Inactivation of Na+, K+ -ATPase from cattle brain by sodium fluoride

The influence of the physiological ligands and modifiers on the plasma membrane Na+, K+ -ATPase from calf brain inactivation by sodium fluoride (NaF) is studied. ATP-hydrolyzing activity of the enzyme was found to be more stable as to NaF inhibition than its K+ -pNPPase activity. The activatory ions of Na+, K+ -ATPase have different effects on the process of the enzyme inhibition by NaF. K+ intensifies inhibition, but Na+ does not affect it. An increase of [Mg2+free] in the incubation medium (from 0.5 to 3.0 mM) rises the sensitivity of Na+, K+ -ATPase to NaF inhibition. But an increase of [ATP] from 0.3 to 1.5 mM has no effect on this process. Ca and Mg ions modify Na+, K+ -ATPase inhibition by fluoride differently. Ca2+free levels this process, and Mg2+free on the contrary increases it. In the presence of Ca ions and in the neutral-alkaline medium (pH 7.0-8.5) the recovery of activity of the transport ATPase inhibited by-NaF takes place. Sodium citrate also protects both ATP-hydrolizing and K-pNPPase activity of the Na+, K+ -ATPase from NaF inhibition. Under the modifing membranous effects (the treatment of plasma membranes by Ds-Na and digitonin) the partial loss of Na+, K+ -ATPase sensitivity to NaF inhibition is observed. It is concluded that Na+, K+ -ATPase inactivation by NaF depends on the influence of the physiological ligands and modifiers as well as on the integrity of membrane structure.     

Functional Significance of the Effects of Neurotransmitters on the Na+,K+-ATPase System

The aim of the present experiments was to study the effects of the neurotransmitters acetylcholine, noradrenaline, 5-hydroxytryptamine, and dopamine on the Na+,K+-ATPase of rat brain synaptosomal fractions. It is shown that dopamine at low concentrations specifically inhibits the Na+,K+-ATPase of synaptic membranes from the brain regions rich in dopaminergic endings, but has no effect on the synaptosomal Na+,K+-ATPase from the other parts of brain. Acetylcholine and noradrenaline have similar specific effects on Na+,K+-ATPase from cholinergic and adrenergic synaptosomes. The Na+,K+-ATPase of synaptic membranes from the different brain regions, characterised by different distributions of cholinergic, adrenergic, and 5-hydroxytryptaminergic endings, show different reactions with neurotransmitters. These data indicate a functional significance of the effects of the neurotransmitters on the synaptosomal Na+,K+-ATPase.     

Substrate inhibition of the (Na+, K+)-ATPase in the presence of excess Mg2+.

1. High concentrations of ATP inhibit completely the activity of (Na+, K+)-ATPase (ATP phosphohydrolase, EC 3.6.1.3) prepared from sheep brain. 2. The inhibition depends on the concentration of total ATP, i.e. complexed ATP+ free ATP. 3. The inhibition by high ATP concentrations persists in the absence of K+, and is then independent of the Na+ concentration between 2 and 140 mM Na+. 4. Raising the K+ concentration at 20 mM Na+ increases the ATP concentration required for the maximal hydrolysis rate. 5. The Hill number for the inhibition process is about three. 6. The inhibition by ATP is temperature-dependent, in that as the temperature is increased, higher ATP concentrations are required for inhibition.     

3H]dizocilpine binding to N-methyl-D-aspartate (NMDA) receptor is modulated by an endogenous Na+, K+-ATPase inhibitor. Comparison with ouabain

An endogenous Na+, K+-ATPase inhibitor termed endobain E has been isolated from rat brain which shares several biological properties with ouabain. This cardiac glycoside possesses neurotoxic properties attributable to Na+, K+-ATPase inhibition, which leads to NMDA receptor activation, thus supporting the concept that Na+/K+ gradient impairment has a critical impact on such receptor function. To evaluate potential direct effects of endobain E and ouabain on NMDA receptors, we assayed [3H]dizocilpine binding employing a system which excludes ionic gradient participation. Brain membranes thoroughly washed and stored as pellets ('non-resuspended' membranes) or after resuspension in sucrose ('resuspended' membranes) were employed. Membrane samples were incubated with 4 or 10 nM ligand with or without added endobain E or ouabain, in the presence of different glutamate plus glycine combinations, with or without spermidine. [3H]dizocilpine basal binding and Na+, K+- and Mg2+-ATPase activities proved very similar in 'non-resuspended' or 'resuspended' membranes. Endobain E decreased [3H]dizocilpine binding to 'resuspended' membranes in a concentration-dependent manner, attaining roughly 50% binding inhibition with the highest endobain E concentration assayed. Among tested conditions, only in 'resuspended' membranes, with 4 nM ligand and with 1x10(-8) M glutamate plus 1x10(-5) M glycine, was [3H]dizocilpine binding enhanced roughly +24% by ouabain (1 mM). After Triton X-100 membrane treatment, which drastically reduces Na+, K+-ATPase activity, the effect of ouabain on binding was lost whereas that of endobain E remained unaltered. Results indicate that not only membrane preparation but also treatment and storage are crucial to observe direct endobain E and ouabain effects on NMDA receptor, which are not attributable to changes in Na+, K+-ATPase activity or to Na+/K+ equilibrium alteration.     

Na+,K+-ATPase activity of the subcellular fractions of the rat brain in aging

The Na+, K+-ATPase activity in the homogenate and in subcellular fractions of different parts of the brain of adult and old rats was studied in comparison. The content of cholesterol in the above fractions was also determined. In old age the Na+, K+-ATPase activity in the homogenate and microsomal fraction of the cerebral hemispheres' cortex decreases, while the Mg2+-ATPase activity in the cortex microsomal fraction increases. The age-related Na+, K+- and Mg2+-ATPase activity in the myelin of the stem in the synaptic plasma membranes of hemispheres and the brain stem remains unchanged whereas in the myelin fraction of hemispheres it grows. The content of cholesterol in the brain of old rats as compared with adult ones increases in the microsomal fraction and remains unchanged in synaptic membranes. The possible role of age-related modification of lipid component of plasma membranes in the above changes of Na+, K+-ATPase activity is discussed.     

Taurine stimulation of isolated hamster brain Na+,K+-ATPase: activation kinetics and chemical specificity

Epileptic foci are associated with locally reduced taurine (2-aminoethanesulfonic acid) concentration and Na+,K+-ATPase (EC 3.6.1.3) specific activity. Topically applied and intraperitoneally administered taurine can prevent the development and/or spread of foci in many animal models. Taurine has been implicated as a possible cytosolic modulator of monovalent ion distribution, cytosolic "free" calcium activity, and neuronal excitability. Taurine may act in part by modulating Na+,K+-ATPase activity of neuronal and glial cells. We characterized the requirements for in vitro modulation of Na+,K+-ATPase by taurine. Normal whole brain homogenate Na+,K+-ATPase activity is 5.1 +/- 0.4 (4) mumol Pi X h-1 X mg-1 Lowry protein. Partial purification of the plasma membrane fraction to remove cytosolic proteins and extrinsic proteins and to uncouple cholinergic receptors yields a membrane-bound Na+,K+-ATPase activity of 204.6 +/- 5.8 (4) mol Pi X h-1 X mg-1 Lowry protein. Taurine activates the Na+,K+-ATPase at all levels of purification. The concentration dependence of activation follows normal saturation kinetics (K1/2 = 39 mM taurine, activation maximum = +87%). The activation exhibits chemical specificity among the taurine analogues and metabolites: taurine = isethionic acid greater than hypotaurine greater than no activation = beta-alanine = methionine = choline = leucine. Taurine can act as an endogenous activator/modulator of Na+,K+-ATPase. Its action is mediated by a membrane-bound protein.     

Influence of nitric oxide donors on the intrinsic fluorescence of Na+,K+-ATPase and effects on the membrane lipids.

Effects of the nitric oxide donors S-nitroso-glutathione (GSNO) and S-nitroso-N-acetylpenicillamine (SNAP) on Na+,K+-ATPase-rich membrane fragments purified from pig kidney outer medulla were studied using intrinsic fluorescence and ESR of spin-labeled membranes. These S-nitrosothiols differently affected the intrinsic fluorescence of Na+,K+-ATPase: GSNO induced a partial quenching, whereas SNAP produced no alteration. Quenching can be due to a direct modification of exposed tryptophan residues or to an indirect effect caused by reactions of nitrogen oxide reactive species with other residues or even with the membrane lipids. Pre-incubation of Na+,K+-ATPase with 0.4mM GSNO resulted in a modest inhibition of ATPase activity (about 24%) measured under optimal conditions. Stearic acid spin-labeled at the 14th carbon atom (14-SASL) was used to investigate membrane fluidity and the protein-lipid interface. SNAP slightly increased the mobility of bulk lipids from Na+,K+-ATPase-rich membranes, but did not change the fraction of bulk to protein-interacting lipids. Conversely, treatment with GSNO extinguished the ESR signals from 14-SASL, indicating generation of free radicals with high affinity for the lipid moiety. Our results demonstrated that membranes influence bioavailability of reactive nitrogen species and bias the activity of different S-nitrosothiols.     

Sodium-potassium adenosine triphosphatase activity of human lymphocyte membrane vesicles: kinetic parameters, substrate specificity, and effects of phytohemagglutinin.

We have prepared human blood lymphocyte membrane vesicles of high purity in sufficient quantity for detailed enzyme analysis. This was made possible by the use of plateletpheresis residues, which contain human lymphocytes in amounts equivalent to thousands of milliliters of blood. The substrate specificity and the kinetics of the cofactor and substrate requirements of the human lymphocyte membrane Na+, K+-ATPase activity were characterized. The Na+, K+-ATPase did not hydrolyze ADP, AMP, ITP, UTP, GTP or TTP. The mean ATPase stimulated by optimal concentrations of Na+ and K+ (Na+, K+-ATPase) was 1.5 nmol of P(i) hydrolyzed, microgram protein-1, 30 min-1 (range 0.9-2.1). This activity was completely inhibited by the cardiac glycoside, ouabain. The K(m) for K+ was approximately 1.0 mM and the K(m) for Na+ was approximately 15 mM. Active Na+ and K+ transport and ouabain-sensitive ATP production increase when lymphocytes are stimulated by PHA. Na+, K+-ATPase activity must increase also to transduce energy for the transport of Na+ and K+. Some studies have reported that PHA stimulates the lymphocyte membrane ATPase directly. We did not observe stimulation of the membrane Na+, K+-ATPase when either lymphocytes or lymphocyte membranes were treated with mitogenic concentrations of PHA. Moreover, PHA did not enhance the reaction velocity of the Na+, K+-ATPase when studied at the K(m) for ATP, Na+, K+ OR Mg++, indicating that it does not alter the affinity of the enzyme for its substrate or cofactors. Thus, our data indicate that the increase in ATPase activity does not occur as a direct result of PHA action on the cell membrane.     

Biochemical characterization of the neuron. ATPase and acetylcholinesterase activities of neuronal cell bodies isolated in bulk from the pig brain stem.

Nerve cell bodies, large and multipolar, were isolated in bulk with the least possible contamination from the pig brain stem. The activities of two neurobiologically important membrane enzymes, Na+, K+-ATPase, and acetylcholinesterase, in the isolated cell bodies were estimated. Na+, K+-ATPase [EC 3.6.1.4], more accurately called ouabain-sensitive ATPase of the nerve cell body, hydrolyzed 94 micronmoles of ATP per h per 100 mg of protein. This activity was one-fourth that in the brain stem. Nerve cell bodies contained a large amount of Ca2+, 275 micronmoles per 100 mg of protein, about half of which was calculated to exist as compounds other than calcium orthophosphate. However, the Na+, K+-ATPase of the nerve cell bodies was not stimulated by EGTA, in contrast to that of the brain stem. Acetylcholinesterase [EC 3.1.1.7] and cholinesterase [EC 3.1.1.8] activities were estimated separately by the use of the specific inhibitors Persidol and BW 284C51 dibromide. Acetylcholinesterase was almost completely responsible for the hydrolysis of acetylcholine in the nerve cell bodies isolated from the brain stem and little cholinesterase activity was detected. 1300-1400 micronmoles of acetylcholine was hydrolyzed per h per 100 mg of protein of the neuronal cell bodies; this activity was about four times higher than that in the brain stem. The differences between the specific activities of Na+, K+-ATPase, and acetylcholinesterase in theneuronal cell bodies and the brain stem are discussed in the light of electron microscopic analysis of the distribution of these enzymes and the preservation of the plasma membrane of the isolated cell bodies.     

Selective inhibition of [3H]nitrendipine binding to brain and cardiac membranes by bacitracin

The effects of bacitracin were investigated on [3H]nitrendipine binding to rat brain and cardiac membranes in a low ionic strength (5 mM Tris-HCl) buffer. Bacitracin inhibited [3H]nitrendipine binding to rat brain and cardiac membranes with IC50 values of 400 +/- 100 and 4600 +/- 400 micrograms/mL, respectively. Scatchard analysis in brain membranes revealed that bacitracin inhibited [3H]nitrendipine binding primarily by reducing the Bmax but also by producing a small increase in the Kd. In brain membranes, Na+ (100 mM) and Ca2+ (2 mM) reduced the potency of bacitracin to inhibit [3H]nitrendipine binding by approximately sixfold with IC50 values of 2600 +/- 300 and 2100 +/- 400 micrograms/mL observed for bacitracin in the presence of 100 mM Na+ and 2 mM Ca2+, respectively. The EC50 values for the effects of Na+ and Ca2+ were 800 +/- 200 microM and 25 +/- 5 mM. K+, Mg2+, choline, and increasing the assay buffer of Tris-HCl to 50 mM also decreased the inhibition of [3H]nitrendipine binding by bacitracin. These results suggest that bacitracin specifically modulates [3H]nitrendipine binding in a cation-dependent manner and that brain and cardiac dihydropyridine binding sites are either biochemically different or exist in a different membrane environment.     

Possible mechanism for the frequency-dependent increases in the cardiac contraction in monkey.

The effects of various interventions on the frequency-dependent increases in the contractility of the papillary muscles of monkeys were investigated. Ouabain (10(-6)M) and KCl-free Krebs-Ringer solution, which are known to inhibit membrane Na+,K+-ATPase (EC 3.6.1.3), abolished the frequency-dependent increases in the contractility of the papillary muscles. Epinephrine (4.5 X 10(8)M) or quinidine (1.3 X 10(-5)M), which are known not to inhibit the membrane Na+,K-ATPase at these concentrations, did not alter the frequency-dependent increases in the contractility. These results indicate that the frequency-dependent increases in the contractility might be mediated through an inhibition of the sarcolemmal Na+,K+-ATPase.     

Protective effect of L-cysteine and glutathione on rat brain Na+,K+-ATPase inhibition induced by free radicals

The aim of this study was to investigate whether the preincubation of brain homogenates with L-phenylalanine (Phe), L-cysteine (Cys) or reduced glutathione (GSH) could reverse the free radical effects on Na+,K+-ATPase activity. Two well established systems were used for the production of free radicals: 1) FeSO4 (84 microM) plus ascorbic acid (400 microM) and 2) FeSO4, ascorbic acid and H2O2 (1 mM) for 10 min at 37 degrees C in homogenates of adult rat whole brain. Changes in brain Na+,K+-ATPase activity and total antioxidant status (TAS) were studied in the presence of each system separately, with or without Phe, Cys or GSH. TAS value reflects the amount of free radicals and the capacity of the antioxidant enzymes to limit the free radicals in the homogenate. Na+,K+-ATPase was inhibited by 35-50% and TAS value was decreased by 50-60% by both systems of free radical production. The enzymatic inhibition was completely reversed and TAS value increased by 150-180% when brain homogenates were preincubated with 0.83 mM Cys or GSH. However, this Na+,K+-ATPase inhibition was not affected by 1.80 mM Phe, which produced a 45-50% increase in TAS value. It is suggested that the antioxidant action of Cys and GSH may be due to the binding of free radicals to sulfhydryl groups of the molecule, so that free radicals cannot induce Na+,K+-ATPase inhibition. Moreover, Cys and GSH could regulate towards normal values the neural excitability and metabolic energy production, which may be disturbed by free radical action on Na+,K+-ATPase.     

Effects of cholesterol on (Na+,K+)-ATPase ATP hydrolyzing activity in bovine kidney

The (Na+,K+)-ATPase ATP hydrolyzing activity from rabbit kidney medulla basolateral membrane vesicles was studied as a function of the cholesterol content of the basolateral membranes. The cholesterol content of the membranes was modified by incubation with phospholipid vesicles. When the cholesterol content was increased above that found in the native membrane, the (Na+,K+)-ATPase ATP hydrolyzing activity was inhibited. When the cholesterol content was decreased from that found in the native membranes, the (Na+,K+)-ATPase ATP hydrolyzing activity was inhibited. Analogous effects were found with the K+-activated phosphatase activity of the same membrane vesicles. Therefore, at low cholesterol contents, cholesterol was stimulatory, and at high cholesterol contents, cholesterol was inhibitory. The structural specificity of this effect was tested by introducing lanosterol and ergosterol as 50% of the membrane sterol. Ergosterol was the least effective at supporting (Na+,K+)-ATPase ATP hydrolyzing activity, while lanosterol was more effective, but still not as effective as cholesterol.     

Partial Purification of a Na+ -ATPase from the Plasma Membrane of the Marine Alga Heterosigma akashiwo

A Na+ -ATPase was partially purified from plasma membranes of the marine alga Heterosigma akashiwo. The plasma membranes of H. akashiwo cells were collected by differential centrifugation with subsequent discontinuous gradient centrifugation. Na+ -ATPase activity was associated with the resultant plasma membrane fraction and was stimulated to the greatest extent in the presence of 100 to 200 mM Na+, 10 mM K+, and 5 mM Mg2+ ions, pH 8.0. The Km value for Na+ ions was 12.2 mM. An apparent Km value for ATP was 880 [mu]M. A 140-kD phosphorylated intermediate was also detected in the same fraction in the presence of both Mg2+ and Na+ ions, and this protein was dephosphorylated upon the addition of K+ ions. We could partially purify the 140-kD protein after solubilization by Suc monolaurate and fractionation by sequential column chromatography on Sephacryl S-300, DEAE-Sepharose CL-6B, and Mono-Q columns. The purified 140-kD polypeptide could also be phosphorylated and be detected after acid sodium dodecyl sulfate-polyacryl-amide gel electrophoresis in the presence of Na+ and Mg2+ ions.     

(Na+ + K+)- and Na+-stimulated Mg2+-dependent ATPase activities in kidney of sea bass (Dicentrarchus labrax L.)

1. Sea bass kidney microsomal preparations contain two Mg2+ dependent ATPase activities: the ouabain-sensitive (Na+ + K+)-ATPase and an ouabain-insensitive Na+-ATPase, requiring different assay conditions. The (Na+ + K+)-ATPase under the optimal conditions of pH 7.0, 100 mM Na+, 25 mM K+, 10 mM Mg2+, 5 mM ATP exhibits an average specific activity (S.A.) of 59 mumol Pi/mg protein per hr whereas the Na+-ATPase under the conditions of pH 6.0, 40 mM Na+, 1.5 mM MgATP, 1 mM ouabain has a maximal S.A. of 13.9 mumol Pi/mg protein per hr. 2. The (Na+ + K+)-ATPase is specifically inhibited by ouabain and vanadate; the Na+-ATPase specifically by ethacrynic acid and preferentially by frusemide; both activities are similarly inhibited by Ca2+. 3. The (Na+ + K+)-ATPase is specific for ATP and Na+, whereas the Na+-ATPase hydrolyzes other substrates in the efficiency order ATP greater than GTP greater than CTP greater than UTP and can be activated also by K+, NH4+ or Li+. 4. Minor differences between the two activities lie in the affinity for Na+, Mg2+, ATP and in the thermosensitivity. 5. The comparison between the two activities and with what has been reported in the literature only partly agree with our findings. It tentatively suggests that on the one hand two separate enzymes exist which are related to Na+ transport and, on the other, a distinct modulation in vivo in different tissues.     

The effect of galactose metabolic disorders on rat brain Na+,K+-ATPase activity

To evaluate the effect of galactose metabolic disorders on the brain Na+,K+-ATPase in suckling rats. Separate preincubations of various concentrations (1-16 mM) of the compounds galactose-1-phosphate (Gal-1-P) and galactitol (galtol) with whole brain homogenates at 37 degrees C for 1 h resulted in a dose dependent inhibition of the enzyme whereas the pure enzyme (from porcine cerebral cortex) was stimulated. Glucose-1-phosphate (Glu-1-P) or galactose (Gal) stimulated both rat brain Na+,K+-ATPase and pure enzyme. A mixture of Gal-1-P (2 mM), galtol (2 mM) and Gal (4 mM), concentrations commonly found in untreated patients with classical galactosemia, caused a 35% (p < 0.001) rat brain enzyme inhibition. Additionally, incubation of a mixture of galtol (2 mM) and Gal (1 mM), which is usually observed in galactokinase deficient patients, resulted in a 25% (p < 0.001) brain enzyme inactivation. It is suggested that: a) The indirect inhibition of the brain Na+,K+-ATPase by Gal-1-P should be due to the presence of the epimer Gal and phosphate and that the pure enzyme direct activation by Gal-1-P and Glu-1-P to the presence of phosphate only. b) The observed brain Na+,K+-ATPase inhibitions in the presence of toxic concentrations of Gal-1-P and/or galtol could modulate the neural excitability, the metabolic energy production and the catecholaminergic and serotoninergic system.     

A comparative study on the effects of different bile salts on mucosal ATPase and transport in the rat jejunum in vivo.

The effects of deoxycholate, taurocholate and cholate on transport and mucosal ATPase activity have been investigated in the rat jejunum in vivo using closed-loop and perfusion techniques. In the closed-loops, 5 mM deoxycholate selectively inactivated (Na+ + K+)-ATPase, and net secretion of Na+ induced by 2.5 mM deoxycholate was due to reduced lumen to plasma flux of the ion; deoxycholate (2.5 mM) produced marked inhibition of 3-0-methylglucose transport. Luminal disappearance rates of deoxycholate (60.5 plus or minus 2.9% per g wet st of gut) greatly exceeded those of taurocholate (4.3 plus or minus 1.0). In the perfusion studies 1 mM deoxycholate induced net secretion of water, Na+ and C1-, and inhibited active glucose transport; concomitantly "total" ATPase, (Na+ + K+)-ATPase, and Mg-2+-ATPase were inhibited. At higher concentrations (5 mM) deoxycholate stimulated Mg-2+-ATPase activity. Taurocholate and cholate at 1mM had no effect on transport of (Na+ + K+)-ATPase. Mucosal lactase, sucrase and maltase activities were not affected by 1 mM deoxycholate, taurocholate or cholate. These results suggest that deoxycholate inhibits sodium-coupled glucose transport by inhibition of (Na+ + K+)-ATPase at the lateral and basal membranes of the epithelial cell, rather than from an effect at the brush-border membrane level.     

The evaluation of the role of endogenous Ca-dependent regulators and protein kinases in activating and inhibiting ion-transport ATPases]

The data on hormonal regulation of ATP-driving ion pumps are contradictory depending on the object used: whether native cells or isolated membranes. To eliminate this contrariety, we studied the ion transporting ATPases in saponin-permeabilized cells in the presence of all endogenous regulators. In permeabilized erythrocytes we obtained the presence of Ca(2+)-dependent activation of Ca(2+)-ATPase by factor(s) not affected by calmodulin antagonist R24571. We obtained also Ca(2+)-dependent activation and inhibition of Na+,K(+)-ATPase. At a concentration of Mg(2+)-ions corresponding to the intracellular level (370 microM), the 0.5-0.7 microM Ca(2+)-activated Na+,K(+)-ATPase (up to 3-fold), whereas the 1-5 microM Ca2+ inhibited it. The cyclic AMP (10(-5) M) inhibited or eliminated Ca(2+)-dependent activation. The decrease in Mg(2+)-ion concentration to 50 microM eliminated the activation and strengthened the inhibition, which reached 100% at the 1-2 microM Ca2+ concentration. The washing of membranes with EGTA eliminated Ca2+ effects on Na+,K(+)-ATPase. These data suggest that the ion-transporting ATPases are activated or inhibited by Ca(2+)-dependent regulators whose activities may be changed by protein kinase catalysed phosphorylation.     

Effects of L-phenylalanine on acetylcholinesterase, (Na+,K+)-ATPase and Mg2+-ATPase activities in adult rat whole brain and frontal cortex

The effect of different L-phenylalanine (Phe) concentrations (0.12-12.1 mM) on acetylcholinesterase (AChE), (Na+,K+)-ATPase and Mg2+-ATPase activities was investigated in homogenates of adult rat whole brain and frontal cortex at 37 degrees C. AChE, (Na+,K+)-ATPase and Mg2+-ATPase activities were determined after preincubation with Phe. AChE activity in both tissues showed a decrease up to 18% (p<0.01) with Phe. Whole brain Na+,K+-ATPase was stimulated by 30-35% (p<0.01) with high Phe concentrations, while frontal cortex Na+,K+-ATPase was stimulated by 50-55% (p<0.001). Mg2+-ATPase activity was increased only in frontal cortex with high Phe concentrations. It is suggested that: a) The inhibitory effect of Phe on brain AChE is not influenced by developmental factors, while the stimulation of Phe on brain Na+,K+-ATPase is indeed affected; b) The stimulatory effect of Phe on rat whole brain Na+,K+-ATPase is decreased with age; c) Na+,K+-ATPase is selectively more stimulated by high Phe concentrations in frontal cortex than in whole brain homogenate; d) High (toxic) Phe concentrations can affect Mg2+-ATPase activity in frontal cortex, but not in whole brain, thus modulating the amount of intracellular Mg2+.