The role of Mg2+ in the hydrolytic activity of the isolated chloroplast ATPase: Study by high-performance liquid chromatography

The influences of total magnesium ion concentration at different total ATP concentrations, and of total ATP concentration, for different total magnesium ion concentrations, on the enzymatic rate of the isolated chloroplast F₁ ATPase, have been followed by a chromatographic method consisting in the separation and determination of ADP. From the various series of curves, it is concluded that the experimental results (position of the maxima,K _m values) are better fitted by a mechanism involving the activation of the enzyme by magnesium ion and hydrolysis of free ATP, rather than by the classical mechanism, for which the enzyme hydrolyzes the MgATP complex and is inhibited by Mg²⁺. Although the equations giving the reaction rate are similar in the two cases, the calculated values ofK _m are widely different. The value obtained from the classical mechanism does not agree withK _D , the dissociation constant of the enzyme-substrate complex, measured by the Hummel and Dreyer method. Moreover, when the total ATP concentration tends toward the total magnesium ion concentration, the nucleotide binding to the enzyme tends toward zero, although it should be maximum if MgATP were the true substrate. Finally, the inhibitory effect of Na⁺ is more easily explained as a competition between this ion and the activating Mg²⁺, than by the classical mechanism.     

Sodium-translocating adenosine triphosphatase inStreptococcus faecalis

Sodium-transloating ATPase in the fermentative bacteriumStreptococcus faecalis exchanges sodium for potassium ions. Sodium ions stimulate its activity, but K⁺ ions have no significant effect at present. Although the molecular nature of the sodium ATPase is not clear, the enzyme is distinct from other ion-motive ATPases (E₁E₂ type and F₁F₀ type) as judged by its resistance to vanadate as well as dicyclohexylcarbodiimde. The sodium ATPase is induced when cells are grown on media rich in sodium, particularly under conditions that limit the generation of a proton potential or block the constitutive sodium/proton antiporter, indicating that an increase in the cytoplasmic sodium level serves as the signal. The enzyme is not induced in response to K⁺ deprivation. The sodium ATPase may have evolved to cope with a sodium-rich environment under conditions that limit the magnitude of the proton potential.     

Characterization of K+-dependent and K+-independentp-nitrophenylphosphatase activity of synaptosomes

These experiments examined effects of several ligands on the K⁺ p-nitrophenylphosphatase activity of the (Na⁺,K⁺)-ATPase in membranes of a rat brain cortex synaptosomal preparation. K⁺-independent hydrolysis of this substrate by the synaptosomal preparation was studied in parallel; the rate of hydrolysis in the absence of K⁺ was approximately 75% less than that observed when K⁺ was included in the incubation medium. The response to the H⁺ concentrations was different: K⁺-independent activity showed a pH optimum around 6.5–7.0, while the K⁺-dependent activity was relatively low at this pH range. Ouabain (0.1 mM) inhibited K⁺-dependent activity 50%; a concentration 10 times higher did not produce any appreciable effect on the K⁺-independent activity. Na⁺ did not affect K⁺-independent activity at all, while the same ligand concentration inhibited sharply the K⁺-dependent activity; this inhibition was not competitive with the substrate,p-nitrophenyl phosphate. K⁺-dependent activity was stimulated by Mg²⁺ with low affinity (millimolar range), and 3 mM Mg²⁺ produced a slight stimulation of the activity in absence of K⁺, which could be interpreted as Mg²⁺ occupying the K⁺ sites. Ca²⁺ had no appreciable effect on the activity in the absence of K⁺. However, in the presence of K⁺ a sharp inhibition was found with all Ca²⁺ concentrations studied. ATP (0.5 mM) did not affect the K⁺-independent activity, but this nucleotide behaved as a competitive inhibitor top-nitrophenylphosphate. Pi inhibited activity in the presence of K⁺, competively to the substrate, so it could be considered as the second product of the reaction sequence.Abbreviations used p-NPP p-nitrophenylphosphate - p-NPPase rho-nitrophenylphosphatase activity     

Gill (Na+,K+)-ATPase from the blue crab Callinectes danae: modulation of K+-phosphatase activity by potassium and ammonium ions

The kinetic properties of a microsomal gill (Na⁺,K⁺)-ATPase from the blue crab Callinectes danae were analyzed using the substrate p-nitrophenylphosphate. The (Na⁺,K⁺)-ATPase hydrolyzed PNPP obeying cooperative kinetics (n=1.5) at a rate of V=125.4±7.5 U mg⁻¹ with K_0.5=1.2±0.1 mmol l⁻¹; stimulation by potassium (V=121.0±6.1 U mg⁻¹; K_0.5=2.1±0.1 mmol l⁻¹) and magnesium ions (V=125.3±6.3 U mg⁻¹; K_0.5=1.0±0.1 mmol l⁻¹) was cooperative. Ammonium ions also stimulated the enzyme through site–site interactions (n_H=2.7) to a rate of V=126.1±4.8 U mg⁻¹ with K_0.5=13.7±0.5 mmol l⁻¹. However, K⁺-phosphatase activity was not stimulated further by K⁺ plus NH₄⁺ ions. Sodium ions (K_I=36.7±1.7 mmol l⁻¹), ouabain (K_I=830.3±42.5 μmol l⁻¹) and orthovanadate (K_I=34.0±1.4 nmol l⁻¹) completely inhibited K⁺-phosphatase activity. The competitive inhibition by ATP (K_I=57.2±2.6 μmol l⁻¹) of PNPPase activity suggests that both substrates are hydrolyzed at the same site on the enzyme. These data reveal that the K⁺-phosphatase activity corresponds strictly to a (Na⁺,K⁺)-ATPase in C. danae gill tissue. This is the first known kinetic characterization of K⁺-phosphatase activity in the portunid crab C. danae and should provide a useful tool for comparative studies.     

Acid tolerance mediated by membrane ATPases in Lactobacillus acidophilus

The acid tolerance response in Lactobacillus acidophilus CRL 639, induced at pH 4.2 for 15 min, is mediated by the cell membrane F ₁-F ₀ ATPase. The specific activity of the enzyme was induced 1.6-fold after acid adaptation compared to a non-adapted control. The ATPase was optimal at pH 6 with a K _m=0.8 mM and a V _max=100 mM.     

Nested cooperativity and salt dependence of the ATPase activity of the archaeal chaperonin Mm-cpn

The properties of the ATPase activity of the type II chaperonin from Methanococcus maripaludis (Mm-cpn) were examined. Mm-cpn can hydrolyze not only ATP, but also CTP, UTP, and GTP, albeit with different effectiveness. The ATPase activity is dependent on magnesium and potassium ions, and is effectively inhibited by sodium ions. Maximal rates of ATP hydrolysis are achieved at 600 mM potassium. Initial rates of ATP hydrolysis by Mm-cpn were determined at various ATP concentrations, revealing for the first time the presence of both positive intra-ring and negative inter-ring cooperativity in the archaeal chaperonin.     

Inhibition by trifluoperazine of ATP synthesis and hydrolysis by particulate and soluble mitochondrial F1: Competition with H2PO 4 −

The effect of trifluoperazine (TFP) on the ATPase activity of soluble and paniculate F₁ATPase and on ATP synthesis driven by succinate oxidation in submitochondrial particles from bovine heart was studied at pH 7.4 and 8.8. At the two pH. TFP inhibited ATP hydrolysis. Inorganic phosphate protected against the inhibiting action of TFP. The results on the effect of various concentrations of phosphate in the reversal of the action of TFP on hydrolysis at pH 7.4 and 8.8 showed that H₂PO ₄ ^– is the species that competes with TFP. The effect of TFP on oxidative phosphorylation was studied at concentrations that do not produce uncoupling or affect the aerobic oxidation of succinate (<15M). TFP inhibited oxidative phosphorylation to a higher extent at pH 8.8 than at pH 7.4; this was through a diminution in theV _max, and an increase in theK _m for phosphate. Data on phosphate uptake during oxidative phosphorylation at several pH showed that H₂PO ₄ ^– is the true substrate for oxidative phosphorylation. Thus, in both synthesis and hydrolysis of ATP, TFP and H₂PO ₄ ^– interact with a common site. However, there is a difference in the sensitivity to TFP of ATP synthesis and hydrolysis; this is more noticeable at pH 8.8, i.e. ATPase activity of soluble F₁ remains at about 40% of the activity of the control in a concentration range of TFP of 40–100M, whereas in oxidative phosphorylation 14M TFP produces a 60% inhibition of phosphate uptake.     

Na+,K+-ATPase in developing fetal guinea pig brain and the effect of maternal hypoxia

Na⁺,K⁺-ATPase activity was determined in fetal guinea pig brain at 35, 40, 45, 50, 55, and 60 days of gestation. The activity remained at a constant level during the early periods (35–45 days) of gestation and increased significantly during 45–60 days. Following maternal hypoxia, the activity of Na⁺,K⁺-ATPase in the term (60 days) fetal brain was reduced by 50% whereas the preterm (50 days) brain activity was unaffected. Under identical hypoxic conditions, the enzymatic activity of adult brain was significantly reduced by 20%. Na⁺,K⁺-ATPase obtained from fetal brain (50 days of gestation) has both a low and a high affinity for ATP (K _m values =0.50 and 0.053 mM and correspondingV _max values =10.77 and 2.82 umoles Pi/mg protein/hr), whereas the enzyme in the adult brain has only a low affinity (K _m=1.67 mM andV _max=20.32 umoles Pi/mg protein/hr). The high and low affinity sites for ATP in the fetal brain suggests a mechanism essential for the maintenance of cellular ionic gradients at low concentrations of ATP and which would provide the fetal brain with a greater tolerance to hypoxia. The high sensitivity of Na⁺,K⁺-ATPase activity to hypoxia in guinea pig brain at term suggests that the cell membrane functions of the fetal brain may be more susceptible to hypoxia at term than it is earlier in gestation.     

Sulfate activation in Rhodobacter sulfidophilus and other species of the Rhodospirillaceae

Rhodobacter sulfidophilus, R. capsulatus, R. sphaeroides, Rhodospirillum rubrum, Rhodopseudomonas palustris, R. viridis and Rhodocyclus gelatinosus were found to be able to synthesize adenylylsulfate and 3-phosphoadenylylsulfate from sulfate and ATP. The presence of ATP sulfurylase was proven for the soluble protein fractions of all these species. ADP sulfurylase was not found. ATP sulfurylase was purified from R. sulfidophilus. Its molecular weight was 290,000. The enzyme is stabilized by magnesium ions and elevated salinities. The optimal pH was 8.0, activity was found between pH 6.8 and 9.4. The enzyme is inactivated at temperatures above 40°C. Kinetic studies resulted in K _m(ATP)=0.26 mM, K _m(sulfate)=0.33 mM; K _i(AMP)-2.1 mM, K _i(ADP)=1.15 mM; K _i(APS)=0.8 M; K _i(sulfite)=0.4. mM; K _i(sulfide)=0.66mM.Uncommon abbreviations APS adenylylsulfate - PAPS 3-phosphoadenylylsulfate - PEP phosphoenolpyruvate Dedicated to Professor Gerhart Drews on the occasion of his 60th birthday     

Mathematical modelling of ATP, K and Na interactions with (Na + K)-ATPase occurring under equilibrium conditions

The controlling effect of ATP, K⁺ and Na⁺ on the rate of (Na⁺ + K⁺)-ATPase inactivation by 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl) is used for the mathematical modelling of the interaction of the effectors with the enzyme under equilibrium conditions.

1. 1. Of a series of conceivable interaction models, designed without conceptual restrictions to describe the effector control of inactivation kinetics, only one fits the experimental data described in a preceding paper.

2. 2. The model is characterized by the coexistence of two binding sites for ATP and the coexistence of two separate binding sites for K⁺ and Na⁺ on the enzyme-ATP complex. On the basis of this model, the effector parameters fitting the experimental data most closely are estimated by means of nonlinear least-squares fits.

3. 3. The apparent dissociation constants for ATP of the enzyme-ATP complex or of the enzyme-(ATP)₂ complex are computed to lie near 0.0024 mM and 0.34 mM, respectively, irrespective of whether K⁺ and Na⁺ were absent or K⁺ and K⁺ plus Na⁺, respectively, were present in the experiments.

4. 4. The origin of the high and the low affinity site for binding of ATP to the (Na⁺ + K⁺)-ATPase molecule is traced back to the coexistence of two catalytic centres which, although primarily equivalent as to the reactivity of their thiol groups with NBD-Cl, are induced into anticooperative communication by ATP binding and thus show an induced geometric asymmetry.

Nitration as a Mechanism of Na+, K+-ATPase Modification During Hypoxia in the Cerebral Cortex of the Guinea Pig Fetus

Previous studies have shown that hypoxia induces nitric oxide synthase-mediated generation of nitric oxide free radicals leading to peroxynitrite production. The present study tests the hypothesis that hypoxia results in NO-mediated modification of Na⁺, K⁺-ATPase in the fetal brain. Studies were conducted in guinea pig fetuses of 58-days gestation. The mothers were exposed to FiO₂ of 0.07% for 1 hour. Brain tissue hypoxia in the fetus was confirmed biochemically by decreased ATP and phosphocreatine levels. P₂ membrane fractions were prepared from normoxic and hypoxic fetuses and divided into untreated and treated groups. The membranes were treated with 0.5 mM peroxynitrite at pH 7.6. The Na⁺, K⁺-ATPase activity was determined at 37°C for five minutes in a medium containing 100 mM NaCl, 20 mM KCl, 6.0 mM MgCl₂, 50 mM Tris HCl buffer pH 7.4, 3.0 mM ATP with or without 10 mM ouabain. Ouabain sensitive activity was referred to as Na⁺, K⁺-ATPase activity. Following peroxynitrite exposure, the activity of Na⁺, K⁺-ATPase in guinea pig brain was reduced by 36% in normoxic membranes and further 29% in hypoxic membranes. Enzyme kinetics was determined at varying concentrations of ATP (0.5 mM-2.0 mM). The results indicate that peroxynitrite treatment alters the affinity of the active site of Na⁺, K⁺-ATPase for ATP and decreases the Vmax by 35% in hypoxic membranes. When compared to untreated normoxic membranes Vmax decreases by 35.6% in treated normoxic membranes and further to 52% in treated hypoxic membranes. The data show that peroxynitrite treatment induces modification of Na⁺, K⁺-ATPase. The results demonstrate that peroxynitrite decreased activity of Na⁺, K⁺-ATPase enzyme by altering the active sites as well as the microenvironment of the enzyme. We propose that nitric oxide synthase-mediated formation of peroxynitrite during hypoxia is a potential mechanism of hypoxia-induced decrease in Na⁺, K⁺-ATPase activity.     

Relevance of Divalent Cations to ATP-Driven Proton Pumping in Beef Heart Mitochondrial F0F1-ATPase

The ATP hydrolysis rate and the ATP hydrolysis-linked proton translocation by the F₀F₁-ATPase of beef heart submitochondrial particles were examined in the presence of several divalent metal cations. All Me–ATP complexes tested sustained ATP hydrolysis, although to a different extent. However, only Mg- and Mn-ATP-dependent hydrolysis could sustain a high level of proton pumping activity, as determined by acridine fluorescence quenching. Moreover, the K _m of the Me-ATP hydrolysis-induced proton pumping activity was very similar to the K _m value of Me-ATP hydrolysis. Both oligomycin and DCCD caused the full recovery of the fluorescence, providing clear evidence for the association of Mg-ATP hydrolysis with proton translocation through the F₀F₁-ATPase complex. In contrast, with other Me-ATP complexes, including Ca-ATP as substrate, the proton pumping activity was undetectable, implicating an uncoupling nature for these substrates. Attempts to demonstrate the involvement of the subunit of the enzyme in the coupling mechanism failed, suggesting that the participation of at least the N-terminal segment of the subunit in the coupling mechanism of the mitochondrial enzyme is unlikely.     

Fluorescent labeling of (Na + K)-ATPase by 5-iodoacetamidofluorescein

5-Iodoacetamidofluorescein (5-IAF) covalently labels dog kidney (Na⁺ + K⁺)-ATPase with approximately 2 moles incorporated per mole of enzyme. ATPase and K⁺-phosphatase activities are fully retained after reaction, and the kinetic parameters for Na⁺, K⁺, Mg²⁺, ATP and p-nitrophenyl phosphate are likewise not significantly affected. The fluorescence of the bound 5-IAF is increased by ATP, Na⁺, and Mg²⁺, and decreased by K⁺. These fluorescence changes likely reflect ligand-induced stabilization of the E₁ or E₂ states of the enzyme.     

Identification and characterization of high-affinity Ca2+-ATPase associated with axonal plasma membranes of dog mesenteric nerves

The microsomal fraction isolated from dog mesenteric nerve fibres was found to contain ATPase activity stimulated by micromolar concentrations of Ca ions. Such a high-affinity Ca²⁺-ATPase (hereafter referred to as HA Ca-ATPase) followed a Michaelis-Menten kinetics with K_m for Ca ions of 0.4 M and V_max=12.5±2.4 mol P_i.mg^–1h^–1. The examination of the subcellular origin of HA Ca-ATPase revealed that this enzyme is associated with axonal plasma membranes as documented by its co-purification with several plasma membrane marker enzymes and with tetrodotoxin-sensitive³H-saxitoxin binding. The addition of exogenous magnesium ions (Mg) resulted in a non-competitive inhibition of HA Ca-ATPase with K_i=0.5 mM. The reaction velocity of HA Ca-ATPase was also inhibited by other divalent ions with the order of potency Mg>Mn >ZnCo>Ni. In contrast to low affinity (high K_m) Mg- and Ca-ATPase, the HA Ca-ATPase was insensitive to the inhibition by sodium azide (10 mM) and sodium fluoride (10 mM). Similarly, the specific activity of HA Ca-ATPase was unaffected by vanadate (100 M) and N-ethylmaleinimide (100 M). It is concluded that axonal plasma membranes of dog mesenteric nerves contain HA Ca-ATPase which seems to be unrelated to calcium-transporting Mg-dependent, Ca-stimulated ATPase.Abbreviations used BSA bovine serum albumin - HA Ca-ATPase high-affinity Ca²⁺-ATPase - K-pNPPase onabain-sensitive, K⁺-stimulated p-nitrophenyl phosphatase - NEM N-ethylmaleinimide - SIM 250 mM sucrose, 10 mM imidazole-HCl pH 7.4 - TRIS tris (hydroxymethyl) aminomethane     

The plasma membrane Ca pump catalyzes the hydrolysis of ATP at low rate in the absence of Ca

The plasma membrane Ca²⁺ ATPase catalyzed the hydrolysis of ATP in the presence of millimolar concentrations of EGTA and no added Ca²⁺ at a rate near 1.5% of that attained at saturating concentrations of Ca²⁺. Like the Ca-dependent ATPase, the Ca-independent activity was lower when the enzyme was autoinhibited, and increased when the enzyme was activated by acidic lipids or partial proteolysis. The ATP concentration dependence of the Ca²⁺-independent ATPase was consistent with ATP binding to the low affinity modulatory site. In this condition a small amount of hydroxylamine-sensitive phosphoenzyme was formed and rapidly decayed when chased with cold ATP. We propose that the Ca²⁺-independent ATP hydrolysis reflects the well known phosphatase activity which is maximal in the absence of Ca²⁺ and is catalyzed by E₂-like forms of the enzyme. In agreement with this idea pNPP, a classic phosphatase substrate was a very effective inhibitor of the ATP hydrolysis.     

A 70-kDa molecular chaperone,DnaK, from the industrial bacterium <Emphasis Type="Italic">Bacillus licheniformis</Emphasis>: gene cloning,purification and molecular characterization of the recombinant protein

The heat shock protein 70 (Hsp70/DnaK) gene of Bacillus licheniformis is 1,839 bp in length encoding a polypeptide of 612 amino acid residues. The deduced amino acid sequence of the gene shares high sequence identity with other Hsp70/DnaK proteins. The characteristic domains typical for Hsps/DnaKs are also well conserved in B. licheniformis DnaK (BlDnaK). BlDnaK was overexpressed in Escherichia coli using pQE expression system and the recombinant protein was purified to homogeneity by nickel-chelate chromatography. The optimal temperature for ATPase activity of the purified BlDnaK was 40°C in the presence of 100 mM KCl. The purified BlDnaK had a V _max of 32.5 nmol Pi/min and a K _M of 439 μM. In vivo, the dnaK gene allowed an E. coli dnaK756-ts mutant to grow at 44°C, suggesting that BlDnaK should be functional for survival of host cells under environmental changes especially higher temperature. We also described the use of circular dichroism to characterize the conformation change induced by ATP binding. Binding of ATP was not accompanied by a net change in secondary structure, but ATP together with Mg²⁺ and K⁺ ions had a greater enhancement in the stability of BlDnaK at stress temperatures. Simultaneous addition of DnaJ, GrpE, and NR-peptide (NRLLLTG) synergistically stimulates the ATPase activity of BlDnaK by 11.7-fold.     

Kinetic characterization of Na,K-ATPase from rabbit outer renal medulla: properties of the (alpha beta)(2) dimer

We describe and compare the main kinetic characteristics of the (alpha beta)(2) form of rabbit kidney Na,K-ATPase. The dependence of ATPase activity on ATP concentration revealed high (K(0.5)=4 microM) and low (K(0.5)=1.4 mM) affinity sites for ATP, exhibiting negative cooperativity and a specific activity of approximately 700 U/mg. For p-nitrophenylphosphate (PNPP) as substrate, a single saturation curve was found, with a smaller apparent affinity of the enzyme for this substrate (K(0.5)=0.5 mM) and a lower hydrolysis rate (V(M)=42 U/mg). Stimulation of ATPase activity by K(+) (K(0.5)=0.63 mM), Na(+) (K(0.5)=11 mM) and Mg(2+) (K(0.5)=0.60 mM) all showed V(M)'s of approximately 600 U/mg and negative cooperativity. K(+) (K(0.5)=0.69 mM) and Mg(2+) (K(0.5)=0.57 mM) also stimulated PNPPase activity of the (alpha beta)(2) form. Ouabain (K(0.5)=0.01 microM and K(0.5)=0.1 mM) and orthovanadate (K(0.5)=0.06 microM) completely inhibited the ATPase activity of the (alpha beta)(2) form. The kinetic characteristics obtained constitute reference values for diprotomeric (alpha beta)(2)-units of Na,K-ATPase, thus contributing to a better understanding of the biochemical mechanisms of the enzyme.     

Characterisation of a salt-stimulated ATPase activity associated with vacuoles isolated from storage roots of red beet (Beta vulgaris L.)

Ion stimulation and some other properties of an ATPase activity associated with vacuoles isolated from storage roots of red beet (Beta vulgaris L.) have been determined. The ATPase had a specific requirement for Mg²⁺ and in the presence of Mg²⁺ it was stimulated by salts of monovalent cations. The degree of stimulation by monovalent salts was influenced mainly by the anion and the order of effectiveness of the anions tested was Cl^->HCO ₃ ^- >Br^->malate>acetate>SO ₄ ^2- . For any given series of anions the magnitude of the stimulation obtained was influenced by the accompanying cation (NH ₄ ⁺ Na⁺>K⁺). This cation effect was abolished by 0.01% (v/v) Triton X-100 and it is suggested that it is the result of different permeabilities of membrane vesicles to the cations. There was no evidence of synergistic stimulation of the ATPase by mixtures of Na⁺ and K⁺. KCl- and NaCl-stimulation was maximal with salt concentrations in the range 60–150 mM. The true substrate of the enzyme was shown to be MgATP. It was shown that KCl stimulation was the result of an increase in V_max rather than a change in the affinity of the enzyme for MgATP. The ATPase was inhibited by N,N-dicyclohexylcarbodiimide, diethylstilbestrol, mersalyl and KNO₃ but other inhibitors tested (azide, oligomycin, orthovanadate, K₃[Cr(oxalate)₆] and ethyl-3-[3-dimethylaminopropyl]carbodiimide) were without effect or caused only partial inhibition at the highest concentration tested. The ATPase activity was equally distributed between pellet and supernatant fractions obtained after the subfractionation of vacuoles but the properties of the ATPase in each fraction were the same. It is suggested that beet vacuoles possess only one ATPase. The properties of the ATPase are compared with those of ATPases associated with other plant membranes and organelles and its possible role in transport at the tonoplast is discussed.Abbreviations ATP_F free ATP - ATP_T total ATP - BSA bovine serum albumen - DCCD N,N-dicyclohexylcarbodiimide - DES diethylstilbestrol - DNP 2,4-dinitrophenol - EDAC ethyl-3-(3-dimethylaminopropyl)carbodiimide - K_m apparent Michaelis constant - MgATP complex of Mg²⁺ and ATP - Mg _F ²⁺ free Mg²⁺ - Mg _T ² total Mg²⁺ - MES 2-(N-Morpholino)ethanesulphonic acid - Na₂EDTA disodium ethylenediaminetetraacetic acid - NEM N-ethylmaleimide - P_i inorganic phosphate - TCA trichloroacetic acid - Tris tris(hydroxymethyl)methylamine - V_max maximum velocity     

Elevating intracellular free Mg2+ preserves sensitivity of Na+−K+ pump to ATP at reduced temperatures in guinea pig red blood cells

Red cells of hibernating species have a higher relative rate of Na⁺–K⁺ pump activity at low temperature than the red cells of a mammal with a typical sensitivity to cold. The kinetics of ATP stimulation of the Na⁺–K⁺ pump were determined in guinea pig and ground squirrel red cells at different temperatures between 5 and 37°C by measuring ouabain-sensitive K⁺ influx at different levels of ATP. In guinea pig cells, elevation of intracellular free Mg²⁺ to 2 mmol·l^-1 by use of the divalent cation ionophore A23187 caused the apparent affinity of the pump for ATP to increase with cooling to 20°C, rather than to decrease, as occurs in cells not loaded with Mg²⁺. In ground squirrel cells raising intracellular free Mg²⁺ had little effect on apparent affinity of the pump for ATP at 20°C. ATP affinity rose slightly with cooling both in Mg²⁺-enriched and in control ground squirrel cells. Increased intracellular free Mg²⁺ in guinea pig cells stimulated Na⁺–K⁺ pump activity so that at 20°C the pump rate was the same in the Mg²⁺-enriched guinea pig and control ground squirrel cells. Pump activity in Mg²⁺-enriched guinea pig cells at 5°C was significantly improved but still lower than pump activity in control cells from ground squirrel. Thus, loss of affinity of the Na⁺–K⁺ pump for ATP that occurs with cooling in cold-sensitive guinea pig red cells can be, at least partially, prevented by elevating cytoplasmic free Mg²⁺. Conversely, in ground squirrel red cells natural rise of free Mg²⁺ may in part account for the preservation of the ATP affinity of their Na⁺–K⁺ pump with cooling.Abbreviations K _m Michaelis-Menten constant for apparent affinity - MOPS 3-(N-morpholino)-propanesulphonic acid - [Mg²⁺]_i intracellular concentration of free Mg²⁺ - OD optical density - RBC red blood cell(s) - T _b body temperature     

The origin of kinetic cooperativity in prehiotic catalysts

A polyallylamine carrying long hydrophobic dodecyl groups and adenine residues as side chains (PALAD C₁₂) may be able to catalyze the hydrolysis ofN-carbobenzoxy-l-alaninep-nitrophenyl ester (N-Cbz-Ala) as well asp-nitrophenyl acetate (pNPA). The progress curve of hydrolysis of the former displays a long lag and apparently no steady state. After this transient the rate falls off due to the accumulation of the products. Conversely, the hydrolysis ofp-nitrophenyl acetate displays classical burst kinetics followed by a slow decline of the reaction rate. Theoretical considerations show that a steady state may be expected to occur only if the concentration of the free catalyst is very small during the reaction. This condition is sufficient to allow the rate of disappearance of the substrate to be equal to the rate of appearance of the products, which is precisely a condition for the existence of a steady state. If the catalyst is poorly active and has a loose affinity for its substrate and product, the measurement of a significant reaction rate will require a much larger concentration of the catalyst. Therefore, under these conditions, one cannot expect a steady state to occur. The mathematical expression of the error made in the steady-state assumption has been derived. This error increases with the catalyst concentration and decreases if the affinity of the substrate for the catalyst is high. Therefore the lack of steady state is associated with the affinity (or the dissociation) of the substrate and the product for the catalyst. When this affinity is low, the free concentration of the catalyst during the reaction is high and one cannot expect a steady state to occur. This is precisely what takes place with N-Cbz-Ala. A mathematical expression of the rate of hydrolysis of N-Cbz-Ala and of any reactant that displays this type of kinetics may be derived at the end of the transient when the rate is close to its maximum value. Under these conditions the rate cannot follow classical Michaelis-Menten kinetics and displays positive cooperativity. It may therefore be speculated that primordial template-like catalysts that were displaying a poor affinity for their substrates and products were already exhibiting apparent positive cooperativity in the kinetic reactions they were able to catalyze. Correspondence to: J. Ricard