Increase of (Na+ + K+)-activated ATPase activity of basolateral plasma membranes from intestinal mucosa of diabetic rats

A significant increase of the (Na+ + K+)-activated ATPase was found in mucosal homogenates of rat small intestine under conditions of alloxan and streptozotocin diabetes. From studies with isolated plasma membranes it has been shown that the activity changes were caused by that part of the (Na+ + K+)-activated ATPase only which is localized in the basolateral plasma membranes, whereas the enzyme activity in the brush border region remains unchanged. In connection with the enhanced capacity of ion, nonelectrolyte and water absorption in experimental diabetes, our findings support a concept of intestinal transport mechanism which suggest that the basolateral part of the (Na+ + K+)-activated ATPase is responsible for metabolic energy supply. The luminal part of the enzyme may be involved in regulation of passive Na+ influx.     

Detection of a Na+-activated, furosemide-inhibited ATPase activity in rat intestinal mucosa

An ouabain-insensitive, Mg++-dependent, Na+-stimulated ATPase activity which is inhibited by furosemide was found in mucosal homogenate of rat small intestine. The subcellular localization of this ATPase activity was studied by means of isolated purified brush borders and basolateral plasma membranes. The results suggest a nearly identical distribution of Na+-activated and (Na+K+)-activated ATPase within the epithelial cells. Under conditions of alloxan and streptozotocin diabetes an increase of both ATPase activities can be found only in the basolateral plasma membranes. These observations agree well with the convective model of intestinal absorption.     

Subcellular localization of (Na+ + K+)-activated ATPase in the brush border membrane of the mucosal cell of the rat small intestine.

The localization of (Na+ + K+)-activated ATPase was investigated in isolated brush borders of rat small intestinal mucosa. The purity of the fractions has been checked by morphological and enzymatic criteria. The brush borders were found to contain a significant quantity of (Na+ + K+)-activated ATPase. Separation of isolated brush borders into their substructures suggests that (Na+ + K+)-activated ATPase is localized deeper within the brush border region than invertase. These findings are discussed in relation to active monosaccharide transport in the intestine.     

Partial purification of (Ca2+ + Mg2+)-dependent ATPase from pig smooth muscle and reconstitution of an ATP-dependent Ca2+-transport system.

(CaMg)ATPase [(Ca2+ + Mg2+)-dependent ATPase] was partially purified from a microsomal fraction of the smooth muscle of the pig stomach (antrum). Membranes were solubilized with deoxycholate, followed by removal of the detergent by dialysis. The purified (CaMg)ATPase has a specific activity (at 37 degrees C) of 157 +/- 12.1 (7)nmol.min-1.mg-1 of protein, and it is stimulated by calmodulin to 255 +/- 20.9 (7)nmol.min.mg-1. This purification of the (CaMg)ATPase resulted in an increase of the specific activity by approx. 18-fold and in a recovery of the total enzyme activity of 55% compared with the microsomal fraction. The partially purified (CaMg)ATPase still contains some Mg2+-and (Na+ + K+)-dependent ATPase activities, but their specific activities are increased relatively less than that of the (CaMg)ATPase. The ratios of the (CaMg)ATPase to Mg2+- and (Na+ + K+)-dependent ATPase activities increase from respectively 0.14 and 0.81 in the crude microsomal fraction to 1.39 and 9.07 in the purified preparation. During removal of the deoxycholate by dialysis, vesicles were reconstituted which were capable of ATP-dependent Ca2+ transport.     

Effect of ovarian steroids on membrane ATPase activities in microsomes (microsomal fractions) from rat myometrium. Inhibition of a component of the Mg2+-activated ATPase by Ca2+-calmodulin and by oxytocin.

The activities of Mg2+-ATPase (Mg2+-activated ATPase), (Ca2+ + Mg2+)-activated ATPase and (Na+ + K+)-activated ATPase have been determined in microsomes (microsomal fractions) obtained from rat myometrium under different hormonal conditions. Animals were either ovariectomized and treated for a prolonged period of time with 17 beta-oestradiol or progesterone, or myometria were obtained at day 21 of pregnancy. In each case the endometrium was carefully removed. The Mg2+-ATPase consists of two components: an inactivating labile component and a second constant component. The rate of ATP hydrolysis by the labile component of the Mg2+-ATPase declines exponentially as a function of time after adding the membranes to the assay medium; this inactivation is caused by the presence of ATP in the medium. This ATPase activity inhibited by ATP is catalysed by a labile enzyme and hence it gradually diminishes within a few hours, even when the microsomes are kept on ice. This labile component has the highest activity in microsomes from pregnant rats, a lower activity in progesterone-treated rats, and the lowest in 17 beta-oestradiol-treated rats. This component of the Mg2+-ATPase is not affected by 90 nM-oxytocin. The constant component of the Mg2+-ATPase must be ascribed to a different enzyme, which, in contrast with the labile component, is very stable and not affected by the hormonal status of the animal. This constant component of the Mg2+-ATPase is inhibited both by Ca2+-calmodulin, and by oxytocin in microsomes from pregnant and from progesterone-treated animals, whereas such inhibition does not occur in microsomes from 17 beta-oestradiol-treated animals. The activity of the (Na+ + K+)-activated ATPase is not dependent on the hormonal status of the animal. Myometrial microsomes present an ATP-dependent Ca2+ transport, irrespective of the hormonal condition, but only in microsomes obtained from rats treated with 17 beta-oestradiol, can a (Ca2+ + Mg2+)-activated ATPase activity be demonstrated. This activity can be stimulated by calmodulin.     

Properties of (Na+ plus K+)-activated ATPase in rat liver plasma membranes enriched with bile canaliculi.

Liver plasma membranes enriched in bile canaliculi were isolated from rat liver by a modification of the technique of Song et al. (J. Cell Biol. (1969) 41, 124-132) in order to study the possible role of ATPase in bile secretion. Optimum conditions for assaying (Na+ plus K+)-activated ATPase in this membrane fraction were defined using male rats averaging 220 g in weight. (Na+ plus K+)-activated ATPase activity was documented by demonstrating specific cation requirements for Na+ and K+, while the divalent cation, Ca(2+), and the cardiac glycosides, ouabain and scillaren, were inhibitory. (Na+ plus K+)-activated ATPase activity averaged 10.07 plus or minus 2.80 mumol Pi/mg protein per h compared to 50.03 plus or minus 11.41 for Mg(2+)-activated ATPase and 58.66 plus or minus 10.07 for 5'-nucleotidase. Concentrations of ouabain and scillaren which previously inhibited canalicular bile secretion in the isolated perfused rat liver produced complete inhibition of (Na+ plus K+)-activated ATPase without any effect on Mg(2+)-activated ATPase. Both (Na+ plus K+)-activated ATPase and Mg(2+)-activated ATPase demonstrated temperature dependence but differed in temperature optima. Temperature induced changes in specific activity of (Na+ plus K+)-activated ATPase directly paralleled previously demonstrated temperature optima for bile secretion. These studies indicate that (Na+ plus K+)-activated ATPase is present in fractions of rat liver plasma membranes that are highly enriched in bile canaliculi and provide a model for further study of the effects of various physiological and chemical modifiers of bile secretion and cholestasis.     

Disulfide of thionosine triphosphate, an ATP-analog inactivating (Na+ + K+)-ATPase.

(Na+ + K+)-activated ATPase in beef brain microsomes is inactivated by the disulfide of thionosine tri[gamma-32P]phosphate, an ATP analog. The inactivation of the enzyme, which is accompanied by an incorporation of radioactivity into the membrane protein, is abolished by ATP or dithiothreitol. Since dithiothreitol restores the activity of (Na+ + K+)-ATPase, which had previously been inactivated by this ATP analog, it is concluded that thionosine triphosphate disulfide reacts with a sulfhydryl group in the ATP binding site of (Na+ + K+)-activated ATPase.     

Axonal transport of [3H]serotonin in an identified neuron of Aplysia californica

The distribution of (Na+ + K+) ATPase over the plasma membranes of the proximal convoluted tubule from canine renal cortex has been determined. Ultrathin frozen sections of this tissue were stained with rabbit antibodies to this enzyme and ferritin-conjugated goat antirabbit gamma-globulin. It is demonstrated that high concentrations of this enzyme uniformly line the intercellular spaces of this epithelium. The consequences of this observation are discussed in terms of the low resistant tight junctions of these tubules and the isotonic fluid transport which they support. Furthermore, antibodies to (Na+ + K+) ATPase recognize an antigen on the luminal surfaces of the tubules within the brush border. It is proposed that the enzyme is present in this region of the plasma membrane as well, although at much lower concentration. To further substantiate this conclusion, a brush border fraction has been purified from rabbit kidney and been shown to contain significant (Na+ + K+) ATPase. These results contradict earlier conclusions about the location of (Na+ + K+) ATPase in this tissue.     

Characterization of Na+/K+-ATPase from Xenopus laevis kidney. Preliminary results

In Xenopus laevis, the renal Na+/K+-dependent ATPase is a very important enzyme involved in osmoregulatory processes and active transport. The enzyme was obtained from a microsome fraction purified by sucrose discontinuous gradient (10%, 15%, 29.4%) ultracentrifugation after SDS treatment, and concentrated in the denser layer. The assayed biochemical parameters and their values are: 1) Km (ATP): 0.24 mM; 2) K1/2 (Na+): 20.6 mM; 3) K1/2 (K+) 1.6 mM; 4) Ki (ouabain): 0.025 micrometer; 5) optimum pH: 7.2; 6) optimum temperature:" two peaks at 37 degrees C and 45 degrees C.     

Functionally distinct classes of K+ sites on the (Na+ + K+)-dependent ATPase.

K+ interactions with a rat brain (Na+ + K+)-dependent ATPase and the associated K+-dependent nitrophenyl phosphatase activity were examined. Classes of sites for K+ were distinguished, initially, on the basis of affinity estimated by kinetic analysis in terms of KO.5 (the concentration for half-maximal activation), and by K+-accelerated enzyme inactivation by F-minus, which permits evaluation of a dissociation constant for K+, KD. Moderate-affinity sites ("alpha sites"), with a KD near 1 mM, were demonstrable for the phosphatase activity and for the "free" enzyme. High-affinity sites ("beta sites"), with a KD near 0.1 mM, were seen for the overall ATPase activity and under conditions in which enzyme phosphorylation by substrate also occurs. Further differentiation between alpha and beta sites was made in terms of (i) the characteristic changes in affinity with pH, and (ii) the efficacy of Li+ relative to K+, Rb+, Cs+, and Tl+ at these two classes of sites. Low-affinity sites ("gamma sites") through which K+ inhibits enzymatic activity were also detectable, with a KD around 140 mM. These data are incorporated into a model for the reaction sequence to accommodate both transport processes and certain K+/ATP antagonisms.     

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.     

Changes in the levels of chloride cells and (Na+ + K+)-dependent ATPase in the gills of yellow and silver eels adapting to seawater.

Changes were measured in the numbers of chloride cells and the levels of (Na+ + K+)-DEPENDENT ATPase in the gills of immature, yellow eels and mature, silver eels during adaptation from freshwater to seawater. The percentage of chloride cells in yellow eels more than doubled after six days in seawater; at this time the specific activity and concentration of (Na+ + K+)-dependent ATPase in gills start to increase in parallel to reach maxima after two weeks that are 2.5 times the starting values. It is concluded that adaptation of yellow eels to seawater involves an increase in the numbers of chloride cells in gills as well as an increased amount of (Na+ + K+)-dependent ATPase per chloride cell. Mature silver eels in freshwater had essentially the same numbers of chloride cells and the same specific activity of the enzyme in the gills as yellow eels fully adapted to seawater. Transferring silver eels to seawater did not alter the percentage of chloride cells in gills although the level of (Na+ + K+)-dependent ATPase and its specific activity increased slightly. Thus, although the silver eel is better prepared for life in seawater than the yellow eel, it still has to attain an increased level of (Na+ + K+)-dependent ATPase in its chloride cells to be fully adapted to seawater.     

No major thermogenic role for (Na+ + K+)-dependent adenosine triphosphatase apparent in hepatocytes from hyperthyroid rats.

(Na+ + K+)-dependent ATPase activity, heat production and oxygen consumption were increased by 59%, 62% and 75% respectively in hepatocytes from tri-iodothyronine-treated rats. Ouabain at concentrations of 1 and 10 mM decreased oxygen uptake by 2--8% in hepatocytes from euthyroid rats and by 5--15% in hepatocytes from hyperthyroid animals. Heat output was decreased by 4--9% with the glycoside in isolated liver parenchymal cells from the control animals and by 11% in the cells from the tri-iodothyronine-treated animals. These results do not support the hypothesis that hepatic (Na+ + K+)-ATPase plays a major role in increased heat production in hepatocytes from hyperthyroid rats.     

AlF4- reversibly inhibits ''P''-type cation-transport ATPases, possibly by interacting with the phosphate-binding site of the ATPase.

The only known cellular action of AlF4- is to stimulate the G-proteins. The aim of the present work is to demonstrate that AlF4- also inhibits 'P'-type cation-transport ATPases. NaF plus AlCl3 completely and reversibly inhibits the activity of the purified (Na+ + K+)-ATPase (Na+- and K+-activated ATPase) and of the purified plasmalemmal (Ca2+ + Mg2+)-ATPase (Ca2+-stimulated and Mg2+-dependent ATPase). It partially inhibits the activity of the sarcoplasmic-reticulum (Ca2+ + Mg2+)-ATPase, whereas it does not affect the mitochondrial H+-transporting ATPase. The inhibitory substances are neither F- nor Al3+ but rather fluoroaluminate complexes. Because AlF4- still inhibits the ATPase in the presence of guanosine 5'-[beta-thio]diphosphate, and because guanosine 5'-[beta gamma-imido]triphosphate does not inhibit the ATPase, it is unlikely that the inhibition could be due to the activation of an unknown G-protein. The time course of inhibition and the concentrations of NaF and AlCl3 required for this inhibition differ for the different ATPases. AlF4- inhibits the (Na+ + K+)-ATPase and the plasmalemmal (Ca2+ + Mg2+)-ATPase noncompetitively with respect to ATP and to their respective cationic substrates, Na+ and Ca2+. AlF4- probably binds to the phosphate-binding site of the ATPase, as the Ki for inhibition of the (Na+ + K+)-ATPase and of the plasmalemmal (Ca2+ + Mg2+)-ATPase is shifted in the presence of respectively 5 and 50 mM-Pi to higher concentrations of NaF. Moreover, AlF4- inhibits the K+-activated p-nitrophenylphosphatase of the (Na+ + K+)-ATPase competitively with respect to p-nitrophenyl phosphate. This AlF4- -induced inhibition of 'P'-type cation-transport ATPases warns us against explaining all the effects of AlF4- on intact cells by an activation of G-proteins.     

Inhibition of Na+,K+-ATPase in Penaeus indicus postlarvae by lead

The plasma membrane/mitochondrial fractions of Penaeus indicus postlarvae contain Mg2+-dependent ATPase, Na+,K+-stimulated ATPase, Na+-stimulated ATPase and K+-stimulated ATPase. The Na+,K+-activated, Mg2+-dependent ATPase was investigated further in relation to different pH and temperature conditions, and at various concentrations of protein, ouabain, ATP and ions in the incubation medium. In vitro and in vivo effects of lead were studied on the enzyme activity. In vitro lead inhibited the enzyme activity in a concentration-dependent manner with an IC50 value of 204.4 microM. In correlation with in vitro studies, in vivo investigations (both concentration and time dependent) of lead also indicated a gradual inhibition in enzyme activity. A maximum decrease of 85.3% was observed at LC50 (7.2 ppm) of lead for concentration-dependent experiments. In time-dependent studies, the decrease was maximal (81.7%) at 30 days of sublethal exposure (1.44 ppm). In addition, the substrate- and ion-dependent kinetics of Na+,K+-ATPase was studied in relation to in vitro exposure of lead; these studies suggest a non-competitive type of inhibition.     

Effects of N-acetylimidazole on human erythrocyte ATPase activity. Evidence for a tyrosyl residue at the ATP binding site of the (Na+ plus K+)-dependent ATPase.

1. Acetylation of human erythrocytes by N-acetylimidazole alters the structure of stroma prepared from these cells and the degree of alteration appears to be dependent upon the level of the initial treatment. These changes do not occur when stroma are acetylated. 2. Deacetylation by hydroxylamine or mild alkaline treatment causes a complete recovery of the (Na+ plus K+)-dependent and the Ca2+ -stimulated ATPase activities and indicates that the inhibition is due to the acetylation of a tyrosyl residue. There is only partial recovery of the Mg2+ -dependent ATPase after deacetylation. 3. ATP or Mg-ATP completely protect the (Na+ plus K+)-dependent ATPase, but not the Ca2+ -stimulated system. 4. The results indicate that the (Na+ plus K+)-dependent and the Ca2+ -stimulated ATPase activities have separate substrate binding sites and most likely are separate enzyme systems. 5. Acetylation of human erythrocytes has no effect on D-glucose transport.     

Mechanisms of detergent effects on membrane-bound (Na+ + K+)-ATPase

Because the nonionic detergent octaethylene glycol dodecyl ether has been used extensively for studies on active solubilized preparations of (Na+ + K+)-ATPase, we tried to see if the detergent alters the properties of the membrane-bound enzyme prior to solubilization. Addition of the detergent, at concentrations below its critical micellar concentration, to reaction mixtures containing the highly purified membrane-bound enzyme reduced the K0.5 of ATP for (Na+ + K+)-dependent ATPase activity without affecting the maximal velocity or abolishing the negative cooperativity of the substrate-velocity curve. Under these conditions, however, the enzyme was not solubilized as evidenced by complete sedimentation of the membrane fragments containing the enzyme upon centrifugation at 100,000 X g for 30 min. Other nonsolubilizing effects of the detergent included an increase in K0.5 of K+, inhibition of Na+-dependent ATPase with no effect on K0.5 of ATP for this activity, and reductions in the spontaneous decomposition rates of the K+-sensitive phosphoenzyme obtained from ATP and the phosphoenzyme obtained from Pi. The nonsolubilizing effects of the detergent on the purified enzyme were obtained with no detectable lag, were readily reversible, and could be distinguished from its vesicle-opening effects on crude membrane preparations. Several other nonionic and ionic detergents had similar effects on the enzyme. The findings indicate (a) detergent binding to hydrophobic sites on extramembranous segments of enzyme subunits; (b) that occupation of these sites mimics the effects of ATP at a low-affinity regulatory site with no effect on high-affinity ATP binding to the catalytic site; and (c) that in studies on detergent-solubilized preparations, it is necessary to distinguish between the effects of solubilization per se and detergent effects at the regulatory site.     

Regulation of rat brain (Na+ +K+)-ATPase activity by cyclic AMP

The interaction between the (Na+ +K+)-ATPase and the adenylate cyclase enzyme systems was examined. Cyclic AMP, but not 5'-AMP, cyclic GMP or 5'-GMP, could inhibit the (Na+ +K+)-ATPase enzyme present in crude rat brain plasma membranes. On the other hand, the cyclic AMP inhibition could not be observed with purified preparations of (Na+ +K+)-ATPase enzyme. Rat brain synaptosomal membranes were prepared and treated with either NaCl or cyclic AMP plus NaCl as described by Corbin, J., Sugden, P., Lincoln, T. and Keely, S. ((1977) J. Biol. Chem. 252, 3854-3861). This resulted in the dissociation and removal of the catalytic subunit of a membrane-bound cyclic AMP-dependent protein kinase. The decrease in cyclic AMP-dependent protein kinase activity was accompanied by an increase in (Na+ +K+)-ATPase activity. Exposure of synaptosomal membranes containing the cyclic AMP-dependent protein kinase holoenzyme to a specific cyclic AMP-dependent protein kinase inhibitor resulted in an increase in (Na+ +K+)-ATPase enzyme activity. Synaptosomal membranes lacking the catalytic subunit of the cyclic-AMP-dependent protein kinase did not show this effect. Reconstitution of the solubilized membrane-bound cyclic AMP-dependent protein kinase, in the presence of a neuronal membrane substrate protein for the activated protein kinase, with a purified preparation of (Na+ +K+)-ATPase, resulted in a decrease in overall (Na+ +K+)-ATPase activity in the presence of cyclic AMP. Reconstitution of the protein kinase alone or the substrate protein alone, with the (Na+ +K+)-ATPase has no effect on (Na+ +K+)-ATPase activity in the absence or presence of cyclic AMP. Preliminary experiments indicate that, when the activated protein kinase and the substrate protein were reconstituted with the (Na+ +K+)-ATPase enzyme, there appeared to be a decrease in the Na+-dependent phosphorylation of the Na+-ATPase enzyme, while the K+-dependent dephosphorylation of the (Na+ +K+)-ATPase was unaffected.     

A model for the reaction pathways of the K+-dependent phosphatase activity of the (Na+ + K+)-dependent ATPase

(Na+ + K+)-dependent ATPase preparations from rat brain, dog kidney, and human red blood cells also catalyze a K+ -dependent phosphatase reaction. K+ activation and Na+ inhibition of this reaction are described quantitatively by a model featuring isomerization between E1 and E2 enzyme conformations with activity proportional to E2K concentration: (formula; see text) Differences between the three preparations in K0.5 for K+ activation can then be accounted for by differences in equilibria between E1K and E2K with dissociation constants identical. Similarly, reductions in K0.5 produced by dimethyl sulfoxide are attributable to shifts in equilibria toward E2 conformations. Na+ stimulation of K+ -dependent phosphatase activity of brain and red blood cell preparations, demonstrable with KCl under 1 mM, can be accounted for by including a supplementary pathway proportional to E1Na but dependent also on K+ activation through high-affinity sites. With inside-out red blood cell vesicles, K+ activation in the absence of Na+ is mediated through sites oriented toward the cytoplasm, while in the presence of Na+ high-affinity K+ -sites are oriented extracellularly, as are those of the (Na+ + K+)-dependent ATPase reaction. Dimethyl sulfoxide accentuated Na+ -stimulated K+ -dependent phosphatase activity in all three preparations, attributable to shifts from the E1P to E2P conformation, with the latter bearing the high-affinity, extracellularly oriented K+ -sites of the Na+ -stimulated pathway.     

Effect of starvation, alloxan diabetes and adrenalectomy on Na+ K+-ATPase of the mucosa of the small intestine of rat.

The dietary stress conditions such as starvation influenced Na+K+-ATPase activity which increased steadily above normal fed levels between the starvation periods of 24--48 hr. Also, an increased enzyme level was observed in alloxan diabetic rats and administration of insulin to diabetic rats led to a tendency towards a lowering of Na+K+-ATPase. Adrenalectomy brought about a lowering of Na+K+-ATPase activity from those of normals while the administration of hydrocortisone induced an enhancement. The results indicate that both starvation and diabetic conditions might cause a stress-like activation of adrenal cortex resulting in increased levels of glucocorticoids which in turn activate the intestinal Na+K+-ATPase activity.