Detection of ouabain-insensitive H(+)-transporting, K(+)-stimulated p-nitrophenylphosphatase activity in rat gastric glands by cerium-based cytochemistry

We employed a modification of our previously reported cerium-based cytochemical method for ouabain-sensitive, K-dependent p-nitrophenylphosphatase (Na-K ATPase) activity to detect ouabain-insensitive, K-stimulated p-nitrophenylphosphatase (K-pNPPase) activity in rat gastric glands. Biochemically, the enzyme activity of gastric glands incubated in a medium containing 50 mM Tricine buffer (pH 7.5), 50 mM KCl, 10 mM MgCl2, 2 mM CeCl3, 2 mM p-nitrophenylphosphate (pNPP), 2.5 mM levamisole, 10 mM ouabain, and 0.00015% Triton X-100, was optimal at pH 7.5-8.0 and decreased above pH 8.5. The amount of p-nitrophenol after incubation increased linearly in proportion to the amount of tissue in the medium. The enzyme activity was inhibited by omeprazole, sodium flouride (NaF), N-ethylmaleimide (NEM), and dicyclohexylcarbodiimide (DCCD). Heat-treated specimens had no enzyme activity. The enzyme activity increased with addition of K ions up to the concentration of 50 mM, and became constant above 50 mM. Cytochemically, the parietal cells of the gastric glands reacted positively for ouabain-insensitive K-pNPPase activity. Intense reaction was observed at the microvilli of the luminal surface and the intracellular canaliculi. The tubulovesicular system showed weak enzyme activity. The reaction products were found as fine, granular, electron-dense deposits in the cytoplasm just beneath the plasma membrane. The ouabain-insensitive K-pNPPase activity detected in this study appears, therefore, to be associated with that of H-transporting, K-stimulated adenosine triphosphatase (H-K ATPase).     

Effect of diamide on proton translocation by the mitochondrial H+-ATPase

Treatment of sonic submitochondrial particles with the bifunctional thiol reagent, diamide, results in an enhancement of proton conductivity and ATPase activity, which is reversed by the reducing agent dithiothreitol, is suppressed by Fo inhibitors like oligomycin and is absent in particles that are deprived of peripheral Fo polypeptides. The effect of diamide is apparently due to oxidation of dithiols to disulfides in peripheral polypeptide(s) of Fo.     

Dynamic changes of red cell membrane thiol groups followed by bimane fluorescent labeling

Monobromobimane labels red cell membrane protein thiol groups; bands exhibit fluorescence after sodium dodecyl sulfate acrylamide gel electrophoresis and correspond to almost all of those staining with Coomassie blue. The response of membrane protein thiol groups to oxidative challenge and the dynamics of recovery of the thiol groups may be followed. Diminished labeling is found after oxidation with diamide, with both intrachain and interchain disulfide bond formation demonstrated by sodium dodecyl sulfate acrylamide gel electrophoresis. Regeneration of thiol groups under physiological conditions (incubation with glucose) after a moderate degree of diamide oxidation is shown to be complete (with respect to thiol group content and degree and distribution of bimane label) in normal human red blood cell membranes. Even after oxidation of almost half of the membrane protein thiol groups (maximum degree of oxidation achieved), regeneration of thiol groups is almost complete; a minor fraction resides in the form of disulfide-linked high molecular weight proteins (demonstrated by the electrophoretic profile) which may be reduced completely with dithiothreitol.Bimane fluorescent labeling provides a convenient and sensitive method for following membrane thiol group status under physiological conditions.     

A water-soluble Mg2+-ATPase from erythrocyte membranes.

A ouabain-insensitive ATPase activity associated with the water-soluble proteins of the human and bovine erythrocyte membrane is demonstrated by means of activity-staining in polyacrylamide gels. The ATPase activity from both sources had an absolute requirement for Mg2+, activity becoming easily detectable at 0.2 mM Mg2+. At low Mg2+ concentrations added Ca2+ appeared to decrease the intensity of the ATPase stain. The activity is unaffected by monovalent cations, does not hydrolyse p-nitrophenyl phosphate and is not inhibited by 2 : 4 dinitrophenol. The ATPase has an apparent molecular weight of approximately 100 000 as determined by electrophoresis in acrylamide gels containing dodecyl sulphate.     

Leishmania amazonensis: characterization of an ouabain-insensitive Na+-ATPase activity

We characterized ouabain-insensitive Na+-ATPase activity present in the plasma membrane of Leishmania amazonensis and investigated its possible role in the growth of the parasite. An increase in Na+ concentration in the presence of 1mM ouabain, increased the ATPase activity with a V(max) of 154.1+/-13.5nmol Pi x h(-1) x mg(-1) and a K0.5 of 28.9+/-7.7mM. Furosemide and sodium orthovanadate inhibited the Na+-stimulated ATPase activity with an IC(50) of 270microM and 0.10microM, respectively. Furosemide inhibited the growth of L. amazonensis after 48h incubation, with maximal effect after 96h. The IC50 for furosemide was 840. On the other hand, ouabain (1mM) did not change the growth of the parasite. Taken together, these results show that L. amazonensis expresses a P-type, ouabain-insensitive Na+-ATPase that could be involved with the growth of the parasite.     

A water-soluble Mg2+-ATPase from erythrocyte membranes

A ouabain-insensitive ATPase activity associated with the water-soluble proteins of the human and bovine erythrocyte membrane is demonstrated by means of activity-staining in polyacrylamide gels. The ATPase activity from both sources had an absolute requirement for Mg²⁺, activity becoming easily detectable at 0.2 mM Mg²⁺. At low Mg²⁺ concentrations added Ca²⁺ appeared to decrease the intensity of the ATPase stain. The activity is unaffected by monovalent cations, does not hydrolyse p-nitrophenyl phosphate and is not inhibited by 2 : 4 dinitrophenol. The ATPase has an apparent molecular weight of approximately 100 000 as determined by electrophoresis in acrylamide gels containing dodecyl sulphate.     

Inhibition of erythrocyte Ca2+-ATPase by activated oxygen through thiol- and lipid-dependent mechanisms

We have studied erythrocyte Ca2+-ATPase as a model target for elucidating effects of activated oxygen on the erythrocyte membrane. Either intracellular or extracellular generation of activated oxygen causes parallel decrements in Ca2+-ATPase activity and cytoplasmic GSH, oxidation of membrane protein thiols, and lipid peroxidation. Subsequent incubation with either dithiothreitol or glucose allows only partial recovery of Ca2+-ATPase, indicating both reversible and irreversible components which are modeled herein using diamide and t-butyl hydroperoxide. The reversible component reflects thiol oxidation, and its recovery depends upon GSH restoration. The irreversible component is largely due to lipid peroxidation, which appears to act through mechanisms involving neither malondialdehyde nor secondary thiol oxidation. However, some portion of the irreversible component could also reflect oxidation of thiols which are inaccessible for reduction by GSH, since we demonstrate existence of different classes of thiols relevant to Ca2+-ATPase activity. Activated oxygen has an exaggerated effect on Ca2+-ATPase of GSH-depleted cells. Sickle erythrocytes treated with dithiothreitol show a heterogeneous response of Ca2+-ATPase activity. These findings are potentially relevant to oxidant-induced hemolysis. They also may be pertinent to oxidative alteration of functional or structural membrane components in general, since many components share with Ca2+-ATPase both free thiols and close proximity to unsaturated lipid.     

Short-term exercise preserves myocardial glutathione and decreases arrhythmias after thiol oxidation and ischemia in isolated rat hearts

The purpose of this study was to determine if exercise (Ex) protects hearts from arrhythmias induced by glutathione oxidation or ischemia-reperfusion (I/R). Female Sprague-Dawley rats were divided into two experimental groups: sedentary controls (Sed) or short-term Ex (10 days of treadmill running). Twenty-four hours after the last session, hearts were excised and exposed to either perfusion with the thiol oxidant diamide (200 μM) or global I/R. Ex significantly delayed the time to the onset of ventricular arrhythmia after irreversible diamide perfusion. During a shorter diamide perfusion protocol with washout, Ex significantly decreased the incidence of arrhythmia, as evidenced by a delayed time to the first observed arrhythmia, lower arrhythmia scores, and lower incidence of ventricular fibrillation. Ex hearts exposed to I/R (30-min ischemia/30-min reperfusion) also showed lower arrhythmia scores and incidence of ventricular fibrillation compared with Sed counterparts. Our finding that Ex protected intact hearts from thiol oxidation was corroborated in isolated ventricular myocytes. In myocytes from Ex animals, both the increase in H(2)O(2) fluorescence and incidence of cell death were delayed after diamide. Although there were no baseline differences in reduced-to-oxidized glutathione ratios (GSH/GSSG) between the Sed and Ex groups, GSH/GSSG was better preserved in Ex groups after diamide perfusion and I/R. Myocardial glutathione reductase activity was significantly enhanced after Ex, and this was preserved in the Ex group after diamide perfusion. Our results show that Ex protects the heart from arrhythmias after two different oxidative stressors and support the hypothesis that sustaining the GSH/GSSG pool stabilizes cardiac electrical function during conditions of oxidative stress.     

Diamide induced shift in protein and glutathione thiol: disulfide status delays DNA rejoining after X-irradiation of human cancer cells

By treating a human tumor cell line with various concentrations of diamide, we explored the relationship between extent and duration of protein and nonprotein thiol oxidation, initiation of DNA double-strand break rejoining after X-rays, and the degree of radiosensitization. We also examined the relationship between protein thiol status and the non-protein thiol, glutathione (GSH). A549 cells were irradiated and incubated postirradiation with 0, 100, 300 or 500 microM diamide for 1 h. The dose of radiation required to give 10% survival decreased from 4.8 Gy to 3.2 Gy with 300 microM and to 2.7 Gy with 500 microM diamide (enhancement ratios of 1.5 and 1.8, respectively) but was not significantly affected by 100 microM diamide. The time of initiation of double-stranded DNA rejoining after X-irradiation (DNA repair) was delayed by 300 and 500 microM diamide. Furthermore, DNA rejoining began only after total cellular protein thiol content recovered to 55% of pretreatment levels for both concentrations. Intracellular GSH/GSSG ratios decreased immediately after diamide addition to less than 1. Large decreases in GSH/GSSG ratio preceded significant loss of protein thiols, but protein-glutathione mixed disulfides accounted for a minor percentage of the total protein thiol oxidized (up to 20%). We believe that diamide-induced protein thiol loss, and not GSH oxidation, is related to the cessation of DNA strand rejoining after X-irradiation, thereby affecting survival.     

Inhibition of sarcoplasmic reticulum Ca2+-ATPase by 2-mercaptoethanol

The Ca²⁺-dependent ATPase activity of sarcoplasmic reticulum was inhibited when membrane vesicles were incubated at 0°C in presence of thiols. 2-mercaptoethanol was the most effective inhibitor from the thiols tested. The effect of 2-mercaptoethanol on the ATPase activity was biphasic; enzyme inhibition originally increased and then decreased with increasing thiol concentration. The inhibitory action of this thiol was significantly higher at low membrane concentrations and the rate of inactivation at 22°C was considerably lower than that at 0°C. Ca²⁺-ATPase previously inhibited by 2-mercaptoethanol was partially reactivated by incubation with periodate.     

The common occurrence of ATP diphosphohydrolase in mammalian plasma membranes

In the plasma membranes from several mammalian tissues (including normal and tumor tissues), a Mg2+ (or Ca2+)-dependent ATP phosphohydrolase activity is present in much greater amount than the (Na+ + K+)-ATPase. The ouabain-insensitive activity can be attributed to at least two enzymes, an ATPase (EC 3.6.1.3) and an ATP diphosphohydrolase (EC 3.6.1.5). The ATPase hydrolyzes ATP and other nucleoside triphosphates and is not inhibited by azide. The ATP diphosphohydrolase hydrolyzes both ATP and ADP (and other nucleoside tri- and diphosphates) and the hydrolysis of adenine nucleotides is strongly inhibited by 10 mM azide. The ratios of these two enzymes in the various membranes (as determined by the extent of azide inhibition) vary widely. The ATP diphosphohydrolase accounts for most of the Mg2+ (or Ca2+)-dependent ATP hydrolysis activity of the plasma membranes of liver (mouse), kidney (dog), two mouse sarcomas, and a human astrocytoma (xenograft in athymic mice). The ATPase is more dominant in the plasma membranes from mouse brain and human oat cell carcinoma. The widespread presence of the ATP diphosphohydrolase in plasma membrane from various types of tissues is demonstrated for the first time and is of particular interest in view of its relatively high activity in the plasma membranes of two sarcomas. The membrane-bound ATP diphosphohydrolase is characterized with respect to its metal ion activators, substrates, and inhibitors. These results should facilitate the distinction of this enzyme from other ATP hydrolyzing enzymes of plasma membranes in future investigations.     

Reversible elimination of myofibrillar Ca2+ sensitivity by diamide and other sulfhydryl reagents: comparison with reversible contracture produced in intact cells

Cardiac contractile activity is usually controlled by intracellular Ca2+, but it can also be modified by oxidizing agents. Incubation of guinea pig heart myofibrils with diamide (3 mM, 1 h) increased basal (no Ca2+) ATPase activity by 580% and abolished Ca2+ dependence. The effect was proportional to diamide concentration (0.01-1 mM) and duration of preincubation (up to 2 h). Dithiothreitol (5 mM, 1 h) reversed most of the basal ATPase activation and restored Ca2+ sensitivity. Other sulfhydryl reagents produced a similar effect but also produced inhibition of total ATPase. In intact cell preparations, diamide produced a slow tonic contraction, consistent with myofibril activation. In the perfused rat heart, 1 mM diamide slowly increased diastolic ventricular pressure; this increase was partially reversed by dithioerythritol. In isolated rat heart myocytes, 1 mM diamide produced a slow tonic contraction, increased contractility in response to stimulation. Cardiocytes superfused for 1 h with buffer containing EGTA to deplete Ca2+ did not contract in response to stimulation but showed a slow tonic contraction with diamide. This contraction could be slowly and only partially reversed by dithioerythritol. Response to stimulation was restored by addition of Ca2+. The results show that diamide can produce contraction in viable cells. This contraction does not require extracellular Ca2+ and is unlikely to involve intracellular Ca2+. The direct activation of myofibrillar ATPase may contribute to the increased myocardial stiffness seen in ischemia and to ischemic contracture.     

Membrane protein thiol cross-linking associated with the permeabilization of the inner mitochondrial membrane by Ca2+ plus prooxidants

In a previous report (Macedo, D.V., Ferraz, V. L., Pereira-da-Silva, L., and Vercesi, A. E. (1988) in Integration of Mitochondrial Functions (Lemasters, J. J., et al., eds) pp. 535-542, Plenum Publishing Corp., New York), we proposed that the alterations in the inner mitochondrial membrane permeability caused by Ca2+ plus prooxidants could be the consequence of membrane protein sulfhydryl-disulfide transitions. In this study, we show that Ca2+ plus diamide, a thiol oxidant, significantly decrease the ability of beef heart submitochondrial particles to build up and sustain a membrane potential generated by succinate oxidation. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of solubilized membrane proteins indicates that these effects on the membrane potential are associated with the production of protein aggregates due to thiol cross-linking. Evidence is also presented that these protein aggregates can be produced in mitoplasts previously loaded with Ca2+ and that this is potentiated by the presence of either diamide or t-butylhydroperoxide. Furthermore, dithiothreitol, a disulfide reductant, was found to be much more effective than NAD(P)+ reductants in reversing Ca2+ efflux induced by prooxidants. It is concluded that the perturbation of the inner mitochondrial membrane caused by Ca2+ plus prooxidants is associated with protein polymerization due to thiol cross-linking, resulting in the production of high molecular mass protein aggregates.     

ATP synthase complex from beef heart mitochondria. Role of the thiol group of the 25-kDa subunit of Fo in the coupling mechanism between Fo and F1

In order to assess the role of thiol groups in the Fo part of the ATP synthase in the coupling mechanism of ATP synthase, we have treated isolated Fo, extracted from beef heart Complex V with urea, with thiol reagents, primarily with diazenedicarboxylic acid bis-(dimethylamide) (diamide) but also with Cd2+ and N-ethylmaleimide. FoF1 ATP synthase was reconstituted by adding isolated F1 and the oligomycin-sensitivity-conferring-protein (OSCP) to Fo. The efficiency of reconstitution was assessed by determining the sensitivity to oligomycin of the ATP hydrolytic activity of the reconstituted enzyme. Contrary to Cd2+, incubation of diamide with Fo, before the addition of F1 and OSCP, induced a severe loss of oligomycin sensitivity, due to an inhibited binding of F1 to Fo. This effect was reversed by dithiothreitol. Conversely, if F1 and OSCP were added to Fo before diamide, no effect could be detected. These results show that F1 (and/or OSCP) protects Fo thiols from diamide and are substantiated by the finding that the oligomycin sensitivity of ATP hydrolysis activity of isolated Complex V was also unaltered by diamide. Gel electrophoresis of FoF1 ATP synthase, reconstituted with diamide-treated Fo, revealed that the loss of oligomycin sensitivity was directly correlated with diminution of band Fo 1 (or subunit b). Concomitantly a band appeared of approximately twice the molecular weight of subunit Fo 1. As this protein contains only 1 cysteine residue (Walker, J. E., Runswick, M. J., and Poulter, L. (1987) J. Mol. Biol. 197, 89-100), the effect of diamide is attributed to the formation of a disulfide bridge between two of these subunits. These results offer further evidence for the proposal, based on aminoacid sequence and structural analysis, that subunit Fo 1 of mammalian Fo is involved in the binding with F1 (Walker et al. (1987]. N-Ethylmaleimide affects oligomycin sensitivity to a lesser extent than diamide, suggesting that the mode of action of these reagents (and the structural changes induced in Fo) is different.     

Changes in the glutathione level induced by transplasma membrane electron transport in maize (Zea mays L.)

Summary We investigated changes of thiols (GSH, GSSG, and cysteine) induced by transplasma membrane electron transport after addition of artificial electron acceptors and the influence of the thiol level on redox activity. GSH, GSSG, and cysteine content of maize (Zea mays L. cv. Golden Bantam) roots and coleoptile segments was determined by high performance liquid chromatography with a fluorescence detector. GSSG increased after treatment with 0.8 mM diamide, an SH-group oxidizer. GSH level of roots increased after treatment with diamide, while GSH levels of coleoptiles decreased. Incubation of roots with the GSH biosynthesis inhibitor buthionine-D,L-sulfoximine for 6 days lowered the glutathione level up to 80%. However, the GSH/GSSG ratio of maize roots remained constant after treatment with both effectors. The GSH/GSSG ratio and the glutathione level were changed by addition of artificial electron acceptors like hexacyanoferrate (III) or hexabromoiridate (IV), which do not permeate the plasma membrane. Hexacyanoferrate (III) reduction was inhibited up to 25% after the cellular glutathione level was lowered by treatment with diamide or buthionine-D,L-sulfoximine. Proton secretion induced by reduction of the electron acceptors was not affected by both modulators. The change in glutathione level is different for roots and coleoptiles. Our data are discussed with regard to the role of GSH in electron donation for a plasma membrane bound electron transport system.Abbreviations Buthionine-D,L-sulfoximine s-n-butyl-homocysteine sulfoximine - cys cysteine - diamide 1,1-azobis (N,N-dimethyl-formamide) - DTE dithioerythritol - EDTA ethylenediaminetetraacetic acid - GSH reduced glutathione - GSSG oxidizied glutathione, glutathione disulfide - HBI IV hexabromoiridate (IV) (K₂[IrBr₆]) - HCF III hexacyanoferrate (III) (K₃[Fe(CN)₆] - NEM N-ethylmaleimide - PM plasma membrane - Tris Tris(hydroxymethyl)aminomethane     

Ouabain-insensitive Na(+)-ATPase activity in Trypanosoma cruzi epimastigotes

In the present paper, the presence of a ouabain-insensitive Na(+)-stimulated, Mg(2+)-dependent ATPase activity in T. cruzi epimastigotes CL14 clone and Y strain was investigated. The increase in Na+ concentration (from 5 to 170 mM), in the presence of 2 mM ouabain, increases the ATPase activity in a saturable manner along a rectangular hyperbola. The Vmax was 18.0 +/- 1.0 and 21.1 +/- 1.1 nmoles Pi x mg-1 x min-1 and the half-activation value (K50) for Na+ was 34.3 +/- 5.8 mM and 37.7 +/- 5.3 in CL14 clone and in Y strain, respectively. The Na(+)-stimulated ATPase activity was inhibited by 5-[aminosulfonyl]-4-chloro-2-[(2-furanylmethyl)-amino] benzoic acid (furosemide) in a dose-dependent manner. The half-inhibition value (I50) was 0.22 +/- 0.03 and 0.24 +/- 0.07 mM, and the Hill number (n) was 0.99 +/- 0.2 and 2.16 +/- 0.29 for CL14 clone and Y strain, respectively. These data indicate that both cell types express the ouabain-insensitive Na(+)-ATPase activity, which might be considered the biochemical expression of the second Na+ pump.     

Role of membrane-associated thiol groups in the functional regulation of gastric microsomal (H+ + K+)-transporting ATPase system.

The distribution of free thiol groups associated with the membrane proteins of the purified pig gastric microsomal vesicles was quantified, and the relation of thiol groups to the function of the gastric (H+ + K+)-transporting ATPase system was investigated. Two different thiol-specific agents, carboxypyridine disulphide (CPDS) and N-(1-naphthyl)maleimide (NNM) were used for the study. The structure-function relationship of the membrane thiol groups was studied after modification by the probes under various conditions, relating the inhibition of the (H+ + K+)-transporting ATPase to the ATP-dependent H+ accumulation by the gastric microsomal vesicles. On the basis of the extent of stimulation of the microsomal (H+ + K+)-transporting ATPase in the presence and absence of valinomycin (val) about 85% of the vesicles were found to be intact. CPDS at 1 mM completely inhibits the valinomycin-stimulated ATPase and the associated p-nitrophenyl phosphatase with a concomitant inhibition of vesicular H+ uptake. Both the enzyme and dye-uptake activities were fully protected against CPDS inhibition when the treatment with CPDS was carried out in the presence of ATP. ATP also offered protection (about 65%) against NNM inhibition of the (H+ + K+)-transporting ATPase system and vesicular H+ uptake. Under similar conditions ATP also protected about 10 and 6 nmol of thiol groups/mg of protein respectively from CPDS and NNM reaction. Our data suggest that the thiol groups on the outer surface of the vesicles are primarily involved in gastric (H+ + K+)-transporting ATPase function. Furthermore, at least about 15% of the total microsomal thiol groups appear to be associated with the ATPase system. The data have been discussed in terms of the structure-function relationship of gastric microsomes.     

Studies on the subcellular distribution of (Na+ + K+)-ATPase,K+-stimulated ATPase and HCO3−-stimulated ATPase activities in malpighian tubules of Locusta migratoria L.

The present study confirms previous reports of the presence of (Na⁺ + K⁺)-ATPase and anion-stimulated ATPase activity in Malpighian tubules of Locusta. In addition, the presence of a K⁺-stimulated, ouabain-insensitive ATPase activity has been identified in microsomal fractions. Differential and sucrose density-gradient centrifugation of homogenates has been used to separate membrane fractions which are rich in mitochondria, apical membranes and basolateral membranes; as indicated by the presence of succinate dehydrogenase and the presence or absence of non-specific alkaline phosphatase activity, respectively. Relatively high specific (Na⁺ + K⁺)-ATPase activity was associated with the basolateral membrane-rich fractions with only low levels of this activity being associated with the apical membrane-rich preparation. K⁺-stimulated ATPase activity was also associated, predominantly, with the basolateral membrane-rich fractions. However, comparison of the distribution of this activity with that of the (Na⁺ + K⁺)-ATPase suggests that the two enzymes did not co-separate. The possibility that the K⁺-stimulated ATPase was not associated with the basolateral plasma membrane is discussed.Anion-stimulated ATPase activity was found in the apical and basolateral membrane-rich fractions and in the fraction contaning mainly mitochondria. Nevertheless, the fact that this bicarbonate-stimulated activity did not co-separate with succinate dehydrogenase activity suggests that it was not exclusively mitochondrial in origin. These results are consistent with physiological studies indicating a basolateral (Na⁺ + K⁺)-ATPase but do not support the K⁺-stimulated ATPase as a candidate for the apical electrogenic pump. The possible role of the bicarbonate-stimulated ATPase activity in ion transport across both the basolateral and apical cell membranes is discussed.     

Oxidative damages in erythrocytes of patients with metabolic syndrome

The aim of the study was to estimate the changes caused by oxidative stress in structure and function of membrane of erythrocytes from patients with metabolic syndrome (MS). The study involved 85 patients with MS before pharmacological treatment and 75 healthy volunteers as a control group. Cholesterol level, lipid peroxidation, glutathione level (GSH), and antioxidant enzyme activities in erythrocytes were investigated. The damage to erythrocyte proteins was also indicated by means of activity of ATPase (total and Na⁺,K⁺ ATPase) and thiol group level. The membrane fluidity of erythrocytes was estimated by the fluorescent method. The cholesterol concentration and the level of lipid peroxidation were significantly higher, whereas the concentration of proteins thiol groups decreased in the patient group. ATPase and GSH peroxidase activities diminished compared to those in the control group. There were no differences in either catalase or superoxide dismutase activities. The membrane fluidity was lower in erythrocytes from patients with MS than in the ones from control group. These results show changes in red blood cells of patients with MS as a consequence of a higher concentration of cholesterol in the membrane and an increased oxidative stress.     

Characterization of the ATPase activities of myosins isolated from the membrane and the cytoplasmic fractions of human platelets

Myosin was purified from the membrane fraction and the cytoplasm of human platelets, and the K+(EDTA)- and Ca2+-dependent ATPase activities were studied under various experimental conditions. The ATPase activity of the myosin from the membrane fraction was slightly lower than that of its cytoplasmic counterpart, regardless of the different assay conditions (pH, ionic strength, and temperature). Both myosins showed the same pH optima and a similar ionic strength dependence for the two ATPase activities measured. In addition, they exhibited the same substrate specificity using ATP, CTP, and GTP as substrates. The activation energy of the Ca2+-dependent ATPase activity was essentially the same for the two myosins, while the activation energy of the K+(EDTA)-dependent ATPase activity of the membrane myosin was higher than that of the cytoplasmic myosin. The ATPase activity of the membrane myosin was found to be more sensitive to freezing and thawing than the cytoplasmic myosin. The alkylation of the thiol groups by N-ethylmaleimide or N-iodoacetyl-N-(5-sulfo-1-naphtyl)ethylenediamine, and the trinitrophenylation of the lysyl residues by 2,4,6-trinitrobenzenesulfonate caused a significant decrease in the K+(EDTA)-dependent ATPase activity of the two myosins. However, the membrane myosin was much less affected than the cytoplasmic myosin. Actin induced inhibition of the K+ (EDTA) ATPase of both myosins, and much smaller quantities of actin were needed to inhibit the cytoplasmic myosin ATPase compared to quantities needed to inhibit the myosin ATPase from the membrane fraction. This indicates that the membrane myosin has a lower affinity toward actin. The observed variations in the ATPase activity of the myosins isolated from the membrane and the cytoplasm fractions of human platelets may reflect differences in their respective physiological functions.