Positive Homotropic Cooperativity Research Articles

Regulation of skeletal muscle AMP deaminase: lysine residues are critical for the pH-dependent positive homotropic cooperativity behaviour of the rabbit enzyme

Reaction of rabbit skeletal muscle AMP deaminase with a low molar excess of trinitrobenzene sulfonic acid (TNBS) results in conversion of the enzyme into a species with about six trinitrophenylated lysine residues per molecule which no longer manifests positive homotropic cooperativity at pH 7.1 or at the optimal pH value of 6.5 in the presence of low K + concentrations. Substitution of the reactive thiol groups with 5,5′-dithiobis-(2-nitrobenzoic acid) does not protect the enzyme from the TNBS-induced changes of the catalytic properties, indicating that cysteine residues modification is not at the basis of the effects of TNBS treatment on AMP deaminase and strongly suggesting the obligatory participation of lysine residues to the constitution of a regulatory anionic site to which AMP must bind to stimulate the enzyme at alkaline pH. The TNBS-treated enzyme is also completely desensitized to inhibition by ATP, but not to inhibition by GTP and stimulation by ADP. This observation suggests a connection between the operation of the hypothesized anionic activating site, responsible for positive homotropic cooperativity, and the inhibition exerted by anionic compounds that compete for the same site, among them the most efficient metabolite being probably ATP.

Steady-state kinetics and inhibitor binding of 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase (tryptophan sensitive) from Escherichia coli.

The tryptophan-inhibited 3-deoxy-d-arabino-heptulosonate-7-phosphate synthase [DAHPS(Trp)] of Escherichia coli was analyzed with respect to steady-state kinetics and tryptophan binding. DAHPS(Trp) is one of three differentially regulated isoforms that catalyze the first step of aromatic biosynthesis, the condensation of phosphoenolpyruvate and erythrose-4-phosphate to form 3-deoxy-D-arabino-heptulosonate-7-phosphate. The DAHP synthase isozymes are metalloproteins, being activated in vitro by a variety of divalent metals. Both catalytic activity and substrate affinity are dependent on the species of activating metal ion. We report here kinetic and binding studies of metal-homogeneous (Mn2+-activated) DAHPS(Trp). The homodimeric enzyme had an apparent kcat of 21 s-1 and displayed sigmoidal kinetics with respect to both substrates. The S0.5 was 35 microM for erythrose-4-phosphate and 5.3 microM for phosphoenolpyruvate. Equilibrium binding studies with radiolabeled tryptophan demonstrated two independent inhibitor binding sites per enzyme dimer, with KdTrp of 1 microM. L-Tryptophan binding decreased kcat, increased affinity for both substrates, decreased positive homotropic cooperativity for both substrates and activated the enzyme at low concentrations of erythrose-4-phosphate. The results suggest an inhibition mechanism analogous to system C5 hyperbolic mixed-type inhibition with respect to erythrose-4-phosphate and partial noncompetitive inhibition with respect to phosphoenolpyruvate.

Purification and characterization of guinea-pig liver microsomal deacetylase involved in the deacetylation of the O-glucoside of N-hydroxyacetanilide.

A microsomal deacetylase that catalyses the deacetylation of the O-glucoside of N-hydroxyacetanilide (GHA) was purified from guinea-pig liver. The activity was located exclusively in the microsomes and not detected in the cytosol. The purified GHA deacetylase was a trimeric protein with a molecular mass of 160+/-10 (S.D.) kDa composed of subunits of 53+/-2 kDa; its pI was 4.7. The N-terminal amino acid sequence of GHA deacetylase was similar to those reported for guinea-pig and rat liver microsomal carboxylesterases. The GHA deacetylase showed a comparable hydrolytic activity towards p-nitrophenyl acetate (PNPA), although the activities towards N-hydroxyacetanilide, acetanilide and some endogenous acylated compounds were very low or not detectable. The deacetylase activity towards GHA was inhibited by organophosphates but not by p-chloromercuribenzoate, suggesting that GHA deacetylase can be classified as a B-esterase. The enzyme exhibited a positive homotropic co-operativity towards GHA. The values of the Hill coefficient, the half-saturating concentration ([S]0.5) for GHA, and Vmax were 1.59+/-0.03, 5.51+/-0.07 mM and 32.5+/-1.4 micromol/min per mg respectively, at the optimum pH of 8.5. The bell-shaped pH dependence of the Vmax/[S]0.5 profile indicated pKa values attributed to histidine and lysine residues. The study of stoichiometric inhibition by di-isopropyl fluorophosphate and kinetic analysis with the Monod-Wyman-Changeux model suggests that GHA deacetylase has six substrate binding sites and three catalytically essential serine residues per enzyme molecule.

Influence of sarmesin on the cardiac and vascular actions of angiotensin II

The angiotensin II (ANG II) receptor blocker properties of sarmesin and its influence on the homotropic cooperativity of ANG II receptors were studied in two experimental models: isolated rabbit aorta and isolated rabbit atria. The results show that: (i) sarmesin is a specific competitive antagonist of ANG II receptors, with high affinity (pA2=8.93 in the isolated aorta and 8.66 in the isolated atria); and (ii) the slope of the concentration-response curves to ANG II and the Hill coefficient increased in the presence of sarmesin, the latter suggesting an enhancement of the positive homotropic cooperativity of ANG II receptors. These results may be explained overall by the reciprocal negative modulation of receptor affinity between sarmesin and ANG II, due, possibly, to opposite effects on the binding of G-proteins to ANG II receptors.

Methionine-321 in the C-terminal α-helix of catabolic ornithine carbamoyltransferase from Pseudomonas aeruginosa is important for positive homotropic cooperativity

Methionine-321 in the C-terminal α-helix of catabolic ornithine carbamoyltransferase from Pseudomonas aeruginosa is important for positive homotropic cooperativity

Purification and characterization of glucosamine-6-phosphate deaminase from dog kidney cortex

Glucosamine-6-phosphate deaminase (EC 5.3.1.10) from dog kidney cortex was purified to homogeneity, as judged by several criteria of purity. The purification procedure was based on two biospecific affinity chromatography steps, one of them using N-ϵ-amino- n-hexanoyl- d-glucosamine-6-phosphate agarose, an immobilized analog of the allosteric ligand, and the other by binding the enzyme to phosphocellulose followed by substrate elution, which behaved as an active-site affinity chromatography. The enzyme is an hexameric protein of about 180 kDa composed of subunits of 30.4 kDa; its isoelectric point was 5.7. The sedimentation coefficient was 8.3S, and its frictional ratio was 1.28, indicating that dog deaminase is a globular protein. The enzyme displays positive homotropic cooperativity toward d-glucosamine-6-phosphate (Hill coefficient = 2.1, pH 8.8). Cooperativity was completely abolished by saturating concentrations of GlcNAc6P; this allosteric modulator activated the reaction with a typical K-effect. Under hyperbolic kinetics, a K m value of 0.25 ± 0.02 m m for d-glucosamine-6-phosphate was obtained. Assuming six catalytic sites per molecule, k cat is 42 s −1. Substrate-velocity data were fitted to the Monod's allosteric model for the exclusive-binding case for both substrate and activator, with two interacting substrate sites. The K dis for N-acetyl- d-glucosamine-6-phosphate was estimated at 14 μ m.

Analysis of the metal requirement of 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase from Escherichia coli.

The three isozymes of 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase from Escherichia coli were overproduced, purified, and characterized with respect to their requirement for metal cofactor. The isolated isozymes contained 0.2-0.3 mol of iron/mol of enzyme monomer, variable amounts of zinc, and traces of copper. Enzymatic activity of the native enzymes was stimulated 3-4-fold by the addition of Fe2+ ions to the reaction mixture and was eliminated by treatment of the enzymes with EDTA. The chelated enzymes were reactivated by a variety of divalent metal ions, including Ca2+, Cd2+, Co2+, Cu2+, Fe2+, Mn2+, Ni2+, and Zn2+. The specific activities of the reactivated enzymes varied widely with the different metals as follows: Mn2+ greater than Cd2+, Fe2+ greater than Co2+ greater than Ni2+, Cu2+, Zn2+ much greater than Ca2+. Steady state kinetic analysis of the Mn2+, Fe2+, Co2+, and Zn2+ forms of the phenylalanine-sensitive isozyme (DAHPS(Phe)) revealed that metal variation significantly affected the apparent affinity for the substrate, erythrose 4-phosphate, but not for the second substrate, phosphoenolpyruvate, or for the feedback inhibitor, L-phenylalanine. The tetrameric DAHPS(Phe) exhibited positive homotropic cooperativity with respect to erythrose 4-phosphate, phophoenolpyruvate, and phenylalanine in the presence of all metals tested.

Purification and characterization of a cyclic GMP-stimulated cyclic nucleotide phosphodiesterase from the cytosol of human platelets

A cyclic GMP-stimulated cyclic nucleotide phosphodiesterase was purified to near homogeneity from the 150,000 g supernatant fraction of human platelets by a combination of DEAE-cellulose chromatography and cyclic GMP affinity chromatography. Overall purification was about 7400-fold with a 10% to 15% recovery of activity. On NaDodSO4-containing polyacrylamide gels, the purified enzyme migrates as a single band M r = 105,000. Phosphodiesterase activity co-migrates with the protein band on native polyacrylamide gels. Both Mg 2+ and Mn 2+ support the activity of this phosphodiesterase. The enzyme hydrolyzes both cyclic AMP and cyclic GMP with similar maximal rates. The hydrolysis of both nucleotides exhibits positive homotropic cooperativity with S 0.5 values of 50 ± 12 μM for cyclic AMP and 35 ± 15 μM for cyclic GMP and Hill coefficients of 1.2 to 1.5 for both nucleotides. Low levels of cyclic GMP stimulate the rate of cyclic AMP hydrolysis from 3- to 10-fold. The activity of this phosphodiesterase is not stimulated by the calcium binding protein, calmodulin. The cyclic GMP stimulation of cyclic AMP hydrolysis by this phosphodiesterase may provide a possible regulatory link between the metabolism of these two nucleotides in platelets.

The relationship between homotropic and heterotropic cooperativity for angiotensin receptors in smooth muscle

1. 1. Angiotensin-induced contraction of smooth muscle is accompanied by both homotropic (receptor-receptor) and heterotropic (receptor-G protein) cooperativity. 2. 2. Binding constants for angiotensins II and III at uterine smooth muscle receptors have been compared in bioassays and binding assays, using the competitive antagonist Sarmesin to verify the binding assay/bioassay interrelationship. 3. 3. Agonist affinities determined from binding studies in the presence of GTPγS were found to be similar to the affinities observed in responding rat uterine tissues under conditions which eliminate positive homotropic cooperativity, suggesting that heterotropic cooperativity and homotropic cooperativity are interdependent events for smooth muscle contraction. 4. 4. The data are consistent with an allosteric or autosteric mechanism of receptor function involving cooperativity between two agonist binding sites on the receptor. 5. 5. The model has been used to calculate homotropic efficacies for angiotensins II and III from bioassay data and binding data, respectively.

Methods for analyzing and interpreting cooperativity in dose-response curves—II. Partial agonists acting on muscarinic receptors in smooth muscle

1. 1. Dose-response curves for eight muscarinic agonists contracting guinea pig ileum were transformed into Hanes-Woolf plots and Hill plots respectively. 2. 2. Hill plots for three full agonists were linear with slopes of 1.6, illustrating positive homotropic cooperativity involving two or more agonist binding sites. 3. 3. Five partial agonists gave curved Hill plots with Hill coefficients of 1.2–1.7 at low agonist concentration and Hill coefficients of 0.3–0.5 at high agonist concentrations. 4. 4. Hanes-Woolf transformations were used to delineate the transition from positive to negative cooperativity and to assist in the interpolation of Hill plots. 5. 5. The results show that all of the agonists studied induce positive cooperativity, and that the difference between full agonists and partial agonists results from the ability of the latter to induce pronounced negative cooperativity at higher concentrations.

Methods for analyzing and interpreting cooperativity in dose-response curves—I. Antagonist effects on angiotensin receptors in smooth muscle

1. 1. Dose-response curves for angiotensins II and III in the rat isolated uterus, portal vein and aorta, in the absence and presence of the antagonist [Sar 1Ile 8]ANG II, have been analyzed by methods which detect cooperativity. 2. 2. Hanes-Woolf transformations of dose-response data were used to define phases of positive homotropic cooperativity, and this information was applied to the interpretation of Hill plots. 3. 3. The level of positive cooperativity was found to be higher for ANG II ( n H = 1.35−2.00) than ANG III ( n H = 1.02−1.43) in the three tissues studied. 4. 4. In the presence of the antagonist [Sar 1Ile 8]ANG II a decrease in the level of cooperativity produced by both agonists was observed, and high concentrations of the antagonist could induce negative cooperativity. 5. 5. These findings have been interpreted on the basis of a 2-site agonist binding mechanism involving receptor dimers, in which angiotensin agonists induce an increase in their own receptor binding affinity, whereas the antagonist [Sar 1Ile 8]ANG II decreases the affinity of angiotensin receptors for ANG II and ANG III.

A study of the allosteric kinetics of Phycomyces pyruvate kinase as judged by the effect of l-alanine and fructose 1,6-bisphosphate

The influence of fructose 1,6-bisphosphate and l-alanine on the kinetics of pyruvate kinase (ATP: pyruvate O 2-phosphotransferase, EC 2.7.1.40) from Phycomyces blakesleeanus NRRL 1555 (−) was studied at pH 7.5. By addition of fructose 1,6-bisphosphate the sigmoid kinetics with respect to phospho enolpyruvate and Mg 2− were abolished and the velocity curves became hyperbolic. In the presence of l-alanine the positive homotropic cooperativity with respect to phospho enolpyruvate increased with Hill coefficient values close to 4, while the sigmoid kinetics with respect to Mg 2+ became hyperbolic. Fructose 1,6-bisphosphate overcomes the inhibition produced by l-alanine, the antagonism between phospho enolpyruvate and l-alanine also being evident. Inhibition has been found at high Mg 2+ concentrations, compatible with the binding of the magnesium ions to an inactive conformational state of the enzyme. The data were analysed on the basis of the two-states concerted-symmetry model of Monod, Wyman and Changeux, and the parameters of the model were calculated. Phospho enolpyruvate and fructose 1,6-bisphosphate appeared to show exclusive binding to the active conformational state (R), whereas magnesium ions bind preferentially, by a factor of 45, to the R state. l-Alanine binds more readily to the inactive T state of the enzyme.

Purification, molecular and kinetic properties of glucosamine-6-phosphate isomerase (deaminase) from Escherichia coli

Glucosamine-6-phosphate isomerase (deaminase), (2-amino-2-deoxy- d-glucose-6-phosphate ketol isomerase (deaminating), EC 5.3.1.10) has been purified to homogeneity from Escherichia coli B as judged by several criteria of purity. The procedure included ammonium sulfate fractionation, anion-exchange chromatography and a biospecific affinity chromatography step with N-ϵ- amino-n- caproyl- d-glucosamine 6-phosphate bound to agarose as the ligand, the elution being performed with GlcNAc6 P. The enzyme appears to be an hexamer of about 178 kDa, composed of six subunits of 29 700 ± 300 Da; the isoelectric point was 6.0–6.1 and the sedimentation constant 9.0 S. The amino-acid composition of the enzyme was determined and a value for E 275 1% of 4.55 was calculated. The molecular activity was 1800 s −1 for the deamination reaction and 455 s −1 for the reaction of GlcN6 P formation. A positive homotropic cooperativity was found for both sugar substrates; it was stronger for GlcN6 P in the deamination reaction (Hill number 2.7 at pH 7.7). Ammonia behaved as a Michaelian substrate. Cooperativity was abolished by 0.1 mM GlcNAc6 P; this allosteric modulator activated the reaction in both directions, with a positive K-effect upon both sugar phosphates, but had no effect on K m for ammonia. The initial velocity patterns for the amination reaction were obtained under conditions of hyperbolic kinetics produced by GlcNAc6 P; the K m values for the allosteric substrates were determined under the same conditions, and their dependence upon pH was studied.

Purification and characterization of a cyclic GMP-stimulated cyclic nucleotide phosphodiesterase from bovine tissues.

A cGMP-stimulated cyclic nucleotide phosphodiesterase has been purified to near homogeneity from bovine adrenal and heart tissues. The purification procedure utilizes chromatography on DEAE-cellulose and cGMP affinity resin. The procedure can be completed within 2 days and is easily adapted to large scale. To obtain pure enzyme, an 8,000-9,000-fold increase in specific activity was required in adrenal tissues and 15,000-30,000-fold in cardiac muscle. A single band of protein having an apparent Mr = 105,000-107,000 was seen on sodium dodecyl sulfate gel electrophoresis. At equilibrium, native polyacrylamide gradient gel electrophoresis revealed a single major band having an apparent Mr = 240,000. Cyclic GMP binding and phosphodiesterase activity co-migrated with the protein band on native polyacrylamide gradient gels. The enzyme bound cGMP with high affinity reaching a maximum binding of 1.02 mol of cGMP bound/mol of enzyme dimer. Titration curves of the binding data indicated at least two classes of binding sites with 10% maximal binding occurring at 7 nM and 90% maximal binding at 4 microM cGMP. Kinetic analysis indicated the enzyme can hydrolyze both cAMP and cGMP with similar maximal rates. The nucleotide concentration at half-maximal velocity were 30 and 10 microM for cAMP and cGMP, respectively. The hydrolyses of both nucleotides exhibit positive homotropic cooperativity with Hill coefficients of 1.9 for cAMP and 1.3 for cGMP. The rate of cAMP hydrolysis by the purified enzyme when measured at 10 microM cAMP was enhanced 5- to 6-fold by low levels of cGMP.

Mechanism and function of the Ca2+-gated cation channel in sarcoplasmic reticulum vesicles.

The mechanism and function of the Ca 2$ -gated cation channel in sarcoplasmic reticulum (SR) vesicles were studied by measuring both choline influx and Ca 2$ efflux. The choline influx was measured by the light scattering method using a stopped flow apparatus. The Ca 2$ efflux was measured by the Millipore filtration method using 46 Ca. Kinetic analysis of the rates of choline influx and Ca 2$ efflux led to the following results. 1. The Ca 2$ -activated choline influx was dependent on the choline concentration. It increased with increase of choline concentration in a cooperative manner ( nH = 2.6) and was saturable at high choline concentrations. The apparent dissociation constant for choline was 0.39 M. 2. Caffeine increased the Ca 2$ -activated choline influx according to the Michaelis-Menten equation at low concentrations of choline, whereas the action of caffeine became cooperative ( nH = 1.9) at higher concentrations of choline, suggesting that there is a cooperative interaction between channels. 3. The Ca 2$ -activated choline influx was inhibited competitively by Ca 2$ . One Ca 2$ competes with 2 cholines. The inhibition constant for Ca 2$ was 22 μ m and the dissociation constant of choline was 0.28 m , suggesting that the competition site is the transport site of choline. 4. Various polyvalent cations also blocked the channel. The apparent inhibition constants for the inhibitors were as follows: La 2$ 0.01 μ m , Co 2$ 7μ m , Mn 2$ 8 μ m , Mg 2$ 14 μ m , Ni 2$ 19 μ m , Ca 2$ 22 μ m , Ba 2 30 μ m , and Sr 2$ 40 μ m . 5. The inhibition of Ca 2$ -activated choline influx by Mg 2$ or Co 2$ was competitive with respect to choline. One Mg 2$ or Ca 2$ competes with 2 cholines. The apparent dissociation constant of choline was 0.31 m , suggesting that the competition site is also the transport site of choline. 6. An organic Ca 2$ antagonist, nicardipine, blocked the Ca 2$ -gated cation channel with a dissociation constant of 5.4 μ m . Ruthenium red and arsenazo III also blocked the channel with apparent dissociation constants of 0.07 μ m and 16 μ m , respectively. 7. The channel was also blocked by a fluorescent cyanine dye, diS-C 2 -(5), and by quinine, which are potent inhibitors of the Ca 2$ -activated K $ channel in red blood cell membrane; the apparent inhibition constants were 4.4μm and 240 μ m , respectively. However, blockers of the K $ channel in nerve membrane, such as TEA and 3,4-diaminopyridine, were not effective. 8. When SR vesicles were modified with N , N ′-dicyclohexylcarbodiimide (DCCD), both Ca 2$ -activated choline influx and Ca 2$ -activated Ca 2$ release from SR vesicles were blocked. The apparent inhibition constant of DCCD was 80 μ m . The blockade was prevented by Ca 2$ . The Ca 2$ concentration required for half-maximal protection was about 1 m m . These results suggest that two cholines are simultaneously transported by one channel and there exists a positive homotropic cooperativity between some channels. In addition, from the pharmacological data, we concluded that the so-called Ca 2$ -gated cation channel is the Ca 2$ channel existing in sarcoplasmic reticulum.

Evidence that tetrahydrofolate does not bind to serine hydroxymethyltransferase with positive homotropic cooperativity.

Previous experiments suggesting that tetrahydrofolate binds to serine hydroxymethyltransferase with positive homotropic cooperativity have been reinvestigated. Our results show that the sigmoid-shaped tetrahydrofolate saturation curve, previously obtained by several other investigators, is due to the instability of tetrahydrofolate in the assay solution. Using a different assay method, we have shown that tetrahydrofolate gives a hyperbolic saturation curve with serine hydroxymethyltransferase. We could find no evidence, as suggested by other investigators, that heating the enzyme during purification destroyed its allosteric properties or that NADH binds to the enzyme as an allosteric effector. Evidence is presented that the loss of tetrahydrofolate during the assay period is due to oxidation by dissolved molecular oxygen.

Demonstration of two classes of opiate binding sites in the nervous tissue of the marine mollusc Mytilus edulis. Positive homotropic cooperativity of lower affinity binding sites.

Demonstration of two classes of opiate binding sites in the nervous tissue of the marine mollusc Mytilus edulis. Positive homotropic cooperativity of lower affinity binding sites.

Relationships between the magnitude of Hill plot slopes, apparent binding constants and factorability of binding polynomials and their hessians

An algebraic analysis of the factorability of binding polynomials in several variables is given. The relationship between factorability and site-site linkage is explored and the special case of ligands competing for the same site is analysed. Such cases always show heterotropic linkage. Existing knowledge concerning the relationship between factorability and statistical ratios between binding constants is summarised and a number of problems related to co-operativity coefficients are clarified. The theory of multiplicity and distribution of roots of binding polynomials is discussed and it is shown how the use of Sylvester resultants and subresultants facilitates the calculation of Sturm functions. A new bigradient algorithm for counting positive roots of polynomials is presented. The various possible definitions of apparent binding constants are investigated and the relationship of these to Hill plot slope is developed. It is shown that the Hill plot slope can never be greater than that given by separate factors of a binding polynomial. Using this theorem, it follows that a binding polynomial leading to a Hill slope greater than two must have at least one irreducible quadratic factor with roots having positive real parts. This substantiates a postulate of Wyman and Gill concerning physically meaningless binding constants. It is shown that the MWC model leads to a binding polynomial that can always be factored. However, the roots of the irreducible quadratic factors can have either positive or negative real parts and this contradicts a second posulate of Wyman and Gill that molecular models with unambiguous linkage always give rise to quadratic factors with negative linear coefficients. The nature of the roots of binding polynomials for higher analogues of the MWC model involving more than two conformational forms are given and it is shown that the Hessian has no positive roots. Hence this model, like the simple MWC one, always gives positive homotropic co-operativity.

Properties of the interaction of the sodium channel with permeant monovalent cations.

The use of sea anemone toxin, veratridine and scorpion toxin which specifically interact with the gating system of the sodium channel and maintain the channel in an open conformation has permitted a study of the mechanism of transport of monovalent cations through the selectivity filter of this channel. The initial rate of 22Na+ influx through the tetrodotoxin-sensitive Na+ channels of excitable cells is dependent upon the external concentrations of Na+ and Na+-substitutes with the following properties. (a) It is saturable at high Na+ concentrations and increases with the external Na+ concentration in a cooperative manner (nH = 1.6). (b) At low external Na+ concentrations (1 mM), it is activated and then inhibited by increasing external concentrations of monovalent cations such as Li+, guanidinium, hydrazinium, hydroxylamine and K+. The activating effect of these cations disappears at higher external Na+ concentrations (10 mM). The experimental data are consistent with a model involving at least two allosteric cation-binding sites per Na+ channel. The binding of monovalent cations to Na+ sites is characterized by a high positive homotropic cooperativity. Most of the work describes the properties of the Na+ channel in neuroblastoma cells. The mechanism has also been shown to be valid for excitable cells of other types and origins.

Kinetic studies of a (Na++ K++ Mg2+)ATPase in sugar beet roots. III. A proposed model for the (Na++ K+) activation and its significance for field properties

AbstractA microsomal (Na++ K++ Mg2+)ATPase preparation from sugar beet roots was used. The activation by simultaneous addition of Na+ and K+ at different levels was examined in terms of steady state kinetics. The observed data can be summarized in the following way:1. The apparent affinity between the enzyme and the substrate MgATP depends on the ratio between Na+ and K+. At low Na+ concentration (below 5 mM), the apparent Km decreases with increasing concentrations of K+ (1–20 mM). At 5 mM Na+, the K+ level does not change the apparent Km, while at Na+ levels above 10 mM, the apparent Km between enzyme and substrate increases with increasing concentration of K+.2. When the MgATP concentration is kept constant, homotropic cooperativity (concerning one type of ligand) and heterotropic cooperativity (concerning different types of ligands) exist in the activation by Na+ and K+. The Na+ binding is cooperative with different Km values and Hill coefficients (n) in the presence of low and high concentration of K+. At low Na+ level (< 5 mM). a negative cooperativity exists for Na+ (nNa < 1) which is more pronounced in the presence of high [K+]. When the concentration of Na+ is raised the negative cooperativity disappears and turns into a positive one (nNa > 1).Only K+ binding in the presence of low [Na+] shows cooperativity with a Hill coefficient that reflects changes from negative to positive homotropic cooperativity with increasing concentrations of K+ (nK < 1 → nK > 1). In the presence of [Na+] > 10 mM, the changes in nk are insignificant.3. A model is proposed in which one or two different K sites and one or two Na sites control the catalytic activity, with multiple interactions between Na+, K+ and MgATP.4. In the presence of Na+ (< 10 mM), K+ is probably bound to two K sites, one of which translocates K+ through the membrane by an antiport Na+/K+ mechanism. This could be connected with an elevated K+ uptake in the presence of Na+ and could therefore explain some field properties of sugar beets.