Cytochemical demonstration of NPPase activity for detecting proton-translocating ATPase of Golgi complex in rat pancreatic acinar cells

An attempt at cytochemical demonstration of acidification proton-translocating ATPase (H(+)-ATPase) of Golgi complex in rat pancreatic acinar cells has been made by using p-nitrophenylphosphatase (NPPase) cytochemistry which is used for detecting of Na(+)-K(+)-ATPase (Mayahara et al. 1980) and gastric H(+)-K(+)-ATPase (Fujimoto et al. 1986). K(+)-independent NPPase activity was observed on the membrane of the trans cisternae of Golgi complex, but not inside of cisternae. The localization of NPPase activity is different from that of acid phosphatase activity where reaction products were seen on the inside of the trans Golgi cisternae. Since this activity was insensitive to vanadate, ouabain and independent of potassium ions, it was distinct from plasma membranous ATPases such as Na(+)-K(+)-ATPase and Ca2(+)-ATPase. The K(+)-independent NPPase activity was diminished by the inhibitors of H(+)-ATPase such as N-ethylmaleimide (NEM) and 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS). The NPPase reaction products were also seen on the membranes of other acidic organelles, i.e., lysosomes, endosomes, autophagosomes and coated vesicles. These results suggest that NPPase activity on the membrane of the Golgi complex and other acidic organelles corresponds with H(+)-ATPase which plays a role in acidification.     

Development of (Na+ -K+)-ATPase in rat hindbrain: increments in parallel with Na+-dependent phosphorylation and K+-pnitrophenylphosphatase.

Rat hindbrain NaI-enriched microsomal (Na⁺-K⁺)-ATPase activity, K⁺-pNPPase activity, and Na⁺-dependent steady-state phosphorylation levels all increase approx 10-fold relative to microsomal protein between 5 days prenatally and 60 days postnatally. These activities, as well as the mean wet weight of the hindbrain, are at half of their 60 day values shortly after the 10th postnatal day. For all ages, these hindbrain activities average over twice those found in the forebrain in a companion paper (Bertoni & Siegel , 1978). Increases during development in the amount of the related phosphorylatable polypeptide, estimated by densitometry of stained polyacrylamide gels containing fixed amounts of microsomal protein dissolved in SDS, are in agreement with increases in steady state levels of Na⁺-dependent phosphorylation. The fraction of total phosphorylation that is Na⁺-dependent rises steadily during development consistent with, but not obligatorily due to, a conversion of some of the previously Na⁺-independent portion. Mg2⁺-ATPase and Mg²⁺ -pNPPase activities and steady-state Na⁺,-independent phosphorylation levels do not increase in parallel during development. These observations add further support to the proposed partial reaction scheme for (Na⁺- K⁺)-ATPase. The major increments in (Na ⁺-K⁺)-ATPase occur simultaneously with the deposition of specialized plasma membranes, particularly in the molecular layer of the cerebellum, as described in previous studies of rat hind brain.     

Development of (Na+-K+)-ATPase in rat cerebrum: correlation with Na+-dependent phosphorylation and K+-paranitrophenylphosphatase.

Abstract— The activities of (Na⁺ K⁺)-ATPase and its proposed partial reactions, K ⁺-pNPPase and Na ⁺-dependent phosphorylation, all increase tenfold relative to microsomal protein between 5 days prior to birth and 60 days postnatally in NaI-treated rat cerebral microsomes, and all reach half of their adult values between the fifth and tenth postnatal day. These increases are concurrent with the most rapid changes in cerebral wet weight. Increases in the amount of the related phosphorylatable polypeptide during development. as estimated by densitometry of Coomassie-stained polyacrylamide gels after electrophoresis of constant amounts of microsomal protein dissolved in sodium dodecylsulfate, parallel the increments in levels of Na ⁺-dependent phosphorylation. The fraction of total phosphorylation that is Na ⁺-dependent increases steadily during development. suggesting a precursor role for some of the Na ⁺-independent fraction. The results are consistent with a single biosynthetic control for the enzymatic sites critical to the partial reactions of (Na ⁺-K ⁺)-ATPase. No changes in turnover number or affinity for substrate or ligands were found during development. Little similarity was noted among the age-related changes of Mg ²⁺ -ATPase activity. Mg ²⁺ -paranitrophenylphosphatase activity, and Na⁺-independent phosphorylation levels. The most rapid changes in (Na⁺-K⁺)-ATPase take place during the period corresponding to glial proliferation and neuronal arborization.     

Sulfatide and Na<Superscript>+</Superscript>-K<Superscript>+</Superscript>-ATPase: A Salinity-sensitive Relationship in the Gill Basolateral Membrane of Rainbow Trout

We investigated the effect of salinity on the relationship between Na⁺-K⁺-ATPase and sulfogalactosyl ceramide (SGC) in the basolateral membrane of rainbow trout (Oncorhynchus mykiss) gill epithelium. SGC has been implicated as a cofactor in Na⁺-K⁺-ATPase activity, especially in Na⁺-K⁺-ATPase rich tissues. However, whole-tissue studies have questioned this role in the fish gill. We re-examined SGC cofactor function from a gill basolateral membrane perspective. Nine SGC fatty acid species were quantified by tandem mass spectrometry (MS/MS) and related to Na⁺-K⁺-ATPase activity in trout acclimated to freshwater or brackish water (20 ppt). While Na⁺-K⁺-ATPase activity increased, the total concentration and relative proportion of SGC isoforms remained constant between salinities. However, we noted a negative correlation between SGC concentration and Na⁺-K⁺-ATPase activity in fish exposed to brackish water, whereas no correlation existed in fish acclimated to freshwater. Differential Na⁺-K⁺-ATPase/SGC sensitivity is discussed in relation to enzyme isoform switching, the SGC cofactor site model and saltwater adaptation.This revised version was published online in June 2005 with a corrected cover date.     

The expression of gastric H+-K+-ATPase mRNA and protein in developing rat fundic gland

The proton pump H⁺-K⁺-ATPase is the final common pathway mediating the production and secretion of hydrochloric acid by gastric parietal cells. The present studies were undertaken to examine whether the expression of gastric H⁺-K⁺-ATPase mRNA and protein changes are associated with the development of H⁺-K⁺-ATPase activity in the rat fundic gland. H⁺-K⁺-ATPase activity was examined in rat fundic gland at different stages from gestational day 18.5 to postnatal 8 weeks. The expression of H⁺-K⁺-ATPase mRNA was detected by in situ hybridization using a digoxigenin-labelled RNA probe with a tyramide signal amplification system. The expression of H⁺-K⁺-ATPase protein was evaluated by immunoblotting and immunohistochemistry using antibodies against H⁺-K⁺-ATPase - and -subunits. We found that H⁺-K⁺-ATPase enzyme activity was detectable from the onset of gland formation (day 19.5 of gestation) and increased with age in the developing rat fundic gland. Expression of mRNA and protein was also discernible at the same time, and a progressive increase in expressions was observed as rats developed. Our results suggested that in developing rat fundic gland, the expression of both mRNA and protein of H⁺-K⁺-ATPase increased with age in a manner that parallels the development of H⁺-K⁺-ATPase enzymebreak activity.     

Rat hypothalamic extract inhibits vasopressin-stimulated Na+-K+-ATPase activity in the rat renal medulla

Arginine vasopressin stimulates Na⁺-K⁺-ATPase activity located in the rat thick ascending limb of s'Henle loop. Mammalian hypothalamus appears to produce a factor capable of inhibiting Na⁺-K⁺-ATPase activity in a variety of tissues. The effect of a purified rat hypothalamic extract with and without AVP on rat renal Na⁺-K⁺-ATPase activity was evaluated by a cytochemical technique. The hypothalamic extract alone failed to affect basal Na⁺-K⁺-ATPase activity throughout renal segments after 10 min exposure. Na⁺-K⁺-ATPase activity stimulated by AVP (1–10 fmol l^?1) for 10 min was inhibited by rat hypothalamic extract over the concentration range 10^?7–10^?3 U ml^?1 in a dose-dependent manner. Complete inhibition of AVP-stimulated Na⁺-K⁺-ATPase activity occurred at a hypothalamic extract concentration of 10^?3 U ml^?1. Only Na⁺-K⁺-ATPase activity located in the renal medullary thick ascending limb was influenced by the rat hypothalamic extract.     

Modification of the erythrocyte membrane by sulfhydryl group reagents

Summary In this study, the consequences of modification of human erythrocyte membrane sulfhydryl groups by N-ethyl maleimide (NEM), 5,5dithiobis-(2-nitrobenzoic acid) (DTNB) andp-hydroxymercuriphenyl sulfonate (PHMPS) were investigated. These reagents differ in chemical reactivity, membrane penetrability and charge characteristics.Results of sulfhydryl modification were analyzed in terms of inhibitory effects on activities of five membrane enzymes; Mg⁺⁺- and Na⁺, K⁺-ATPase, K⁺-dependent and independentp-nitrophenyl phosphatase (NPPase) and DPNase. Structural considerations involved in the sulfhydryl-mediated inhibition were evaluated by studying the changes in susceptibility to sulfhydryl alteration produced by shearing membranes into microvesicles and by the addition of the membrane modifiers, Mg⁺⁺ and ATP.Conclusions from the data suggest that the effects of NEM appeared to result from modification of a single class of sulfhydryls; DTNB interacted with two different sulfhydryl classes. Increasing concentrations of PHMPS resulted in the sequential modification of many types of sulfhydryls, presumably as a result of increasing membrane structural disruption. DTNB and PHMPS caused solubilization of about 15% of membrane protein at concentrations giving maximal enzyme inhibition.In contrast to the usually observed parallels between Na⁺, K⁺-ATPase and K⁺-dependent NPPase, activities of Mg⁺⁺-ATPase, Na⁺, K⁺-ATPase and K⁺-dependent NPPase varied independently as a result of sulfhydryl modification. We suggest complex structural and functional relationships exist among these components of the membrane ATP-hydrolyzing system.Our studies indicate that the effects of sulfhydryl group reagents on these membrane systems should not be ascribed to sulfhydryl modificationper se, but rather to the resulting structural perturbations. These effects depend upon the structural characteristics of the particular membrane preparation studied and on the chemical characteristics of the sulfhydryl group reagent used.     

Effect of Hypoxia on Na+-K+-Cl− Cotransport in Cultured Brain Capillary Endothelial Cells of the Rat

Abstract: The effect of hypoxia on Na⁺,K⁺-ATPase and Na⁺-K⁺-Cl^? cotransport activity in cultured rat brain capillary endothelial cells (RBECs) was investigated by measuring ⁸⁶Rb⁺ uptake as a tracer for K⁺. RBECs expressed both Na⁺,K⁺-ATPase and Na⁺-K⁺-Cl^? cotransport activity (4.6 and 5.5 nmol/mg of protein/min, respectively). Hypoxia (24 h) decreased cellular ATP content by 43.5% and reduced Na⁺,K⁺-ATPase activity by 38.9%, whereas it significantly increased Na⁺-K⁺-Cl^? cotransport activity by 49.1% in RBECs. To clarify further the mechanism responsible for these observations, the effect of oligomycin-induced ATP depletion on these ion transport systems was examined. Exposure of RBECs to oligomycin led to a time-dependent decrease of cellular ATP content (by ～65%) along with a complete inhibition of Na⁺,K⁺-ATPase and a coordinated increase of Na⁺-K⁺-Cl^? cotransport activity (up to 100% above control values). Oligomycin augmentation of Na⁺-K⁺-Cl^? cotransport activity was not observed in the presence of 2-deoxy-d -glucose (a competitive inhibitor of glucose transport and glycolysis) or in the absence of glucose. These results strongly suggest that under hypoxic conditions when Na⁺,K⁺-ATPase activity is reduced, RBECs have the ability to increase K⁺ uptake through Na⁺-K⁺-Cl^? cotransport.     

Carotenoid oxidative degradation products inhibit Na+-K+-ATPase

This study investigates the biological significance of carotenoid oxidation products using inhibition of Na⁺-K⁺-ATPase activity as an index. β-Carotene was completely oxidized by hypochlorous acid and the oxidation products were analyzed by capillary gasliquid chromatography and high performance liquid chromatography. The Na⁺-K⁺-ATPase activity was assayed in the presence of these oxidized carotenoids and was rapidly and potently inhibited. This was demonstrated for a mixture of β-carotene oxidative breakdown products, β-Apo-10′-carotenal and retinal. Most of the β-carotene oxidation products were identified as aldehydic. The concentration of the oxidized carotenoid mixture that inhibited Na⁺-K⁺-ATPase activity by 50% (IC₅₀) was equivalent to 10μM non-degraded β-carotene, whereas the IC₅₀ for 4-hydroxy-2-nonenal, a major lipid peroxidation product, was 120 μM. Carotenoid oxidation products are more potent inhibitors of Na⁺-K⁺-ATPase than 4-hydroxy-2-nonenal. Enzyme activity was only partially restored with hydroxylamine and/or β-mercaptoethanol. Thus, in vitro binding of carotenoid oxidation products results in strong enzyme inhibition. These data indicate the potential toxicity of oxidative carotenoid metabolites and their activity on key enzyme regulators and signal modulators.     

Studies on (Na+-K+)-activated ATPase XXXIV. Phosphatidylserine not essential for (Na+-K+)-ATPase activity

An (Na⁺-K⁺)-ATPase preparation, consisting of NaI-treated microsomes from cattle brain, was incubated with a phosphatidylserine decarboxylase preparation from Escherichia coli. This led to a reduction in the phosphatidylserine content from 10.1 % to less than 0.1%, accompanied by an equimolar formation of phosphatidylethanolamine. Since the (Na⁺-K⁺)-ATPase activity was not reduced, it can be concluded that phosphatidylserine is not essential for the Na⁺-K⁺)-ATPase activity.     

Effects of batrachotoxin on electroplax Na + -K + -ATPase and levels of ATP in rat muscle

—Batrachotoxin (BTX) in low concentrations (20 nm ) depolarizes electrically excitable membranes (Albuquerque , Daly and Witkop , 1971). At these levels, BTX does not inhibit Na⁺-K⁺-ATPase. At much higher concentrations (60 μm ) BTX partially inhibits Na⁺-K⁺-ATPase from electroplax of Electrophorus electricus. In contrast to inhibition by cardiac glycosides, the inhibition of Na⁺-K⁺-ATPase by batrachotoxin is not antagonized by KCl. BTX had no effect on ATP levels in stimulated nerve muscle preparations at the time when sustained contracture was initiated by the drug. Phosphocreatine levels were decreased and levels of glucose-6-phosphate and 6-phosphogluconate were increased, while levels of fructose-1,6-diphosphate and α-ketoglutarate were unchanged. It is concluded that the inhibition of Na⁺-K⁺-ATPase or lowering of ATP levels by BTX is not significantly involved in the membrane depolarization produced by the toxin.     

Effects of salt-adaptation and salt-stress on extracellular acidification and microsome phosphohydrolase activities in tomato cell suspensions

The effects of NaCl-adaptation and NaCl-stress on in vivo H⁺ extrusion and microsomal vanadate- and bafilomycin-sensitive ATPase and PPase activities were studied in tomato cell suspensions. Acidification of the external medium by 50 mM NaCl-adapted and non-adapted (control) tomato cells was similar. Extracellular acidification by both types of cells during the first hour of incubation with 2 μM fusicoccin (FC) in the presence of 100 mM NaCl was lightly increased while in the presence of 100 mM KCl it was increased by 3 (control)- and 6.5 (adapted)-fold. Extracellular alkalinization after 2 h of cell incubation in 100 mM NaCl indicated the possibility that a Na⁺/H⁺ exchange activity could be operating in both types of cells. Moreover, acidification induced by adding 100 mM NaCl + FC to non-adapted cells was relatively less affected by vanadate than that induced by 5 mM KCl + FC, which suggested that salt stress could induce some component other than H⁺ extrusion by H⁺-ATPase. In addition, no differences were observed in microsomal vanadate-sensitive ATPase activity among control, NaCl-adapted and NaCl-stressed cells, while K⁺-stimulated H⁺-PPase and bafilomycin-sensitive H⁺-ATPase activities were higher in microsomes from NaCl-adapted than in those from control cells. Likewise, the stimulation of in vivo H⁺ extrusion in NaCl adapted cells under NaCl or KCl stress in the presence of FC occurred with an inhibition of H⁺-PPase and bafilomycin-sensitive H⁺-ATPase activities and without changes in the vanadate-sensitive H⁺-ATPase activity. These results suggest that the stimulation of tonoplast proton pumps in NaCl-adapted cells, without changes in plasmalemma H⁺-ATPase, could serve to energize Na⁺ efflux across the plasmalemma and Na⁺ fluxes into vacuoles catalyzed by the Na⁺/H⁺ antiports. This revised version was published online in June 2006 with corrections to the Cover Date.     

In vitro binding of inorganic mercury to the plasma membrane of rat platelet affects Na+-K+-ATPase activity and platelet aggregation

Hg²⁺ binding to ouabain-sensitive Na⁺-K⁺-ATPase of rat platelet membrane was specific with a K_a of 1.3×10⁹ moles and B_max of 3.8 nmoles/mg protein. The binding of mercury to Na⁺-K⁺-ATPase also inhibits the enzyme significantly (P<0.001), which is greater than its ouabain sensitivity. Further in the cytosol of washed platelets conjugation of reduced glutathione (GSH) to Hg²⁺ is correlated dose dependently (25, 50 and 100 pmoles) to enhanced GSH-S-transferase (GST) activity. It may be concluded from the present in vitro experiments that mercury binds specifically to thiol groups present in the platelet membrane Na⁺-K⁺-ATPase, inhibits the enzyme and induces changes in platelet function, namely, platelet aggregation by interfering with the sodium pump.     

Caffeine prevents high-intensity exercise-induced increase in enzymatic antioxidant and Na+-K+-ATPase activities and reduction of anxiolytic like-behaviour in rats

Objective: Here we investigated the impact of chronic high-intensity interval training (HIIT) and caffeine consumption on the activities of Na⁺-K⁺-ATPase and enzymes of the antioxidant system, as well as anxiolytic-like behaviour in the rat brain.

Methods: Animals were divided into groups: control, caffeine (4?mg/kg), caffeine (8?mg/kg), HIIT, HIIT plus caffeine (4?mg/kg) and HIIT plus caffeine (8?mg/kg). Rats were trained three times per week for 6 weeks, and caffeine was administered 30 minutes before training. We assessed the anxiolytic-like behaviour, Na⁺-K⁺-ATPase, superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPx) activities, levels of reduced glutathione (GSH) and thiobarbituric acid reactive substances (TBARS) in the brain.

Results and discussion: HIIT-induced anxiolytic-like behaviour increased Na⁺-K⁺-ATPase and GPx activities and TBARS levels, altered the activities of SOD and CAT in different brain regions, and decreased GSH levels. Caffeine, however, elicited anxiogenic-like behaviour and blocked HIIT effects. The combination of caffeine and HIIT prevented the increase in SOD activity in the cerebral cortex and GPx activity in three brain regions. Our results show that caffeine promoted anxiogenic behaviour and prevented HIIT-induced changes in the antioxidant system and Na⁺-K⁺-ATPase activities.     

Endothelin 1 Stimulates Na+,K+-ATPase and Na+-K+-Cl− Cotransport Through ETA Receptors and Protein Kinase C-Dependent Pathway in Cerebral Capillary Endothelium

Abstract: The effect of endothelins (ET-1 and ET-3) on ⁸⁶Rb⁺ uptake as a measure of K⁺ uptake was investigated in cultured rat brain capillary endothelium. ET-1 or ET-3 dose-dependently enhanced K⁺ uptake (EC₅₀ = 0.60 ± 0.15 and 21.5 ± 4.1 nM, respectively), which was inhibited by the selective ET_A receptor antagonist BQ 123 (cyclo-d -Trp-d -Asp-Pro-d -Val-Leu). Neither the selective ET_B agonists IRL 1620 [N-succinyl-(Glu⁹,-Ala^11,15)-ET-1] and sarafotoxin S6c, nor the ET_B receptor antagonist IRL 1038 [(Cys¹¹,Cys¹⁵)-ET-1] had any effect on K⁺ uptake. Ouabain (inhibitor of Na⁺,K⁺-ATPase) and bumetanide (inhibitor of Na⁺-K⁺-Cl^? cotransport) reduced (up to 40% and up to 70%, respectively) the ET-1-stimulated K⁺ uptake. Complete inhibition was seen with both agents. Phorbol 12-myristate 13-acetate (PMA), activator of protein kinase C (PKC), stimulated Na⁺,K⁺-ATPase and Na⁺-K⁺-Cl^? cotransport. ET-1-but not PMA-stimulated K⁺ uptake was inhibited by 5-(N-ethyl-N-isopropyl)amiloride (inhibitor of Na⁺/H⁺ exchange system), suggesting a linkage of Na⁺/H⁺ exchange with ET-1-stimulated Na⁺,K⁺-ATPase and Na⁺-K⁺-Cl^? cotransport activity that is not mediated by PKC.     

Evolution of acidocalcisomes and their role in polyphosphate storage and osmoregulation in eukaryotic microbes

Acidocalcisomes are acidic electron-dense organelles, rich in polyphosphate (poly P) complexed with calcium and other cations. While its matrix contains enzymes related to poly P metabolism, the membrane of the acidocalcisomes has a number of pumps (Ca²⁺-ATPase, V-H⁺-ATPase, H⁺-PPase), exchangers (Na⁺/H⁺, Ca²⁺/H⁺), and at least one channel (aquaporin). Acidocalcisomes are present in both prokaryotes and eukaryotes and are an important storage of cations and phosphorus. They also play an important role in osmoregulation and interact with the contractile vacuole complex in a number of eukaryotic microbes. Acidocalcisomes resemble lysosome-related organelles (LRO) from mammalian cells in many of their properties. They share similar morphological characteristics, acidic properties, phosphorus contents and a system for targeting of their membrane proteins through adaptor complex-3 (AP-3). Storage of phosphate and cations may represent the ancestral physiological function of acidocalcisomes, with cation and pH homeostasis and osmoregulatory functions derived following the divergence of prokaryotes and eukaryotes.     

Comparison of lipid effects on K+-Mg2+ activated p-nitrophenyl phosphatase and Na+-K+-Mg2+ activated adenosine triphosphatase of membrane

Abstract— A comparison was made between K⁺-Mg²⁺ activated p-nitrophenyl phosphatase and Na⁺-K⁺-Mg²⁺ activated adenosine triphosphatase with a solubilized enzyme preparation from a membrane fraction of cerebral cortex. The NPPase showed activity even in the absence of phospholipid, whereas the ATPase required the lipid for its activity. More varied types of phospholipids were effective in activating the NPPase than the ATPase, and with each phospholipid the extent and the pattern of the NPPase activation differed from that of the ATPase. By deoxycholate treatment the pH optimum of the NPPase was shifted independently from the pH optimum shift of the ATPase. The specific activity ratio of the NPPase to the ATPase was not constant during purification. These two enzymes were, however, not separable with ammonium sulphate fractionation, and their thermo-lability was identical regardless of the presence of phospholipid. The results suggested two possibilities: (1) the NPPase is a separate enzyme entity from the ATPase; (2) although the NPPase is a part of the ATPase system, the mechanism of action of lipids on the former part differs from that on the rest of the system.     

Age-related responses of hepatic ATPases to starvation and cold stress in male garden lizards

Hepatic Na⁺-K⁺-ATPase and Mg²⁺-ATPase activities of male green lizards declined during the maturation phase (juvenile to 1-year-old) and stabilized thereafter. On the other hand, the Ca²⁺-ATPase activity of the liver declined during the later half of the life span (1-year-old to 2–4-year-old). Starvation stress induced a decline in hepatic Na⁺-K⁺-ATPase and Mg²⁺-ATPase activities of juvenile lizards and caused an increase in 1-year-old and 2–4-year-old counterparts. The Ca²⁺-ATPase activity declined only in starved 1-year-old lizards. Following cold stress, the hepatic Na⁺-K⁺-ATPase activity of juvenile lizards showed a higher degree of decline than 2–4-year-old counterparts. The Mg²⁺-ATPase activity declined in cold-stressed juvenile lizards, but the parameter was elevated in similarly treated 1-year-old lizards. On the other hand, the increase in Ca²⁺-ATPase activity in response to cold stress was confined only to 2–4-year-old lizards.     

Activation of Na+-K+-adenosine triphosphatase by spermine.

Spermine activated Na⁺-K⁺-ATPase when the concentrations of K⁺ and ATP were low, whereas it inhibited K⁺-dependent and ouabain-inhibitable monophosphatase. The activating effect of sperimine was not due to the substitution for K⁺ or Na⁺. Excess K⁺ inhibited Na⁺-K⁺-ATPase partially, and reduced the spermine activation. When 1 mM ATP was used, spermine at higher concentrations inhibited Na⁺-K⁺-ATPase, and did not activate at all. It is suggested that the K⁺-sites essential to Na⁺-K⁺-ATPase and the K⁺-phosphatase co-exist at different places of the enzyme.     

The role of glutathione in renal cortical tissue. effects of diamide on Na+ and GSSG levels,amino acid transport and Na+-K+-ATPase activity

Summary The effects of diamide were studied in rat kidney cortical tissue. It was found that diamide increased oxidized glutathione levels and inhibited Na⁺-K⁺-ATPase activity. Consistent with this finding was the observation that diamide compromised the sodium gradients maintained in renal cortical slices. Amino acid transport studies with ouabain or a sodium-free buffer indicated that diamide interferes with both Na⁺-dependent and Na⁺-independent transport systems. These results indicate that diamide has a number of different effects on renal cortical tissue and emphasize the important role of glutathione in maintaining control of a number of key metabolic pathways.