Kinetics of (Na+ + K+)-ATPase: analysis of the influence of Na+ and K+ by steady-state kinetics

The influence of Na+ and K+ on the steady-state kinetics at 37 degrees C of (Na+ + K+)-ATPase was investigated. From an analysis of the dependence of slopes and intercepts (from double-reciprocal plots or from Hanes plots) of the primary data on Na+ and K+ concentrations a detailed model for the interaction of the cations with the individual steps in the mechanism may be inferred and a set of intrinsic (i.e. cation independent) rate constants and cation dissociation constants are obtained. A comparison of the rate constants with those obtained from an analogous analysis of Na+-ATPase kinetics (preceding paper) provides evidence that the ATP hydrolysis proceeds through a series of intermediates, all of which are kinetically different from those responsible for the Na+-ATPase activity. The complete model for the enzyme thus involves two distinct, but doubly connected, hydrolysis cycles. The model derived for (Na+ + K+)-ATPase has the following properties: The empty, substrate free, enzyme form is the K+-bound form E2K. Na+ (Kd = 9 mM) and MgATP (Kd = 0.48 mM), in that order, must be bound to it in order to effect K+ release. Thus Na+ and K+ are simultaneously present on the enzyme in part of the reaction cycle. Each enzyme unit has three equivalent and independent Na+ sites. K+ binding to high-affinity sites (Kd = 1.4 mM) on the presumed phosphorylated intermediate is preceded by release of Na+ from low-affinity sites (Kd = 430 mM). The stoichiometry is variable, and may be Na:K:ATP = 3:2:1. To the extent that the transport properties of the enzyme are reflected in the kinetic ATPase model, these properties are in accord with one of the models shown by Sachs ((1980) J. Physiol. 302, 219-240) to give a quantitative fit of transport data for red blood cells.     

Kinetics of inhibition of green crab (Scylla serrata) alkaline phosphatase by vanadate

Green crab (Scylla serrata) alkaline phosphatase is a metalloenzyme that catalyzes the nonspecific hydrolysis of phosphate monoesters. The kinetics of inhibition of the enzyme by vanadate has been studied. The time course of the hydrolysis of p-nitrophenyl phosphate catalyzed by the enzyme in the presence of different Na3VO4 concentrations showed that, at each Na3VO4 concentration, the rate decreased with increasing time until a straight line was approached, the slopes of the straight lines being the same for all concentrations. The results suggest that the inhibition of the enzyme by Na3VO4 is a slow, reversible reaction with fractional residual activity. The microscopic rate constants were determined for the reaction of the inhibitor with the enzyme. As compared with Na2HPO4 (Ki = 0.95 mM), Na2HAsO4 (Ki = 1.10 mM), and Na2WO4 (Ki = 1.55 mM), the results suggest that Na3VO4 (Ki = 0.135 mM) is a considerably more potent inhibitor than other inhibitors.     

The effects of external sodium and potassium concentration on the membrane potential of atrioventricular fibers of the toad

Intracellular records were made from fibers in the A-V conducting system of isolated toad hearts. The A-V region was perfused with Ringer's solution of various K and Na concentrations. Resting potential in 2.8 mM [K]o was about 60 mv. Over the range 0.28 to 28 mM, resting potential diminished with increasing [K]o. Spontaneous action potentials appeared when [K]o was increased to 11.2 mM, and when resting potential had fallen to about 40 to 50 mv. Changes in [Na]o over the range 22 to 110 mM had a little effect on resting potential, but there was a linear relation between the peak value of the action potential and log [Na]o. Wenckebach periodicity was observed when [Na]o was lowered.     

Effects of choline on Na(+)- and K(+)-interactions with the Na+/K(+)-ATPase.

Choline chloride, 100 mM, stimulates Na+/K(+)-ATPase activity of a purified dog kidney enzyme preparation when Na+ is suboptimal (9 mM Na+ and 10 mM K+) and inhibits when K+ is suboptimal (90 mM Na+ and 1 mM K+), but has a negligible effect at optimal concentrations of both (90 mM Na+ and 10 mM K+). Stimulation occurs at low Na+ to K+ ratios, but not at those same ratios when the actual Na+ concentration is high (90 mM). Stimulation decreases or disappears when incubation pH or temperature is increased or when Li+ is substituted for K+ or Rb+. Choline+ also reduces the Km for MgATP at the low ratio of Na+ to K+ but not at the optimal ratio. In the absence of K+, however, choline+ does not stimulate at low Na+ concentrations: either in the Na(+)-ATPase reaction or in the E1 to E2P conformational transition. Together, these observations indicate that choline+ accelerates the rate-limiting step in the Na+/K(+)-ATPase reaction cycle, K(+)-deocclusion; consequently, optimal Na+ concentrations reflect Na+ accelerating that step also. Thus, the observed K0.5 for Na+ includes high-affinity activation of enzyme phosphorylation and low-affinity acceleration of K(+)-deocclusion. Inhibition of Na+/K(+)-ATPase and K(+)-nitrophenylphosphatase reactions by choline+ increases as the K(+)-concentration is decreased; the competition between choline+ and K+ may represent a similar antagonism between conformations selected by choline+ and by K+.     

The reactions of oxicam and sulfoanilide non steroidal anti-inflammatory drugs with hypochlorous acid: determination of the rate constants with an assay based on the competition with para-aminobenzoic acid chlorination and identification of some oxidation products

Hypochlorous acid (HOCl) is an oxygen-derived species involved in physiological processes related to the defence of the organism that may cause adverse effects when its production is insufficiently controlled. In order to examine its reactivity with potential scavenging molecules from the non steroidal anti-inflammatory drugs (NSAIDs) family, a competition assay based on para-aminobenzoic acid (PABA) chlorination was developed. The original optimised in vitro fluorimetric procedure offered the possibility to determine rate constants (k_s) for the reaction with HOCl in physiologically relevant conditions. The specificity of the system was improved by a liquid chromatography (LC) which allows the separation of the drugs and their oxidation products. After determination of the rate constant for PABA chlorination by HOCl (mean±SD in M_-1 s_-1: 4.3±0.3×10³), the applied mathematical model for a chemical competition permits to obtain linear curves from competition studies between several NSAIDs and PABA. Their slopes provided the following rate constants for the different studied drugs: tenoxicam: 4.0±0.7×10³, piroxicam: 3.6±0.7×10³, lornoxicam: 4.3±0.7×10³, meloxicam: 1.7±0.3×10⁴, nimesulide: 2.3±0.6×10². Meloxicam therefore reacted significantly faster than the other oxicams and nimesulide, which is the weakest scavenger of the studied series. The identification of some of the oxidation products by NMR or MS permitted to explore the reaction mechanism and to examine some aspects of the structure/activity relationships for the molecules of the same chemical family.     

Linear free energy relationship for 4-substituted (o-phenylenediamine)platinum(II) dichloride derivatives using quantum mechanical descriptors

In the present work quantum mechanical methods were used to calculate the rate constants for the first step of the aquation of a set of 4-substituted (o-phenylenediamine)platinum(II) dichloride derivatives containing electron-donating and withdrawing substituents at the 4-position of the aromatic ring. A linear free energy relationship was obtained for log(k(X)/k(H)), k being the rate constant for the first step of hydrolysis, and the electronic Hammett constants sigma(m) and sigma(p). The results showed that electron-donating groups promote the hydrolysis reaction. The quantitative models described here may be useful for the rational design of new, less mutagenic drugs based on platinum complexes.     

Purification and some properties of phosphoenolpyruvate carboxylase from Brevibacterium flavum and its aspartate-overproducing mutant

Phosphoenolpyruvate (PEP) carboxylases (PC) were purified from a wild strain and an aspartate-producing mutant of Brevibacterium flavum to electrophoretic homogeneity. The molecular weights of the enzymes were determined to be 4.1 X 10(5) by the gel-filtration technique. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the enzyme gave only one protein band with a molecular weight of 1.07 X 10(5). The enzyme was labile and stabilized by substrate PEP, activators, metallic cofactors, an allosteric inhibitor and ammonium sulfate. The mechanism for the PC reaction was rapid equilibrium random Bi Bi with a dead end complex, enzyme-bicarbonate-Pi. The KmS for PEP and bicarbonate were 2.5 and 0.63 mM, respectively, and the apparent KmS were not affected by the secondary substrate concentrations. Dissociation constants for Pi of enzyme-Pi and the dead end complex were 5.0 and 16 mM, respectively. Aspartate inhibition was completely competitive with both the substrates, PEP and bicarbonate, with an inhibitor constant of 0.044 mM. An activator, acetyl-CoA, did not alter the apparent Km for bicarbonate but decreased that for PEP. The activator constants for the enzyme-PEP complex and free enzyme were 6.3 and 40 microM, respectively. Double reciprocal plots of reaction rate against PEP concentration were not linear at lower PEP concentrations. Hill coefficients for PEP were 1.6 in the absence of any effectors, 1.0 in the presence of acetyl-CoA, and 2.3 in the presence of aspartate. As to the mutant enzyme, only the inhibitor constant for aspartate was increased, being 0.18 mM, but other constants, coefficients, as described above, and specific activity were almost the same as those of the wild-type enzyme.     

Detection and characterization of an additional site for binding of substrate and its analogs by inorganic pyrophosphatase

Phosphate, pyrophosphate, imidodiphosphate, EDTA and tripolyphosphate increase the rate constant for dissociation of the inorganic pyrophosphatase-substrate intermediate formed after cessation of the reaction by fluoride. The effect is enhanced in the given order 19-fold, the dependence of this effect on ligand concentration being hyperbolic. The values of the dissociation constants of the enzyme-ligand complexes lie within the concentration range of 0.16-1.0 mM. At high concentrations of Na2+ added simultaneously with the ligands this effect is decreased. The value of tau 1/2 for Pi binding to the enzyme-substrate compound is 0.15 min. The data obtained suggest that pyrophosphatase contains an anion ligand binding site, differing from that of the active one. This site does not affect the hydrolytic function of pyrophosphatase, as can be evidenced from the fact that Pi (9.5 mM) does not change the rate of enzymatic cleavage of PPi.     

Kinetics of Ethanol Oxidation Catalyzed by Yeast Alcohol Dehydrogenase in Aqueous Solutions of Sodium Dodecylsulfate

A study has been made of the effect of sodium dodecylsufate (SDS) addition on the oxidation of ethanol catalyzed by yeast alcohol dehydrogenase. Experiments were performed at pH = 8.1 and SDS concentrations employed were below and above the surfactant critical micelle concentration (CMC). The double reciprocal plots obtained in the absence and in the presence of the surfactant were compatible with a sequential bi-bi ordered mechanism. In the presence of the surfactant the initial reaction rates were consistently lower than in pure buffer at all the surfactant concentrations considered (0.5-50 mM). This effect is mainly due to an increase in the dissociation constant of beta-NAD(+) which reaches its maximum value (7,100 +/- 1,700 microM) at the CMC. Above the CMC the effect of the surfactant is mainly due to an increase in the Michaels constants of the alcohol, with values of 41 +/- 1 mM for 15 mM SDS and 50 +/- 1 mM for 50 mM SDS. The catalytic rate constant was found to be practically independent of the presence of the surfactant in the range of concentrations considered (up to 50 mM).     

Distinction between the intermediates in Na+-ATPase and Na+,K+-ATPase reactions. I. Exchange and hydrolysis kinetics at millimolar nucleotide concentrations

Parallel measurements in steady-state of ATP hydrolysis rate (vhydr) and the simultaneous reverse reaction, i.e., the ADP-ATP exchange rate (vexch), allowed the determination of a kinetic parameter, KE, containing only the four rate constants needed to characterize the enzyme intermediates involved in the sequence (Formula: see text). In order to compare the properties of these enzyme intermediates under different sets of conditions, KE was measured at varying K+ and Na+ concentrations in the presence of millimolar concentrations of ATP, ADP and MgATP, using an enzyme preparation that was partially purified from bovine brain. (1) In the presence of Na+ (150 mM), K+ (20-150 mM) was found to increase the exchange rate and decrease the ATP hydrolysis rate at steady-state. As a result, KE increased at increasing K+. However, the value of KE found by extrapolation to K+ = 0 was 7-times lower than the value actually measured in the absence of K+. This finding indicates that one of the intermediates, EATP or EP, or both, when formed in the presence of Na+ alone, are different from the corresponding intermediate(s) formed in the presence of Na+ + K+ (at millimolar substrate concentration). (2) In the presence of 150 mM K+, Na+ (5-30 mM) was found to increase the ADP/ATP exchange as well as the ATP hydrolysis rate at steady-state. The ratio of the two rates was constant. This finding, when interpreted in terms of KE, indicates that Na+ does not have to leave the enzyme for ATP release to be accelerated by K+ in the backward reaction. This also is in opposition to the usual versions of the Albers-Post model, which does not have simultaneous presence of Na+ and K+.     

Kinetic studies on the (Na+ + K+)-dependent ATPase evidence for coexisting sites for Na+, K+ and Mg2+

Na+-ATPase activity of a dog kidney (Na+ + K+)-ATPase enzyme preparation was inhibited by a high concentration of NaCl (100 mM) in the presence of 30 microM ATP and 50 microM MgCl2, but stimulated by 100 mM NaCl in the presence of 30 microM ATP and 3 mM MgCl2. The K0.5 for the effect of MgCl2 was near 0.5 mM. Treatment of the enzyme with the organic mercurial thimerosal had little effect on Na+ -ATPase activity with 10 mM NaCl but lessened inhibition by 100 mM NaCl in the presence of 50 microM MgCl2. Similar thimerosal treatment reduced (Na+ + K+)-ATPase activity by half but did not appreciably affect the K0.5 for activation by either Na+ or K+, although it reduced inhibition by high Na+ concentrations. These data are interpreted in terms of two classes of extracellularly-available low-affinity sites for Na+: Na+-discharge sites at which Na+-binding can drive E2-P back to E1-P, thereby inhibiting Na+-ATPase activity, and sites activating E2-P hydrolysis and thereby stimulating Na+-ATPase activity, corresponding to the K+-acceptance sites. Since these two classes of sites cannot be identical, the data favor co-existing Na+-discharge and K+-acceptance sites. Mg2+ may stimulate Na+-ATPase activity by favoring E2-P over E1-P, through occupying intracellular sites distinct from the phosphorylation site or Na+-acceptance sites, perhaps at a coexisting low-affinity substrate site. Among other effects, thimerosal treatment appears to stimulate the Na+-ATPase reaction and lessen Na+-inhibition of the (Na+ + K+)-ATPase reaction by increasing the efficacy of Na+ in activating E2-P hydrolysis.     

Conformational transitions of the H,K-ATPase studied with sodium ions as surrogates for protons

Following a recent demonstration that H,K-ATPase can active transport Na+ at a low rate (Polvani, C., Sachs, G., and Blostein, R. (1989) J. Biol. Chem. 264, 17854-17859), we have looked for and found effects of Na+ ions on the conformational state of gastric H,K-ATPase labeled with fluorescein isothiocyanate. Na+ ions reverse the K(+)-induced quench of the fluorescein fluorescence and somewhat enhance fluorescence in the absence of K+ ions. Equilibrium titrations of the cation effects show that Na+ and K+ ions are strictly competitive with apparent dissociation constants of KNa+ = 62 mM (n = 2) and KK+ = 6.6 mM (n = 2). The observations demonstrate that Na+ ions bind to and stabilize the high fluorescence E1 form of the protein while K+ ions stabilize the low fluorescence E2 form. Elevation of pH from 6.4 to 8.0 increased the apparent affinity of the Na+ ions from approximately 62 to 10.2 mM, consistent with competition between protons and Na+. The action of Na+ to stabilize the E1 form was used to measure the rate of the E2K----E1Na transition with a stopped-flow fluorimeter. The rate at pH 6.4 and 20 degrees C is 18.1 s-1. In addition the rate of the reverse conformational transition E1K----E2K has been measured at several K+ concentrations. From the hyperbolic dependence on K+ concentration a maximal rate of 211 +/- 32 s-1 and intrinsic K+ dissociation constant on E1 of 64.6 +/- 3.3 mM have been estimated. The kinetic and equilibrium data are self-consistent and thus support the proposed action of Na+ and K+ ions. Compared with Na,K-ATPase, the H,K-ATPase exhibits a lower affinity for Na+ on E1 and a much faster rate of the E2K----E1Na transition, but a similar affinity for K+ ions on E1 and rate of the transition E1K----E2K. The significance of the similarities and differences in cation specificity and rates of conformational changes of Na,K- and H,K-ATPases is discussed.     

Effect of monovalent cations on the pre-steady-state kinetic parameters of the plasma protease bovine activated protein C

Activated bovine plasma protein C (APC) was not reactive with the substrate p-nitrophenyl p-guanidinobenzoate (NPGB) in the absence of cations. In the presence of increasing concentrations of Na+, the acylation rate constant, k2,app, at 7 degrees C, progressively increased from 0.32 +/- 0.03 s-1 at 12.5 mM Na+ to 1.15 +/- 0.10 s-1 at 62.5 mM Na+. A linear dependence of the reciprocal of k2,app with [Na+]-2 was observed, indicating that at least two monovalent cation sites, or classes of sites, are necessary for the catalytic event to occur. From this latter plot, the k2,max for APC catalysis of NPGB hydrolysis, at saturating [Na+] and [NPGB], was calculated to be 1.21 +/- 0.10 s-1, and the Km for Na+ was found to be 21 +/- 3 mM. The dissociation constant, Ks, for NPGB to APC, at 7 degrees C, was not altered as [Na+] was increased, yielding a range of values of 18.5 X 10(-5) to 19.9 X 10(-5) M as [Na+] was varied from 12.5 to 62.5 mM. The deacylation rate constant, k3, for p-guanidinobenzoyl-APC hydrolysis was also independent of [Na+], with a value of (3.8 +/- 1.0) X 10(-3) s-1 in the absence of Na+ or in the presence of concentrations of Na+ up to 200 mM. Identical kinetic behavior was observed when Cs+ was substituted for Na+ in the above enzymic reaction. The pre-steady-state kinetic parameters were calculated according to the same methodology as described above.(ABSTRACT TRUNCATED AT 250 WORDS)     

Kinetics and thermodynamics of ouabain binding by intact turkey erythrocytes: effects of external sodium ion, potassium ion, and temperature

The kinetics of association and dissociation for the ouabain-Na+,K+- dependent ATPase complex have been studied in intact turkey erythrocytes as a function of external Na+ concentration, K+ concentration, and temperature. At free ligand concentrations substantially exceeding the concentration of available binding sites, the association reaction exhibits pseudo-first-order kinetics with an association rate constant (k1) that is conveniently determined over a wide range of temperatures (5-37 degrees C). The dissociation reaction exhibits strict first-order kinetics with a dissociation rate constant (k-1) that has the unusual property, in the turkey cell, of being sufficiently great to permit its direct determination even at temperatures as low as 5 degrees C. Values for the equilibrium binding constant for the ouabain-ATPase complex (KA) predicted from the ratio of the association and dissociation rate constants agree closely with independently measured values of KA determined directly under conditions of equilibrium binding. KA is a sensitive function of the composition of the external ionic environment, rising with increasing Na+ concentration and falling with increasing K+ concentration. These changes in KA are shown to be quantitatively attributable to changes in the rate constant k1, k-1 in contrast being unaffected at any given temperature by even very large changes in Na+ or K+ concentration. Arrhenius plots of k1 and k-1 both yield straight lines over the entire temperature range corresponding to activation energies for association and dissociation of 29.5 and 24.2 kcal/mol, respectively. These observations have made it possible to calculate the following standard values for the ouabain binding reaction in the presence of 150 mM Na+: delta G degree = -9.8 kcal/mol; delta H degree = +5.3 kcal/mol; delta S degree = +48.7 cal/degree/mol. The large positive value of delta S degree presumably reflects a highly ordered configuration of the ouabain-free ATPase molecule that is lost upon ouabain binding and that "drives" the reaction despite the positive value of delta H degree.     

The amiloride-sensitive Na+/H+ exchange system in skeletal muscle cells in culture

Chick skeletal muscle cells in culture have an amiloride-sensitive Na+-transporting system that has the following properties. Na+ uptake is dependent on the extracellular Na+ concentration. The Km value for Na+ is 25 mM and remains constant between pH 7.5 and 8.5. The maximal rate of Na+ transport is higher at alkaline pH. An ionizable group with a pK of 7.6 is essential for the system to be functional. The activity of the amiloride-sensitive Na+ uptake system is controlled by internal Na+ and H+ concentrations. Amiloride inhibition of Na+ uptake is competitively antagonized by increasing Na+ concentration. The dissociation constant for amiloride is 5 microM in Na+-free conditions and is constant between pH 7.5 and 8.5. The Km value for Na+ found from competition experiments is 13 mM. The amiloride-sensitive Na+ influx occurs in parallel with an amiloride-sensitive H+ efflux. This H+ efflux is stimulated by increasing external Na+ concentrations, the Km for Na+ being 15 mM. It is inhibited by amiloride with the same concentration dependence as Na+ influx.     

Studies of the thermal inactivation of cardiac adenylyl cyclase: evidence for a conformational change in the reaction mechanism

Membrane bound cardiac adenylyl cyclase was shown to undergo a spontaneous and irreversible thermal inactivation with a t1/2 of approximately 10 min. The loss of activity could not be explained by the action of endogenous proteases. Repeated freeze-thaw of membrane preparations resulted in a much increased rate of thermal inactivation (t1/2 = approx. 2 min). ATP, adenylimidodiphosphate, ADP, and PPi protected the enzyme from thermal inactivation with dissociation constants (Kd) of 193, 5.04, 84.4, and 6.3 microM, respectively. 5'-AMP and cyclic AMP were ineffective as protectors at concentrations as high as 3 mM. Activators of adenylyl cyclase such as Mn2+, forskolin, 5-guanylylimidodiphosphate, and NaF and 9 mM Mg2+ protected against thermal inactivation with Kd of 16.8 microM, 8.81 microM, 0.23 microM and 1.04 mM, respectively. Mg2+ alone was without effect. Thermal inactivation was first order under all conditions tested. Arrhenius plots of the rate constants for inactivation vs temperature were linear. The increased stability of ligand bound adenylyl cyclase was shown to be associated with an increased free energy of activation (delta G 0). These data provide evidence for the existence of two distinct conformations of cardiac adenylyl cyclase based on different susceptibilities to thermal inactivation. These enzyme conformations, termed E1 and E2, may be important reaction intermediates. The thermal stability of E1 was highly influenced by the enzyme's membrane lipid environment. The formation of E2 from E1 was enhanced by interaction with substrate, PPi, activators of adenylyl cyclase, and by interaction with dissociated stimulatory guanine nucleotide binding protein-alpha beta gamma heterotrimers.     

Characterization of the plasma membrane ATPase of Candida tropicalis

1) Plasma membrane vesicles from Candida tropicalis were isolated from protoplasts by differential centrifugation and purified in a continuous sucrose gradient. 2) The plasma membrane bound ATPase was characterized. It is highly specific for ATP and requires Mg2+. It is stimulated by K+, Na+ and NH4+. Lineweaver-Burk plots for ATPase activity are linear with a Vmax of 4.2 mumoles of ATP hydrolyzed min-1.mg-1 protein and a Km for ATP of 0.76 mM. The ATPase activity is inhibited competitively by ADP with a Ki of 1.7 mM and non competitively by vanadate with a Ki of 3 microM. The activity is unaffected by oligomycin or azide but is sensitive to DCCD.     

Role of calcium as an inhibitor of rat liver carbamylphosphate synthetase I

The mechanism of Ca2+ inhibition of carbamylphosphate synthetase I has been investigated using purified enzyme obtained from livers of rats fed a high protein diet. Binding of Mn2+ to the enzyme was measured by EPR techniques at pH 7.8, and Scatchard plots of the data indicated one Mn2+-binding site with a K'd of 13 microM. From competition studies between Mn2+ and Ca2+ or Mg2+ binding, values of 180 microM were obtained for K'd (Mg) and 193 microM for K'd (Ca). A nonlinear least squares curve fitting program was used to calculate the K'm for MgATP2- at the metal-nucleotide binding sites using a simplified rate equation of the enzyme reaction mechanism. Values of 140 and 2420 microM were obtained for K'm (MgATP) at the first and second sites, respectively, at pH 7.8, with a free Mg2+ of 1 mM and other substrates and activators present at saturating concentrations. Variations of the bicarbonate, N-acetylglutamate, and ammonia concentrations in the absence and presence of different amounts of total calcium, from which free Ca2+, free Mg2+, MgATP2-, and CaATP2- concentrations were calculated, permitted values for K'i (CaATP) to be obtained by graphic procedures. Mean values of 375 and 120 microM were obtained for K'i (CaATP) at the first and second sites, respectively. Using the above kinetic constants, a computer model of the enzyme reaction was constructed and tested using two further sets of kinetic data obtained by varying the concentrations of Mg2+, Ca2+, MgATP2-, and CaATP2-. Poor fits were obtained unless the formation of a mixed complex involving CaATP2- competition with MgATP2- at the second metal-nucleotide-binding site was incorporated into the rate equation. Nonlinear least squares curve fitting of both sets of experimental data gave a well determined value of 124 microM for this final CaATP2- inhibitory constant. Sensitivity tests for variation of the primary kinetic constants with the computer model showed that the inhibitory effect of free Ca2+ was weak and that the observed calcium inhibition of carbamylphosphate synthetase can be accounted for primarily by competitive interaction of CaATP2- at the second MgATP2- binding site. With 1 mM free Mg2+ and 5 mM MgATP2-, half-maximal inhibition of enzyme activity was obtained with 0.2 mM CaATP2-.     

The steady-state kinetics of yeast phosphoglycerate kinase. Anomalous kinetic plots and the effects of salts on activity.

1. A re-investigation of the kinetics of yeast phosphoglycerate kinase in the direction of 1,3-bisphosphoglycerate formation has been carried out, covering a 1000-fold range in substrate concentrations. A variety of improved spectrophotometric and fluorimetric assay procedures have been used. 2. Kinetic plots proved to be non-linear for each variable substrate. A variety of checks have been carried out to show that this is not due to artifacts in the assay procedures or heterogeneity of the enzyme preparation. 3. The effects of a variety of salts on the activity of the enzyme have been examined. Most salts, especially those with multivalent anions, can cause activation of the enzyme, but inhibit at high concentration. 4. The salt effect is shown to be principally due to anions rather than cations, and not to ionic strength changes. Sulphate, as one of the most effective anions has been used in most comparisons. 5. Salt activation is steepest when the substrate concentrations are low; maximum activation has been about 5-fold with 0.2 mM MgATP and 0.2 mM 3-phosphoglycerate. Inhibition at the higher salt concentrations is strongest at the same substrate concentrations as when activation is steepest, indicating a link between the two effects. 6. The presence of 20 mM or more Na2SO4 converted non-linear kinetic plots to linear ones. A study of the kinetics in the presence of 40 mM Na2SO4 was interpreted in terms of a random sequential binding mechanism, with sulphate acting as a competitive inhibitor. 7. Possible explanations for these anomalous results are discussed in terms of several mechanisms which have been shown to apply in other systems.     

Kinetics of uptake of glucose in rabbit jejunum: influence of sodium, unstirred layers, and passive permeation

Failure to account for the effect of the unstirred water layer and the contribution of passive permeation will lead to errors in the estimation of the kinetic constants of glucose uptake into the intestine. It is widely accepted that variations in the concentration of sodium in the bulk phase profoundly influence the rate of uptake of glucose in the intestine, but the kinetic basis for this effect remains in dispute. Accordingly, a previously validated in vitro technique was used to assess the effect of Na+ on the uptake of glucose into rabbit jejunum under conditions selected to reduce the unstirred layer resistance. Varying Na+ had no effect on the uptake of lauryl alcohol and therefore on unstirred layer resistance. The passive permeability coefficient for glucose uptake was estimated from the uptake of L-glucose, of D-glucose at 4 degrees C, or in the presence of 1 mM phlorizin or 40 mM galactose. The permeability for glucose increased as Na+ rose. The values of both the maximal transport rate and the Michaelis constant (Km) were influenced by Na+. A linear relationship was noted between Na+ and the maximal transport rate; the value of Km fell as Na+ was increased to 75 mequiv./L, but Km did not decline further with higher values of Na+. These results support the theoretical predictions of the presence of both an affinity and a velocity effect of the sodium gradient on the intestinal transport system for glucose.