Na+,K+-ATPase activity in cultured C6 glioma cells

Rat C₆ glioma cells were cultured for 4 days in MEM medium supplemented with 10% bovine serum and Na⁺,K⁺-ATPase activity was determined in homogenates of harvested cells. Approximately 50% of enzyme activity was attained at 1.5 mM K⁺ and the maximum (2.76±0.13 mol Pi/h/mg protein) at 5 mM K⁺. The specific activity of Na⁺,K⁺-ATPase was not influenced by freezing the homogenates or cell suspensions before the enzyme assay. Ten minutes' exposure of glioma cells to 10^–4 or 10^–5 M noradrenaline (NA) remained without any effect on NA⁺,K⁺-ATPase activity. Neither did the presence of NA in the incubation medium, during the enzyme assay, influence the enzyme activity. The nonresponsiveness of Na⁺,K⁺-ATPase of C₆ glioma cells to NA is consistent with the assumption that (+) form of the enzyme may be preferentially sensitive to noradrenaline. Na⁺,K⁺-ATPase was inhibited in a dose-dependent manner by vanadate and 50% inhibition was achieved at 2×10^–7 M concentration. In spite of the fact that Na⁺,K⁺-ATPase of glioma cells was not responsive to NA, the latter could at least partially reverse vanadate-induced inhibition of the enzyme. Although the present results concern transformed glial cells, they suggest the possibility that inhibition of glial Na⁺,K⁺-ATPase may contribute to the previously reported inhibition by vanadate of Na⁺,K⁺-ATPase of the whole brain tissue.     

Effect of L-arginine on Na+,K+-ATPase activity in rat aorta endothelium

The effect of L-arginine on the Na⁺,K⁺-ATPase activity in rat aorta endothelium was studied at its physiological concentrations in the range of 10^–6-10^–3 M. The enzyme activity was 35.5% increased by low concentrations of L-arginine (10^–5 M) and its activity was 32.3-37.1% decreased at the L-arginine concentrations of 10^–4-10^–3 M. A similar inhibition (by 34.5-42.8%) was also found in the presence of a NO-donor nitroglycerol (10^–4-10^–3 M). An optical isomer of L-arginine, D-arginine, at the concentrations of 10^–5 M also increased the enzyme activity by 37.1%, but its inhibiting effect was much less pronounced and was 15.7% at the D-arginine concentration of 10^–3 M. An inhibitor of NO-synthase, L-NAME (N^G-nitroarginine, methyl ester), failed to inhibit Na⁺,K⁺-ATPase. However, the presence of L-NAME abolished the inhibition of Na⁺,K⁺-ATPase by high concentrations of L-arginine. Thus, the effect of L-arginine on the endothelial Na⁺-pump depended on its concentration, and it is suggested that the enzyme inhibition by high concentrations of L-arginine should be associated with activation of the endogenous synthesis of NO.     

Role of heme oxygenase in heme-mediated inhibition of rat brain Na+−K+-ATPase: Protection by tin-protoporphyrin

Hemoglobin has been shown to inhibit brain Na⁺–K⁺-ATPase through an iron-dependent mechanism. Both hemoglobin and iron cause spontaneous peroxidation of brain lipids. Release of iron from the heme molecule in animal tissues is dependent on the activity of heme oxygenase. We hypothesized that inhibition of heme catabolism by heme oxygenase prevents the iron-mediated inhibition of Na⁺–K⁺-ATPase and might subsequently reduce the tissue damage. Therefore, we studied the effect of heme and tin-protoporphyrin, an inhibitor of heme oxygenase, on the activity of partially purified Na⁺–K⁺-ATPase from rat brain in the presence and absence of purified hepatic heme oxygenase. Heme alone at a concentration of 30 M did not inhibit Na⁺–K⁺-ATPase. However, in the presence of heme oxygenase, heme inhibited Na⁺–K⁺-ATPase by 75%. Pretreatment of rats with SnCl₂, a known inducer of heme oxygenase, reduced the basal activity of the brain Na⁺–K⁺-ATPase by 50%. Inhibition of heme oxygenase by tin-protoporphyrin (30 M) prevented the inhibition of Na⁺–K⁺-ATPase which occurred in the presence of heme and heme oxygenase. It is concluded that suppression of heme oxygenase by tin-protoporphyrin might be a therapeutic approach to management of hemoglobin-associated brain injury following CNS hemorrhage.     

Pressure adaptation of teleost gill Na+/K+-adenosine triphosphatase: role of the lipid and protein moieties

Summary The effects of temperature and pressure on Na⁺/K⁺-adenosine triphosphatases (Na⁺/K⁺-ATPases) from gills of marine teleost fishes were examined over a range of temperatures (10–25°C) and pressures (1–680 atm). The relationship between gill membrane fluidity and Na⁺/K⁺-ATPase activity was studied using the fluorescent probe 1,6-diphenyl-1,3,5-hexatriene (DPH). The increase in temperature required to offset the membrane ordering effects of high pressure was 0.015–0.025°C·atm^-1, the same coefficient that applied to Na⁺/K⁺-ATPase activities. Thus, temperature-pressure combinations yielding the same Na⁺/K⁺-ATPase activity also gave similar estimates of membrane fluidity. Substituion of endogenous lipids with lipids of different composition altered the pressure responses of Na⁺/K⁺-ATPase. Na⁺/K⁺-adenosine triphosphatase became more sensitive to pressure in the presence of chicken egg phosphatidylcholine, but phospholipids isolated from fish gills reduced the inhibition by pressure of Na⁺/K⁺-ATPase. Cholesterol increased enzyme pressure sensitivity. Membrane fluidity and pressure sensitivity of Na⁺/K⁺-ATPase were correlated, but the effects of pressure also dependent on the source of the enzyme. Our results suggest that pressure adaptation of Na⁺/K⁺-ATPase is the result of both changes in the primary structure of the protein and homeoviscous adaptation of the lipid environment.Abbreviations EDTA; DPH 1,6-diphenyl-1,3,5-hexatriene - PC phosphatidylcholine - PL phospholipid - SDH succinate dehydrogenase     

Functional Consequences of Various Leucine Mutations in the M3/M4 Loop of the Na<Superscript>+</Superscript>,K<Superscript>+</Superscript>-ATPase α-Subunit

Leucines were mutated within the sequence L³¹¹ILGYTWLE³¹⁹ of the extracellular loop flanking the third (M3) and fourth (M4) transmembrane segments (M3/M4 loop) of the Torpedo Na⁺,K⁺-ATPase α-subunit. Replacement of Leu³¹¹ with Glu resulted in a considerable loss of Na⁺,K⁺-ATPase activity. Replacement of Leu³¹³ with Glu shifted the equilibrium of E₁P and E₂P toward E₁P and reduced the rate of the E₁P to E₂P transition. The reduction of the transition rate and stronger inhibition of Na⁺,K⁺-ATPase activity by Na⁺ at higher concentrations together suggest that there is interference of Na⁺ release on the extracellular side in the Leu³¹³ mutant. Thus, Leu³¹³ could be in the pathway of Na⁺ exit. Replacement of Leu³¹⁸ with Glu yielded an enzyme with significantly reduced apparent affinity for both vanadate and K⁺, with an equilibrium shifted toward E₂P and no alteration in the transition rate. The reduced vanadate affinity is due to the lower rate of production of vanadate-reactive [K⁺ ₂]E₂ caused by inhibition of dephosphorylation through reduction of the K⁺ affinity of E₂P. Thus, Leu³¹⁸ may be a critical position in guiding external K⁺ to its binding site.     

The influence of potassium ion (K+) on digoxin-induced inhibition of porcine cerebral cortex Na+/K+-ATPase

The in vitro influence of potassium ion modulations, in the concentration range 2 mM–500 mM, on digoxin-induced inhibition of porcine cerebral cortex Na⁺/K⁺-ATPase activity was studied. The response of enzymatic activity in the presence of various K⁺ concentrations to digoxin was biphasic, thereby, indicating the existence of two Na⁺/K⁺-ATPase isoforms, differing in the affinity towards the tested drug. Both isoforms showed higher sensitivity to digoxin in the presence of K⁺ ions below 20 mM in the medium assay. The IC₅₀ values for high/low isoforms 2.77 × 10^{? 6} M / 8.56 × 10^{? 5} M and 7.06 × 10^{? 7} M /1.87 × 10^{? 5} M were obtained in the presence of optimal (20 mM) and 2 mM K⁺, respectively. However, preincubation in the presence of elevated K⁺ concentration (50 – 500 mM) in the medium assay prior to Na⁺/K⁺-ATPase exposure to digoxin did not prevent the inhibition, i.e. IC₅₀ values for both isoforms was the same as in the presence of the optimal K⁺ concentration. On the contrary, addition of 200 mM K⁺ into the medium assay after 10 minutes exposure of Na⁺/K⁺-ATPase to digoxin, showed a time-dependent recovery effect on the inhibited enzymatic activity. Kinetic analysis showed that digoxin inhibited Na⁺/K⁺-ATPase by reducing maximum enzymatic velocity (V_max) and K_m, implying an uncompetitive mode of interaction.     

Inhibition of red cell Ca-ATPase by vanadate

1. 1. The Mg²⁺- plus Ca²⁺-dependent ATPase (Ca²⁺-ATPase) in human red cell membranes is susceptible to inhibition by low concentrations of vanadate.

2. 2. Several natural activators of Ca²⁺-ATPase (Mg²⁺, K⁺, Na⁺ and calmodulin) modify inhibition by increasing the apparent affinity of the enzyme for vanadate.

3. 3. Among the ligands tested, K⁺, in combination with Mg²⁺, had the most pronounced effect on inhibition by vanadate.

4. 4. Under conditions optimal for inhibition of Ca²⁺-ATPase, the K for vanadate was 1.5 μM and inhibition was nearly complete at saturating vanadate concentrations.

5. 5. There are similarities between the kinetics of inhibition of red cell Ca²⁺-ATPase and (Na⁺ + K⁺)-ATPase prepared from a variety of sources; however, (Na⁺ + K⁺)-ATPase is approx. 3 times more sensitive to inhibition by vanadate.

The effects of transmitters on the affinity of the insoluble fraction of Ox brain for Na+ and K+

The insoluble fraction of ox-brain, which had previously been shown to have a non-linear affinity for Na⁺ and K⁺, was prepared. Acetylcholine (1×10^–8 mol/l and 1×10^–7 mol/l) reduced the affinity of the fraction for Na⁺ and K⁺ to zero, while at 1×10^–6 mol/l, the affinity for the cations was almost as high as in the absence of the transmitter; the affinities for Na⁺ and K⁺ were particularly high, when the supernatant concentrations of these ions exceeded 80–100 mM. Addition of eserine (3×10^–5 mol/l) considerably modified the response of the fraction to acetylcholine (1×10^–5 mol/l). Atropine (1×10^–8 mol/l) in the absence or presence of acetylcholine (1×10^–5, or 1×10^–4 mol/l) reduced the affinity of the fraction for Na⁺ and K⁺ to zero. Epinephrine (3×10^–10 mol/l) lowered the affinity for Na⁺ and K⁺, while ergotamine itself (1×10^–5 mol/l) reduced it to zero. The addition of both epinephrine and ergotamine at the latter concentrations restored the affinity of the fractions for Na⁺ and K⁺ to what it had been in the absence of the transmitter or antagonist, previously reported. Norepinephrine (3×10^–10 mol/l), or ouabain (1×10^–7 mol/l) reduced the affinity of the fraction for Na⁺ and K⁺ to zero. Thus, the transmitters and antagonists altered the affinity of the insoluble fraction for Na⁺ and K⁺ nonlinearity, dependent upon their concentrations, the concentrations of the cations, and the interaction of transmitter and antagonist.     

Characterization of Syrian hamster gastric mucosal H+,K+-ATPase

Employing a simple one-step sucrose gradient fractionation method, gastric mucosal membrane of Syrian hamster was prepared and demonstrated to be specifically enriched in H⁺,K⁺-ATPase activity. The preparation is practically devoid of other ATP hydrolyzing activity and contains high K⁺-stimulated ATPase, activity of at least 4–5 fold compared to basal ATPase activity. The H⁺,K⁺-ATPase showed hydroxylamine-sensitive phosphorylation and K⁺-dependent dephosphorylation of the phospho-enzyme, characteristic inhibition by vanadate, omeprazole and SCH 28080, and nigericin-reversible K⁺-dependent H⁺-transport — properties characteristic of gastric proton pump One notable difference with H⁺,K⁺-ATPase of other species has been the observation of valinomycin-independent H⁺ transport in such membrane vesicles. It is proposed that such H⁺,K⁺-ATPase-rich hamster gastric mucosal membrane preparation might provide a unique model to study physiological aspects of H⁺,K⁺-ATPase-function in relation to HCl secretion.     

Preconditioning Prevents the Inhibition of Na+,K+-ATPase Activity after Brain Ischemia

Application of single transient forebrain ischemia (ISC) in adult Wistar rats, lasting 2 or 10 min, caused inhibition of Na⁺,K⁺-ATPase activity in cytoplasmic membrane fractions of hippocampus and cerebral cortex immediately after the event. In the 2-min ISC group followed by 60 min of reperfusion, the enzyme inhibition was maintained in the cortex, while there was an increase in hippocampal enzyme activity; both effects were over 1 day after the event. However, in the 10-min ISC group enzyme inhibition had been maintained for 7 days in both cerebral structures. Interestingly, ischemic preconditioning (2-min plus 10-min ISC, with a 24-hour interval in between) prevented the inhibitory effect of ischemia/reperfusion on Na⁺,K⁺-ATPase activity observed either after a single insult of 2 min or 10 min ischemia. We suggest that the maintenance of Na⁺,K⁺-ATPase activity afforded by preconditioning be related to cellular neuroprotection.     

Evidence That Antioxidants Prevent the Inhibition of Na+,K+-ATPase Activity Induced by Octanoic Acid in Rat Cerebral Cortex in Vitro

The objective of the present study was to investigate the in vitro effects of octanoic acid, which accumulates in medium-chain acyl-CoA dehydrogenase (MCAD) deficiency and in Reye syndrome, on key enzyme activities of energy metabolism in the cerebral cortex of young rats. The activities of the respiratory chain complexes I–IV, creatine kinase, and Na⁺, K⁺-ATPase were evaluated. Octanoic acid did not alter the electron transport chain and creatine kinase activities, but, in contrast, significantly inhibited Na⁺, K⁺-ATPase activity both in synaptic plasma membranes and in homogenates prepared from cerebral cortex. Furthermore, decanoic acid, which is also increased in MCAD deficiency, and oleic acid strongly reduced Na⁺, K⁺-ATPase activity, whereas palmitic acid had no effect. We also examined the effects of incubating glutathione and trolox (-tocopherol) alone or with octanoic acid on Na⁺, K⁺-ATPase activity. Tested compounds did not affect Na⁺, K⁺-ATPase activity by itself, but prevented the inhibitory effect of octanoic acid. These results suggest that inhibition of Na⁺, K⁺-ATPase activity by octanoic acid is possibly mediated by oxidation of essential groups of the enzyme. Considering that Na⁺, K⁺-ATPase is critical for normal brain function, it is feasible that the significant inhibition of this enzyme activity by octanoate and also by decanoate may be related to the neurological dysfunction found in patients affected by MCAD deficiency and Reye syndrome.     

Effect of tissue specificity of brain soluble fractions on Na+, K+-ATPase activity

Previous evidence from this laboratory indicated that catecholamines and brain endogenous factors modulate Na⁺, K⁺-ATPase activity of the synaptosomal membranes. The filtration of a brain total soluble fraction through Sephadex G-50 permitted the separation of two fractions-peaks I and II-which stimulated and inhibited Na⁺, K⁺-ATPase, respectively (Rodríguez de Lores Arnaiz and Antonelli de Gomez de Lima, Neurochem. Res.11, 1986, 933). In order to study tissue specificity a rat kidney total soluble was fractionated in Sephadex G-50 and kidney peak I and II fractions were separated; as control, a total soluble fraction prepared from rat cerebral cortex was also processed. The UV absorbance profile of the kidney total soluble showed two zones and was similar to the profile of the brain total soluble. Synaptosomal membranes Na⁺, K⁺- and Mg²⁺-ATPases were stimulated 60–100% in the presence of kidney and cerebral cortex peak I; Na⁺, K⁺-ATPase was inhibited 35–65% by kidney peak II and 60–80% by brain peak II. Mg²⁺-ATPase activity was not modified by peak II fractions. ATPases activity of a kidney crude microsomal fraction was not modified by kidney peak I or brain peak II, and was slightly increased by kidney peak II or brain peak I. Kidney purified Na⁺, K⁺-ATPase was increased 16–20% by brain peak I and II fractions. These findings indicate that modulatory factors of ATPase activity are not exclusive to the brain. On the contrary, there might be tissue specificity with respect to the enzyme source.     

Kinetics of Na+, K+-ATPase Inhibition by a Rat Brain Endogenous Factor (II-E)

Previous work from this laboratory led to the isolation by gel filtration and anionic exchange HPLC of a rat brain fraction named II-E, which highly inhibits synaptosomal membrane Na⁺, K⁺-ATPase activity. In this study we evaluated the kinetics of such inhibition and found that inhibitory potency was independent of Na⁺(1.56–200 mM), K⁺(1.25–40 mM), or ATP (1–8 mM) concentration. Hanes-Woolf plots indicated that II-E decreases Vmax but does not alter K_Mvalue, and suggested uncompetitive inhibition for Na⁺, K⁺or ATP. However, II-E became a stimulator at 0.5 mM ATP concentration. It is postulated that this brain factor may modulate ionic transport at synapses, thus participating in central neurotransmission.     

Effect of Cd and Hg on the Activity of Na/K-ATPase and Mg-ATPase Adsorbed on Polystyrene Microtiter Plates

In the present study a polystyrene microtiter plate was tested as a support material for synaptic plasma membrane (SPM) immobilization by adsorption. The adsorption was carried out by an 18-h incubation at +4°C of SPM with a polystyrene matrix, at pH 7.4. Evaluation of the efficiency of the applied immobilization method revealed that 10% protein fraction of initially applied SPM was bound to the support and that two SPM enzymes, Na⁺/K⁺-ATPase and Mg²⁺-ATPase, retained 70–80% activity after the adsorption. In addition, adsorption stabilizes Na⁺/K⁺-ATPase and Mg²⁺-ATPase, since the activities are substantial 3 weeks after the adsorption. Parallel kinetic analysis showed that adsorption does not alter significantly the kinetic properties of Na⁺/K⁺-ATPase and Mg²⁺-ATPase and their sensitivity to and mechanism of Cd²⁺- or Hg²⁺-induced inhibition. The only exception is the “high affinity” Mg²⁺-ATPase moiety, whose affinity for ATP and sensitivity toward Cd²⁺ were increased by the adsorption. The results show that such system may be used as a practical and comfortable model for the in vitro toxicological investigations.     

Freshwater to seawater acclimation of juvenile bull sharks (<Emphasis Type="Italic">Carcharhinus leucas</Emphasis>): plasma osmolytes and Na<Superscript>+</Superscript>/K<Superscript>+</Superscript>-ATPase activity in gill,rectal gland,kidney and intestine

This study examined the osmoregulatory status of the euryhaline elasmobranch Carcharhinus leucas acclimated to freshwater (FW) and seawater (SW). Juvenile C. leucas captured in FW (3 mOsm l^–1 kg^–1) were acclimated to SW (980–1,000 mOsm l^–1 kg^–1) over 16 days. A FW group was maintained in captivity over a similar time period. In FW, bull sharks were hyper-osmotic regulators, having a plasma osmolarity of 595 mOsm l^–1 kg^–1. In SW, bull sharks had significantly higher plasma osmolarities (940 mOsm l^–1 kg^–1) than FW-acclimated animals and were slightly hypo-osmotic to the environment. Plasma Na⁺, Cl^–, K⁺, Mg²⁺, Ca²⁺, urea and trimethylamine oxide (TMAO) concentrations were all significantly higher in bull sharks acclimated to SW, with urea and TMAO showing the greatest increase. Gill, rectal gland, kidney and intestinal tissue were taken from animals acclimated to FW and SW and analysed for maximal Na⁺/K⁺-ATPase activity. Na⁺/K⁺-ATPase activity in the gills and intestine was less than 1 mmol Pi mg^–1 protein h^–1 and there was no difference in activity between FW- and SW-acclimated animals. In contrast Na⁺/K⁺-ATPase activity in the rectal gland and kidney were significantly higher than gill and intestine and showed significant differences between the FW- and SW-acclimated groups. In FW and SW, rectal gland Na⁺/K⁺-ATPase activity was 5.6±0.8 and 9.2±0.6 mmol Pi mg^–1 protein h^–1, respectively. Na⁺/K⁺-ATPase activity in the kidney of FW and SW acclimated animals was 8.4±1.1 and 3.3±1.1 Pi mg^–1 protein h^–1, respectively. Thus juvenile bull sharks have the osmoregulatory plasticity to acclimate to SW; their preference for the upper reaches of rivers where salinity is low is therefore likely to be for predator avoidance and/or increased food abundance rather than because of a physiological constraint.     

The chlorpromazine inhibition of transport ATPase and acetylcholinesterase activities in the microsomal membranes of rat in vitro and in vivo

Chlorpromazine, an antipsychotic drug, is found to inhibit Na⁺,K⁺-ATPase activity in rat brain microsomal membranes in vitro in concentration and time dependent manner but some inconsistency is observed when the effect was studied with respect to different temperatures. Various ligands and/or substrate affect the inhibition by chlorpromazine in different ways. The in vivo study with this drug shows that the activities of Na⁺,K⁺-ATPase, Ca^–2-ATPase and acetylcholinesterase in the microsomal membranes of different organs are inhibit with increases in concentration or lengths of time of treatment and then levels off.     

Demonstration of the electrogenicity of proton translocation during the phosphorylation step in gastric H+K+-ATPase

Summary Membrane fragments containing the H⁺K^–-ATPase from parietal cells have been adsorbed to a planar lipid membrane. The transport activity of the enzyme was determined by measuring electrical currents via the capacitive coupling between the membrane sheets and the planar lipid film. To initiate the pump currents by the ATPase a light-driven concentration jump of ATP from caged ATP was applied as demonstrated previously for Na⁺K⁺-ATPase (Fendler, K., Grell, E., Haubs, M., Bamberg, E. 1985.EMBO J. 4:3079–3085). Since H⁺K⁺-ATPase is an electroneutrally working enzyme no stationary pump currents were observed in the presence of K⁺. By separation of the H⁺ and K⁺ transport steps of the reaction cycle, however, the electrogenic step of the phosphorylation could be measured. This was achieved in the absence of K⁺ or at low concentrations of K⁺. The observed transient current is ATP dependent which can be assigned to the proton movement during the phosphorylation. From this it was conclueded that the K⁺ transport during dephosphorylation is electrogenic, too, in contrast to the Na⁺K⁺-ATPase where the K⁺ step is electroneutral. The transient current was measured at different ionic conditions and could be blocked by vanadate and by the H⁺K⁺-ATPase specific inhibitor omeprazole. An alternative mechanism for activation of this inhibitor is discussed.     

Kinetics of Na+-, K+-ATPase Inhibition by an Endogenous Modulator (II-A)

We have previously reported the isolation by gel filtration and anionic exchange HPLC of two brain Na⁺, K⁺-ATPase inhibitors, II-A and II-E, and kinetics of enzyme interaction with the latter. In the present study we evaluated the kinetics of synaptosomal membrane Na⁺, K⁺-ATPase with II-A and found that inhibitory activity was independent of ATP (2–8 mM), Na⁺ (3.1–100 mM), or K⁺ (2.5–40 mM) concentration. Hanes-Woolf plots showed that II-A decreases V_max in all cases; K_M value decreased for ATP but remained unaltered for Na⁺ and K⁺, indicating respectively uncompetitive and noncompetitive interaction. However, II-A became a stimulator at 0.3 mM K⁺ concentration. It is postulated that brain endogenous factor II-A may behave as a sodium pump modulator at the synaptic region, an action which depends on K⁺ concentration.     

Inhibition of Rat Brain Na+, K+-ATPase Activity Induced by Homocysteine Is Probably Mediated by Oxidative Stress

The objective of the present study was to investigate the effects of preincubation of hippocampus homogenates in the presence of homocysteine or methionine on Na⁺, K⁺-ATPase and Mg²⁺-ATPase activities in synaptic membranes of rats. Homocysteine significantly inhibited Na⁺, K⁺-ATPase activity, whereas methionine had no effect. Mg²⁺-ATPase activity was not altered by the metabolites. We also evaluated the effect of incubating glutathione, cysteine, dithiothreitol, trolox, superoxide dismutase and GM1 ganglioside alone or incubation with homocysteine on Na⁺, K⁺-ATPase activity. Tested compounds did not alter Na⁺, K⁺-ATPase and Mg²⁺-ATPase activities, but except for trolox, prevented the inhibitory effect of homocysteine on Na⁺, K⁺-ATPase activity. These results suggest that inhibition of this enzyme activity by homocysteine is possibly mediated by free radicals and may contribute to the neurological dysfunction found in homocystinuric patients.     

The inhibitory activity of a brain extract on synaptosomal Na+,K+-ATPase is sensitive to carboxypeptidase A and to chelating agents

In the present study some properties of an inhibitory extract of synaptosomal membrane Na⁺,K⁺-ATPase were investigated. This extract (peak II) was prepared by gel filtration in Sephadex G-50 of a soluble fraction of the rat cerebral cortex. Ultrafiltration of peak II through Amicon membranes indicated that the inhibitor has a low MW (<1000). The inhibitory activity was not modified by heating in neutral pH at 95°C for 20 min but it was destroyed by charring in acid pH at 200°C for 120 min. The inhibitory activity decreased by incubation of peak II with carboxypeptidase A. These findings suggest that the factor responsible for the inhibition of Na⁺,K⁺-ATPase activity is probably a polypeptide. On the other hand, the inhibition was reverted by the chelators EDTA and EGTA, indicating the participation of an ionic compound as well. The increase of Mg²⁺ concentration during the enzyme assay did not increase the inhibition, indicating that the ion involved might not be vanadate. It is suggested that both a polypeptide and an ionic compound coparticipate in the inhibitory effect of peak II on Na⁺,K⁺-ATPase activity.