Hemoglobin degradation lipid peroxidation,and inhibition of na+/k+-atpase in rat erythrocytes exposed to acrylonitrile

The effect of acrylonitrile (VCN) on erythrocyte lipid metabolism was investigated in vitro in metabolically active red cells from male Sprague-Dawley rats containing three types of hemoglobins: oxyhemoglobin, methemoglobin, and carbon monoxyhemoglobin. VCN at the concentration of 10 mM rapidly depleted erythrocyte glutathione (GSH) (75% of control) and induced lipid peroxidation (274% of control). Degradation of oxy- and methemoglobin was directly proportional to the extent of lipid peroxidation (r = 0.89). Addition of glucose to the incubation medium decreased hemoglobin degradation while it slightly increased VCN-induced lipid peroxidation. The highest amount of lipid peroxidation occurred in erythrocytes containing carbon monoxyhemoglobin and glucose. In the isolated red cell membranes incubated with 10 mM VCN, the lipid peroxidation was 400% of controls. VCN (25 mM) noncompetitively inhibited erythrocyte membrane Na⁺/K⁺-ATPase activity and the degree of inhibition was inversely proportional to the reaction temperature (r = ?0.88). These findings indicate that the VCN induced hemoglobin degradation and lipid peroxidation are two extremes of a spectrum of oxidative damage in red cells leading to a change in physical state of membrane structure causing inhibition of adenosine triphosphatase (ATPase) activity.     

Kinetics of oligomycin inhibition and activation of Na+/K(+)-ATPase.

Oligomycin inhibition of the maximal hydrolysis activity of ox brain Na+/K(+)-ATPase was studied at varying NaCl concentrations and it was found that for a given amount of live enzyme, the observed inhibition of a particular total oligomycin concentration decreased as the amount of added, (heat-) denatured enzyme increased. In the present article we derive a scale factor for the oligomycin concentration, i.e., the fraction of the total concentration of oligomycin which is free in solution, as a function of the enzyme concentration used. This fraction decreased linearly with the protein concentration and may attain quite small values. We also study the Na(+)-dependence of the hydrolysis rate at saturating substrate concentrations ([Mg2+] = [ATP] = 3 mM), in the presence as well as the absence of KCl, at various concentrations of oligomycin. These data may be explained if it is assumed that the sole effect of oligomycin is to confer upon the enzyme an increased affinity for Na+, i.e., oligomycin merely enhances the inhibitory effect of Na+ on the (maximal) activity seen at high Na(+)-concentrations. The increased Na(+)-affinity in the presence of oligomycin should result in activation of the hydrolysis rate measured under conditions where Na(+)-activation is predominant, i.e., at low Na(+)-concentration and sub-saturating substrate concentrations. This prediction is verified for both Na(+)-ATPase and for Na+/K(+)-ATPase. This proposed action of oligomycin seems to be corroborated also by other evidence discussed in the text.     

Na+/K+-ATPase activity in rat erythrocytes after prolonged starvation

Na⁺/K⁺-ATPase (sodium, potassium adenosine triphosphatase, EC 3.6.3.9) activity has been studied in whole erythrocytes from rats over time of total food deprivation for 1, 3, 5, 7–8, and 10–12 days with free access to water. Changes in Na⁺/K⁺-ATPase activity have been found to be phase-specific, i.e., associated with periods of certain metabolism level. After the hunger state and accommodation to endogenous nutrition (phases 0-I), from the 3rd to the 7th–8th day a period of compensated accommodation begins (phase II characterized by a stable euglycemic state, while the level of plateau of protein losses and hormonal stimulation are achieved). The Na⁺/K⁺-ATPase activity changes during the phase II were insignificant (p > 0.05), but potassium loss was observed in erythrocytes and blood plasma from the 5th day of starvation onwards. The phase III (the 10th–12th days) is an onset of the terminal period characterized by the lower activities of Na⁺/K⁺-ATPase (ouabain-sensitive activity) and Mg²⁺-ATPase (ouabain-independent activity) and by reduced sodium plasma levels that previously had remained virtually unchanged. There are considered possible causes of the observed decrease in the Na⁺/K⁺-ATPase activity during prolonged starvation, such as aging of the circulating erythrocyte population (the absence of reticulocytes and young erythrocytes), depletion of cell energy resources (hypoglycemia and glycopenia), effect of endogenous ouabain, and endotoxemia.     

Na+/K(+)-ATPase: modes of inhibition by Mg2+.

Adding 15 mM free Mg2+ decreased Vmax of the Na+/K(+)-ATPase reaction. Mg2+ also decreased the K0.5 for K+ activation, as a mixed inhibitor, but the increased inhibition at higher K+ concentrations diminished as the Na+ concentration was raised. Inhibition was greater with Rb+ but less with Li+ when these cations substituted for K+ at pH 7.5, while at pH 8.5 inhibition was generally less and essentially the same with all three cations: implying an association between inhibition and ion occlusion. On the other hand, Mg2+ increased the K0.5 for Na(+)-activation of the Na+/K(+)-ATPase and Na(+)-ATPase reactions, as a mixed inhibitor. Changing incubation pH or temperature, or adding dimethylsulfoxide affected inhibition by Mg2+ and K0.5 for Na+ diversely. Presteady-state kinetic studies on enzyme phosphorylation, however, showed competition between Mg2+ and Na+. In the K(+)-phosphatase reaction catalyzed by this enzyme Mg2+ was a (near) competitor toward K+. Adding Na+ with K+ inhibited phosphatase activity, but under these conditions 15 mM Mg2+ stimulated rather than inhibited; still higher Mg2+ concentrations then inhibited with K+ plus Na+. Similar stimulation and inhibition occurred when Mn2+ was substituted for Mg2+, although the concentrations required were an order of magnitude less. In all these experiments no ionic substitutions were made to maintain ionic strength, since alternative cations, such as choline, produced various specific effects themselves. Kinetic analyses, in terms of product inhibition by Mg2+, require Mg2+ release at multiple steps. The data are accommodated by a scheme for the Na+/K(+)-ATPase with three alternative points for release: before MgATP binding, before K+ release and before Na+ binding. The latter alternatives necessitate two Mg2+ ions bound simultaneously to the enzyme, presumably to divalent cation-sites associated with the phosphate and the nucleotide domains of the active site.     

Kinetic studies on Na+/K+-ATPase and inhibition of Na+/K+-ATPase by ATP

Na+/K+-ATPase (EC 3.6.1.3) is an important membrane-bound enzyme. In this paper, kinetic studies on Na+/K+-ATPase were carried out under mimetic physiological conditions. By using microcalorimeter, a thermokinetic method was employed for the first time. Compared with other methods, it provided accurate measurements of not only thermodynamic data (deltarHm) but also the kinetic data (Km and Vmax). At 310.15K and pH 7.4, the molar reaction enthalpy (deltarHm) was measured as -40.514 +/- 0.9kJmol(-1). The Michaelis constant (Km) was determined to be 0.479 +/- 0.020 mM and consistent with literature data. The reliability of the thermokinetic method was further confirmed by colorimetric studies. Furthermore, a simple and reliable kinetic procedure was presented for ascertaining the true substrate for Na+/K+-ATPase and determining the effect of free ATP. Results showed that the MgATP complex was the real substrate with a Km value of about 0.5mM and free ATP was a competitive inhibitor with a Ki value of 0.253 mM.     

Na+/K(+)-ATPase regulation by neurotransmitters.

A long period of experimental work has led to the conclusion that Na+/K(+)-ATPase is the enzymatic version of the Na+/K+ pump. This enzymatic system is in charge of various important cell functions. Among them cationic equilibrium and recovering of resting membrane potential in neurons is relevant. A tetrameric ensemble of peptides conform the system known as alpha and beta subunits. The alpha subunit is subdivided in alpha 1, alpha 2 and alpha 3, according to different location and properties. Regulatory factors intrinsic to the Na+/K(+)-ATPase system are: ATP, Na+ and Mg2+ concentrations inside the cell, and K+ outside. The enzyme activity is also regulated by extrinsic factors like some hormones (insulin and thyroxine). Induction of gene expression or post-translational modifications of the preexisting pool of the enzyme are the basic mechanisms of regulation proposed. Other extrinsic factors that seem to regulate the enzyme activity are some neurotransmitters. Among them the most extensively studied are catecholamines, mainly norepinephrine (NE) and lately serotonin (5-HT). The mechanism suggested for NE activation of the enzyme seems to involve specific receptors or a non-specific chelating action related to the catechol group that would relieve the inhibition by divalent cations. Another possibility is that NE removes an endogenous inhibitory factor present in the cytoplasm. The Na+/K(+)-ATPase is activated also by 5-HT. In vivo pharmacological and nutriological manipulations of brain 5-HT are accompanied by parallel responses of Na+/K(+)-ATPase activity. Serotonin agonists do activate the enzyme and antagonists neutralize the activation. In vitro there is a different dose dependent activation, according to the brain region. The mechanism involved seems to implicate a specific receptor system. Serotonin-Na+/K(+)-ATPase interaction in the rat brain is probably of functional relevance because it disappears in amygdaloid kindling. Also it seems to influence the ionic regulation of the pigment transport mechanism in crayfish photoreceptors. In relation to other neurotransmitters, a weak response to histamine was observed with acetylcholine, GABA and glutamic acid, the results were negative.     

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+.     

Effect of lipid peroxidation on Na+,K(+)-ATPase, 5'-nucleotidase and CNPase in mouse brain myelin

In the presence of H2O2, solutions of Fe2+ were applied to brain homogenate and isolated myelin from adult SWV control mice and the shiverer dysmyelinating mutant mouse as a source of a reactive oxygen species (Fenton reaction). Under these conditions, lipid peroxidation was initiated and measured as thiobarbituric acid-reactive oxidation products (TBAR). This was accompanied by 85% inhibition of myelin-associated Na+,K(+)-ATPase and 25% inhibition of 5'-nucleotidase. In contrast, CNPase activity was not altered. Studies on the shiverer mutant brain revealed that in spite of hypomyelination and prevalence of premature, myelin-like membranes in the homogenate, the myelin-related enzymes reacted as normal enzymes to peroxidation. Differences in the resistance of Na+,K(+)-ATPase to peroxidation in the brain homogenate and myelin suggest that the myelin enzyme is extremely sensitive to reactive oxygen toxicity.     

Cis-allosteric effects of cytoplasmic Na+/K+ discrimination at varying pH. Low-affinity multisite inhibition of cytoplasmic K+ in reconstituted Na+/K(+)-ATPase engaged in uncoupled Na(+)-efflux.

In liposomes with reconstituted shark Na+/K(+)-ATPase the effect of cytoplasmic K+ was investigated in the absence of extracellular alkali ions. During such conditions the Na+/K(+)-ATPase is engaged in the so called uncoupled Na+ efflux mode in which cytoplasmic Na+ activates and binds to the enzyme and becomes translocated without countertransport of K+ as in the physiological Na+/K+ exchange mode. In this uncoupled flux mode only low-affinity inhibition by K+cyt is found to be present. The inhibition pattern is consistent with a model in which cytoplasmic K+ exhibit mixed inhibition of Na+ activation, probably by binding at the three cytoplasmic loading sites on E1ATP (E1A). With determined intrinsic binding constants for cytoplasmic Na+ to this form of KS1, KS2, KS3 = 40 mM, 2 mM, 2 mM the inhibition pattern can be simulated assuming three K+cyt sites with equal affinity for Ki = 40 mM, similar to KS1 for the first Na+cyt site. The discrimination between cytoplasmic Na+ and K+ is therefore enhanced by allosteric interaction initiated from the cis-side due to binding of the first Na+, as opposed to K+, which induces the positive cooperatively in the successive Na+ bindings. pH is found to influence the pattern of K+cyt inhibition: A lowering of the pH potentiates the K+cyt inhibition, whereas at increased pH the inhibition is decreased and transformed into a pure competitive competition.     

Pump current and Na+/K+ coupling ratio of Na+/K+-ATPase in reconstituted lipid vesicles

A method is described for studying the coupling ratio of the Na+/K+ pump, i.e., the ratio of pump-mediated fluxes of Na+ and K+, in a reconstituted system. The method is based on the comparison of the pump-generated current with the rate of K+ transport. Na+/K+-ATPase from kidney is incorporated into the membrane of artificial lipid vesicles; ATPase molecules with outward-oriented ATP-binding site are activated by addition of ATP to the medium. Using oxonol VI as a potential-sensitive dye for measuring transmembrane voltage, the pump current is determined from the change of voltage with time t. In a second set of experiments, the membrane is made selectively K+-permeable by addition of valinomycin, so that the membrane voltage U is equal to the Nernst potential of K+. Under this condition, dU/dt reflects the change of intravesicular K+ concentration and thus the flux of K+. Values of the Na+/K+ coupling ratio determined in this way are close to 1.5 in the experimental range (10-75 mM) of extravesicular (cytoplasmic) Na+ concentrations.     

Mechanism of Na+/K(+)-ATPase activation by trypsin and kallikrein

The mechanism of the Na+/K(+)-ATPase activation by trypsin (from bovine pancreas) and kallikrein (from human plasma) was investigated on enzyme preparations from different sources (beef heart and dog kidney) and at different degrees of purification (beef heart). Kallikrein was effective on both beef and dog enzymes, whereas trypsin stimulated only the beef-heart Na+/K(+)-ATPase. The extent of activation by the proteinases was inversely related to the degree of purification (maximal enzyme activation about 60 and 20% on the partially purified and the more purified enzymes, respectively). Enzyme activation was observed up to 0.5-0.6 microgram/ml of proteinase. At higher concentrations the activation decreased and was converted into inhibition at proteinase concentrations above 1.0 micrograms/ml. Na+/K(+)-ATPase stimulation was due to an increase in the Vmax of the enzyme reaction. Km for ATP remained unaffected. The activating effect was favoured by sodium and counteracted by potassium. Accordingly, Na(+)-ATPase activity was stimulated to a greater extent (up to 350%), whereas K(+)-dependent p-nitrophenylphosphatase activity proved to be insensitive to the actions of the proteinases. The Na+/K(+)-ATPase stimulation by both proteinases was antagonized by either ouabain or canrenone, two drugs that bind on the extracellular side of the Na+/K(+)-ATPase molecule. On the contrary, the enzyme inactivation observed at high proteinase concentrations was not counteracted by these two drugs. The stimulation of either Na+/K(+)- or Na(+)-ATPase activity was shown to be an irreversible effect without any significant protein degradation detectable by SDS gel electrophoresis. The results obtained suggest that proteinases exert their stimulatory effects by interacting preferentially with the E2 conformation of Na+/K(+)-ATPase at site(s) located on the extracellular moiety of the enzyme.     

Binding of ethylenediamine to phosphatidylserine is inhibitory to Na+/K(+)-ATPase.

Covalent linkage of ethylenediamine with the Na+/K(+)-ATPase complex from rabbit kidney outer medulla by the use of the water-soluble carbodiimide, N-ethyl,N'-(3-dimethylaminopropyl)carbodiimide, resulted in a 73% reaction with phosphatidylserine and only 27% with carboxylic groups in the proteic component of the enzyme. Condensation products from the reaction between phosphatidylserine and ethylenediamine, N-(O-phosphatidylseryl)ethylenediamine, N,N'-bis(O-phosphatidylseryl)ethylenediamine and its intermediary product O-phosphatidyl-[N,N'-bis(seryl)]ethylenediamine, were synthesised. Symmetrically substituted ethylenediamine was the most likely condensation product of ethylenediamine with endogenous phosphatidylserine. The synthesised lipids were incorporated in proteoliposomes containing Na+/K(+)-ATPase and only the addition of the phospholipid phosphatidylcholine. The ratio of phospholipid to protein was 52 (w/w). These proteoliposomes were perforated by the addition of 0.5% cholate and both the Na(+)-dependent phosphorylation level and its dependence on Na+, Mg2+ and ATP were measured. Phosphatidylcholine alone increased the half-maximal activation concentration for Na+ ([Na+]0.5) from 0.2 to 1-2 mM, for Mg2+ from 0.1 to 0.8 microM and for ATP from 0.02 to 0.3 microM. The Ki for K+ (in the absence of Na+) was unaffected: 12.8 microM vs. 12.5 microM in the non-reconstituted system. Replacing 10 mol% of phosphatidylcholine by phosphatidylethanolamine: or phosphatidylserine had no significant effect on [Na+]0.5: 1.1 and 0.7 mM, respectively. Replacing 5 mol% phosphatidylcholine by the bis(phosphatidylseryl) substituent of ethylenediamine further increased [Na+]0.5 to 13.7 mM, while half-maximal activation concentrations for Mg2+ and ATP were unaltered. The mono-phosphatidylseryl derivatives of ethylenediamine, each 5 mol%, also increased [Na+]0.5, but to a lesser extent (3.2-3.8 mM). In addition to their competitive effects, the phosphatidylseryl-substituted ethylenediamine compounds exerted a slowly-increasing non-competitive inhibition, not only in phosphorylation, but also in overall ATPase activity, which was reduced, although not abolished, by exogenous protein (bovine serum albumin). A detergent-like action in the usual sense is unlikely since liposomes containing these lipids remained intact. These studies prove that phospholipids are not only required for optimal activity of this transport enzyme, but in excess or in compositions deviating from the normal, may also be inhibitory.     

Assembly of a hybrid from the alpha subunit of Na+/K(+)-ATPase and the beta subunit of H+/K(+)-ATPase.

Messenger RNA for the alpha subunit of Torpedo californica Na+/K(+)-ATPase was injected into Xenopus oocytes together with that of the beta subunit of rabbit H+/K(+)-ATPase. The Na+/K(+)-ATPase alpha subunit was assembled in the microsomal membranes with the H+/K(+)-ATPase beta subunit, and became resistant to trypsin. These results suggest that the H+/K(+)-ATPase beta subunit facilitates the stable assembly of the Na+/K(+)-ATPase alpha subunit in microsomes.     

Na+/K(+)-ATPase as an effector of synaptic transmission.

Evidence is presented in support of the hypothesis that transmitter monoamines can exert their post-synaptic effects by stimulation or inhibition of Na+/K(+)-ATPase in neuronal or glial cell plasma membranes. Stimulation of electrogenic sodium pumping, causing a hyperpolarization with an increase in membrane resistance, could account for the depression of neuronal spontaneous firing and the signal/noise enhancing actions of these amines. Conversely, inhibition of an electrogenic sodium pump in neuronal plasma membranes would lead to depolarization and enhanced excitability.     

Early inhibition of the Na+/K+-ATPase ion pump during acetaminophen-induced hepatotoxicity in rat

The status of Na+ regulation was examined during early stages of alkylation insult to rat liver. Na+/K+-ATPase activity in plasma membranes declined by 52% within 3 hr of treatment with 850 mg/kg acetaminophen. This loss preceded the release of alanine aminotransferase (2880 +/- 1550 U/ml) and necrosis (2+) seen at 24 hr. Activities of 5'-nucleotidase and Mg2+-ATPase and recovery of plasma membranes were comparatively unchanged at 3 hr. Because damage to Na+/K+-ATPase appeared early in the pathogenesis of acetaminophen hepatotoxicity, loss of hepatocellular Na+ regulation could represent one of the critical molecular consequences of lethal alkylation by acetaminophen.     

Kinetics of phosphorylation of Na+/K(+)-ATPase by protein kinase C

The kinetics of phosphorylation of an integral membrane enzyme, Na+/K(+)-ATPase, by calcium- and phospholipid-dependent protein kinase C (PKC) were characterized in vitro. The phosphorylation by PKC occurred on the catalytic alpha-subunit of Na+/K(+)-ATPase in preparations of purified enzyme from dog kidney and duck salt-gland and in preparations of duck salt-gland microsomes. The phosphorylation required calcium (Ka approximately 1.0 microM) and was stimulated by tumor-promoting phorbol ester (12-O-tetradecanoylphorbol 13-acetate) in the presence of a low concentration of calcium (0.1 microM). PKC phosphorylation of Na+/K(+)-ATPase was rapid and plateaued within 30 min. The apparent Km of PKC for Na+/K(+)-ATPase as a substrate was 0.5 microM for dog kidney enzyme and 0.3 microM for duck salt-gland enzyme. Apparent substrate inhibition of PKC activity was observed at concentrations of purified salt-gland Na+/K(+)-ATPase greater than 1.0 microM. Phosphorylation of purified kidney and salt-gland Na+/K+ ATPases occurred at both serine and threonine residues. The 32P-phosphopeptide pattern on 15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis after hydroxylamine cleavage of pure 32P-phosphorylated alpha subunit was the same for the two sources of enzyme, which suggests that the phosphorylation sites are similar. The results indicate that Na+/K(+)-ATPase may serve as a substrate for PKC phosphorylation in intact cells and that the Na+/K(+)-ATPase could be a useful in vitro model substrate for PKC interaction with integral membrane proteins.     

钠钾ATP酶的信号转导功能新进展

钠钾ATP酶不仅是主动跨膜转运钠钾离子的载体蛋白,而且可能作为内源性洋地黄物质的受体参与信号转导。它通过在质膜上与小窝蛋白、Src激酶之间的相互作用,并在细胞内借助Src激酶反式激活上皮生长因子受体,组装信号转导的复合物激活信号转导的级联反应,从而介导内源性洋地黄物质增加心脏和血管的收缩性、促进正常细胞肥大或增殖、促进肿瘤细胞的凋亡等作用。研究内源性洋地黄物质与钠钾ATP酶结合后激活的信号转导通路将有助于明确它在病理生理条件下的重要性,有可能为治疗高血压和肿瘤提供新思路,并深化对细胞内信号转导机制的认识。     

Modulation of reconstituted pig kidney Na+/K(+)-ATPase activity by cholesterol in endogenous lipid vesicles: role of lipid domains

Diverse experimental and theoretical evidence suggests that plasma membranes contain cholesterol-induced segregated domains that could play a key role in the modulation of membrane functions, including intrinsic enzyme activity. To gain insight into the role of cholesterol, we reconstituted pig kidney Na+/K+-ATPase into unilamellar vesicles of endogenous lipids mimicking the natural membrane and addressed the question of how modification of the cholesterol content could affect the ATPase activity via changes in the membrane lipid phase and in the protein structure and dynamics. We used steady-state and time-resolved fluorescence spectroscopy with the lipid phase probes DPH and Laurdan and the protein probe fluorescein and also used infrared spectroscopy using attenuated total reflectance. Upon modification of membrane cholesterol content, the ATPase activity did not change monotonically but instead exhibited abrupt changes resulting in two peaks at or close to critical cholesterol mole fractions (25 and 33.3 mol %) predicted by the superlattice or regular distribution model. Fluorescence parameters associated with the membrane probes also showed abrupt changes with peaks, coincident with the cholesterol concentrations associated with the peaks in the enzyme activity, while parameters associated with the protein probes also showed slight but abrupt changes resulting in dips at the same cholesterol concentrations. Notably, the IR amide I band maximum also showed spectral shifts, characterized by a frequency variation pattern with peaks at the same cholesterol concentrations. Overall, these results indicate that the lipid phase had slightly lower hydration, at or near the two critical cholesterol concentrations predicted by the superlattice theory. However, in the protein domains monitored there was a slight but significant hydration increase along with increased peptide backbone flexibility at these cholesterol concentrations. We propose that in the vicinity of the critical mole fractions, where superlattice formation can occur, minute changes in cholesterol concentration produce abrupt changes in the membrane organization, increasing interdomain surfaces. These changes, in turn, induce small changes in the protein's structure and dynamics, therefore acting to fine-tune the enzyme.     

Lipoic acid decreases lipid peroxidation and protein glycosylation and increases (Na(+) + K(+))- and Ca(++)-ATPase activities in high glucose-treated human erythrocytes

Lipoic acid supplementation has been found to be beneficial in preventing neurovascular abnormalities in diabetic neuropathy. Insufficient (Na(+) + K(+))-ATPase activity has been suggested as a contributing factor in the development of diabetic neuropathy. This study was undertaken to test the hypothesis that lipoic acid reduces lipid peroxidation and glycosylation and can increase the (Na(+) + K(+))- and Ca(++)-ATPase activities in high glucose-exposed red blood cells (RBC). Washed normal human RBC were treated with normal (6 mM) and high glucose concentrations (45 mM) with 0-0.2 mM lipoic acid (mixture of S and R sterioisomers) in a shaking water bath at 37 degrees C for 24 h. There was a significant stimulation of glucose consumption by RBC in the presence of lipoic acid both in normal and high glucose-treated RBC. Lipoic acid significantly lowered the level of glycated hemoglobin (GHb) and lipid peroxidation in RBC exposed to high glucose concentrations. High glucose treatment significantly lowered the activities of (Na(+) + K(+))- and Ca(++)-ATPases of RBC membranes. Lipoic acid addition significantly blocked the reduction in activities of (Na(+) + K(+))- and Ca(++)-ATPases in high glucose- treated RBC. There were no differences in lipid peroxidation, GHb and (Na(+) + K(+))- and Ca(++)-ATPase activity levels in normal glucose-treated RBC with and without lipoic acid. Thus, lipoic acid can lower lipid peroxidation and protein glycosylation, and increase (Na(+) + K(+))- and Ca(++)-ATPase activities in high-glucose exposed RBC, which provides a potential mechanism by which lipoic acid may delay or inhibit the development of neuropathy in diabetes.