The distribution of ATPase activities in purified transverse tubular membranes

Vesiculated fragments of transverse tubules (TT) and sarcoplasmic reticulum (SR) membranes were purified from heterogeneous microsomal membrane fractions of chicken breast muscle by a modification of an iterative calcium-oxalate loading technique. The distribution of ATPase activities were determined for the TT and SR and were compared to enriched fractions of sarcolemma (SL) membranes. The TT membranes were characterized by high rates of magnesium-stimulated ATPase (Mg-ATPase) and 5′-nucleotidase activities but were virtually devoid of calcium-stimulated, magnesium-dependent ATPase (Ca,Mg-ATPase) activity. Moderate levels of a latent sodium and potassium-stimulated ATPase (Na,K-ATPase) were observed for TT membranes when unmasked with valinomycin and monensin. In contrast to the behavior of TT membranes, highly purified SR membranes displayed an active Ca,Mg-ATPase but negligible Na,K-ATPase, Mg-ATPase, and 5′-nucleotidase activities. High levels of Na,K-ATPase and 5′-nucleotidase activities were observed for SL membranes; however, the SL displayed no appreciable Ca,Mg-ATPase and Mg-ATPase activities. The lack of significant Mg-ATPase activity in the SR and SL fractions suggested that the Mg-ATPase was uniquely associated with the TT membranes. The TT Mg-ATPase was further characterized by its pH and temperature dependences, and its sensitivity to pharmacologic agents. The Mg-ATPase of the TT was insensitive to inhibition by sodium azide and oligomycin in concentrations shown to exert maximum inhibition on the F₁ ATPase of submitochondrial particles. The Mg-ATPase was also resistant to the effects of ouabain and orthovanadate in concentrations which abolished the Na,K-ATPase and Ca,Mg-ATPase activities of the SL and SR, respectively. The Mg-ATPase displayed temperature and pH optima (25 °C, pH 7.3) which were distinguishable from the Ca,Mg-ATPase (45 °, pH 7.0) of highly purified SR fractions but which were very similar to the temperature and pH dependencies of the mixed microsomal fractions (MMF) from which the TT membranes were derived. Similarities in the pH and temperature dependencies of the TT and MMF Mg-ATPases plus the absence of appreciable Mg-ATPase activity in highly purified SR membranes suggests that the “basic” Mg-ATPase often seen in crude SR fractions may originate from TT membrane contamination. The resistance of the TT Mg-ATPase to inhibition by the pharmacologic agents tested plus its unique temperature and pH dependences indicate that this ATPase is distinguishable from other ATPases and may, therefore, be of value as a specific biochemical marker for TT membranes.     

Changes in the membrane ATPase activity of erythrocytes and Candida guilliermondii under the action of roseofungin

Changes in the Mg-ATPase and Na, K-ATPase activity of the rat erythrocyte and Candida guilliermondii membranes under the effect of roseofungin were studied. The antibiotic was totally bound to the isolated plasmatic membranes of Candida guilliermondii, up to 3 micrograms of the antibiotic per 1 microgram of the yeast protein. The Mg-APTase activity of these membranes was slightly inhibited by the antibiotic. The activity of Na, K-ATPase was almost completely inhibited even at 0.04 mg of roseofungin per 1 mg of protein. Much higher concentrations of the antibiotic inhibited the Mg-ATPase and Na, K-ATPase activity of the erythrocyte membranes to a less extent.     

Differential Effect of w3 PUFA Supplementations on Na,K-ATPase and Mg-ATPase Activities: Possible Role of the Membrane w6/w3 Ratio

Several functional properties of Na,K-ATPase are strongly dependent on membrane fatty acid composition, but the underlying mechanism is still not well defined. We have studied the effects of two types of supplementations enriched in the w3 polyunsaturated fatty acids on the Na,K-ATPase and Mg-ATPase activities in sciatic nerve (SN) and red blood cells (RBC). Eight groups of rats, controls and diabetics, received a standard diet, supplemented or not with 30 or 60 mg/kg/day of docosahexaenoic acid (DHA) or with soybean for eight weeks. Diabetes induced significant decrease of Na,K-ATPase activity in SN (-23%) and RBC (-25%), without affecting Mg-ATPase activity. In RBC, soybean and DHA supplementations caused significant increases in Na,K-ATPase activity (in various range, +13% to +145%) in all groups, and in Mg-ATPase activity in control soybean (+65%), control and diabetic DHA high dose (+39%, +53%) and diabetic DHA low dose (+131%) groups. In SN, the soybean caused a significant decrease in Na,K-ATPase activity (-26%) and still more in the diabetic group (-53%). The DHA diet induced a slight decrease in activity in control groups, whilst during diabetes, at high dose, we noted an aggravation of this decrease (-36%). Mg-ATPase activity was not modified by supplementations except for the low dose of DHA where the activity was slightly decreased in the control group (-16%). The supplementations induced multiple tissue-specific modifications in the membrane fatty acid composition of RBC and of SN homogenates. Several specific correlations have been found between variations in fatty acids amounts and Na,K-ATPase activity in these tissues but only in RBC for Mg-ATPase activity. Indeed, we observed that the variations in Na,K-ATPase activity are positively and significantly correlated with changes in the omega6/omega3 ratio in SN as well as in RBC. These data clearly show, for the first time, that the diet could modulate the Na,K-ATPase activity via the omega6/omega3 ratio in the membranes. A similar correlation was observed with Mg-ATPase activity in RBC, suggesting also a dietary regulation of the enzyme; but for the SN, this activity might be regulated by a different omega6/omega3 ratio or by another pathway.     

Increased brain Na, K-ATPase activity of rats under stress

The activities of Na, K- and Mg-dependent ATPases were measured in crude synaptosomal fractions isolated from the rat brain gray matter. Prolonged (6 h) exposure to emotional painful stress stimulated Na, K-ATPase activity by 40% without affecting that of Mg-ATPase. Preliminary injection of the free radical scavenger ionol presented Na, K-ATPase activation, thus suggesting the involvement of lipid peroxidation initiated in brain tissues under stress in acceleration of NA-pump function. However, model studies with lipid peroxidation induced in vitro by an ascorbate-dependent system in a membranous suspension demonstrated an opposite effect, i. e. fast inhibition of Na, K-ATPase. Possible reasons for the different effects of lipid peroxidation in vivo under stress and on Na, K-ATPase activity in vitro are discussed. It is concluded that activation of Na K-ATPase is a mechanism which is responsible for acceleration of reflex conditioning and for the maintenance of the conditioned reflexes in stress-exposed animals.     

Bile pigments and bilirubin UDP-glucuronyl transferase during the metamorphosis of Rana catesbeiana tadpoles

1. Cold-acclimated (1 degree C) goldfish (Carassius auratus) branchial Na/K-ATPase activity was elevated 100% while renal Na/K-ATPase activity was not significantly affected compared with warm-acclimated (20 degrees C) goldfish. 2. Cold-acclimated goldfish branchial and renal Mg-ATPase activity was reduced 18 and 30% on a per mg protein basis, respectively. 3. Renal Na/K-ATPase activity was 4.6- and 1.6-fold greater than gill in cold- and warm-acclimated fish, respectively. 4. The elevated branchial Na/K-ATPase activity and the unchanged renal Na/K-ATPase activity are consistent with the maintenance of the reduced blood ion level in cold-acclimated goldfish.     

Effect of acetylcholine on the cerebral microsomal Na, K-ATPase of rats of different ages

Na, K- and Mg-ATPase activity of the cerebral cortex microsomal fraction has been studied and compared in adult and old rats. The activity of Na, K-ATPase decreases while that of Mg-ATPase increases with age. The total ATPase activity remains unchanged. The effect of acetylcholine on ATPase activity has been found to be age-dependent.     

Effect of carbacholine and serotonin on the ATPase activity in synaptosomes of the projection and association areas of the feline cerebral cortex]

Na, K-ATPase and Mg-ATPase activities were measured in the synaptosomes of the temporal auditory projection area and the frontal association area. Moreover, the effects of carbacholine and serotonin on those activities were investigated. Na, K-ATPase activity in the synaptosomes of the association area was shown to be reliably higher that in the synaptosomes of the projection area (11.02 +/- 0.45 vs 8.40 +/- 0.55 microM Pi/mg of protein hr; P less than 0.05). Mg-ATPase activity was higher in the second case as compared to the first one (11.40 +/- 0.38 vs 9.04 +/- 0.35; p less than 0.05). Carbacholine and serotonin (10(-8)-10(-3) M) were found to induce equal inhibition of Na, K-ATPase activity in the synaptosomes of both cortices (1 max = 25-30%, 1C50 = 0.2-0.3 microM) which is blocked respectively with atropine (10(-6) M) and methysergide (10(-6) M) and enhanced in presence of GTP (5.10(-5) M). The enzyme activity is also inhibited by the non-hydrolysable guanine nucleotide, GTP gamma S (10(-8)-10(-4) M), in the absence of the antagonists (1 max = 35-40%, 1 C50 = 0.02 microM). In the methysergide-containing medium serotonin exerts a dose-dependent stimulatory effect on Na, K-ATPase which is more pronounced in the synaptosomes of the association area (A max = 25%, A C50 = 0.05 microM). Mg-ATPase activity of membrane preparations is liable to be stimulated by both serotonin and carbacholine, stimulation being more pronounced in the synaptosomes of the association cortex as well (A max = 35%, A C50 = 0.2-0.3 microM). This effect is insensitive either to the antagonists of the corresponding receptors or to GTP. GTP gamma S does not cause alterations in the enzymatic activity. Na, K-ATPase is suggested to be coupled to muscarine and serotonin receptors in the synaptic membranes of both projection and association cortical areas via a GTP-binding protein. At the same time, the agonists of receptors mentioned above are presumably also capable to effect Mg-ATPase activity by the receptor-independent way.     

Activity of erythrocyte membrane Na,K-ATPase in rats with experimental botulism

The effect of type C botulinum toxin on Na, K, Mg-ATPase activities of erythrocyte membranes of white rats was studied in experiments in vivo and in vitro. The activity of Na, K, Mg-ATPase was found to be markedly inhibited in the preclinical period of poisoning, 2 hours after intraperitoneal injection of the toxin. In this case Mg-ATPase activity noticeably increased. In the presence of the development of a grave paralytic syndrome one day after intraperitoneal injection of the toxin, the activity of Na, K-ATPase of the erythrocyte membrane remained decreased as was the case in the preclinical period of poisoning, whereas the activity of Mg-ATPase returned to normal. The experiments in vitro with preincubation of erythrocyte membranes with botulinum toxin in the concentrations corresponding to the mean calculated ones in the experiments in vivo demonstrated inhibition of Na, K-ATPase. The magnitude of Mg-ATPase activity remained virtually unchanged in all the modifications of the experiments with boiled and native botulinum toxin. The in-vivo experiments with intraperitoneal injection of glutathione and unithiol to the pretreated animals attested to normalization of Na, K-ATPase in the preclinical period of poisoning, with this normalization being brought about by unithiol. In the in-vitro experiments with addition of unithiol or glutathione into the incubation medium, each of the donators of sulphhydryl groups prevented Na, K-ATPase inhibition with botulinum toxin.     

Large-scale preparation of Na, K-ATPase from ox brain.

A method for the isolation of membrane-bound Na,K-ATPase in quantity from brain gray matter is described. The method permits a large amount of enzyme to be obtained rather quickly with about 60% of the original activity of Na,K-ATPase of the tissue being recovered. The enzyme is stable, it has a specific activity of about 200 μmoles ATP split per mg protein per hour. Mg-ATPase comprises about 1% of the total ATPase activity. The enzymatic properties of this Na,K-ATPase do not differ from those in the literature; the turnover number is about 9300 min^?1.     

Activity and thermal stability of the enzymes of energy support in the common frog

The activity and heat resistance of succinate dehydrogenase (SDG), Na, K-ATPase and Mg-ATPase of the grass frog (39 specimens) have been determined. No correlation was found between individual levels of heat resistance both of either enzyme examined and of the same enzyme but taken from different tissues (SDG of liver and muscles). The average level of heat resistance of SDG in liver is significantly higher than that in m. gastrocnemius. A statistically significant correlation was observed between individual levels of enzyme activity in the internal organs (SDG of liver, Na, K-ATPase and Mg-ATPase of kidney). The activity of SDG of muscles does not correlate with that of either partner.     

The Na, K-ATPase activity of the separate structural components of the guinea-pig visual system in hypoxia

Different degree of sensitivity to acute hypoxia in vivo has been shown in guinea-pig pigmented epithelium, retina and visual cortex Na, K-ATPase. The highest degree of the enzyme activity inhibition has been noted in the pigmented epithelium (more than 3 times), lower inhibition-in visual cortex (26%) and the lowest-in the retina (18%). In contrast to Na, K-ATPase, hypoxia effect on Mg-ATPase resulted in both inhibition and activation of the enzyme in all 3 structures. Reoxygenation following acute hypoxia increases Na, K-ATPase activity inhibition in the retina and visual cortex. But under reoxygenation the enzyme activity is recovered in the pigmented epithelium. Preliminary administrations of vitamin E completely prevented Na, K-ATPase activity inhibition in all studied structures.     

Some properties of hog thyroidal membrane-bound adenosine tri-phosphatase: proteolytic activation of Mg-dependent activity.

The ATPase preparations from the hog thyroid was preincubated with various amounts of trypsin. The activity of Mg-ATPase was consistently elevated. On the contrary, the Na, K-ATPase activity decreased with increasing amounts of trypsin. The effects were similar to those which were observed in the enzyme preparations treated with basis polyamino acids as previously reported. This phenomenon seemed to be specific in the preparations from the thyroid. The Mg-dependent activity was increased after pretreatment with trypsin or poly-L-lysine (PLL) when CTP, ITP and UTP were used as substrate. Thus the substrate specificity of Mg-ATPase was low. The enzyme-kinetics using ATP as substrate showed that the increase in activity was due to an increase in Vmax and not to a change in Km. The activity of Mg-ATPase was increased even after 30 min of preincubation with trypsin, while the Na, K-ATPase activity was almost diminished. These results suggest that the activity of Mg-ATPase in the preparation from the thyroid is specifically changed by the modification of the molecular environment of the enzyme with trypsin or basic polyamino acids.     

Changes in the properties of the enzymes of energy allowance resulting from heat shock in lake frogs

A study was made of the activity and heat resistance of preparations of Na, K-ATPase, Mg-ATPase and succinate dehydrogenase of the lake frog R. ridibunda caused by the heat shock of animals. A decrease in the activity and an increase in the heat resistance of all the three enzymes studied were observed. The level of individual correlations between these parameters remained unchanged. An increase in the heat resistance of Na, K-ATPase occurs at the expense of a higher threshold of its thermal inactivation without changes in the affinity of this enzyme to Na+ and K+. Under discussion is the question of the functional significance of the changes observed.     

Adenosine triphosphatase from plasma membranes of cattle intestinal epithelium

The activities of Na,K-, Ca,Mg- and Mg-ATPases in the membrane fractions of plasma membranes of intestinal enterocytes of cattle, brush border and basolateral membranes, were studied. The activities were estimated under conditions of alkaline phosphatase activity inhibition by theophylline to exclude the nonspecific hydrolysis of ATP as well as to establish the orientation of vesicles with the use of alamethicine. 98% of the Na,K-ATPase activity (0.99 +/- 0.031 mumol/mg protein/min) was found to be localized in basolateral membranes. Both the brush border and basolateral membranes were found to possess the Ca,Mg-ATPase (0.193 +/- 0.018 and 0.795 +/- 0.025 mumol/mg protein/min) and Mg-ATPase (0.22 +/- 0.013 and 0.403 +/- 0.022 mumol.mg protein/min) activities.     

Phospholipids and ATPase activities in the developing chick brain.

During the embryogenesis of chick brain an increase in the specific activities of Na,K- and Mg-ATPase takes place. We determined the quantities of the phospholipids phosphatidylserine, phosphatidylcholine, phosphatidylinositol, phosphatidylethanolamine and sphingomyelin in the developing chicken brain by quantitative thin-layer chromatography and found changes in their composition during the first 18 days after incubation. The increase in the Na,K-ATPase activity was proportional to the increase in phosphatidylserine and phosphatidylcholine; the increase in the Mg-ATPase activity was approximately proportional to the increase in phosphatidylethanolamine.     

Participation of Na, K-ATPase in the mechanism of isotonic fluid absorption by the epithelium of the frog gallbladder]

In experiments on the isolated frog gallbladders it was shown that addition of ouabain (1.10(-4) M) or noradrenaline 3.10(-6) M) into the incubation Ringer solution from the serous surface of the gallbladder and also replacement of K+ in the solution for the equivalent quantity of Na+ ions caused a reduction of the absorption of isotonic fluid by the epithelium and a fall of the Na,K-ATPase activity in its cells. Noradrenaline also cased a reduction of Mg-ATPase activity. A significant positive correlation was found between the transport rate of the isotonic fluid by the epithelium and the Na,K-ATPase activity in its cells.     

Effect of thyroid hormone on the distribution and activity of Na, K-ATPase in ventricular myocardium

Employing detergent-free sucrose-density gradient fractionation method we isolated cholesterol-rich lighter membrane fractions containing ∼10% of protein, ∼30% of cholesterol in membranes of ventricular myocardium. Cholesterol-rich lighter membrane fractions contain >70% of Na, K-ATPase and caveolins 1 and 3 and <10% of β-actin. Treatment of hypothyroid rats with T₃ increased the relative abundance of both α1 and β1 Na, K-ATPase subunits in total membranes by 4- to 5-fold (with no change in caveolin-3), and resulted in 1.9-fold increase in enzyme activity. T₃-induced Na, K-ATPase subunits were preferentially distributed to the lighter fractions (#s 4, 5 and 6); and increased abundance of α1 and β1 were 34-70% and 43-68%, respectively. We conclude that the activity of Na, K-ATPase is not uniform in cardiac membranes, and while a significant amount of Na, K-ATPase is present in cardiac cholesterol-rich membrane fractions, the intrinsic activity is significantly less than the enzyme present in relatively cholesterol-poor membranes.     

Effect of adrenaline on the ATPase activity of suslik brain synaptosomes]

Action of adrenaline on ATPase activity of ground squirrel synaptosomes in vitro at 37 degrees and 17 degrees C was studied. It has been shown in experiments in vitro at 37 degrees C that adrenaline in a concentration of 5.10(-4) M influenced Mg and Na, K-ATPase of the synaptosomes in ground squirrel brain. The inhibition (42-72%) of Na, K-ATPase in the synaptosomes of the brain was seen during hibernation and in summer. The inhibition of Mg-ATPase (50%) was observed only in summer. The effect of adrenaline on the activity of Na, K-ATPase of synaptosome was seen in vitro as well as at 17 degrees (a 50% inhibition). It was shown that adrenaline in vitro at a concentration of 5.10(-4) M inhibited ATPases more than noradrenaline.     

Differential Response of Adenylate Cyclase and ATPase Activities After Chronic Ethanol Exposure of PC12 Cells

The direct effects of chronic ethanol administration on adenylate cyclase, Na,K-ATPase, and Mg-ATPase activities in a cell containing neuronal characteristics were investigated using PC12 pheochromocytoma cells. Exposure of PC12 cells to 0, 75, and 150 mM ethanol for 4 days caused a dose-dependent increase in the stimulation of adenylate cyclase by in vitro ethanol without altering activation of the enzyme by GTP, NaF, MnCl2, or 2-chloroadenosine. Conversely, a 4-day treatment with 150 mM ethanol increased Na,K-ATPase and Mg-ATPase activities without altering the inhibitory effects of in vitro ethanol. The increase in Na,K-ATPase activity was associated with an increase in Vmax without any change in the Km for KCl. Chronic ethanol exposure also increased the amount of [3H]ouabain specifically bound to PC12 cell membranes. Except for the increase in Mg-ATPase activity, the above results were also observed when chronic ethanol treatment was carried out in the presence of pyrazole. Although ethanol slowed PC12 cell growth, observed changes were not due to an ethanol-induced reduction in cellular density. A 4-day exposure of a nonneuronal cell line (Madin Darby canine kidney cell) to 150 mM ethanol did not alter adenylate cyclase or ATPase activities. The present study indicates that the direct effects of chronic ethanol exposure of a neuronal-like cell involve an increase in the density of sodium pumps per cell and an enhanced sensitivity of adenylate cyclase to activation by ethanol.     

Characterization of Mg-ATPase and (Na+ + K+)-stimulated Mg-ATPase in smooth muscular cells of the sheep's common carotid artery.

The Mg-ATPase and (Na+ + K+)-stimulated Mg-ATPase in the mitochondrial and microsomal fraction of smooth muscular cells of the sheep's common carotid artery have been characterized in more detail. Optimal enzyme activities were found for all ATPases to be at pH 7.5-8.0 and 45 degrees C-50 degrees C. The energies of activation were found to be at 5-9 kcal/mole for both ATPases. Two-thirds of the (Na+ + K+)-stimulated Mg-ATPase were found to be ouabain-sensitive and thus attributed to the coupled (Na, K)-transport system. The pI 50 values of ouabain for microsomal and mitochondrial fractions are 6.3 and 6.0, respectively. The highest activity of (Na+ + K+)-stimulated Mg-ATPase is at 5-10 mM K+ and more than 50 mM Na+. One-third of the (Na+ + K+)-stimulated Mg-ATPase activity was found to be due to a stimulation of Mg-ATPase by Na+ alone, which is not inhibited by ouabain. The relationship of this activity to the ouabain-sensitive part of the (Na+ + K+)-stimulated Mg-ATPase and to Na+-transport is discussed. For the Mg-ATPases apparent KM(ATP) values were determined to be 1.4 and 1.0 mM, resp., and for the (Na+ + K+)-stimulated Mg-ATPases 0.15 and 0.14 mM, resp.