Spin-label studies on the specificity of interaction of cardiolipin with beef heart cytochrome oxidase

The selectivity of interaction of various cardiolipin analogues with beef heart cytochrome oxidase in reconstituted complexes with dimyristoylphosphatidylcholine has been studied by electron spin resonance spectroscopy, using lipids spin-labeled in the acyl chains. No difference in selectivity is observed between cardiolipin and its monolyso derivative, and similarly no selectivity is observed between phosphatidylcholine and lysophosphatidylcholine. Removal of the cardiolipin charge by methylation of the phosphate groups reduces but does not eliminate selectivity relative to phosphatidylcholine. The dependence of the lipid selectivity on head group and chain composition is in the order cardiolipin approximately equal to monolysocardiolipin greater than acylcardiolipin greater than dimethylcardiolipin greater than phosphatidylcholine approximately equal to lysophosphatidylcholine, where acylcardiolipin has the spin-label chain attached at the center -OH of the head group. The degree of association of the negatively charged cardiolipin derivatives with cytochrome oxidase decreases with increasing salt concentration, to a level comparable to that for dimethylcardiolipin. At high ionic strength there is still a marked selectivity relative to phosphatidylcholine. Li+ ions are more effective in screening the interaction than are Na+ ions, and divalent ions are more effective than monovalent ions. The selectivity for cardiolipin is only slightly reduced on titrating the protein to high pH. Alkylation of the protein with N-ethylmaleimide has little effect on the titration behavior. Covalent modification of the protein by reaction with citraconic anhydride decreases the selectivity of interaction with cardiolipin. It is concluded that cardiolipin possesses an additional specificity of interaction with cytochrome oxidase other than that of purely electrostatic origin.     

ESR spin-label studies of lipid-protein interactions in membranes

Lipid spin labels have been used to study lipid-protein interactions in bovine and frog rod outer segment disc membranes, in (Na+, K+)-ATPase membranes from shark rectal gland, and in yeast cytochrome oxidase-dimyristoyl phosphatidylcholine complexes. These systems all display a two component ESR spectrum from 14-doxyl lipid spin-labels. One component corresponds to the normal fluid bilayer lipids. The second component has a greater degree of motional restriction and arises from lipids interacting with the protein. For the phosphatidylcholine spin label there are effectively 55 +/- 5 lipids/200,000-dalton cytochrome oxidase, 58 +/- 4 mol lipid/265,000 dalton (Na+, K+)-ATPase, and 24 +/- 3 and 22 +/- 2 mol lipid/37,000 dalton rhodopsin for the bovine and frog preparations, respectively. These values correlate roughly with the intramembrane protein perimeter and scale with the square root of the molecular weight of the protein. For cytochrome oxidase the motionally restricted component bears a fixed stoichiometry to the protein at high lipid:protein ratios, and is reduced at low lipid:protein ratios to an extent which can be quantitatively accounted for by random protein-protein contacts. Experiments with spin labels of different headgroups indicate a marked selectivity of cytochrome oxidase and the (Na+, K+)-ATPase for stearic acid and for cardiolipin, relative to phosphatidylcholine. The motionally restricted component from the cardiolipin spin label is 80% greater than from the phosphatidylcholine spin label for cytochrome oxidase (at lipid:protein = 90.1), and 160% greater for the (Na+, K+)-ATPase. The corresponding increases for the stearic acid label are 20% for cytochrome oxidase and 40% for (Na+, K+)-ATPase. The effective association constant for cardiolipin is approximately 4.5 times greater than for phosphatidylcholine, and that for stearic acid is 1.5 times greater, in both systems. Almost no specificity is found in the interaction of spin-labeled lipids (including cardiolipin) with rhodopsin in the rod outer segment disc membrane. The linewidths of the fluid spin-label component in bovine rod outer segment membranes are consistently higher than those in bilayers of the extracted membrane lipids and provide valuable information on the rate of exchange between the two lipid components, which is suggested to be in the range of 10(6)-10(7) s-1.     

Influence of sterols and phospholipids on sarcolemmal and sarcoplasmic reticular cation transporters

We have examined the influence of different sterols and phospholipids on the activities of the cardiac sarcolemmal Na+-Ca2+ exchanger and Na+,K+-ATPase and the sarcoplasmic reticular Ca2+-ATPase in reconstituted proteoliposomes. When either the solubilized Na+-Ca2+ exchanger or the Na+,K+-ATPase is reconstituted into phosphatidylcholine (PC):phosphatidylserine (30:50 by weight) vesicles, high cholesterol levels (20% by weight) are required for activity to be expressed. This sterol requirement is highly specific for cholesterol. Several cholesterol analogues with minor structural changes are unable to support Na+-Ca2+ exchange or Na+,K+-ATPase activities. When solubilized sarcolemma is reconstituted into PC:cardiolipin vesicles, however, the requirement for cholesterol is lost. Substantial activity can be obtained in the complete absence of cholesterol or in the presence of several cholesterol analogues. Thus, sterol/protein interactions can be highly dependent on the phospholipid environment. In contrast, the skeletal muscle sarcoplasmic reticular Ca2+-ATPase functions equally well in the presence or absence of cholesterol after reconstitution into either PC:phosphatidylserine or PC:cardiolipin proteoliposomes. Phospholipid requirements of the transporters were also examined. The sarcolemmal Na+-Ca2+ exchanger, Na+,K+-ATPase, and the sarcoplasmic reticular Ca2+-ATPase all function optimally in the presence of phosphatidylserine or cardiolipin after reconstitution. Thus, the sarcolemmal cation transporters have similar sterol and phospholipid requirements and may have structural similarities in their hydrophobic regions. The sarcoplasmic reticular Ca2+ pump evolved in a low cholesterol membrane and has different lipid interactions. These findings may have general applicability to other plasma membrane and endoplasmic reticular enzymes.     

Salt intake and intestinal dopaminergic activity in adult and old Fischer 344 rats

We have earlier shown that the renal dopaminergic system failed to respond to high salt (HS) intake in old (24-month-old) Fisher 344 rats (Hypertension 1999;34:666-672). In the present study, intestinal Na+,K+-ATPase activity and intestinal dopaminergic tonus were evaluated in adult and old Fischer 344 rats during normal salt (NS) and HS intake. Basal intestinal Na+,K+-ATPase activity (nmol Pi/mg protein/min) in adult rats (142+/-6) was higher than in old Fischer 344 rats (105+/-7). HS intake reduced intestinal Na+,K+-ATPase activity by 20% (P<0.05) in adult, but not in old rats. Dopamine (1 microM) failed to inhibit intestinal Na+,K+-ATPase activity in both adult and old Fischer 344 rats (NS and HS diets). In adult animals, co-incubation of pertussis toxin with dopamine (1 microM) produced a significant inhibitory effect in the intestinal Na+,K+-ATPase activity. L-DOPA and dopamine tissue levels in the intestinal mucosa of adult rats were higher (45+/-9 and 38+/-4 pmol/g) than those in old rats (27+/-9 and 14+/-1 pmol/g). HS diet did not change L-DOPA and DA levels in both adult and old rats. DA/L-DOPA tissue ratios, an indirect measure of dopamine synthesis, were higher in old (1.1+/-0.2) than in adult rats (0.6+/-0.1). Aromatic L-amino acid decarboxylase (AADC) activity in the intestinal mucosa of old rats was higher than in adult rats. HS diet increased the AADC activity in adult rats, but not in old rats. It is concluded that intestinal dopaminergic tonus in old Fisher 344 rats is higher than in adult rats and is accompanied by lower basal intestinal Na+,K+-ATPase activity. In old rats, HS diet failed to alter the intestinal dopaminergic tonus or Na+,K+-ATPase activity, whereas in adult rats increases in AADC activity were accompanied by decreases in Na+,K+-ATPase activity. The association between salt intake, increased dopamine formation and inhibition of Na+,K+-ATPase at the intestinal level was not as straightforward as that described in renal tissues.     

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.     

Characterization of gill (Na+ + K+)-ATPase in the sea bass (Dicentrarchus labrax L.)

Bass gill microsomal preparations contain both a Na+, K+ and Mg2+-dependent ATPase, which is completely inhibited by 10(-3)M ouabain and 10(-2)M Ca2+, and also a ouabain insensitive ATP-ase activity in the presence of both Mg2+ and Na+. Under the optimal conditions of pH 6.5, 100 mM Na+, 20 mM K+, 5 mM ATP and 5 mM Mg2+, (Na+ + K+)-ATPase activity at 30 degrees C is 15.6 mumole Pi hr/mg protein. Bass gill (Na+ + K+)-ATPase is similar to other (Na+ + K+)-ATPases with respect to the sensitivity to ionic strength, Ca2+ and ouabain and to both Na+/K+ and Mg2+/ATP optimal ratios, while pH optimum is lower than poikilotherm data. The enzyme requires Na+, whereas K+ can be replaced efficiently by NH+4 and poorly by Li+. Both Km and Vm values decrease in the series NH+4 greater than K+ greater than Li+. The break of Arrhenius plot at 17.7 degrees C is close to the adaptation temperature. Activation energies are scarcely different from each other and both lower than those generally reported. The Km for Na+ poorly decreases as the assay temperature lowers. The comparison with literature data aims at distinguishing between distinctive and common features of bass gill (Na+ + K+)-ATPase.     

Adenosine diphosphate binding to sodium-plus-potassium ion-dependent adenosine triphosphatase. The role of lipid in the nucleotide-potassium ion interplay.

Delipidated dogfish rectal-gland Na++K+-ATPase (Na++K+-dependent adenosine triphosphatase), almost devoid of hydrolytic activity, is able to bind about 2nmol of ADP/mg of protein. The "affinity" of delipidated enzyme for ADP is not affected by K+ in concentrations that greatly decrease the "affinity" of native Na++K+-ATPase. The K+-sensitivity of the ADP binding is in part restored by relipidation with dioleoyl phosphatidylcholine.     

(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.     

Monovalent cation dependence of tissue plasminogen activator synthesis by HeLa cells

HeLa cells synthesize and secrete increased levels of tissue plasminogen activator (tPA) when incubated for 18 h with 10-20 nM phorbol myristate acetate. This response was inhibited by a number of conditions which affect intracellular Na+ and K+ concentrations. Removing extracellular Na+, while maintaining isotonicity with choline+, reduced the secretion of both functional and antigenic tPA in a linear fashion. A series of cardiac glycosides and related compounds strongly inhibited tPA secretion with the following rank order of potency: digitoxin = ouabain greater than digoxin greater than digitoxigenin greater than digoxigenin greater than digitoxose greater than digitonin. These compounds also inhibited cellular Na+/K+-ATPase activity over an identical concentration range. Two compounds which selectively increase cellular permeability to K+, valinomycin, and nigericin, strongly inhibited tPA secretion, with IC50 values of approximately 50 nM. In contrast, monensin, which selectively increases cellular permeability to Na+, was much less active. Valinomycin, but not nigericin, also inhibited cellular Na+/K+-ATPase activity. Phorbol myristate acetate, 5-20 nM, increased Na+/K+-ATPase activity up to 2-fold and tPA secretion up to 15-fold. We conclude that the secretion of tPA by HeLa cells treated with phorbol myristate acetate proceeds via a mechanism which requires extracellular Na+ and a functional Na+/K+-ATPase ("sodium pump") enzyme.     

Properties and subcellular localization of elastase-like activities of arterial smooth muscle cells in culture

The interaction of cardiolipin with Ca2+ was assessed by measuring the cardiolipin-mediated extraction of 45Ca2+ from an aqueous to an organic (methylene chloride) phase. Cardiolipin binds Ca2+ with high affinity [Kd(apparent) = 0.70 +/- 0.17 microM (S.D.)]. Cation-cardiolipin interactions are selective. Interaction of cardiolipin with Ca2+ is insensitive to Na+, but is inhibited by divalent cations with Mn2+ greater than Zn2+ greater than Mg2+. In addition La3+ and Ruthenium red are particularly potent inhibitors of Ca2+ binding by cardiolipin. Cardiolipin-mediated extraction of Ca2+ into an aqueous phase is also inhibited by phosphatidylcholine. Inhibition of Ca2+-cardiolipin interaction by phosphatidylcholine (a phospholipid known to stabilize the bilayer conformation) may implicate inverted, non-bilayer lipid structures in the binding.     

Ouabain-insensitive Na+ stimulation of a microsomal Mg2+ -ATPase in gills of sea bass (Dicentrarchus labrax L.)

Bass gill microsomal preparations contain a Mg2+-dependent Na+-stimulated ATPase activity in the absence of K+, whose characteristics are compared with those of the (Na+ + K+)-ATPase of the same preparations. The activity at 30 degrees C is 11.3 mumol Pi X mg-1 protein X hr-1 under optimal conditions (5 mM MgATP, 75 mM Na+, 75 mM HEPES, pH 6.0) and exhibits a lower pH optimum than the (Na+ + K+)-ATPase. The Na+ stimulation of ATPase is only 17% inhibited by 10-3M ouabain and completely abolished by 2.5 mM ethacrinic acid which on the contrary cause, respectively, 100% and 34% inhibition of the (Na+ + K+)-ATPase. Both Na+-and (Na+ + K+)-stimulated activities can hydrolyze nucleotides other than ATP in the efficiency order ATP greater than CTP greater than UTP greater than GTP and ATP greater than CTP greater than GPT greater than UTP, respectively. In the presence of 10(-3)M ouabain millimolar concentrations of K+ ion lower the Na+ activation (90% inhibition at 40 mM K+). The Na+-ATPase is less sensitive than (Na+ + K+)-ATPase to the Ca2+ induced inhibition as the former is only 57.5% inhibited by a concentration of 1 X 10(-2)M which completely suppresses the latter. The thermosensitivity follows the order Mg2+--greater than (Na+ + K+)--greater than Na+-ATPase. A similar break of the Arrhenius plot of the three enzymes is found. Only some of these characteristics do coincide with those of a Na+-ATPase described elsewhere. A presumptive physiological role of Na+-ATPase activity in seawater adapted teleost gills is suggested.     

Comparative study of properties of Na+, K+-ATPase and Mg2+-ATPase of the myometrium plasma membrane

The comparative research of catalytic properties of two ATP-hydrolases of the sarcolemma of the smooth muscle of the uterus--ouabaine-sensitive Na+,K+-ATPase and ouabaine-resistent Mg2+-ATPase is carried out. The specific enzymatic activity of Na+,K+-ATPase and Mg2+-ATPase makes 10.2 +/- 0.7 and 18.1 +/- 1.2 mmol P/mg of protein for 1 hour, accordingly. The action of ouabaine on Na+,K+-ATPase is characterized by magnitude of quotient of inhibition I0.5=21.3 +/- 1.5 mkM. Processing of the sarcolemma fraction by digitonin in concentrations 0.001 +/- 0.1% promotes an activation of Na+,K+ATPase and Mg2+- ATPase, and in the first case much more efficiently than in the second. The kinetics of accumulation of the product of ATP-hydrolase reactions of phosphate satisfies the laws of the zero order reaction (incubation time--about 10 min). Na+,K+-ATPase is highly specific concerning the univalent cations--Na+, K+, however Li+ can partially substitute K+. Activity of Mg2+-ATPase is not specific concerning univalent cations. The dependence of Na+,K+-ATPase activity on pH in the range of 6.0-8.0 is characterized by the bell-shaped curve, at the same time the linear dependence on pH is peculiar to Mg2+-ATPase. The functioning of Na+,K+-ATPase is provided only by ATP, in the case of Mg2+-ATPase ATP can be successfully replaced with other nucleotidetriphosphates. It is supposed that the obtained experimental data can be beneficial in further research of membranous mechanisms underlying the cation exchange in the smooth muscles, in particular when studying the role of the plasma membrane in the maintenance of electromechanical coupling in them, and also in the regulation of ionic homeostasis in myocytes.     

Lipid-protein interactions in ADP-ATP carrier/egg phosphatidylcholine recombinants studied by spin-label ESR spectroscopy

The stoichiometry and specificity of lipid-protein interaction, as well as the lipid exchange rates at the protein interface, have been determined from the electron spin resonance spectra of spin-labeled lipids in reconstituted complexes of the mitochondrial ADP-ATP carrier with egg phosphatidylcholine. With the exception of cardiolipin and phosphatidic acid, the lipids studied are found to compete for approximately 50 sites at the intramembranous surface of the protein dimer. This number of first-shell lipid sites is unusually large for a protein of this size. The specificity for the protein is in the order stearic acid approximately phosphatidic acid approximately cardiolipin greater than phosphatidylserine greater than phosphatidylglycerol approximately phosphatidylcholine, with the maximum association constant relative to phosphatidylcholine being approximately 4. The selectivity for anionic lipids was partially screened with increasing ionic strength, but to a lesser extent for cardiolipin and phosphatidic acid than for stearic acid. Only in the case of phosphatidylserine was the selectivity reduced at high ionic strength to a level close to that for phosphatidylcholine. The off rates for lipid exchange at the protein surface were independent of lipid/protein ratio and correlated in a reciprocal fashion with the different lipid selectivities, varying from 5 x 10(6) s-1 for stearic acid at low ionic strength to 2 x 10(7) s-1 for phosphatidylcholine and phosphatidylglycerol. The off rates for cardiolipin were unusually low in comparison with the observed selectivity, and indicated the existence of a special population of sites (ca. 30% of the total) for cardiolipin, at which the exchange rate was very low.(ABSTRACT TRUNCATED AT 250 WORDS)     

Radical-induced inactivation of kidney Na+,K(+)-ATPase: sensitivity to membrane lipid peroxidation and the protective effect of vitamin E

The Na+,K(+)-ATPase is a membrane-bound, sulfhydryl-containing protein whose activity is critical to maintenance of cell viability. The susceptibility of the enzyme to radical-induced membrane lipid peroxidation was determined following incorporation of a purified Na+,K(+)-ATPase into soybean phosphatidylcholine liposomes. Treatment of liposomes with Fenton's reagent (Fe2+/H2O2) resulted in malondialdehyde formation and total loss of Na+,K(+)-ATPase activity. At 150 microM Fe2+/75 microM H2O2, vitamin E (5 mol%) totally prevented lipid peroxidation but not the loss of enzyme activity. Lipid peroxidation initiated by 25 microM Fe2+/12.5 microM H2O2 led to a loss of Na+,K(+)-ATPase activity, however, vitamin E (1.2 mol%) prevented both malondialdehyde formation and loss of enzyme activity. In the absence of liposomes, there was complete loss of Na+,K(+)-ATPase activity in the presence of 150 microM Fe2+/75 microM H2O2, but little effect by 25 microM Fe2+/12.5 microM H2O2. The activity of the enzyme was also highly sensitive to radicals generated by the reaction of Fe2+ with cumene hydroperoxide, t-butylhydroperoxide, and linoleic acid hydroperoxide. Lipid peroxidation initiated by 150 microM Fe2+/150 microM Fe3+, an oxidant which may be generated by the Fenton's reaction, inactivated the enzyme. In this system, inhibition of malondialdehyde formation by vitamin E prevented loss of Na+,K(+)-ATPase activity. These data demonstrate the susceptibility of the Na+,K(+)-ATPase to radicals produced during lipid peroxidation and indicate that the ability of vitamin E to prevent loss of enzyme activity is highly dependent upon both the nature and the concentration of the initiating and propagating radical species.     

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.     

Na+/K+-ATPase activity of different types of striated muscles

Na+/K+-ATPase activity was determined in striated muscles with different aerobic capacities. The underlying hypothesis was that different aerobic capacities are reflective of different contractile activity which imposes greater demands on sarcolemmal ion translocation and may thus set Na pumping capacity. The added ion translocation demands required during exercise-training on Na+/K+-ATPase activity in different muscle fiber types may require an adaptation of this enzyme. The highest and lowest Na+/K+-ATPase activity was in the heart and white gastrocnemius muscle (WG), respectively. A high linear correlation existed between Na+/K+-ATPase activity and succinate dehydrogenase activity in the six muscles studied. Exercise-training did not increase Na+/K+-ATPase activity in any of the muscles, but did increase the aerobic capacity, except in the heart and WG. It was concluded that Na+/K+-ATPase activity has a high positive correlation with the aerobic capacity of striated muscles in the rat and that the Na pump capacity does not adapt to exercise-training of 1 hr X day-1 as does aerobic capacity.     

Potassium binding to the (Na+ + K+)-ATPase

The number of K+ bound to the (Na+ + K+)-ATPase has been measured under equilibrium conditions by a differential-titration technique (Hastings, D.F. (1977) Anal. Biochem. 83, 416-432). 5.1 K+ were bound per 32P-labelling site. The K'D for K+ was dependent on the concentration of choline, which was included to give ionic strength. K'D was 59 +/- 2.5 microM with 97 mM choline, 26 +/-1.9 microM with 30 mM choline. The K+ : choline selectivity was 2564 : 1 and the calculated K'D for K+ with zero choline was 11 microM and for choline with zero K+ was 28 mM. 20 microM ATP in the presence of 97 mM choline incresed the K'D for potassium 3-fold to 177 +/- 14 microM. The K'D for K+ with 3 mM Na+ in the presence of 27 mM choline was 81 +/- 10 microM and with 30 mM Na+ without choline 700 +/- 250 microM. The calculated K'D for Na+ at zero K+ and zero choline was 0.6 +/- 0.2 mM. The K+ : Na+ selectivity was 54 : 1.     

Tonoplast Na+/H+ Antiport Activity and Its Energization by the Vacuolar H+-ATPase in the Halophytic Plant Mesembryanthemum crystallinum L

Tonoplast vesicles were isolated from leaf mesophyll tissue of the inducible Crassulacean acid metabolism plant Mesembryanthemum crystallinum to investigate the mechanism of vacuolar Na+ accumulation in this halophilic species. In 8-week-old plants exposed to 200 mM NaCl for 2 weeks, tonoplast H+-ATPase activity was approximately doubled compared with control plants of the same age, as determined by rates of both ATP hydrolysis and ATP-dependent H+ transport. Evidence was also obtained for the presence of an electroneutral Na+/H+ antiporter at the tonoplast that is constitutively expressed, since extravesicular Na+ was able to dissipate a pre-existing transmembrane pH gradient. Initial rates of H+ efflux showed saturation kinetics with respect to extravesicular Na+ concentration and were 2.1-fold higher from vesicles of salt-treated plants compared with the controls. Na+-dependent H+ efflux also showed a high selectivity for Na+ over K+, was insensitive to the transmembrane electrical potential difference, and was more than 50% inhibited by 200 [mu]M N-amidino-3,5-diamino-6-chloropyrazinecarboxamide hydrochloride. The close correlation between increased Na+/H+ antiport and H+-ATPase activities in response to salt treatment suggests that accumulation of the very high concentrations of vacuolar Na+ found in M. crystallinum is energized by the H+ electrochemical gradient across the tonoplast.     

Effect of phospholipases A2 from bee and cobra venom on the phospholipid composition and Na+-,K+-ATPase activity of synaptosomes

The bee and cobra venom phospholipases A2 as well as partially acetylated cobra venom phospholipase A2 are studied for their effect on phospholipid composition of synaptosomes and their Mg2+- and Na+,K+-ATPase activity. It is established that these phospholipases induce the splitting of phosphatidylethanolamine, phosphatidylcholine and phosphatidylserine, inhibition of the Na+,K+-ATPase activity and activation of Mg2+-ATPase. Bee venom phospholipase A2 is more effective than cobra venom phospholipase A2, the both phospholipases splitting phosphatidylethanolamine most intensively. The ATPase activity may be partially or completely restored by exogenic phosphatidylcholine and phosphatidylserine; exogenic phosphatidylethanolamine is not efficient in this respect.