A search for an ‘Ouabain-like’ substance from the electric organ of Electrophorus electricus which led to arachidonic acid and related fatty acids

The electric organ of Electrophorus electricus contains substances which inhibit (Na⁺ + K⁺)-ATPase activity, the specific binding of [³H]ouabain to purified (Na⁺ + K⁺)-ATPase and ⁸⁶Rb⁺ uptake by chick cardiac cells in culture. The active organic material was extracted from microsomal membranes. Its purification was carried out by chromatography on Sep-Pak C-18 and thin-layer chromatography. Reverse-phase liquid chromatography and mass spectrometry identified the active material as a mixture of unsaturated fatty acids. Linoleic (18:2), arachidonic (20:4), linolenic (18:3) and docosahexaenoic acids (22:6) contributed to about 60% of the total activity of the active material. The other active substances could be arachidonic analogs, since they have both a lipophilic and carboxylic character. Pure unsaturated fatty acids have been shown to be active in the different biological assays used to analyze the endogenous ‘ouabain-like’ activity. Linolenic, arachidonic and docosahexaenoic acids were the most active, whereas saturated fatty acids and glyceryl esters or methyl esters of unsaturated fatty acids were inactive. It is possible that in pathological situations in which the level of unsaturated fatty acids increases, these molecules may then act as physiological inhibitors of the sodium pump.     

Identification of linoleic and oleic acids as endogenous Na+,K+-ATPase inhibitors from acute volume-expanded hog plasma

Na+,K+-ATPase inhibitors have been found to exist in acutely saline-infused hog plasma, which also inhibit the specific binding of ouabain to Na+,K+-ATPase and the binding of digoxin to specific anti-digoxin antibody. Two of these inhibitors were purified by a combination of Amberlite XAD-2 adsorption chromatography and 3 steps of high-performance liquid chromatography. Reverse phase, high-performance liquid chromatography, mass spectrometry, and nuclear magnetic resonance spectrometry identified these substances as linoleic (18:2) and oleic acids (18:1). A significant increase in the ouabain-displacing activity was observed in hog plasma during saline infusion. The maximal level reached was approximately 10 times higher than that of the preinfusion plasma sample. The two unsaturated fatty acids contributed to approximately 52% of the total ouabain-displacing activity after 120 min of saline infusion. The increased fatty acid levels in volume-expanded plasma are sufficient for an extensive inhibition of Na+,K+-ATPase activity. These results strongly suggest that free unsaturated fatty acids in plasma regulate extracellular fluid volume in a pathological volume-expanded condition through modulation of Na+,K+-ATPase activity.     

Inhibition of gastric (H+ + K+)-ATPase by unsaturated long-chain fatty acids

Arachidonic acid and unsaturated C18 fatty acids at concentrations near 10(-5) M markedly inhibited (H+ + K+)-ATPase in hog or rat gastric membranes. Arachidonic acid was a more potent inhibitor than unsaturated C18 fatty acids, but the involvement of the metabolites of arachidonic acid cascade was ruled out. Linolenic acid inhibited the formation of phosphoenzyme and the K+ -dependent p-nitrophenylphosphatase activity of the hog ATPase. Treatment with fatty acid-free bovine serum albumin abolished only the inhibitory effect of the fatty acid on the phosphatase activity without restoring the overall ATPase action. These data suggest the existence of at least two groups of hydrophobic binding sites in the gastric ATPase for unsaturated long-chain fatty acids which affect differentially the catalytic reactions of the ATPase. (H+ + K+)-ATPase in rat gastric membranes was found more susceptible to the fatty acid inhibition and also more unstable than the ATPase in hog gastric membranes. The presence of a millimolar level of lanthanum chloride or ethylene glycol bis(beta-aminoethyl ether)-N,N'-tetraacetic acid stabilized the rat ATPase probably via the inhibition of Ca2+ -dependent phospholipases in the gastric membranes.     

Long chain fatty acid inhibition of sodium plus potassium-activated adenosine triphosphatase from rat heart.

Long chain fatty acids were found to inhibit (Na+ + K+)-ATPase prepared from rat heart. Unsaturated and polyunsaturated fatty acids were more inhibitory than saturated fatty acids with myristic acid being the most inhibitory saturated fatty acid tested and linoleic the most inhibitory unsaturated fatty acid. As an example of fatty acid modification of the enzyme, inhibition of (Na+ + K+)-ATPase by oleate was examined. When compared to ouabain, inhibition of (Na+ + K+)-ATPase by oleate was found to be similar in that both were dependent on K+ concentration, but, in contrast to the almost instantaneous inhibition by ouabain, oleate inhibition was a slow process requiring over 20 min incubation at 37 degrees to produce maximum inhibition. Inhibition of rat heart (Na+ + K+)-ATPase by oleate was found to be readily reversible by washout. In the presence of albumin an oleate/albumin molar ratio greater than 7.5 was required for inhibition to occur. The activity of rat heart (Na+ + K+)-ATPase had a temperature optimum above 40 degrees and a discontinuous Arrhenius' plot with a transition temperature of 25 degrees. In the presence of oleate, however, the enzyme's optimum temperature decreased to below 40 degrees, the activation energy of the reaction at temperatures below 25 degrees was lowered from 24.7 kcal/mol to 12.6 kcal/mol and the enzyme had a linear Arrhenius' plot. The possibility of in vivo inhibition of cardiac (Na+ + K+)-ATPase under conditions of elevated fatty acids is discussed.     

Free fatty acids and (Na+,K+)-ATPase: effects on cation regulation, enzyme conformation, and interactions with ethanol

Effects of free fatty acids on parameters of (Na+,K+)-ATPase regulation related to enzyme conformation were examined. Sensitivity to inhibition by free fatty acid increased as the number of double bonds increased. Free fatty acids reduced affinity for K+ or Na+ at their regulatory sites without altering apparent K+ affinity at its high-affinity site, and increased apparent affinity for ATP. The apparent E2/E1 ratio and apparent delta H and delta S for the E1-E2 transition were reduced by fatty acid. High K+ or low temperature reduced the sensitivity of enzyme to inhibition by free fatty acid. In the presence of low K+, arachidonic acid potentiated inhibition of phosphatase activity by ethanol. Arachidonic acid alone had little effect on the rate of ouabain binding, but accelerated ouabain binding in the presence of K+. These data suggest that fatty acids alter (Na+,K+)-ATPase by preventing the univalent cation-mediated transition to E2, the K+-sensitive form of enzyme. (Na+,K+)-ATPase could potentially be influenced in vivo by free fatty acids released by phospholipases or during hypoxia, or by changes in membrane lipid saturation.     

Response of rat heart membranes and associated ion-transporting ATPases to dietary lipid

The effects of different dietary fat intake on the lipid composition and enzyme behaviour of sarcolemmal (Na+ + K+)ATPase and sarcoplasmic reticulum Ca2+-ATPase from rat heart were investigated. Rat diets were supplemented with either sunflower seed oil (unsatd./satd. 5.6) or sheep kidney fat (unsatd./satd. 0.8). Significant changes in the phospholipid fatty acid composition were observed in both membranes after 9 weeks dietary lipid treatment. For both membranes, the total saturated/unsaturated fatty acid levels were unaffected by the dietary lipid treatment, however the proportions of the major unsaturated fatty acids were altered. Animals fed the sunflower seed oil diet exhibited an increase in n-6 fatty acids, including linoleic (18:2(n-6] and arachidonic (20:4(n-6] while the sheep kidney fat dietary rats were higher in n-3 fatty acids, principally docosahexaenoic (22:6), with the net result being a higher n-6/n-3 ratio in the sunflower seed oil group compared to sheep kidney fat dietary animals. Fluorescence polarization indicated that the fluidity of sarcoplasmic reticular membrane was greater than that of sarcolemmal membrane, with a dietary lipid-induced decrease in fluidity being observed in the sarcoplasmic reticular membrane from sheep kidney fat dietary animals. Despite these significant changes in membrane composition and physical properties, neither the specific activity nor the temperature-activity relationship (Arrhenius profile) of the associated ATPases were altered. These results suggest that with regard to the parameters measured in this study, the two ion-transporting ATPases are not modulated by changes which occur in the membrane lipid composition as a result of the diet.     

Gastric lipolysis in the developing rat. Ontogeny of the lipases active in the stomach

Two nutritional models, an essential fatty acid deficiency model and the feeding of saturated versus unsaturated fats, were used in a feeding study in order to assess the relationship between tissue fatty acid composition and the activities of some membrane-associated enzymes. Purified diets containing 7% hydrogenated coconut oil, 7% corn oil, 10% safflower oil or butter were fed to rats for a total of 49 weeks (1 week of pregnancy, 3 weeks of lactation and 45 weeks post-weaning). Tissue homogenates from submandibular salivary glands and kidneys were analyzed for fatty acid composition of total lipids and phospholipids. Changes in fatty acid patterns typical of essential fatty acid deficiency such as an increase in the levels of 16:1 and 18:1, a decrease in 18:2 and 20:4 and an accumulation of 20:3 omega 9 were observed in salivary glands and kidneys of rats fed the deficient diet. Tissues of rats fed 10% butter also showed fatty acid compositional changes which were somewhat similar to those in essential fatty acid deficiency, but to a lesser degree. The activities of ouabain-sensitive (Na+ + K+)-ATPase were higher in homogenates of salivary glands and kidneys of the deficient rats and those fed butter as compared with their controls. The results suggest a relationship between the double bond index of fatty acids as an indication of membrane lipid fluidity and allosteric modification of (Na+ + K+)-ATPase activity. However, other explanations for the observed changes in (Na+ + K+)-ATPase activity cannot be ruled out. There were no diet-related differences in the activities of gamma-glutamyltranspeptidase or 5'-nucleotidase.     

Isolation and characterization of a specific endogenous Na+,K+-ATPase inhibitor from bovine adrenal

In order to identify a specific endogenous Na+,K+-ATPase inhibitor which could possibly be related to salt-dependent hypertension, we looked for substances in the methanol extract of bovine whole adrenal which show all of the following properties: (i) inhibitory activity for Na+,K+-ATPase; (ii) competitive displacing activity against [3H]ouabain binding to the enzyme; (iii) inhibitory activity for 86Rb uptake into intact human erythrocytes; and (iv) cross-reactivity with sheep anti-digoxin-specific antibody. After stepwise fractionation of the methanol extract of bovine adrenal glands by chromatography on a C18 open column, a 0-15% acetonitrile fraction was fractionated by high-performance liquid chromatography on a Zorbax octadecylsilane column. One of the most active fractions in 0-15% acetonitrile was found to exhibit all of the four types of the activities. It was soluble in water and was distinct from various substances which have been known to inhibit Na+,K+-ATPase such as unsaturated free fatty acids, lysophosphatidylcholines, vanadate, dihydroxyeicosatrienoic acid, dehydroepiandrosterone sulfate, dopamine, lignan, ascorbic acid, etc. This substance was further purified by using an additional five steps of high-performance liquid chromatography with five different types of columns. Molecular mass was estimated as below 350 by fast atom bombardment mass spectroscopy and ultrafiltration. Heat treatment at 250 degrees C for 2 h and acid treatment with 6 N HCl at 115 degrees C for 21 h almost completely destroyed the inhibitory activity of the purified substance for Na+ pump activity. Additionally, alkaline treatment with 0.2 N NaOH at 23 degrees C for 2 h destroyed approximately 70% of the inhibitory activity, whereas boiling for 10 min and various enzyme digestion did not destroy the activity. The dose dependency for the four types of the activities for this substance paralleled those of ouabain, spanning 2 orders of magnitude in concentration range. The inhibitory potencies of the purified substance for Na+,K+-ATPase, Na+ pump, and ouabain binding activities were diminished with increasing K+ concentration, exhibiting a characteristic typical of cardiac glycosides. This substance had no effect on the Ca2+-ATPase activity or the Ca2+ loading rate into the vesicle prepared from skeletal muscle sarcoplasmic reticulum. These results strongly suggest that this water-soluble nonpeptidic Na+,K+-ATPase inhibitor may be a specific endogenous regulator for the ATPase.     

Reductions of Γ-Aminobutyric Acid and Glutamate Uptake and (Na++ K+)-ATPase Activity in Brain Slices and Synaptosomes by Arachidonic Acid

Arachidonic acid, a major polyunsaturated fatty acid of membrane phospholipids in the CNS, reduced the high-affinity uptake of glutamate and gamma-aminobutyric acid (GABA) in both rat brain cortical slices and synaptosomes. alpha-Aminoisobutyric acid uptake was not affected. Intrasynaptosomal sodium was increased concomitant with decreased (Na+ + K+)-ATPase activity in synaptosomal membranes. The reduction of GABA uptake in synaptosomes could be partially reversed by alpha-tocopherol. The inhibition of membrane-bound (Na+ + K+)-ATPase by arachidonic acid was not due to a simple detergent-like action on membranes, since sodium dodecyl sulfate stimulated the sodium pump activity in synaptosomes. These data indicate that arachidonic acid selectively modifies membrane stability and integrity associated with reductions of GABA and glutamate uptake and of (Na+ + K+)-ATPase activity.     

Characterization of fatty acid interaction with ouabain and vanadate binding to (Na+ + K+)-activated ATPase

The candidateship of unsaturated fatty acids as endogenous ouabain-like factors was studied. Binding of the artificial ligand vanadate at the intracellular phosphorylation epitope of membrane-bound Na+/K+-ATPase was unaffected by linoleic and arachidonic acid. In the (Mg2+ + Pi)-facilitated system for ouabain binding they were characterized as noncompetitive inhibitors of cardiac glycoside binding, however. The ouabain binding capacity as well as the affinity decreased and the ouabain dissociation rate was accelerated by fatty acids. In the presence of vanadate for facilitation of ouabain binding an increase in ouabain affinity was seen. It is concluded that elementary criteria for the characterization of unsaturated fatty acids as ouabain-like factors are not fulfilled. The ratio between E2-subconformations of Na+/K+-ATPase with different ouabain affinities may be changed by incorporation of fatty acids in the lipid membrane.     

Mechanism of the control of (Na+ + K+)-ATPase by long-chain acyl coenzyme A

Long-chain fatty acid esters of CoA activate (Na+ + K+)-ATPase (the sodium pump) when ATP is suboptimal. To explore the nature of the interactions of these CoA derivatives with the pump, reversible effects of palmitoyl-CoA on the purified membrane-bound kidney enzyme were studied under conditions where interference from the irreversible membrane-damaging effect of the compound was ruled out. With 50 microM ATP, while saturating palmitoyl-CoA increased (Na+ + K+)-ATPase activity, it caused partial inhibition of Na+-ATPase activity without affecting the steady-state level of the phosphoenzyme. Palmitoyl-CoA did not change the K0.5 of ATP for Na+-ATPase, but it altered the complex Na+ activation curve to suggest the antagonism of the low-affinity, but not the high-affinity, Na+ sites. At a low ATP concentration (0.5 microM), K+ inhibited Na+-ATPase as expected. In the presence of palmitoyl-CoA and 0.5 microM ATP, however, K+ became an activator, as it is at high ATP concentrations. The activating effect of palmitoyl-CoA on (Na+ + K+)-ATPase activity was reduced with increasing pH (6.5-8.5), but its inhibitory effect on Na+-ATPase was not altered in this pH range. The data show two distinct actions of palmitoyl-CoA: 1) blockade of the extracellular "allosteric" Na+ sites whose exact role in the control of the pump is yet to be determined, and 2) activation of the pump through increased rate of K+ deocclusion. Since in their latter action the fatty acid esters of CoA are far more effective than ATP at a low-affinity regulatory site, we suggest that these CoA derivatives may be the physiological ligands of this regulatory site of the pump.     

Studies on (Na+ + K+)-activated ATPase. XLIX. Content and role of cholesterol and other neutral lipids in highly purified rabbit kidney enzyme preparation

(1) The neutral lipids and the free and bound fatty acids of a highly purified (Na+ + K+)-ATPase preparation from rabbit kidney outer medulla have been analysed. (2) On a dry weight basis, the total lipid content is nearly the same as the total protein content, and consists for 66% of phospholipids and for 34% of neutral lipids and free fatty acids. In the latter category cholesterol is the main component (71%). (3) On a molar basis the enzyme preparation contains 382 mol phospholipids, 67 mol free fatty acids, 9, 16 and 12 mol mono-, di- and triacylglycerols, 249 and 19 mol free and esterified cholesterol per mol enzyme. (4) The fatty acid composition of each lipid and of the free fatty acid fraction, present in the enzyme preparation, is reported. (5) All cholesterol and part of the phospholipids can be removed by hexane extraction, leaving 66% of the (Na+ + K+)-ATPase activity. Oxidation of all cholesterol to cholest-4-en-3-one by cholesterol oxidase leaves 85% of the (Na+ + K+)-ATPase activity. These results indicate that cholesterol is not essential for (Na+ + K+)-ATPase activity.     

Polyunsaturated fatty acids inhibit Mg2+ -ATPase in basolateral membranes from rat enterocytes.

It is known that certain polyunsaturated fatty acids of the n-6 family, for example linoleic and arachidonic acids, can activate both Na+, K+-ATPase and Ca2+-ATPase. These enzymes drive active absorption processes in the duodenal enterocyte. This study presents data which show a 30-50% inhibition of Mg2+-ATPase activity in enterocyte basolateral membrane preparations by linoleic and gamma-linolenic acids (also a member of the n-6 family.) Mg2+-ATPase activity has several possible roles in the enterocyte: involvement in Mg2+ and Ca2+ absorption (as part of Ca2+-ATPase and also myosin I activity) as well as control of phospholipid distribution in the membrane by a class of Mg2+-ATPases called 'flippases'. The action of linoleic and gamma-linolenic acids on basolateral membrane Mg2+-ATPase may thus modulate several cellular transport processes.     

Fractionation of protein components of plasma membranes from the electric organ of Torpedo marmorata

A procedure has been developed for the separation of intrinsic proteins of plasma membranes from the electric organ of Torpedo marmorata. (Na+ + K+)-ATPase, nicotinic acetylcholine receptor and acetylcholinesterase remained active after solubilization with the nonionic detergent dodecyl octaethylene glycol monoether (C12E8). These components could be separated by ion exchange chromatography on DEAE-Sephadex A-25. Fractions enriched in ouabain-sensitive K+-phosphatase or (Na+ + K+)-ATPase activity showed two bands in sodium dodecyl sulphate polyacrylamide gel electrophoresis corresponding to the alpha- and beta-subunits. The (Na+ + K+)-ATPase was shown to have immunological determinants in common with a 93 kDa polypeptide which copurified with the nicotinic acetylcholine receptor, also after solubilization in Triton X-100 and chromatography on Naja naja siamensis alpha-toxin-Sepharose columns. The data suggest that the alpha-subunit of (Na+ + K+)-ATPase associates with the acetylcholine receptor in the membranes of the electric organ.     

Relationship between (Na + K)-ATPase activity, lipid peroxidation and fatty acid profile in erythrocytes of hypertensive and normotensive subjects

Oxidative stress may play a role in the pathogenic mechanism of essential hypertension. Lipid peroxidation can alter the cellular structure of membrane-bound enzymes by changing the membrane phospholipids fatty acids composition. We investigated the relationship between (Na + K)-ATPase activity, lipid peroxidation, and erythrocyte fatty acid composition in essential hypertension. The study included 40 essential hypertensive and 49 healthy normotensive men (ages 35–60 years). Exclusion criteria were obesity, dyslipidemia, diabetes mellitus, smoking, and any current medication. Patients underwent 24-h ambulatory blood pressure monitoring and blood sampling. Lipid peroxidation was measured in the plasma and erythrocytes as 8-isoprostane or malondialdehyde (MDA), respectively. Antioxidant capacity was measured as ferric reducing ability of plasma (FRAP) in the plasma and as reduced/oxidized glutathione (GSH/GSSG ratio) in erythrocytes. (Na + K)-ATPase activity and fatty acids were determined in erythrocyte membranes. Hypertensives had higher levels of plasma 8-isoprostane, erythrocyte MDA, and relative percentage of saturated membrane fatty acids, but lower plasma FRAP levels, erythrocyte GSH/GSSG ratio, (Na + K)-ATPase activity and relative percentage of unsaturated membrane fatty acids, compared with normotensives. Day-time systolic and diastolic blood pressures correlated positively with lipid peroxidation parameters, but negatively with (Na + K)-ATPase activity. These findings suggest that the modulation of (Na + K)-ATPase activity may be associated with changes in the fatty acid composition induced by oxidative stress and provide evidence of a role for this enzyme in the pathophysiology of essential hypertension.     

Increased ouabain-sensitive 86Rb+ uptake and sodium and potassium ion-activated adenosine triphosphatase activity in transformed cell lines.

During the log phase of growth both the active, ouabain-sensitive K+ uptake, measured as 86Rb+, and the sodium and potassium ion-activated ATPase ((Na+ + K+)-ATPase) activity of SV40-transformed 3T3 cells were 2.5-and 5,5-fold higher, respectively, than in untransformed 3T3 cells. A similar higher active K+ uptake was found for Rous sarcoma virus and SV40-transformed baby hamster kidney cells compared with untransformed BHK cells. The active K+ uptake in SV403T3 and normal 3T3 cells decreased when the growth rate of both cell types diminished. Reduction in ouabain-sensitive ATP hydrolysis only occurred later, however, when appreciable decreases in cell viability were seen. Arrhenius plots of the (Na+ + K+)-ATPase activity of SV403T3 cells indicated a discontinuity at 24 degrees, whereas no similar discontinuity was indicated for 3T3 cells. The consequences of elevated K+ transport and (Na+ + K+)-ATPase activity in transformed cells and the possibility that the increased activity might be related to differences inphospholipid fatty acyl chain fluidity are discussed.     

Effect of Fatty Acids on [3H]Ouabain Binding to Cerebromicrovascular (Na++ K+)-ATPase

The effects of short- and long-chain fatty acids on the cerebromicrovascular (Na+ + K+)-ATPase were investigated using specific [3H]ouabain binding to the enzyme. Specific binding increased linearly with total microvessel protein (37-110 micrograms) and was time-dependent with maximum binding obtained by 10 min. Arachidonic acid, but not palmitic acid, stimulated [3H]ouabain binding in a dose-dependent manner, with a 105% increase over basal levels at 100 microM arachidonic acid. Preincubation of the microvessels with arachidonic acid did not alter the stimulation observed. 4-Pentenoic acid stimulated [3H]ouabain binding only at high concentrations (10 mM). Scatchard analysis of [3H]ouabain binding to untreated microvessels yielded a single class of "high-affinity" binding sites with an apparent binding affinity (KD) of 64.7 +/- 2.0 nM and a binding capacity (Bmax) of 10.1 +/- 1.5 pmol/mg protein. In the presence of 100 microM arachidonic acid, a monophasic Scatchard plot also was obtained, but the KD significantly decreased to 51.9 +/- 2.7 nM (p less than 0.01), whereas the Bmax remained virtually unchanged (12.5 +/- 1.2 pmol/mg protein). The stimulation of [3H]ouabain binding in the presence of arachidonic acid was potentiated by 4-pentenoic acid, but not by indomethacin or eicosatetraynoic acid. These data suggest that long-chain polyunsaturated fatty acids may be involved in the regulation of blood-brain barrier (Na+ + K+)-ATPase and may play a role in the cerebral dysfunction associated with diseases in which plasma levels of nonesterified fatty acids are elevated.     

Stimulation of (Ca2+ + Mg2+)-ATPase activity in human erythrocyte membranes by synthetic lysophosphatidic acids and lysophosphatidylcholines. Effects of chain length and degree of unsaturation of the fatty acid groups

Synthetic lysophosphatidic acids and lysophosphatidylcholines were examined for their effects on the (Ca2+ + Mg2+)-ATPase of human erythrocyte membranes. Addition of these compounds to erythrocyte ghosts caused significant changes in ATPase activity. The degree of unsaturation and the length of the sn-1 long chain hydrocarbon moiety were both contributing factors. All lysophosphatidic acids tested stimulated (Ca2+ + Mg2+)-ATPase activity. Of the species having a saturated acyl group, the most active was the myristoyl derivative. Linoleoyllysophosphatidic acid was the most potent of the unsaturated species. Saturated lysophosphatidylcholines with a short chain fatty acyl group (C10 to C14) exhibited only a moderate stimulatory activity, whereas the longer chain homologues, i.e., C16 and C18 were inhibitory at high concentrations. On the other hand, unsaturated lysophosphatidylcholines had stimulatory activities comparable to the unsaturated lysophosphatidic acids. These results suggest that the acidic moiety of lysophosphatidic acid is not an important structural determinant for expressing ATPase stimulatory activity in ghosts. Rather the nature of the hydrocarbon chain as well as the lyso structure of these compounds appear most critical under these conditions. The stimulatory effects of lysophosphatidic acids or lysophosphatidylcholines were additive to that induced with calmodulin, suggesting that these lysophospholipids affect the (Ca2+ + Mg2+)-ATPase by a mechanism which is different from that seen with calmodulin.     

Studies of (Na+ + K+)-sensitive ATPase activity in pig lymphocytes. Effects of concanavalin A.

(Na+ + K+)-ATPase activity is demonstrated in plasma membranes from pig mesenteric lymph nodes. After dodecyl sulfate treatment plasma membranes have an 18-fold higher (Na+ + K+)-ATPase activity, while their ouabain-insensitive Mg2+-ATPase is markedly lowered. A solubilized (Na+ +K+)-ATPase fraction, obtained by Lubrol WX treatment of the membranes, has very high specific activity (21 mumol Pi/h per mg protein). Concanavalin A has no effect on these partially purified (Na+ + K+)-ATPase, while inhibits (40%) this activity in less purified fractions which still contain Mg2+-ATPase activity.     

Activation of (Na++K+)-ATPase by long-chain fatty acids and fatty acyl coenzymes A

Long-chain unsaturated fatty acids and fatty acyl CoA derivatives activated (Na++K+)-ATPase at suboptimal, but not optimal, ATP concentrations. Activation was obtained within a narrow range of fatty acid concentrations; higher acid levels inhibited the enzyme. The various CoA esters, however, activated with K0.5 values in the range of 0.15-10 microM; and with no inhibitory effects at concentrations up to 100 microM. Palmitoyl CoA, binding reversibly to a regulatory site, reduced K0.5 of ATP from 0.37 mM to 0.17 mM; and changed the Hill coefficient of the substrate-velocity curve from 0.86 to 0.63. These compounds may be physiological regulators that desensitize the function of this enzyme to diminishing ATP levels.