The magnitudes of the hydrophobic and internal vibrational contributions to the heat capacity and entropy changes of this complex on Ca2+ binding have been estimated by the empirical method of Sturtevant (Sturtevant, J. M. (1977) Proc. Natl. Acad. Sci. U. S. A. 74, 2236-2240). In the presence of Mg2+, the vibrational as well as hydrophobic entropy is slightly increased in a parallel manner by Ca2+ binding to each of the binding sites. In contrast, when Mg2+ is absent, the hydrophobic entropy gradually increases on Ca2+ binding, but the vibrational entropy decreases. These changes of entropy indicate the assembling of non-polar groups on the surface of the complex and suggest that the overall structure is loosened in the presence of Mg2+, but tightened in the absence of Mg2+.     

Mn 2+-stimulatedATPase in rat brain

Divalent cation ATPases were prepared from rat brain synaptic vesicles, synaptosomal plasma membranes, and plasma membranes from the brain stem and sciatic nerve and tested for optimal stimulation by Mn2+, Mg2+, or Ca2+. ATPase in the synaptic vesicle subfraction was optimally stimulated by Mn2+. All plasma membrane preparations were optimally stimulated by Mg2+. Separate Mn2+ and Mg2+ ATPases could not be distinguished by either chemical inactivation or substrate preference criteria. Mn2+ stimulated ATPase in the micromolar range and it is suggested that Mn2+ interaction with ATPase may be of physiological and/or toxicological importance by being related to the cellular metabolism of this element.     

Phosphorolysis of acetyl phosphate by orthophosphate with energy conservation in the phosphoanhydride linkage of pyrophosphate

The formation of pyrophosphate as a result of nucleophilic attack by orthophosphate at the acylphosphate bond of acetyl phosphate was detectable in completely aqueous media, and was enhanced by dimethyl sulfoxide. The reaction had an absolute requirement for divalent cations, the rate constant of phosphorolysis being dependent on the species and concentration of cations as well as on temperature and pH. The amount of pyrophosphate formed depended on both the acetyl phosphate and orthophosphate concentrations. In purely aqueous media, phosphorolysis was barely detectable in the presence of Mg2+, and its rate increased 40-fold when Mg2+ was replaced by Ca2+ or Sr2+. In the presence of Mg2+ the rate of phosphorolysis increased 400-fold when 50 to 80% of the water was replaced by dimethyl sulfoxide. In the latter case, the rate also increased as the pH was raised from 4.0 to 9.0. The entropy of activation was large and negative in the presence of Mg2+ or Ca2+, indicating that the nucleophile is involved in the rate-limiting step of the reaction. Since this thermodynamic parameter became large and positive in the presence of Ca2+ when dimethyl sulfoxide was omitted, it is inferred that the transition state of the same reaction may be changed by the solvent composition and the solvation of reactants.     

Autophosphorylation of phosphorylase kinase. Divalent metal cation and nucleotide dependency

This study reports on the divalent metal ion specificity for phosphorylase kinase autophosphorylation and, in particular, provides a comparison between the efficacy of Mg2+ and Mn2+ in this role. As well as requiring Ca2+ plus divalent metal ion-ATP2- as substrate, both phosphorylase kinase autoactivation and phosphorylase conversion are additionally modulated by divalent cations. However, these reactions are affected differently by different ions. Phosphorylase kinase-catalyzed phosphorylase conversion is maximally enhanced by a 4- to 10-fold lower concentration of Mg2+ than is autocatalysis and, whereas both reactions are stimulated by Mg2+, autophosphorylation is activated by Mn2+, Co2+, and Ni2+ while phosphorylase a formation is inhibited. This difference may be due to an effect of free Mn2+ on phosphorylase rather than the inability of phosphorylase kinase to use MnATP as a substrate when catalyzing phosphorylase conversion since Mn2+, when added at a level which minimally decreases [MgATP], greatly inhibits phosphorylase phosphorylation. The interactions of Mn2+ with phosphorylase kinase are different from those of Mg2+. Not only are the effects of these ions on phosphorylase activation opposite, but they also provoke different patterns of subunit phosphorylation during phosphorylase kinase autocatalysis. With Mn2+, the time lag of phosphorylation of both the alpha and beta subunits of phosphorylase kinase in autocatalysis is diminished in comparison to what is observed with Mg2+, and the beta subunit is only phosphorylated to a maximum of 1 mol/mol of subunit. With both Mg2+ and Mn2+ the alpha subunit is phosphorylated to a level in excess of 3 mol/mol, a level similar to that obtained for beta subunit phosphorylation in the presence of Mg2+. The support of autophosphorylation by both Co2+ and Ni2+ has characteristics similar to those observed with Mn2+. Although Mn2+ stimulation of autophosphorylation occurs at levels much higher than normal physiological levels, the possible potential of phosphorylase kinase autophosphorylation as a control mechanism is illustrated by the 80- to 100-fold activation that occurs in the presence of Mn2+, a level far in excess of the enzyme activity change normally seen with covalent modification. Autophosphorylation of phosphorylase kinase demonstrates a Km for Mg X ATP2- of 27.7 microM and a Ka for Mg2+ of 3.1 mM. The reaction mechanism of autophosphorylation is intramolecular. This latter observation may indicate that phosphorylase kinase autocatalysis could be of potential physiological relevance and could occur with equal facility in cells containing either constitutively high or low levels of this enzyme.     

Regulation of intracellular magnesium by Mg2+ efflux

Chicken erythrocytes were loaded with Mg2+ by incubation with the cation ionophore A 23187 in the presence of Mg2+. After removing A 23187 by intensive washing with serum albumin and reincubating the Mg2+-loaded cells, Mg2+ was transported out of the cells until the original Mg2+ content was achieved. The net Mg2+ efflux followed Michaelis-Menten-kinetics and was independent of extracellular and intracellular Ca2+ and calmodulin. The net Mg2+ efflux was not affected by adrenalin, isoproterenol, p-chloromercuribenzenesulfonate, ouabain and tetrodotoxin, but was inhibited by dicyclohexylcarbodiimide, KCN, iodoacetate, high extracellular concentrations of Mg2+, Mn2+ and when extracellular Na+ was substituted by choline or K+. The efflux of 1 Mg2+ was coupled with the uptake of 2 Na+. It is concluded that there exists an additional gating process at the inner cell surface becoming active only at increased concentrations of intracellular free Mg2+ regulating the exit of Mg2+ by the efflux system.     

Purification and characterization of cyclic GMP-stimulated cyclic nucleotide phosphodiesterase from calf liver. Effects of divalent cations on activity

Cyclic GMP-stimulated cyclic nucleotide phosphodiesterase purified greater than 13,000-fold to apparent homogeneity from calf liver exhibited a single protein band (Mr approximately 102,000) on polyacrylamide gel electrophoresis under denaturing conditions. Enzyme activity comigrated with the single protein peak on analytical polyacrylamide gel electrophoresis, sucrose density gradient centrifugation, and gel filtration. From the sedimentation coefficient of 6.9 S and Stokes radius of 67 A, an Mr of 201,000 and frictional ratio (f/fo) of 1.7 were calculated, suggesting that the native enzyme is a nonspherical dimer of similar, if not identical, peptides. The effectiveness of Mg2+, Mn2+, and Co2+ in supporting catalytic activity depended on the concentration of cGMP and cAMP present as substrate or effector. Over a wide range of substrate concentrations, optimal concentrations for Mg2+, Mn2+, and Co2+ were about 10, 1, and 0.2 mM, respectively. At concentrations higher than optimal, Mg2+ inhibited activity somewhat; inhibition by Co2+ (and in some instances by Mn2+) was virtually complete. At low substrate concentrations, activity with optimal Mn2+ was equal to or greater than that with Co2+ and always greater than that with Mg2+. With greater than or equal to 0.5 microM cGMP or 20 to 300 microM cAMP and for cAMP-stimulated cGMP or cGMP-stimulated cAMP hydrolysis, activity with Mg2+ greater than Mn2+ greater than Co2+. In the presence of Mg2+, the purified enzyme hydrolyzed cGMP and cAMP with kinetics suggestive of positive cooperativity. Apparent Km values were 15 and 33 microM, and maximal velocities were 200 and 170 mumol/min/mg of protein, respectively. Substitution of Mn2+ for Mg2+ increased apparent Km and reduced Vmax for cGMP with little effect on Km or Vmax for cAMP. Co2+ increased Km and reduced Vmax for both. cGMP stimulated cAMP hydrolysis approximately 32-fold in the presence of Mg2+, much less with Mn2+ or Co2+. In the presence of Mg2+, Mn2+ and Co2+ at concentrations that increased activity when present singly inhibited cGMP-stimulated cAMP hydrolysis. It appears that divalent cations as well as cyclic nucleotides affect cooperative interactions of this enzyme. Whereas Co2+ effects were observed in the presence of either cyclic nucleotide, Mn2+ effects were especially prominent when cGMP was present (either as substrate or effector).