Size-dependent allosteric effects of monovalent cations on rabbit liver fructose-1,6-bisphosphatase.

Effects of monovalent cations on the neutral rabbit liver fructose-1,6-bisphosphatase are multifunctional and dependent on their nonhydrated ionic size. (a) The maximal velocity is increased by addition of monovalent cations with the optimum stimulation occurring with a nonhydrated ionic radius of 1.2 A in the presence of a chelating agent such as EDTA. (B) Activation curves are sigmoidal with n values varying from 1.5 to 2.3 as ionic radius of monovalent cation increases. The apparent Ka values from 16.0 to 180 mM, obtained for various monovalent cations, have a linear relationship to ionic radii of cations. (c) At lower concentrations of fructose 1,6-bisphosphate monovalent cations show the inhibitory effect and the apparent Km for fructose 1,6-bisphosphate is increased as the concentration of monovalent cation is increased. A linear relationship is obtained between the slopes of increase in the Km and the reciprocals of ionic volume of monovalent cations. (d) The apparent Ka for Mg2+ is also increased as the concentration of monovalent cation is increased, and a linear relationship is obtained again between the increases in Ka and the reciprocals of ionic volume of monovalent cations. The cooperative nature for Mg2+ saturation is decreased as the Ka increases. (e) The apparent Ki for AMP is also linearly altered as the concentration of monovalent cation is varied. However, the alteration of the Ki is unusual, that is, the smaller cations than K+ increase the Ki (Li+ greater than Na+ greater than NH4+), whereas the larger cations decrease the value ((CH2CH2OH)3N+ greater than Cs+ greater than Rb+). The effect of K+ is insignificant. Alterations in the Ki are also linearly related to the reciprocals of ionic volume of monovalent cations. The cooperative nature for AMP inhibition is decreased or increased as the Ki increased or decreased. (f) In the absence of the chelating agent, the curves for Mg2+ saturation and AMP inhibition were hyperbolic without monovalent cations. By addition of monovalent cation the Ka for Mg+2+ or Ki for AMP is increased and cooperative natures for binding of both ligands are induced. For nonspherical monovalent cations, the application of "functional ionic radius" is proposed. Functional ionic radii of NH4+, (CH2OH)3CNH3+, and (CH2CH2OH)3N+ are estimated to be 1.17, 2.55, and 2.87 A, respectively. The presence of two distinct sites for the actions of monovalent cations is suggested.     

Interaction of Tl+ with product complexes of fructose-1,6-bisphosphatase

Fructose-1,6-bisphosphatase requires divalent cations (Mg2+, Mn2+, or Zn2+) for catalysis, but a diverse set of monovalent cations (K+, Tl+, Rb+, or NH(4)(+)) will further enhance enzyme activity. Here, the interaction of Tl+ with fructose-1,6-bisphosphatase is explored under conditions that support catalysis. On the basis of initial velocity kinetics, Tl+ enhances catalysis by 20% with a K(a) of 1.3 mm and a Hill coefficient near unity. Crystal structures of enzyme complexes with Mg2+, Tl+, and reaction products, in which the concentration of Tl+ is 1 mm or less, reveal Mg2+ at metal sites 1, 2, and 3 of the active site, but little or no bound Tl+. Intermediate concentrations of Tl+ (5-20 mm) displace Mg2+ from site 3 and the 1-OH group of fructose 6-phosphate from in-line geometry with respect to bound orthophosphate. Loop 52-72 appears in a new conformational state, differing from its engaged conformation by disorder in residues 61-69. Tl+ does not bind to metal sites 1 or 2 in the presence of Mg2+, but does bind to four other sites with partial occupancy. Two of four Tl+ sites probably represent alternative binding sites for the site 3 catalytic Mg2+, whereas the other sites could play roles in monovalent cation activation.     

The interaction of “K+-like” cations with the apical K+ channel in frog skin

The apparent permeability of the apical K+ channel in the abdominal skin of the frog (Rana temporaria) for different monovalent cations was tested by comparing the short-circuit current (SCC) obtained after imposition of serosally directed ionic concentration gradients. Furthermore, the SCC was subjected to noise analysis. Of various cations tested, only the "K+-like" ions NH+4, Rb+ and Tl+, besides K+, were found to permeate the apical K+ channel, as reflected by SCC- and fluctuation analysis: (i) The SCC could be depressed by addition of the K+-channel blocker Ba2+ to the mucosal solution. (ii) With the K+-like ions (Ringer's concentration), a spontaneous Lorentzian noise was observed. Plateau values were similar for K+ and Tl+, and smaller for NH+4 and Rb+. The corner frequencies clearly increased in the order K+ less than NH+4 less than Tl+ much less than Rb+. The SCC dose-response relationships revealed a Michaelis-Menten-type current saturation only for pure K+- or Tl+-Ringer's solutions as mucosal medium, whereas a more complicated SCC behavior was seen with Rb+ and especially, NH+4. For K+-Tl+ mixtures an anomalous mole-fraction relationship was observed: At low [Tl+]/[K+] ratios, Tl+ ions appeared to inhibit competitively the K+ current while, at high [Tl+]/[K+] ratios, Tl+ seemed to be a permeant cation. This feature was also detected in the noise analysis of K+-Tl+ mixtures. Long-term exposure to mucosal Tl+ resulted in an irreversible deterioration of the tissue. The SCC depression by Ba2+ was of a simple saturation-type characteristic with, however, different half-maximal doses (NH+4 less than K+ less than Rb+). Ba2+ induced a "blocker noise" in presence of all permeant cations with corner frequencies that depended on the Ba2+ concentration. A linear increase of the corner frequencies of the Ba2+-induced noise with increasing Ba2+ concentration was seen for NH+4, Rb+ and K+. With the assumption of a pseudo two-state model for the Ba2+ blockade the on- and off-rate constants for the Ba2+ interaction with the NH+4/Rb+/K+ channel were calculated and showed marked differences, dependent on the nature of the permeant ion. The specific problems with Tl+ prevented such an analysis but SCC- and noise data indicated a comparably poor efficiency of Ba2+ as Tl+-current inhibitor. We attempted a qualitative analysis of our results in terms of a "two-sites, three-barriers" model of the apical K+ channel in frog skin.     

Voltage-dependent stimulation of Na+/K(+)-pump current by external cations: selectivity of different K+ congeners.

Currents generated by the endogenous Na+/K+ pump in the oocytes of Xenopus laevis were determined under voltage-clamp as currents activated by different K+ congeners. The voltage dependence of the pump current reflects voltage-dependent steps in the reaction cycle. The decrease of K(+)-activated pump current at positive potentials has been attributed to voltage-dependent stimulation by the external K+ (Rakowski, Vasilets, LaTona and Schwarz (1991) J. Membr. Biol. 121, 177-187). In Na(+)-free solution, activation of the pump by external cations seems to be the dominating voltage-dependent and rate-determining step in the reaction cycle. Under these conditions, the voltage dependence of apparent Km values for pump activation can be analyzed. The dependence suggests voltage-dependent binding of extracellular cations assuming that an effective charge of about 0.4 of an elementary charge is moved in the electrical field during a step associated with the cation binding. The apparent Km values at 0 mV differ for various cations that stimulate pump activity. The values are in mM: 0.10 for Tl+, 0.63 for K+, 0.71 for Rb+, 9.3 for NH4+, and 12.9 for Cs+. The corresponding apparent affinities follow the same sequence as the cation permeability of the K(+)-selective delayed rectifier channel of nerve cells. The results are compatible with the interpretation that the cations have to pass an ion-selective access channel to reach their binding sites in the pump molecule.     

Additional effects of monovalent cations on the lysine-sensitive aspartokinase of E. coli B

The apparent specificity of activation of lysine-sensitive aspartokinase (E.C.2.7.2.4) from E. coli by monovalent cations differs depending on the assay used and on the Mg2+ concentration. Activity is nearly absolutely dependent on and is highly specific for a monovalent cation in the aspartate semialdehyde dehydrogenase coupled assay or the adenosine triphosphate-adenosine diphosphate exchange assay. Little specificity for monovalent cations is observed using the aspartyl hydroxamate assay. Activation and specificity are also altered by Mg2+ concentrations at a constant 5 mM nucleotide concentration. At a low (1.25 or 1.6 mM)Mg2+ concentration, monovalent cation activation and specificity are nearly absolute. Less dependence on monovalent cations and less specificity are observed at a higher Mg2+ concentration (6 mM). Li+ inhibits aspartokinase competitively with respect to either K+ or NH4+. Monovalent cations are also thermoprotective and differential thermal inactivation experiments at 56 degrees C reveal that NH4+ and K+, either of which will produce maximum catalytic activity, interact differently with aspartokinase. K+ interacts with positive cooperativity, whereas NH4+ does not. K+, NH4+, and Na+ are about equally effective in enhancing the dissociation of the aspartokinase-aspartylphosphate complex. Li+ is less effective.     

Effect of environmental factors on inactivation of B12-dependent glycerol dehydratase from Aerobacter aerogenes]

Effect of temperature, pH and univalent cation on kinetics of self-activation of B12-dependent glycerol dehydratase (GD) from Aerobacter aerogenes with Co alpha-[alpha-(5,6-dimethylbenzimidazolyl]-Co beta-adenosylcobamide (AdoCbl) was investigated. The activation energy of the process of GD inactivation is found to be 3.9 kkal/M, the effect of pH on GD inactivation being insignificant. Monovalent cation is not required for the formation of GD-AdoCbl complex, but it protects the complex from selfinactivation. The rate of GD inactivation greatly depends on concentration of monovalent cations. Effect of K+, Rb+, Cs+, Tl+ and NH4+ cations, which are enzyme cofactors, qualitatively differs from the effect of Na+ and Li+, which are inactive in a catalytic reaction. The presence of at least two cation-binding sites in GD molecule is suggested. Possible mechanism of the effect of environmental factors in self-inactivation of GD-AdoCbl complex is discussed.     

Effect of monovalent cations on the catalytic and spectral properties of tyrosine-phenol-lyase from Citrobacter intermedius

The action of monovalent cations Li+, Na+, K+, Rb+, Cs+, NH4+ on catalytic and physico-chemical properties of bacterial tyrosine--phenol-lyase was investigated. It was shown that K+, Rb+, Cs+, NH4+ were the noncompetitive activators of the enzyme, Na+ was an inhibitor, Li+ did not influence the catalytic activity. The values of KA and Vmax were determined for the activators in the reaction of alpha, beta-elimination of L-tyrosine. Monovalent cations affect the absorption and CD spectra of the enzyme and its complex with the quasi-substrate--L-alanine. It was suggested that an activation of tyrosine phenollyase by monovalent cations was connected with the increase of the active protonated form of the holoenzyme (lambda max 420 mm) induced by the cations-activators.     

Functional role and affinity of inorganic cations in stabilizing the tetrameric structure of the KcsA K+ channel

Crystal structures of the tetrameric KcsA K+ channel reveal seven distinct binding sites for K+ ions within the central pore formed at the fourfold rotational symmetry axis. Coordination of an individual K+ ion by eight protein oxygen atoms within the selectivity filter suggests that ion-subunit bridging by cation-oxygen interactions contributes to structural stability of the tetramer. To test this hypothesis, we examined the effect of inorganic cations on the temperature dependence of the KcsA tetramer as monitored by SDS-PAGE. Inorganic cations known to permeate or strongly block K+ channels (K+, Rb+, Cs+, Tl+, NH4+, Ba2+, and Sr2+) confer tetramer stability at higher temperatures (T0.5 range = 87 degrees C to >99 degrees C) than impermeant cations and weak blockers (Li+, Na+, Tris+, choline+; T0.5 range = 59 degrees C to 77 degrees C). Titration of K+, Ba2+, and other stabilizing cations protects against rapid loss of KcsA tetramer observed in 100 mM choline Cl at 90 degrees C. Tetramer protection titrations of K+, Rb+, Cs+, Tl+, and NH4+ at 85 degrees C or 90 degrees C exhibit apparent Hill coefficients (N) ranging from 1.7 to 3.3 and affinity constants (K0.5) ranging from 1.1 to 9.6 mM. Ba2+ and Sr2+ titrations exhibit apparent one-site behavior (N congruent with 1) with K0.5 values of 210 nM and 11 microM, respectively. At 95 degrees C in the presence of 5 mM K+, titration of Li+ or Na+ destabilizes the tetramer with K0.5 values of 57 mM and 109 mM, respectively. We conclude that specific binding interactions of inorganic cations with the selectivity filter are an important determinant of tetramer stability of KscA.     

Effects of monovalent cations on AMP nucleosidase from Azotobacter vinelandii.

The effect of monovalent cations on the purified AMP nucleosidase (AMP phosphoribohydrolase, EC 3.2.2.4) from Azotobacter vinelandii was investigated. All the monovalent cations were activators of the enzyme: Rb+ and Cs+ were the most effective, followed by K+, Na+, NH4+ and Li+ in that order. The apparent Ka for MgATP and nH values (Hill's interaction coefficient) decreased from 0.9 to 0.1 mM, and from 4 to 1, respectively, with the increase in K+ concentration, suggesting that the cation effects are on MgATP binding rather than catalysis. Gel filtration studies have revealed that the enzyme forms a non-dissociable enzyme species with a Stokes radius of 6.0--6.2 nm in the presence of saturating concentrations of monovalent cations, which can be distinguished from the 5.5-nm enzyme species showing temperature-dependent dissociation of the molecule in sulfate or phosphate. These results suggest that these ligands affect the association of the subunits through changes in the environment of the hydrophobic side chains of the enzyme molecules.     

The activity and reaction specificity of tyrosine phenol-lyase regulated by monovalent cations

This work was aimed at studying the effect of monovalent inorganic cations (Li+, Na+, K+, Rb+, Cs+, NH+4) on the catalytic and spectral characteristics of tyrosine phenol-lyase from Citrobacter intermedius. These cations were shown to influence the proportion of the beta-elimination reaction rate to the rate of side transamination reaction. Most of the monovalent cations are non-competitive activators of the beta-elimination reaction; Li+ exerts no effect on the enzyme activity in this reaction; Na+ is an inhibitor of the beta-elimination reaction. The activation of tyrosine phenol-lyase by monovalent cations stems from the creation of an active holoenzyme form (lambda max 420 nm) due to conformational rearrangements of the protein molecule.     

Role of bivalent and monovalent cations in the functioning of myosin ATPase

The effects of bivalent (Mg2+, Ca2+, Sr2+) and monovalent (K+, Na+, NH4+) cations on the ATPase activity of subfragment 1 of myosin (SI) with a decreased Mg2+ content (EDTA-SI) were studied. Mg2+ activate the EDTA-SI ATPase, but only in the absence of other activating cations. K+, NH4+, a2+ and Sr2+ have a much stronger activating effect on EDTA-SI ATPase than on Mg-SI (SI enriched with Mg2+) ATPase. Monovalent cations inhibit Mg2+-ATPase and Ca2+-ATPase of EDTA-SI, while K+ and NH4+ activate Sr2+-ATPase of EDTA-SI. Based on experimental results and literary data, a hypothesis on the participation of the cations in the functioning of myosin ATPase was postulated. This hypothesis entails the existence of two closely interconnected cation-binding sites in the vicinity of the myosin active center (one for bivalent and one for monovalent cations); the ATPase activity of myosin is at any moment dependent on the nature of cations present in these two sites. An attempt to explain the role of the cations in the accomplishment of the ATPase reaction by myosin was made.     

Chemical modification of the most reactive thiol group of rabbit skeletal muscle phosphofructokinase, to reduce its affinity toward substrate ATP and activating monovalent cations.

The most reactive single thiol group of rabbit skeletal muscle phosphofructokinase per protomer was modified with the following thiol reagents: iodoacetamide, iodoacetate, 2-hydroxyethyl disulfide, 3,3'-dithiodipropionate, and glutathione disulfide. As a result of the modification, there was increase in not only the apparent activation constants of activating monovalent cations, NH4+ (about 3-, 9-, 12-, 20-, and 30-fold, respectively) and K+ (about 3-, 10-, 15-, 17-, and 20-fold, respectively), but also the apparent Km for ATP (about 3-, 10-, 15-, 100-, and 20-fold, respectively) without any significant change in maximum velocity or apparent Km for fructose 6-phosphate in the presence of high concentrations of NH4+. These results suggest that modification of the thiol group destabilizes the enzyme-monovalent cation-MgATP complex proposed by Suelter [Science (1970) 168, 789-795], causing an apparent loss in catalytic activity.     

Monovalent cation binding to cubic insulin crystals.

Two localized monovalent cation binding sites have been identified in cubic insulin from 2.8 A-resolution difference electron density maps comparing crystals in which the Na+ ions have been replaced by Tl+. One cation is buried in a closed cavity between insulin dimers and is stabilized by interaction with protein carbonyl dipoles in two juxtaposed alternate positions related by the crystal dyad. The second cation binding site, which also involves ligation with carbonyl dipoles, is competitively occupied by one position of two alternate His B10 side chain conformations. The cation occupancy in both sites depends on the net charge on the protein which was varied by equilibrating crystals in the pH range 7-10. Detailed structures of the cation binding sites were inferred from the refined 2-A resolution map of the sodium-insulin crystal at pH 9. At pH 9, the localized monovalent cations account for less than one of the three to four positive counterion charges necessary to neutralize the negative charge on each protein molecule. The majority of the monovalent counterions are too mobile to show up in the electron density maps calculated using data only at resolution higher than 10 A. Monovalent cations of ionic radius less than 1.5 A are required for crystal stability. Replacing Na+ with Cs+, Mg++, Ca++ or La+++ disrupts the lattice order, but crystals at pH 9 with 0.1 M Li+, K+, NH4+, Rb+ or Tl+ diffract to at least 2.8 A resolution.     

Interaction of sodium and potassium ions with Na+,K+-ATPase. II. General properties of ouabain-sensitive K+ binding

General properties of ouabain-sensitive K+ binding to purified Na+,K+-ATPase [EC 3.6.1.3] were studied by a centrifugation method with 42K+. 1) The affinity for K+ was constant at pH values higher than 6.4, and decreased at pH values lower than 6.4. 2) Mg2+ competitively inhibited the K+ binding. The dissociation constant (Kd) for Mg2+ of the enzyme was estimated to be about 1 mM, and the ratio of Kd for Mg2+ to Kd for K+ was 120 : 1. The order of inhibitory efficiency of divalent cations toward the K+ binding was Ba2+ congruent to Ca2+ greater than Zn2+ congruent to Mn2+ greater than Sr2+ greater than Co2+ greater than Ni2+ greater than Mg2+. 3) The order of displacement efficiency of monovalent cations toward the K+ binding in the presence or absence of Mg2+ was Tl+ greater than Rb+ greater than or equal to (K+) greater than NH4+ greater than or equal to Cs+ greater than Na+ greater than Li+. The inhibition patterns of Na+ and Li+ were different from those of other monovalent cations, which competitively inhibited the K+ binding. 4) The K+ binding was not influenced by different anions, such as Cl-, SO4(2-), NO3-, acetate, and glycylglycine, which were used for preparing imidazole buffers. 5) Gramicidin D and valinomycin did not affect the K+ binding, though the former (10 micrograms/ml) inhibited the Na+,K+-ATPase activity by about half. Among various inhibitors of the ATPase, 0.1 mM p-chloromercuribenzoate and 0.1 mM tri-n-butyltin chloride completely inhibited the K+ binding. Oligomycin (10 micrograms/ml) and 10 mM N-ethylmaleimide had no effect on the K+ binding. In the presence of Na+, however, oligomycin decreased the K+ binding by increasing the inhibitory effect of Na+, whether Mg2+ was present or not. 6) ATP, adenylylimido diphosphate and ADP each at 0.2 mM decreased the K+ binding to about one-fourth of the original level at 10 microM K+ without MgCl2 and at 60 microM K+ with 5 mM MgCl2. On the other hand, AMP, Pi, and p-nitrophenylphosphate each at 0.2 mM had little effect on the K+ binding.     

Cationic modulation of follicle-stimulating hormone binding to granulosa cell receptor

Magnesium (Mg2+) increases binding of follicle-stimulating hormone (FSH) to membrane-bound receptors and increases adenylyl cyclase activity. We examined the effects of divalent and monovalent cations on FSH binding to receptors in granulosa cells from immature porcine follicles. Divalent and monovalent cations increased binding of [125I]iodo-porcine FSH (125I-pFSH). The divalent cations Mg2+, calcium (Ca2+) and manganese, (Mn2+) increased specific binding a maximum of 4- to 5-fold at added concentrations of 10 mM. Mg2+ caused a half-maximal enhancement of binding at 0.6 mM, whereas Ca2+ and Mn2+ had half-maximal effects at 0.7 mM and 0.8 mM, respectively. The monovalent cation potassium (K+) increased binding a maximum of 1.5-fold at an added concentration of 50 mM, whereas the monovalent cation (Na+) did not increase binding at any concentration tested. The difference between K+ and Na+ suggested that either enhancement of binding was not a simple ionic effect or Na+ has a negative effect that suppresses its positive effect. Ethylenediamine tetraacetic acid, a chelator of Mg2+, prevented binding of 125I-pFSH only in the presence of Mg2+, whereas pregnant mare's serum gonadotropin, a competitor with FSH for the receptor, prevented binding in both the absence and the presence of Mg2+. Guanyl-5-ylimidodiphosphate (Gpp[NH]p) inhibited binding of 125I-pFSH in the absence or presence of Mg2+, but only at Gpp(NH)p concentrations greater than 1 mM. We used Mg2+ to determine if divalent cations enhanced FSH binding by increasing receptor affinity or by increasing the apparent number of binding sites.(ABSTRACT TRUNCATED AT 250 WORDS)     

Regulation of connexin hemichannels by monovalent cations

Opening of connexin hemichannels in the plasma membrane is highly regulated. Generally, depolarization and reduced extracellular Ca2+ promote hemichannel opening. Here we show that hemichannels formed of Cx50, a principal lens connexin, exhibit a novel form of regulation characterized by extraordinary sensitivity to extracellular monovalent cations. Replacement of extracellular Na+ with K+, while maintaining extracellular Ca2+ constant, resulted in >10-fold potentiation of Cx50 hemichannel currents, which reversed upon returning to Na+. External Cs+, Rb+, NH4+, but not Li+, choline, or TEA, exhibited a similar effect. The magnitude of potentiation of Cx50 hemichannel currents depended on the concentration of extracellular Ca2+, progressively decreasing as external Ca2+ was reduced. The primary effect of K+ appears to be a reduction in the ability of Ca2+, as well as other divalent cations, to close Cx50 hemichannels. Cx46 hemichannels exhibited a modest increase upon substituting Na+ with K+. Analyses of reciprocal chimeric hemichannels that swap NH2- and COOH-terminal halves of Cx46 and Cx50 demonstrate that the difference in regulation by monovalent ions in these connexins resides in the NH2-terminal half. Connexin hemichannels have been implicated in physiological roles, e.g., release of ATP and NAD+ and in pathological roles, e.g., cell death through loss or entry of ions and signaling molecules. Our results demonstrate a new, robust means of regulating hemichannels through a combination of extracellular monovalent and divalent cations, principally Na+, K+, and Ca2+.     

Gill (Na+ + K+)- and Na+-stimulated Mg2+-dependent ATPase activities in the gilthead bream (Sparus auratus L.)

1. Gilthead gill 10(-3) M ouabain-inhibited (Na+ + K+)-ATPase and 10(-2) M ouabain-insensitive Na+-ATPase require the optimal conditions of pH 7.0, 160 mM Na+, 20 mM K+, 5 mM MgATP and pH 4.8-5.2, 75 mM Na+, 2.5 mM Mg2+, 1.0 mM ATP, respectively. 2. The main distinctive features between the two activities are confirmed to be optimal pH, the ouabain-sensitivity and the monovalent cation requirement, Na+ plus another cationic species (K+, Rb+, Cs+, NH4+) in the (Na+ + K+)-ATPase and only one species (Na+, K+, Li+, Rb+, Cs+, NH4+ or choline+) in the Na+-ATPase. 3. The aspecific Na+-ATPase activation by monovalent cations, as well as by nucleotide triphosphates, opposed to the (Na+ + K+)-ATPase specificity for ATP and Na+, relates gilthead gill ATPases to lower organism ATPases and differentiates them from mammalian ones. 4. The discrimination between the two activities by the sensitivity to ethacrynic acid, vanadate, furosemide and Ca2+ only partially agrees with the literature. 5. Present findings are viewed on the basis of the ATPase's presumptive physiological role(s) and mutual relationship.     

Some properties of the pyruvate carboxylase from Pseudomonas fluorescens.

The pyruvate carboxylase of Pseudonomas fluorescens was purified 160-fold from cells grown on glucose at 20 degrees C. The activity of this purified enzyme was not affected by acetyl-coenzyme A or L-aspartate, but was strongly inhibited by ADP, which was competitive towards ATP. Pyruvate gave a broken double reciprocal plot, from which two apparent Km values could be determined, namely 0-08 and 0-21 mM, from the lower and the higher concentration ranges, respectively. The apparent Km for HCO3 at pH 6-9, in the presence of the manganese ATP ion (MnATP2-), was 3-1 mM. The enzyme reaction had an optimum pH value of 7-1 or 9-0 depending on the use of MnATP2- or MgATP2-, respectively, as substrate. Free Mg2+ was an activator at pH values below 9-0. The enzyme was strongly activated by monovalent cations; NH4+ and K+ were the better activators, with apparent Ka values of 0-7 and 1-6 mM, respectively. Partially purified enzymes from cells grown on glucose at 1 or 20 degrees C had the same properties, including the thermal stability. In both cases 50% of the enzyme activity was lost after pre-incubation for 10 min at 46 degrees C. The molecular weight was estimated to be about 300000 daltons by gel filtration on Sephadex G-200. The regulatory properties and molecular weight are thus similar to those determined for the pyruvate carboxylases from Pseudomonas citronellolis and Azotobacter vinelandii.     

Monovalent cation activation of tryptophanase.

The interaction of monovalent cations with holotryptophanase has been examined by spectral and kinetic methods. Using S-orthonitrophenyl-L-cysteine as a substrate, activation by the following monovalent cations was demonstrated; values of KA (mM, in italics) and Vmax (mumol min-1 mg) aare given in parentheses: Li+ (54 +/- 11.6, 4.3 +/- 0.28), Na+ (40 +/- 0.03, 18) K+ (1.44 +/- 0.06, 41.1 +/- 3.5), Tl+ (0.95 +/- 0.1, 39 +/- 4.4), NH4+ (0.23 +/- 0.01, 57.9 +/- 2.6), Rb+ (3.5 +/- 0.3, 33.5 +/- 1.8), Cs+ (14.6 +/- 2.6, 21 +/- 2.3). It was demonstrated by circular dichroic spectra that the competitive inhibitor, ethionine, interacts with the holoenzyme in the absence of activating monovalent cations, although it does not undergo labilization of the alpha proton. On addition of monovalent cation to the holoenzyme-ethionine complex, a marked increase occurs in absorption of 508 nm resulting from labilization of the alpha proton with formation of the quinoid form of the pyridoxal phosphate moiety of the enzyme-substrate complex at the catalytic center (Morino, Y., and Snell, E.E. (1967) J. Biol. Chem; 242, 2800-2809. The extent of formation of this quinoid intermediate was linearly related to the maximum velocity observed with each cation except NH4+, which was anomalously active. When measured at 500 nm, the change in absorption ranged from deltaA = 0.45 mg-1 of tryptophanase for NH4+ to 0.06 mg-1 for Li+. Two moles of thallium (I) were bound per mole of subunit. The data are most consistent with the interaction of monovalent cation at or near the catalytic center in such a way that it either participates directly in the reaction or is required for the critical alignment of one or more functional groups necessary for catalysis.     

Sodium ion-stimulated alpha-[1-14C]aminoisobutyric acid uptake in alkalophilic Bacillus species.

Alkalophilic Bacillus no. 8-1 grows well in alkaline media containing 2.5 to 5% NaCl. The uptake of alpha-aminoisobutyric acid (AIB) into the cells is stimulated by the addition of NaCl (Na+) up to a concentration of 0.2 M, but other monovalent cations such as K+, Li+, or NH4+ cannot substitute for Na+. The kinetic studies reveal that, when the Na+ concentration increases from 0.02 to 0.2 M in alkaline medium, the Km for transport decreases, whereas Vmax remains almost constant. Competition studies indicate that glycine, L-alanine, L-serine, and AIB share common carriers for the transport of the compounds into cells. Other alkalophilic bacteria are also found to require Na+ for the uptake of AIB into the cells.