Modulation of (Na+,K+)-ATPase activity by the lipid bilayer examined with dansylated phosphatidylserine

The fluorescent probe 8-(dimethylamino)naphthalene-1-sulfonylphosphatidylserine (Dns-PS) was incorporated into purified lamb kidney Na+- and K+-stimulated adenosinetriphosphatase (EC 3.6.1.3) [(Na+,K+)-ATPase] by using a purified phospholipid exchange protein. Phospholipase C was used to reduce phospholipid content. Up to 40% of the phospholipid could be hydrolyzed with only 10% inhibition of the (Na+,K+)-ATPase, but when 67% of the phospholipid was hydrolyzed, the enzyme was inhibited 53%. To examine the effect of protein on the phospholipid bilayer, the fluorescent parameters of the probe incorporated into the enzyme preparation were contrasted with the same parameters for the probe incorporated into the total lipid extract of the preparation. The polarization of fluorescence of the probe in the lipid extract was 0.118 while in the enzyme preparation it was 0.218. This reflected a decrease in fluidity of the glycerol region of the phospholipid bilayer which was mediated by the protein. This effect increased as the phospholipid content of the (Na+,K+)-ATPase preparation was reduced so that with maximal phospholipid reduction the polarization of fluorescence was 0.262. The protein caused a decrease in the transition temperature from gel to fluid states of the bilayer detected by polarization of the probe. The midpoint temperature transition of the enzyme preparation decreased from 33 degrees C when all phospholipids were present to 20 degrees C when 67% of the phospholipids were hydrolyzed. This decrease was not observed for the lipid extract of these samples. A direct correlation between the (Na+,K+)-ATPase specific activity and the polarization of fluorescence of Dns-PS was found. The reduction in phospholipid content did not affect the steady-state level of phosphorylation of the enzyme by ATP but did affect the rate of dephosphorylation which would require conformational changes of the enzymes. The data showed that the fluidity of the phospholipid bilayer can modulate the activity of the (Na+,K+)-ATPase.     

Structural changes in (Na+ + K+)-ATPase accompanying detergent inactivation

Structural changes in the purified (Na+ + K+)-ATPase accompanying detergent inactivation were investigated by monitoring changes in light scattering, intrinsic protein fluorescence, and tryptophan to beta-parinaric acid fluorescence resonance energy transfer. Two phases of inactivation were observed using the non-ionic detergents, digitonin, Lubrol WX and Triton X-100. The rapid phase involves detergent monomer insertion but little change in protein structure or little displacement of closely associated lipids as judged by intrinsic protein fluorescence and fluorescence resonance energy transfer. Lubrol WX and Triton X-100 also caused membrane fragmentation during the rapid phase. The slower phase of inactivation results in a completely inactive enzyme in a particle of 400 000 daltons with 20 mol/mol of associated phospholipid. Fluorescence changes during the course of the slow phase indicate some dissociation of protein-associated lipids and an accompanying protein conformational change. It is concluded that non-parallel inhibition of (Na+ + K+)-ATPase and p-nitrophenylphosphate activity by digitonin (which occurs during the rapid phase of inactivation) is unlikey to require a change in the oligomeric state of the enzyme. It is also concluded that at least 20 mol/mol of tightly associated lipid are necessary for either (Na+ + K+)-ATPase or p-nitrophenylphosphatase activity and that the rate-limiting step in the slow inactivation phase involves dissociation of an essential lipid.     

Uncoupling of ATP binding to Na+,K+-ATPase from its stimulation of ouabain binding: studies of the inhibition of Na+,K+-ATPase by a monoclonal antibody

The effects of a monoclonal antibody, prepared against the purified lamb kidney Na+,K+-ATPase, on the enzyme's Na+,K+-dependent ATPase activity were analyzed. This antibody, designated M10-P5-C11, is directed against the catalytic subunit of the "native" holoenzyme. It inhibits greater than 90% of the ATPase activity and acts as a noncompetitive or mixed inhibitor with respect to the ATP, Na+, and K+ dependence of enzyme activity. It inhibits the Na+- and Mg2+ATP-dependent phosphoenzyme intermediate formation. In contrast, it has no effect on K+-dependent p-nitrophenylphosphatase (pNPPase) activity, the interconversion of the phosphoenzyme intermediates, and ADP-sensitive or K+-dependent dephosphorylation. It does not alter ATP binding to the enzyme nor the covalent labeling of the enzyme at the presumed ATP site by fluorescein 5'-isothiocyanate (FITC), but it prevents the ATP-induced stimulation in the rate of cardiac glycoside [3H]ouabain binding to the Na+,K+-ATPase. M10-P5-C11 binding appears to inhibit enzyme function by blocking the transfer of the gamma-phosphoryl of ATP to the phosphorylation site after ATP binding to the enzyme has occurred. In the presence of Mg2+ATP, it also prevents the ATP-induced transmembrane conformational change that enhances cardiac glycoside binding. This uncoupling of ATP binding from its stimulation of ouabain binding and enzyme phosphorylation demonstrates the existence of an enzyme-Mg2+ATP transitional intermediate preceding the formation of the Na+-dependent ADP-sensitive phosphoenzyme intermediate. These results are also consistent with a model of the Na+,K+-ATPase active site being composed of two distinct but interacting regions, the ATP binding site and the phosphorylation site.     

Photoaffinity labeling of erythrocyte membrane (Na+ + K+)-ATPase with high specific activity [125I]iodoazidogalactosyl digitoxigenin

Photoaffinity labeling of (Na+ + K+)-ATPase in erythrocyte membranes with cardiotonic steroid derivatives, followed by gel electrophoresis, requires a radiolabel of very high specific activity, since the enzyme represents less than 0.05% of the total membrane protein. We report the synthesis of a radioiodinated, photosensitive derivative of the cardiac glycoside, 3-beta-O-(4-amino-4,6-dideoxy-beta-D-galactosyl)digitoxigenin, with very high specific activity. The product, [125I]iodoazidogalactosyl digitoxigenin ([125I]IAGD), is carrier-free with a specific activity of 2200 Ci/mmol. Incubation of [125I]IAGD (1.8 nM) with human erythrocyte membranes (300 micrograms protein), followed by photolysis and analysis by SDS-PAGE, showed specific radiolabeling of a polypeptide that had the same molecular weight as catalytic alpha subunit (100,000 Mr) of (Na+ + K+)-ATPase in eel electroplax microsomes. Photoaffinity labeling of erythrocyte and electroplax membranes by [125I]IAGD was specific for the cardiac glycoside binding site of (Na+ + K+)-ATPase since radiolabeling of the alpha subunit was inhibited when ouabain was included in the pre-photolysis incubation. [125I]IAGD can, therefore, be used as a probe in structural studies of human erythrocyte membrane (Na+ + K+)-ATPase.     

Nitric oxide and peroxynitrite anion modulate liver plasma membrane fluidity and Na(+)/K(+)-ATPase activity.

Free radicals attack membranes and frequently alter their fluidity and function. The aim of the present work was to study the effect of nitric oxide (NO) radical and peroxynitrite anion on basolateral liver plasma membrane fluidity and on the activity of Na(+)/K(+)-ATPase. Basolateral membranes (BM) were isolated by ultracentrifugation in sucrose gradients and characterized enzymatically. BM were incubated with SNAP (a NO donor) or SIN-1 (a peroxynitrite donor). The release of NO or peroxynitrite was monitored by measuring NO(-)(2) + NO(-)(3). Relative fluidity was measured by polarization of fluorescence. NO increased membrane fluidity while peroxynitrite decreased it in a concentration-dependent manner. Na(+)/K(+)-ATPase activity was reduced by NO or peroxynitrite. Peroxynitrite anion inhibits ATPase activity in part by decreasing fluidity. However, it is very likely that both compounds inhibit ATPase activity by oxidation of the thiol groups of the enzyme. Our results suggest that NO may exert part of its biological effects by modulating membrane fluidity and function.     

The role of membrane fluidity changes and thiobarbituric acid-reactive substances production in the inhibition of cerebral cortex Na+/K+-ATPase activity.

Lipid peroxidation of rat cerebral cortex membranes was induced by Fe2+/ADP and ascorbate. The rate of Na+/K(+)-ATPase inhibition was correlated with the increase of thiobarbituric acid-reactive substances (TBARS) and conjugated dienes (CD) and with membrane fluidity changes. Our data showed that membrane fluidity changes (evaluated by fluorescence steady-state anisotropy measurements) can participate in Na+/K(+)-ATPase inhibition during the initial period of lipid peroxidation process, whereas during the following period the enzyme inhibition correlates only with TBARS and CD production.     

A monoclonal antibody against a native conformation of the porcine renal Na+/K(+)-ATPase alpha-subunit protein

A monoclonal antibody (mAb50c) against the native porcine renal Na+/K(+)-transporting adenosinetriphosphatase (EC 3.6.1.37, ATP phosphohydrolase) (Na+/K(+)-ATPase) was characterized. The antibody could be classified as a conformation-dependent antibody, since it did not bind to Na+/K(+)-ATPase denatured by detergent and its binding was affected by the normal conformational changes of the enzyme induced by ligands. The binding was the greatest in the presence of Na+, ATP or Mg2+ (E1 form), slightly less in the presence of K+ (E2K form) and the least when the enzyme was phosphorylated, especially in the actively hydrolyzing form in the presence of Na+, Mg2+ and ATP. The antibody inhibited both the Na+,K(+)-ATPase activity and the K(+)-dependent p-nitrophenylphosphatase activity by 25%, but it had no effect on Na(+)-dependent ATPase activity. The antibody partially inhibited the fluorescence changes of the enzyme labeled with 5'-isothiocyanatofluorescein after the addition of orthophosphate and Mg2+, and after the addition of ouabain. Proteolytic studies suggest that a part of the epitope is located on the cytoplasmic surface of the N-terminal half of the alpha-subunit.     

An essential arginine residue in the ATP-binding centre of (Na+ + K+)-ATPase.

1. Incubation of purified (Na+ + K+)-ATPase (ATP phosphohydrolase EC 3.6.1.3) from rabbit kidney outer medulla with butanedione in borate buffer leads to reversible inactivation of the (Na+ + K+)-ATPase activity. 2. The reaction shows second-outer kinetics, suggesting that modification of a single amino acid residue is involved in the inactivation of the enzyme. 3. The pH dependence of the reaction and the effect of borate ions strongly suggest that modification of an arginine residue is involved. 4. Replacement of Na+ by K+ in the butanedione medium decreases inactivation. 5. ATP, ADP and adenylyl imido diphosphate, particularly in the presence of trans-1,2-diaminocyclohexane-N,N,N',N'-tetraacetic acid to complex Mg2+, protect the enzyme very efficiently against inactivation by butanedione. 6. The (Na+ + Mg2+)-dependent phosphorylation capacity of the enzyme is inhibited in the same degree as the (Na+ + K+)-ATPase activity by butanedione. 7. The K+-stimulated p-nitrophenylphosphatase activity is much less inhibited than the (Na+ + K+)ATPase activity. 8. The ATP stimulation of the K+-stimulated p-nitrophenylphosphatase activity is inhibited by butanedione to the same extent as the (Na+ + K+)-ATPase activity. 9. Modification of sulfhydryl groups with 5,5'-dithiobis(2-nitrobenzoic acid) protects partially against the inactivating effect of butanedione. 10. The results suggest that an arginine residue is present in the nucleotide binding centre of the enzyme.     

Reversible inactivation of (Na+ + K+)-ATPase by use of a cleavable bifunctional reagent.

1. Purified (Na+ + K+)-ATPase, prepared from rabbit kidney outer medulla, is incubated with the bifunctional NH2-directed reagent dimethyl 3,3'-dithiobis-propionimidate. This results in a cross-link between the subunits of the enzyme and a simultaneous reduction of the (Na+ + K+)-ATPase and K+-stimulated p-nitrophenylphosphatase activities. 2. The most abundant cross-link product is a dimer of the two different subunits of the enzyme. 3. Reduction of the disulfide cross-link by dithioerythritol results in partial recovery of the original subunit structure of the enzyme and of the (Na+ + K+)-ATPase and K+-stimulated p-nitrophenylphosphatase activities. 4. These results suggest that a free mobility of the subunits of the (Na+ + K+)-ATPase system relative to each other is essential for proper functioning of both enzyme activities.     

Absence of an endogenous regulator of Na+,K+-ATPase activity in the tissues of cirrhotic rats

Microsomal Na+,K+-ATPase isolated from the renal cortex of rats with CCL4-induced cirrhosis (CIR) showed a higher specific activity than the enzyme obtained from control rats (COR). Kinetic studies showed a lower K0.5 for ATP (0.08 +/- 0.03 vs. 0.24 +/- 0.04 mM; p less than 0.05), a lower Na+ activation constant (9.6 +/- 1.5 vs. 19.0 +/- 1.7 mM; p less than 0.05), and a higher K+ activation constant (1.2 +/- 0.1 vs. 0.6 +/- 0.1 mM; p less than 0.05) for CIR. The optimal pH of the enzyme was 0.5 units higher in CIR than COR. The fluorescence of eosin-treated enzymes indicated a higher ratio of E1/E2 forms of Na+,K+-ATPase in CIR. The K+-activated p-nitrophenylphosphatase (pNPPase) activity of the enzyme was lower in CIR than COR rats (1.5 +/- 0.1 vs. 2.2 +/- 0.1 mU/mg; p less than 0.05). Dialysing (24 h) COR microsomes reproduced most of the changes observed in CIR enzymes (kinetics, optimal pH, and eosin fluorescence). Lyophilized dialysate of COR, but not of CIR microsomes, inhibits Na+,K+-ATPase activity. These results suggest that a dialysable inhibitor modifies the Na+,K+-ATPase activity in the kidney of COR which is almost absent in that of CIR. The absence of this factor may lead to the overall inability to excrete Na+ in the cirrhotic state.     

跨膜Ca~(2+)梯度对肌质网Ca~3+-ATP酶调节的特异性

我们曾报道跨膜Ca~(2+)梯度可通过膜脂影响肌质网Ca~(2+)-ATP 酶的构象和活性。本文就跨膜Ca~(2+)梯度对肌质网Ca~(2+)-ATP 酶的调节是否具有特异性作进一步研究。结果表明这种特异性表现在两方面:一是跨膜Ca~(2+)梯度对肌质网Ca~(2+)-ATP 酶功能的调节不能归结于跨膜Ca~(2+)浓度梯度所导致的膜电位的作用,离子载体FCCP 可消除跨膜电位但并不影响肌质网Ca~(2+)-ATP 酶的活力;二是其它二价金属离子如Sr~(2+)的跨膜梯度对肌质网Ca~(2+)-ATP 酶活力基本无影响。荧光偏振系列探剂n-AS 测定的结果表明跨膜Ca~(2+)与Sr~(2+)梯度对嵌有Ca~(2+)-ATP 酶的脂酶体的中部流动性的影响有较大差异。而Ca~(2+)-ATP 酶的Ca~(2+)结合位点正处于脂双层中部,这进一步提示膜脂参与了跨膜Ca~(2+)梯度对Ca~(2+)-ATP 酶的调节作用。     

Effects of an ATP site affinity analog on some conformational and enzymatic properties of the canine kidney (Na+ + K+)-ATPase

We have shown previously that the canine kidney Na+,K+ pump [Na+ + K+)-ATPase) reacts with the ATP affinity analog p-fluorosulfonylbenzoyladenosine (FSBA). At 20 degrees C, we find the time-course of this reaction to be that predicted for a first-order reaction accompanied by competing solvolysis of the reagent. The FSBA-inactivated (Na+ + K+)-ATPase retains the ability to move between the E1 and E2 conformations that predominate in Na+ and K+ medium, respectively. Therefore, FSBA reaction with the enzyme does not interfere significantly with either its alkali metal cation binding or its conformational freedom. The ability of ATP to influence the enzyme's conformation by binding to the high-affinity nucleotide site is decreased, however, in proportion to the degree of inhibition of enzyme activity by FSBA. In addition, the ability of the enzyme to shift from the E1 to the E2 conformation through the (ATP + Na+)-dependent phosphorylation cycle is inhibited by FSBA treatment, as shown by the decreased ability of these substrates to stimulate the K+-dependent p-nitrophenylphosphatase activity. Both of these effects are consistent with specific reaction of FSBA with the ATP binding site of the enzyme. An additional effect of FSBA treatment is that it causes loss of p-nitrophenylphosphatase activity, but to a lesser extent than (Na+ + K+)-ATPase or Na+-ATPase activity. Binding of p-nitrophenylphosphate to the enzyme is apparently unaffected by FSBA treatment, since the Km for p-nitrophenylphosphate is not changed.     

A monoclonal antibody against horse kidney (Na+ + K+)-ATPase inhibits sodium pump and E2K to E1 conversion of (Na+ + K+)-ATPase from outside of the cell membrane

Monoclonal antibodies against horse kidney outer medulla (Na+ + K+)-ATPase were prepared. One of these antibodies (M45-80), was identified as an IgM, recognized the alpha subunit of the enzyme. M45-80 had the following effects on horse kidney (Na+ + K+)-ATPase: (1) it inhibited the enzyme activity by 50% in 140 mM Na+ and by 80% in 8.3 mM Na+; (2) it increased the Na+ concentration necessary for half-maximal activation (K0.5 for Na+) from 12.0 to 57.6 mM, but did not affect K0.5 for K+; (3) it slightly increased the K+-dependent p-nitrophenylphosphatase (K-pNPPase) activity; (4) it inhibited phosphorylation of the enzyme with ATP by 30%, but did not affect the step of dephosphorylation; and (5) it enhanced the ouabain binding rate. These data are compatible with a stabilizing effect on the E2 form of (Na+ + K+)-ATPase. M45-80 was concluded to bind to the extracellular surface of the plasmamembrane, based on the following evidence: (1) M45-80 inhibited by 50% the ouabain-sensitive 86Rb+ uptake in human intact erythrocytes from outside of the cells; (2) the inhibition of (Na+ + K+)-ATPase activity in right-side-out vesicles of human erythrocytes was greater than that in inside-out vesicles; and (3) the fluorescence intensity due to FITC-labeled rabbit anti-mouse IgM that reacted with M45-80 bound to the right-side-out vesicles was much greater than that in the case of the inside-out vesicles.     

Variations in dietary triacylglycerol saturation alter the lipid composition and fluidity of rat intestinal plasma membranes

Rats were maintained on nutritionally complete diets enriched in unsaturated (corn oil) or saturated (butter fat) triacylglycerols. After 6 weeks, significant differences in the lipid composition and fluidity of a number of intestinal membranes were observed. The corn oil diet (enriched mainly in linoleic acid) increased the overall unsaturation of the acyl chains and enhanced the lipid fluidity, as assessed by the fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatriene, of enterocyte microvillus and basolateral membranes and of colonocyte basolateral membranes. Concomitantly, the cholesterol content and the cholesterol/phospholipid molar ratio were increased in the microvillus but not in the basolateral membranes. The increased cholesterol in ileal microvillus membranes can result from enhanced cellular biosynthesis, since ileal slices from rats fed the unsaturated diet incorporated [14C]octanoate more rapidly into digitonin-precipitable sterol. Increased fluidity of the enterocyte microvillus and basolateral membranes, respectively, enhanced the enzyme specific activities of p-nitrophenylphosphatase and (Na+ + K+)-dependent adenosine triphosphatase. The results indicate that the lipid composition, fluidity and enzyme activities of intestinal plasma membranes can be altered by dietary means. Moreover, rat enterocytes possess regulatory mechanisms which modulate the cholesterol content of the microvillus membranes so as to mitigate changes in lipid fluidity.     

Ethinylestradiol administration selectively alters liver sinusoidal membrane lipid fluidity and protein composition

Administration of high-dose ethinylestradiol to rats decreases bile flow, Na,K-ATPase specific activity, and liver plasma membrane fluidity. By use of highly purified sinusoidal and bile canalicular membrane fractions, the effect of ethinylestradiol administration on the protein and lipid composition and fluidity of plasma membrane fractions was examined. In sinusoidal fractions, ethinylestradiol (EE) administration decreased Na,K-ATPase activity (32%) and increased activities of alkaline phosphatase (254%), Mg2+-ATPase (155%), and a 160-kDa polypeptide (10-fold). Steady-state and dynamic fluorescence polarization was used to study membrane lipid structure. Steady-state polarization of diphenylhexatriene (DPH) was significantly higher in canalicular compared to sinusoidal membrane fractions. Ethinylestradiol (5 mg/kg per day for 5 days) selectively increased sinusoidal polarization values. Similar changes were demonstrated with the probes 2- and 12-anthroyloxystearate. Time-resolved fluorescence polarization measurements indicated that EE administration for 5 days did not change DPH lifetime but increased the order component (r infinity) and decreased the rotation rate (R). However, 1 and 3 days after EE administration and with low doses (10-100 micrograms/kg per day for 5 days) the Na,K-ATPase, bile flow, and order component were altered, but the rotation rate was unchanged. Vesicles prepared from total sinusoidal membrane lipids of EE-treated rats, as well as phospholipid vesicles, demonstrated increased DPH polarization, as did intact plasma membrane fractions. Liver plasma membrane fractions showed no change in free cholesterol or cholesterol/phospholipid molar ratio, while esterified cholesterol content was increased with high-dose but not low-dose ethinylestradiol.(ABSTRACT TRUNCATED AT 250 WORDS)     

S-adenosyl-L-methionine modulates Na+ + K+-ATPase activity in rat colonic basolateral membranes.

Rat colonic basolateral membranes were incubated with S-adenosyl-L-[methyl-3H]methionine (0.3 mM) at 37 degrees C for 2 h at pH 9.0. This resulted in an increase in the specific activity of Na+ + K+-ATPase by 60%. Kinetic parameter analysis revealed a 2-fold increase in the Vmax. of this enzymatic activity, whereas the Km for ATP was unchanged. The methylation inhibitor S-adenosyl-L-homocysteine (2 mM) significantly reduced these S-adenosyl-L-methionine-stimulated increases in specific activity and the Vmax. of Na+ + K+-ATPase. S-Adenosyl-L-methionine treatment of basolateral membranes was also found to significantly increase the fluidity of these preparations, as assessed by steady-state fluorescence polarization techniques using the fluorophore 1,6-diphenyl-1,3,5-hexatriene; S-adenosyl-L-homocysteine (2 mM) again markedly reduced this S-adenosyl-L-methionine-induced increase in fluidity. While transmethylation reactions involving phospholipids, non-polar lipids and proteins were all found to exist in rat colonic basolateral membranes, based on a number of observations, the results of the present studies suggest that transmethylation of membrane phospholipids, but not membrane non-polar lipids or proteins, influenced the fluidity of basolateral membranes which, in turn, modified Na+ + K+-ATPase activity in these membranes.     

Activation of ouabain-sensitive p-nitrophenylphosphatase by carbachol and cGMP in rat submandibular gland

Na+,K+-ATPase activity was monitored by measuring ouabain-sensitive K+-dependent p-nitrophenylphosphatase (p-NPPase) activity in rat submandibular gland slices. Carbachol (carbamylcholine chloride) stimulated the p-NPPase activity in the presence of calcium but not in its absence. Carbachol activation of the enzyme was totally ouabain sensitive and could be blocked by atropine. A minimal requirement of sodium ion extracellularly was required for this carbachol stimulation. cGMP and its dibutyryl analogue was also effective in stimulating the enzyme activity, whereas, cAMP was ineffective. Calcium, however, was not required for cGMP activation of the p-NPPase activity. The result indicates that calcium is the second messenger and cGMP is the tertiary connection between cholinergic stimulation and Na+,K+-ATPase activation in these glands. Activation of Na+,K+-ATPase is postulated to be responsible for primary fluid formation.     

Spatial relationship and conformational changes between the cardiac glycoside site and beta-subunit oligosaccharides in sodium plus potassium activated adenosinetriphosphatase

(Na,K)-ATPase, the enzyme responsible for active transport of Na and K across the plasma membranes of animal cells, consists of a catalytic subunit (alpha) and a glycoprotein subunit (beta) with unknown function. We have determined the distance between fluorescent probes directed to specific sites on the alpha- and beta-subunits and ligand-induced changes in the fluorescence of a probe specifically attached to the beta-subunit. The cardiac glycoside site on the alpha-subunit was labeled with anthroylouabain [Fortes, P. A. G. (1977) Biochemistry 16, 531-540]. The oligosaccharides on the beta-subunit were labeled with lucifer yellow carbohydrazide [Lee, J. A., & Fortes, P. A. G. (1985) Biochemistry 24, 322-330]. Resonance energy transfer from anthroylouabain to lucifer yellow was measured by steady-state and time-resolved fluorescence spectroscopy. The distance between these probes was determined from the efficiency of energy transfer. The average distance between anthroylouabain and lucifer yellow was 47 A and was independent of the number of acceptor molecules attached to the beta-subunit. The measured distance corresponds to the distance between the cardiac glycoside site and the center of the labeled oligosaccharides on the beta-subunit within one alpha beta dimer. The distance was the same (47 A) when anthroylouabain was bound with ATP or Pi as phosphorylating ligands but increased to 49 A in the presence of vanadate. The change in average distance provides quantitative evidence of a conformational difference between the complexes of cardiac glycosides with (Na,K)-ATPase induced by phosphorylating ligands or by vanadate.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of streptozotocin-induced diabetes on rat liver Na+/K+-ATPase.

Na+/K+-ATPase during diabetes may be regulated by synthesis of its alpha and beta subunits and by changes in membrane fluidity and lipid composition. As these mechanisms were unknown in liver, we studied in rats the effect of streptozotocin-induced diabetes on liver Na+/K+-ATPase. We then evaluated whether fish oil treatment prevented the diabetes-induced changes. Diabetes mellitus induced an increased Na+/K+-ATPase activity and an enhanced expression of the beta1 subunit; there was no change in the amount of the alpha1 and beta3 isoenzymes. Biphasic ouabain inhibition curves were obtained for diabetic groups indicating the presence of low and high affinity sites. No alpha2 and alpha3 isoenzymes could be detected. Diabetes mellitus led to a decrease in membrane fluidity and a change in membrane lipid composition. The diabetes-induced changes are not prevented by fish oil treatment. The results suggest that the increase of Na+/K+-ATPase activity can be associated with the enhanced expression of the beta1 subunit in the diabetic state, but cannot be attributed to changes in membrane fluidity as typically this enzyme will increase in response to an enhancement of membrane fluidity. The presence of a high-affinity site for ouabain (IC50 = 10-7 M) could be explained by the presence of (alphabeta)2 diprotomeric structure of Na+/K+-ATPase or an as yet unknown alpha subunit isoform that may exist in diabetes mellitus. These stimulations might be related, in part, to the modification of fatty acid content during diabetes.     

Interactions of lectins with (Na+ + K+)-ATPase of eel electric organ.

Interaction of lectins with a detergent-solubilized ATPase from eel electric organ was studied. Concanavalin A, which binds to alpha-mannosides, altered the rate of enzyme migration in agar and inhibited the formation of an antigen-antibody precipitate: other lectins had no such effects. Concanavalin A similar amounts partially inhibited (Na+ + K+)-ATPase; this inhibition was reversible by alpha-methylglucoside. There was no corresponding effect of concanavalin A on the potassium p-nitrophenylphosphatase. Concanavalin A also did not interfere with ouabain binding. Thus, concanavalin A binds to an antigenic region also involved in Na+ and/or ATP binding, but does not interact with a K+ site.