The effect of monovalent cations on the catalytic activity of thrombin

The effect of monovalent cations on the catalytic action of thrombin has been examined utilizing a variety of substrates. Sodium chloride noncompetitively inhibited the action of thrombin on α-tosyl-l-arginine methyl ester and α-N-benzoyl-l-arginine-p-nitroanilide. No inhibition was noted when α-N-benzoyl-l-arginine ethyl ester was the substrate. The extent of inhibition was considerably less with either potassium chloride or lithium chloride. The rate of inactivation of thrombin by 1-chloro-3-tosylamido-7-amino-l-2-heptanone was reduced in the presence of sodium ions. The results are interpreted to show a specific effect of sodium ions on the ability of the active-site histidine residue to participate in thrombic catalysis.     

Pyruvate kinase: Activation by and catalytic role of the monovalent and divalent cations

Summary This mini review is primarily concerned with the monovalent and divalent cation activation of pyruvate kinase. All preparations of pyruvate kinase from vertebrate tissue which have been examined require monovalent cations such as K⁺ for catalysis. However, several microbial preparations are not activated by monovalent cations. In fact,E. coli synthesizes depending on growth conditions, 2 different forms of the enzyme; one form is not activated while the other is activated by monovalent cations. The monovalent cation was shown by NMR techniques to bind within 4–8 ? of the divalent cation activat or and apparently plays a direct role in the catalytic process. As with all kinases, pyruvate kinase requires a divalent cation for catalysis. Mg⁺² is optimal for the physiological reaction, however, Co⁺², Mn⁺², and Ni⁺² also activate. The divalent cation activation of several non-physiological reactions catalyzed by pyruvate kinase are reviewed. Several lines of evidence suggest that 2 moles of the divalent cation are required in the catalytic event. However, the specific role of both atoms in the catalytic event have not been thoroughly elucidated.     

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.     

Adsorption of monovalent and divalent cations by phospholipid membranes. The monomer-dimer problem.

A generalization of the Stern theory is derived to treat the simultaneous adsorption of monovalent cations and divalent cations by single-component phospholipid membranes, where the ion:phospholipid binding stoichiometries are 1:1 for the monovalent cations and 1:1 and/or 1:2 for the divalent cations. This study treats both the situation in which the monovalent and divalent cations compete for membrane binding sites and that in which they do not compete. The general formalism of the screening/binding problem is reviewed, and it is shown how the adsorption problem can be isolated from the electrostatics. The statistical mechanics of mixed 1:1- and 1:2-stoichiometric adsorption (the monomer-dimer problem) is treated, and the problem of simultaneous 1:1 and 1:2 binding is solved. A simple expression for this solution, given in the Bethe approximation, is combined with the electrostatics to yield an adsorption isotherm encompassing both 1:1 monovalent-cation, and 1:1 and 1:2 divalent-cation, binding to charged membranes. A comparison with the simplified treatment of previous authors is made and the significance of their assumptions clarified in light of the present result. The present and previous treatments are plotted for a representative case of Na+ and Ca++ binding to a phosphatidylserine membrane. Criteria are established to permit unambiguous experimental testing of the present vs. previous treatments.     

Influence of monovalent and divalent cations on the surface area of phosphatidylglycerol monolayers

A theory is presented on the electrostatic properties of the surface area of phosphatidyl-glycerol monolayers spreading at an air-water interface in the presence of monovalent and divalent cations. In the present theory, the adsorption of monovalent and divalent cations to the membranes is taken into account, besides the dissociation of protons, as a possible cause of the change of surface charge density with the variation of pH or ion concentrations. It is also pointed out that, in the presence of structure-making ions such as Li⁺ and Na⁺, the nearest-neighbour interactions between proton dissociation sites become important for the monolayers in the gel state to yield a sharp expansion of the surface area with the increase of pH. The present theory shows quantitative agreements with previously-observed data.     

Corn leaf phosphoenolpyruvate carboxylases: The effect of divalent cations on activity

Differences in the kinetic properties of corn leaf phosphoenolpyruvate (PEP) carboxylase isoenzymes were found, depending on whether Mg²⁺ or Mn²⁺ was used as the metal cofactor of the reaction. Also, differences in kinetic constants with respect to Mg²⁺ and Mn²⁺ were noticed between the two isoenzymes which further differentiates the two proteins. The catalytic activity of the enzyme in the Mg²⁺-activated system was dependent on a PEP-Mg²⁺ complex and not on the concentration of free Mg²⁺ or free PEP. Kinetics in the presence of total Mg²⁺ and those of PEP-Mg²⁺ suggest a negative cooperative effect with respect to ligand binding with concurrent progressive substrate activation. Magnesium ions, thus, have a special regulatory role in the corn leaf PEP carboxylase reaction.     

Inhibition of protein kinase C phosphorylation by mono- and divalent cations

The effect of a matrix of concentrations of Ca2+ (0.01, 0.1, 0.5, 5 mM), Mg2+ (0.2, 0.5, 1, 2, 5, 10 mM), and Na+ (50, 100, 150 mM) on the phosphorylation of histone H-1 by protein kinase C was measured in the presence of 5 mol % diacylglycerol and Mg-ATP in both phosphatidylserine micelles and liposomes formed from a 1:4 mixture of phosphatidylserine and phosphatidylcholine. Monovalent cations (150 mM) reduced activity by 60 and 84% in the micelle and liposome assay systems, respectively. Inhibition was also observed with 5 mM Ca2+ and 10 mM Mg2+. The phosphorylating activity was compared with computer calculations of the negative electrostatic potentials (psi o) of the phospholipid membranes in the presence of the cations.     

Ionic currents of channels that are permeable to monovalent and divalent cations.

The cation-selective channel from Tetrahymena cilia is permeable to both monovalent and divalent cations. The single channel conductance in mixed solutions of K+ and Ca2+ was determined by the Gibbs-Donnan ratio of K+ and Ca2+, and the binding sites of this channel were considered to be always occupied by two potassium ions or by one calcium ion under the experimental conditions: 5-90 mM K+ and 0.5-35 mM Ca2+ (Oosawa and Kasai, 1988). A two-barrier model for the channel was introduced and the values of Michaelis-Menten constants and maximum currents carried by K+ and Ca2+ were calculated using this model. Single channel current amplitudes and reversal potentials were calculated from these values. The calculated single-channel currents were compared with those obtained experimentally. The calculated reversal potentials were compared with the resting potentials of Tetrahymena measured in various concentrations of extracellular K+ and Ca2+. The method of calculation of ionic currents and reversal potentials presented here is helpful for understanding the properties of the channels permeable to both monovalent and divalent cations.     

Comparison of the hammerhead cleavage reactions stimulated by monovalent and divalent cations

Although the hammerhead reaction proceeds most efficiently in divalent cations, cleavage in 4 M LiCl is only approximately 10-fold slower than under standard conditions of 10 mM MgCl2 (Murray et al., Chem Biol, 1998, 5:587-595; Curtis & Bartel, RNA, 2001, this issue, pp. 546-552). To determine if the catalytic mechanism with high concentrations of monovalent cations is similar to that with divalent cations, we compared the activities of a series of modified hammerhead ribozymes in the two ionic conditions. Nearly all of the modifications have similar deleterious effects under both reaction conditions, suggesting that the hammerhead adopts the same general catalytic structure with both monovalent and divalent cations. However, modification of three ligands previously implicated in the binding of a functional divalent metal ion have substantially smaller effects on the cleavage rate in Li+ than in Mg2+. This result suggests that an interaction analogous to the interaction made by this divalent metal ion is absent in the monovalent reaction. Although the contribution of this divalent metal ion to the overall reaction rate is relatively modest, its presence is needed to achieve the full catalytic rate. The role of this ion appears to be in facilitating formation of the active structure, and any direct chemical role of metal ions in hammerhead catalysis is small.     

Shielding effect of monovalent and divalent cations on solid-phase DNA hybridization: surface plasmon resonance biosensor study

Solid-phase hybridization, i.e. the process of recognition between DNA probes immobilized on a solid surface and complementary targets in a solution is a central process in DNA microarray and biosensor technologies. In this work, we investigate the simultaneous effect of monovalent and divalent cations on the hybridization of fully complementary or partly mismatched DNA targets to DNA probes immobilized on the surface of a surface plasmon resonance sensor. Our results demonstrate that the hybridization process is substantially influenced by the cation shielding effect and that this effect differs substantially for solid-phase hybridization, due to the high surface density of negatively charged probes, and hybridization in a solution. In our study divalent magnesium is found to be much more efficient in duplex stabilization than monovalent sodium (15 mM Mg²⁺ in buffer led to significantly higher hybridization than even 1 M Na⁺). This trend is opposite to that established for oligonucleotides in a solution. It is also shown that solid-phase duplex destabilization substantially increases with the length of the involved oligonucleotides. Moreover, it is demonstrated that the use of a buffer with the appropriate cation composition can improve the discrimination of complementary and point mismatched DNA targets.     

The effect of divalent cations on aggregation of Dictyostelium discoideum.

Following the initiation of development, amoebae of Dictyostelium discoideum aggregate chemotactically toward cyclic AMP (cAMP). Adenyl cyclase, cAMP phosphodiesterase, and cAMP binding sites all increase 20--40 fold during the first few hours of development. It has been shown that addition of 1 mM EDTA and 5 mM MgCl2 accelerates the aggregation process. Likewise, the calcium ionophore, A23187, leads to precocious aggregation while 4 X 10(-5) M progesterone considerably delays it. These treatments have now been shown to result in increased accumulation of adenyl cyclase in the case of EDTA and Mg2+ or the ionophore and greatly decreased accumulation in the case of the steroid. Treatment with EDTA and Mg2+ or the ionophore has been shown not only to accelerate aggregation in wild-type amoebae but to overcome complete blocks to aggregation in certain mutant strains. We have found that addition of Mn2+ will also permit aggregation of mutant cells otherwise unable to aggregate. This divalent ion, unlike EDTA and Mg2+ or the ionophore, was shown to directly stimulate adenyl cyclase. Calcium ions were also found to affect the enzyme such that at Ca2+ concentrations found within the cells the great majority of the activity is inhibited. Manganese ions can overcome the inhibition by Ca2+. These findings show that conditions which stimulate aggregation result in increased activity of adenyl cyclase either by increased accumulation of the enzyme or by increased activity of the available enzyme, and support the proposed central role of adenyl cyclase in aggregation.     

Induction of passive monovalent cation-exchange activity in heart mitochondria by depletion of endogenous divalent cations

Depletion of mitochondrial divalent cations by addition of the ionophore A23187 results in a marked increase in passive ${}^{42}\text{K}{}^{\mathrm{+}}\text{K}{}^{\mathrm{+}}$ exchange activity. The exchange is activated by increasing pH and temperature and inhibited by added divalent cations. The reaction is independent of the amount of A23187 present, but depends on the concentration of external K⁺ (K_m = 25 mm). Intramitochondrial ⁴²K⁺ in cation-depleted mitochondria exchanges passively with external Na⁺ and Li⁺, but not with choline⁺. The evidence suggests that removal of mitochondrial divalent cations by A23187 activates the endogenous $\text{K}{}^{\mathrm{+}}\text{H}{}^{\mathrm{+}}$ exchange component of the mitochondrion and that the activated exchanger promotes cation/cation exchange in the absence of a metabolic pH gradient.