Evidence for the presence and role of tightly bound adenine nucleotides in phospholipid-free purifiedMicrococcus lysodeikticus adenosine triphosphatase

[³²P]-labeled ATPase was isolated in a highly purified state fromMicrococcus lysodeikticus strain PNB grown in medium supplemented with [³²P]orthophosphate. Selective extraction procedures allowed us to determine that at least 25% of the firmly bound label belonged to adenine nucleotides, ATP and ADP being present in equimolar amounts. However, no³²P label was found to be part of phospholipids. This was confirmed by purification of the ATPase from cells fed with [2—³H]glycerol. Using the luciferin-luciferase assay we estimated that ATPase freshly isolated by Sephadex chromatography (specific activity 10–14 µmole substrate transformed · min^–1 · mg protein^–1) contained 2 moles ATP/mole of enzyme. The ratio fell with the age of enzyme and its purification by gel electrophoresis and this was paralleled by a loss of ATPase activity. The endogenous nucleotides were readily exchanged by added ADP or ATP. This result suggests that the sites for tight binding of adenine nucleotides are equivalent, although ADP seems to have a higher affinity for them. The last properties represent a peculiar characteristic of this bacterial ATPase as compared with other bacterial and organelle energy-transducing proteins.     

Properties of the chloride-ATPase fromLimonium salt glands: Activation by,and binding to,specific sugars

Summary Attempts to separate membrane fractions enriched in Cl^–-ATPase activity fromLimonium leaf microsomes were hampered because, it seemed, the microsomal membranes were aggregated in clumps. We found hemagglutination activity, specific for N-acetylgalactosamine and to a lesser extent galactose, in the soluble phase of the homogenate, and we were able to prevent membrane aggregation by adding galactose to the microsomes. We discovered that the Cl^–-ATPase activity of the microsomes was increased by galactose and to an even greater extent by N-acetylgalactosamine. We report that the Cl^–-ATPase binds to galactosamine-sepharose, from which it can be eluted in 0.1m galactose, i.e., the enzyme is associated with a saccharide-binding site similar to that of the hemagglutinins. This procedure results in a 100-fold enrichment of the Cl^–-ATPase activity and represents a new way of purifying a membrane-bound enzyme from a heterogeneous membrane preparation in one step. Enzyme isolated by affinity chromatography of Triton-solubilized membranes was essentially free of other ATPase and accounted for a substantial proportion (sometimes all) of the Cl^–-ATPase of the microsomes. This purified preparation of the enzyme shows N-acetylgalactosamine-specific hemagglutination activity. However, we can show that the Cl^–-ATPase and the hemagglutinins are different entities. Thus, material isolated in the same way from salt-free plants showed hemagglutination but not Cl^–-ATPase activity. Also, the hemagglutinins, but not the Cl^–-ATPase, will bind to galactosaminesepharose in the absence of ATP.This is the first report of enzyme activity associated with a carbohydrate receptorspecific protein. Possible roles for saccharide-binding in the control, assembly, and orientation of the chloride-pump are discussed.     

Diabetes induced by streptozotocin causes reduced Na−K ATPase in the brain

Na–K ATPase activity in the brain decreased significantly after diabetes was induced with streptozotocin in rats. Largest decreases were observed in the hippocampus (–30%) and the cerebral cortex (–26%). Smaller decreases were observed in the thalamus (–13%), hypothalamus (–11%) and brain stem (–10%). Na–K ATPase activity in the striatum and the cerebellum were not significantly decreased. The varied decreases suggest that the regional variation of the enzyme is enhanced in the diabetic state. The enzymes of glucose metabolic pathway, namely hexokinase, lactate dehydrogenase and citrate synthase in the brain regions largely remained unchanged although increases in lactate dehydrogenase were observed in some regions. Acetylcholinesterase activity, a marker for the cholinergic system, remains unaltered in the brain during diabetes. The results are discussed with respect to the possible metabolic factors which alter the Na–K ATPase in the brain and its comparison with the peripheral nerve.     

The Ca<Superscript>2+</Superscript>-ATPase of the Scallop Sarcoplasmic Reticulum Is of a Cold-adapted Type

At 0 to 20°C, the Ca²⁺-ATPase activity of the scallop sarcoplasmic reticulum (SR) was observed to be 7–60% of the peak activity at 30°C, while the ATPase activity of the rabbit SR was 0–7% of its peak at 55°C. The relative rabbit ATPase activity (0.7–7.0%) at 7–20°C became higher (6–15 times) and lower (1/4–1/2), respectively, by the solubilization of the rabbit ATPase with a detergent, dodecyloctaethylenglycol monoether, and by the reconstitution of the ATPase with asolectin (soybean lecithin). No activity at 0°C remained irrespective of these treatments. The relative scallop ATPase activity at 0–20°C was, however, scarcely affected by such solubilization and reconstitution. In contrast to the rabbit ATPase, the scallop ATPase seems to be capable of operating independently without the help of the membrane lipid at low temperature.     

Mg-ATPase and CA2+ activated myosin ATPase activity in ventricular myofibrils from non-failing and diseased human hearts – effects of calcium sensitizing agents MCI-154, DPI 201–106, and caffeine

We investigated the effects of two purported calcium sensitizing agents, MCI-154 and DPI 201–106, and a known calcium sensitizer caffeine on Mg-ATPase (myofibrillar ATPase) and myosin ATPase activity of left ventricular myofibrils isolated from non-failing, idiopathic (IDCM) and ischemic cardiomyopathic (ISCM) human hearts (i.e. failing hearts). The myofibrillar ATPase activity of non-failing myofibrils was higher than that of diseased myofibrils. MCI-154 increased myofibrillar ATPase Ca²⁺ sensitivity in myofibrils from non-failing and failing human hearts. Effects of caffeine similarly increased Ca²⁺ sensitivity. Effects of DPI 201–106 were, however, different. Only at the 10^–6 M concentration was a significant increase in myofibrillar ATPase calcium sensitivity seen in myofibrils from non-failing human hearts. In contrast, in myofibrils from failing hearts, DPI 201–106 caused a concentration-dependent increase in myofibrillar ATPase Ca²⁺ sensitivity. Myosin ATPase activity in failing myocardium was also decreased. In the presence of MCI-154, myosin ATPase activity increased by 11, 19, and 24% for non-failing, IDCM, and ISCM hearts, respectively. DPI 201–106 caused an increase in the enzymatic activity of less than 5% for all preparations, and caffeine induced an increase of 4, 11, and 10% in non-failing, IDCM and ISCM hearts, respectively. The mechanism of restoring the myofibrillar Ca²⁺ sensitivity and myosin enzymatic activity in diseased human hearts is most likely due to enhancement of the Ca²⁺ activation of the contractile apparatus induced by these agents. We propose that myosin light chain-related regulation may play a complementary role to the troponin-related regulation of myocardial contractility.     

The enhancement by pervanadate of tyrosine phosphorylation on prostatic proteins occurs through the inhibition of membrane-associated tyrosine phosphatases

The Na⁺–K⁺ ATPase activity and SH group content were decreased whereas malondialdehyde (MDA) content was increased upon treating the porcine cardiac sarcolemma with xanthine plus xanthine oxidase, which is known to generate superoxide and other oxyradicals. Superoxide dismutase either alone or in combination with catalase and mannitol fully prevented changes in SH group content but the xanthine plus xanthine oxidase-induced depression in Na⁺–K⁺ ATPase activity as well as increase in MDA content were prevented partially. The Lineweaver-Burk plot analysis of the data for Na⁺–K⁺ ATPase activity in the presence of different concentrations of MgATP or Na⁺ revealed that the xanthine plus xanthine oxidase-induced depression in the enzyme activity was associated with a decrease in V_max and an increase in K_m for MgATP; however, K_a value for Na⁺ was decreased. Treatment of sarcolemma with H₂O₂ plus Fe²⁺, an hydroxyl and other radical generating system, increased MDA content but decreased both Na⁺–K⁺ ATPase activity and SH group content; mannitol alone or in combination with catalase prevented changes in SH group content fully but the depression in Na⁺–K⁺ ATPase activity and increase in MDA content were prevented partially. The depression in the enzyme activity by H₂O₂ plus Fe²⁺ was associated with a decrease in V_max and an increase in K_m for MgATP. These results indicate that the depressant effect of xanthine plus xanthine oxidase on sarcolemmal Na⁺–K⁺ ATPase may be due to the formation of superoxide, hydroxyl and other radicals. Furthermore, the oxyradical-induced depression in Na⁺–K⁺ ATPase activity may be due to a decrease in the affinity of substrate in the sarcolemmal membrane.     

The action of phloridzin and sugars on (Na+−K+)-Activated ATPase

Summary The action of phloridzin and simple sugars on the (Na⁺–K⁺)-activated ATPase obtained from rabbit kidney has been studied. Phloridzin 10^–4to 10^–3 m was found to inhibit the enzyme at Na⁺:K⁺ ratios less than optimal for enzyme activity, whereas stimulation was noted at Na⁺:K⁺ ratios greater than optimal for enzyme activity. Some sugars in concentrations of 0.1 to 0.5m were found to inhibit the (Na⁺–K⁺)-activated ATPase. The sugars and related compounds could be ranked according to decreasing inhibitory potency as: D-mannose>D-arabinose, D-xylose>L-xylose>D-glucose>fructose, L-arabinose>D-galactose, myo-inositol, mannitol=0. No stimulatory effect or interaction with K⁺ was found with these compounds. The action of these substances on the (Na⁺–K⁺)-activated ATPase suggests an interaction of actively transported sugars and sodium-potassium transport at the level of the sodium pump that may be important in the biological coupling of the two systems.Supported by a Research Career Development Award (K 3-GM-8158) from the U. S. Public Health Service.     

Branchial Cl transport, anion-stimulated ATPase and acid-base balance inAnguilla anguilla adapted to freshwater: Effects of hyperoxia

Summary Freshwater eel gills are notorious for their limited ability to pump chloride. As a result there is a considerable discrepancy between the Na⁺ and Cl^– plasma levels, and plasma HCO₃ ^– and blood pH are relatively high in this species.When eels are kept in tanks aerated with pure oxygen, significant alterations in blood acid-base balance, an increase in plasma pCO₂ and a decrease in blood pH, are observed. In fish studied after 3 weeks hyperoxia, the decrease in blood pH is compensated by an increase in plasma HCO₃ ^–. Such fish exhibit a Cl^– influx 5 times higher than that observed in normoxic fish. This Cl^– influx is readily inhibited by addition of SCN^– to the external medium.An anion-stimulated ATPase activated by HCO₃ ^– and by Cl^– and inhibited by SCN^– was recently described in membrane fractions of the gills ofCarassius auratus, a fish noted for its high Cl^– pumping rate. This enzyme is also found in the gills of the eel. While the maximal rates of enzyme activation by HCO₃ ^– and by Cl^– are similar inCarassius andAnguilla, the affinity of the enzyme for Cl^– is 25 times higher inCarassius. In the microsomal fraction of the hyperoxic eel gills, the maximal anionstimulated ATPase activity remains unchanged but HCO₃ ^– affinity decreases by 50%, while Cl^– affinity increases 5 times. Thus some characteristics of this ATPase seem to be closely related to the Cl^– pump activity exhibited by the gill in fresh water.     

Membrane ATPase of Escherichia coli K 12 Selective solubilization of the enzyme and its stimulation by trypsin in the soluble and membrane-bound states

ATPase (EC 3.6.1.3) of Escherichia coli has been solubilized from two morphologically distinct membranes (vesicles and “ghosts”). Maximum ATPase release is attained with 3 mM EDTA in NH₄HCO₃, pH 9.0, and depends on protein concentration. After solubilization, the total enzyme activity is increased by 300% with respect to the membrane-bound enzyme. The released soluble ATPase accounts for more than 90% of this activity. Its specific activity is at least 10 times higher than the original value. Membrane treatment with buffers of various ionic strengths without EDTA and detergents is less selective. The molecular sieving properties (gel electrophoresis and Sephadex G-200 filtration) confirm the soluble nature of the preparation. A molecular weight close to 300 000 has been estimated for it.The membrane-bound ATPase is stimulated by trypsin by 70–100%. Most of the soluble ATPase maintains a trypsin activation of the same order. Exceptions are the preparations obtained at high protein dilution and extracted with sodium dodecyl sulphate and deoxycholate. The soluble ATPase is more labile than the membrane-bound enzyme. Its sensitivity to different temperatures depends upon protein concentration and pH during storage. Inactivation seems to result from dissociation and/or proteolysis.We suggest an ATPase link to the membrane through ionic divalent cation bridges. We also suggest that the enzyme possesses self-regulatory properties which would account for trypsin stimulation.     

The activation and inactivation of the Dunaliella salina chloroplast coupling factor 1 (CF1) in vivo and in situ

Preillumination of intact cells of the eukaryotic, halotolerant, cell-wall-less green alga Dunaliella salina induces a dark ATPase activity the magnitude of which is about 3–5-fold higher than the ATPase activity observed in dark-adapted cells. The light-induced activity arises from the activation and stabilization in vivo of chloroplast coupling factor 1 (CF₁). This activity, 150–300 μmol ATP hydrolyzed/mg Chl per h, rapidly decays (with a half-time of about 6 min at room temperature) in intact cells but only slowly decays (with a half-time of about 45 min at room temperature) if the cells are lysed by osmotic shock immediately after illumination. The activated form of the ATPase in lysed cells is inhibited if the membranes are treated with ferri- but not ferrocyanide, suggesting that the stabilization of the activated form of CF₁ is due to the reduction of the enzyme in vivo in the light.     

Properties of Plasma Membrane Isolated from Chilling-Sensitive Etiolated Seedlings of Vigna radiata L

Plasma membrane was isolated in a uniform population and with a high purity from chilling-sensitive etiolated young seedlings of Vigna radiata (mung bean) utilizing an aqueous two polymer phase separation system and subsequent sucrose density gradient. The isolated plasma membrane was associated with vanadate-sensitive and KNO₃-insensitive ATPase. The ATPase has high specificities both for substrate and Mg²⁺ ion with optimum pH at 6.5. It was slightly stimulated by monovalent anions, especially Cl⁻. Proton ionophores such as gramicidin D and carbonyl cyanide p-trifluoromethoxyphenylhydrazone did not stimulate the enzyme activity. The ATPase is apparently latent and highly stimulated by the addition of detergents such as Triton X-100. A maximum stimulation was achieved by the addition of 0.02% Triton X-100. After treatment with proteinase K in an isotonic buffer solution, the enzyme activity was less affected, whereas the peptides were specifically digested. Based on these facts, the isolated plasma membrane vesicles appear to be tightly sealed and in a right-side-out orientation. The plasma membrane ATPase had two inflection points at higher (18.9°C) and lower (6.7°C) temperatures on the Arrhenius plots of the activity. The lower inflection temperature apparently coincided with that of the anisotropy parameter of embedded 1,6-diphenyl-1,3,5-hexatriene, indicating that the membrane bound ATPase activity was affected by a phase transition of membrane lipids and/or temperature-dependent conformational changes in the enzyme molecules per se. Considering the fact that the plant material used here is highly sensitive to chilling temperatures and injured severely by exposure to temperatures below 5°C for a relatively short period, the thermotropic properties of membrane molecules are considered to be involved in the mechanism of chilling injury.     

Effect of retinol deficiency and curcumin or turmeric feeding on brain Na+−K+ adenosine triphosphatase activity

The effect of retinol deficiency and curcumin and turmeric feeding on brain microsomal Na⁺-K⁺ ATPase activity was investigated. The brain Na⁺–K⁺ ATPase activity registered an increase of 148.5% as compared to the control group. Upon treating retinol deficient rats with curcumin or turmeric, the abnormally elevated activity showed a decrease of 36.9 and 47.1%, respectively, when compared to the retinol deficient group. An increase in V_max by 67% and K_m by 66% for ATP was observed in the retinol deficient group. Curcumin or turmeric fed retinol-deficient groups reduced the V_max by 25 and 33%, while K_m was reduced by 25 and 31%, respectively, compared to the retinol deficient group. Arrhenius plot of Na⁺–K⁺ ATPase showed a typical bi-phasic pattern in all the groups. Cholesterol: Phospholipid ratio showed a decrease in the retinol-deficient group by 67.8%, which showed a marked increase in curcumin or turmeric treated groups. Detergents could increase the Na⁺–K⁺ ATPase activity more in the control group than in the retinol deficient groups. Curcumin or turmeric improved the detergent action on the enzyme. Subsequent freezing and thawing over a period of 30 min decreased the enzyme activity by 22.8% in the retinol deficient group compared to 15.9% decrease in the control group. Curcumin or turmeric treated groups showed a decrease in the enzyme activity by 22.0 and 19.2%, respectively, when compared to the zero time in each group. In the presence of concanavalin-A (Con-A) there was only 52.4% stimulation in the enzyme activity in retinol deficient groups, compared to 108.0% in the control group. Curcumin or turmeric treated retinol-deficient groups showed a stimulation in the presence of con-A by 70 and 99.5%, respectively.     

Temperature dependence of mitochondrial oligomycin-sensitive proton transport ATPase

The temperature dependence of the oligomycin-sensitive ATPase (complex V) kinetic parameters has been investigated in enzyme preparations of different phospholipid composition. In submitochondrial particles, isolated complex V, and complex V reconstituted in dimirystoyl lecithin vesicles, the Arrhenius plots show discontinuities in the range 18–28°C, while no discontinuity is detected with dioleoyl lecithin recombinant. Van't Hoff plots ofK _m also show breaks in the same temperature interval, with the exception of the dioleoylenzyme vesicles, whereK _m is unchanged. Thermodynamic analysis of the ATPase reaction shows that DMPC-complex V has rather larger values of activation enthalpy and activation entropy below the transition temperature (24°C) than those of the other preparations, while all enzyme preparations show similar free energies of activation (14.3–18.5 kcal/mol). The results indicate that temperature and lipid composition influence to a different extent both kinetic and thermodynamic parameters of ATP hydrolysis catalyzed by the mitochondrial ATPase.     

ATPase Activity of Pea Cotyledon Submitochondrial Particles: ACTIVATION, SUBSTRATE SPECIFICITY, AND ANION EFFECTS

Submitochondrial particles freshly prepared by sonication from pea cotyledon mitochondria showed low ATPase activity. Activity increased 20-fold on exposure to trypsin. The pea cotyledon submitochondrial particle ATPase was also activated by “aging” in vitro. At pH 7.0 addition of 1 millimolar ATP prevented the activation. ATPase of freshly prepared pea cotyledon submitochondrial particles had a substrate specificity similar to that of the soluble ATPase from pea cotyledon mitochondria, with GTPase > ATPase. “Aged” or trypsin-treated particles showed equal activity with the two substrates. NaCl and NaHCO₃, which stimulate the ATPase but not the GTPase activity of the soluble pea enzyme, were stimulatory to both the ATPase and GTPase activities of freshly prepared submitochondrial particles. However, they were stimulatory only to the ATPase activity of trypsin-treated or “aged” submitochondrial particles. In contrast, the ATPase activity of rat liver submitochondrial particles was stimulated by HCO₃⁻, but inhibited by Cl⁻, indicating that Cl⁻ stimulation is a distinguishing property of the pea mitochondrial ATPase complex.     

Renaissance of cytochemical localization of membrane ATPases in the myocardium

ATPases of cardiac cells are known to be among the most important enzymes to maintain the fluxes of vital cations by hydrolysis of the terminal high-energy phosphate of ATP. Biochemically the activities of Ca²⁺-pump ATPase, Ca²⁺/Mg²⁺-ecto ATPase, Na⁺,K⁺-ATPase and Mg²⁺-ATPase are determined in homogenates and isolated membranes as well as in myofibrillar and mitochondrial fractions of various purities. Such techniques permit estimation of enzyme activitiesin vitro under optimal conditions without precise enzyme topography. On the other hand, cytochemical methods demonstrate enzyme activityin situ, but not under optimal conditions. Until recently several cytochemical methods have been employed for each enzyme in order to protect its specific activity and precise localization but the results are difficult to interpret. To obtain more consistent data from biochemical and cytochemical point of view, we modified cytochemical methods in which unified conditions for each ATPase were used. The fixative solution (1% paraformaldehyde –0.2% glutaraldehyde in 0.1 M Tris Base buffer, pH 7.4), the same cationic concentrations of basic components in the incubation medium (0.1 M Tris Base, 2mM Pb(NO₂)₃, 5 mM MgSO₄, 5 mM ATP) and selective stimulators or inhibitors were employed. The results reveal improved localization of Ca²⁺-pump ATPase, Na⁺–K⁺ ATPase and Ca²⁺/Mg²⁺-ecto ATPase in the cardiac membrane.     

Localization of a Mg- or Ca-activated (“basic”) ATPase in skeletal muscle

A chicken pectoralis muscle membrane fraction enriched in a Mg²⁺- or Ca²⁺-activated (‘basic’) ATPase was obtained by sucrose gradient centrifugation. Enzymatic properties of the ‘basic’ ATPase were determined and used to localize its enzymatic activity in situ by ultrastructural cytochemistry. The enzyme was activated by Mg²⁺ or Ca²⁺ but not by Sr²⁺, Ba²⁺, Co²⁺, Ni²⁺ or Pb²⁺. It was present in a membranous fraction with a buoyant density of 1.10-1.12 (24–27.5% (w/w) sucrose). ‘Basic’ ATPase activity had a sedimentation pattern similar to the putative plasma membrane enzymes, 5′-nucleotidase and leucyl β-naphthylamidase, but different from that of sarcoplasmic reticulum Ca²⁺ ATPase. Also unlike sarcoplasmic reticulum Ca²⁺ ATPase, ‘basic’ ATPase was resistant to N-ethylmaleimide and aldehyde fixatives, was active in a medium containing a high Ca²⁺ concentration (3 mM), and was lost when exposed to Triton X-100 or deoxycholate. In cytochemical studies, a low Pb²⁺ concentration was used to capture the enzymatically released phosphate ions. Under conditions which eliminated interfering (Na⁺ + K⁺) ATPase and sarcoplasmic reticulum Ca²⁺ ATPase activities, electron-dense lead precipitates were present at the plasmalemma and T-system membranes. These studies suggest that ‘basic’ ATPase activity is associated with plasmalemma and T-system membranes of skeletal muscle.     

Preparation of a highly active ATPase of the mesophilic cyanobacterium Spirulina maxima

In this work, we report new studies on the ATPase attached to the photosynthetic membranes of the mesophilic cyanobacterium Spirulina maxima. This enzyme does not display persistent latency as had been previously reported for the ATPase of Spirulina platensis. The enzyme is readily activated by the careful application of methods currently used to activate chloroplast CF₁. Photosynthetic membranes of Spirulina maxima show a Mg²⁺-dependent ATPase activity of 195±25 mol Pi (mg chl)^–1 h^–1 after a light plus dithiothreitol (DDT) treatment. Methanol treatment of these membranes elicits Mg²⁺-dependent ATPase activity of 222±18 mol Pi (mg chl)^–1 h^–1.Here, we also describe the purification of the soluble coupling factor AF₁ of Spirulina maxima. This enzyme is unique among mesophilic cyanobacterial F₁ preparations in regard to its high specific Ca²⁺-dependent ATPase activity after heat treatment (14.75±1.91 mol Pi (mg prot)^–1 min^–1) and its room temperature stability.Abbreviations AF₁ cyanobacterial coupling factor - DTT dithiothreitol - DEAE cellulose diethylaminoethyl cellulose - DCCD N,N'-dicyclohexylcarbodiimide - EDTA ethylenediamine tetracetic, sodium salt - PAGE polyacrylamide gel electrophoresis - PMS phenazine methosulfate - PMSF phenylmethylsulfonyl fluoride - MV methylviologen - SDS sodium dodecylsulfate - TPSnCl triphenyltin chloride - Tris tris (hydroxymethyl) aminomethane - tricine N Tris (hydroxymethyl) methylglycine - BAEE N-benzoyl-L-arginine ethyl ester     

Polarographic studies of membrane particles containing Na−K ATPase

Summary The properties of a suspension of membrane particles containing Na–K ATPase have been investigated with the aid of d–c and a–c polarography. In particular, we have studied the interaction of three cations, two very effective enzyme inhibitors and one activator, with the enzyme preparation. Ag⁺ and Cu⁺⁺, which inhibit the enzyme at very low concentrations, bind very strongly. No binding could be found with the activating ion, Tl⁺, however. Adsorption of a substance with an isoelectric point between pH 4 and pH 5.5 occurred at the electrode surface between –0.1 and –1.2 V at pH 7, and was associated with the random currents that appear during the measurements. The random currents arise when the membrane particles collide with the electrode and cause changes in the structure of the electrical double layer. (Added substances that adsorb more strongly at the mercury/water interface eliminate the random currents.) The adsorbed film impedes the flow of the free Ag⁺ and Cu⁺⁺ ions, and to a smaller extent, the flow of Tl⁺ ions. The differences between the binding of inhibiting and activating ions are correlated with their effects on the ATPase enzyme activity.     

The use of a hidden metal-capture reagent for the measurement of Na+−K+-ATPase activity: A new concept in cytochemistry

Summary The original lead-trapping method for demonstrating Na⁺–K⁺-ATPase activity was discredited because of the effect that lead ions can have on the substrate and on the enzyme. Current methods, that measure this activity by the related K⁺-dependent phosphatase activity, do not appear to measure activity that is known, from microchemistry, to occur in proximal convoluted tubules. The disadvantages of using lead appear to have been overcome by the use of a new reagent in which the lead is complexed with ammonium citrate ions; phosphate, liberated enzymatically, successfully competes with these ions. The activities of total ATPase and of the ouabain sensitive Na⁺–K⁺-ATPase have been measured in three regions of the nephron in the guinea-pig and in the rat. The relative activities found, by this method, in the different regions of the latter, appear to be comparable with results found by others, using microchemical methods applied to isolated regions of the nephron.     

Resistance adaptation of the freshwater crayfish and thermal inactivation of membrane-bound enzymes

Summary Heat death and resistance adaptation of freshwater crayfish are thought to be properties of its muscle membranes. The inactivation at high temperatures of a membrane-bound enzyme, the Ca⁺⁺-stimulated ATPase of crayfish abdominal muscle sarcoplasmic reticulum, and the effect of thermal acclimation of crayfish upon the inactivation kinetics have been investigated. In the absence of KCl, the Ca⁺⁺-stimulated ATPase is irreversibly inactivated with pseudo-first order kinetics at temperatures that cause heat death in the whole animal. 0.1–10.0 mM KCl resulted in slower inactivation, while 100 mM KCl activated the enzyme to 120–180% of its original activity. Enzyme activation by KCl and heat involved a shift in the enzyme concentration/activity curve. Thermal acclimation of crayfish had no significant effect upon the kinetics or Arrhenius activation energy for enzyme inactivation (100.6±10.5 and 92.3±14.6 kcal/mole for preparations from 4°C and 25°C acclimated crayfish).Ca⁺⁺-stimulated ATPase isolated from heat dead crayfish exhibited normal in vitro activity due presumably to the high intracellular K⁺ concentration. Nevertheless, the close correspondence between heat death temperatures and inactivation temperatures for several membrane-bound enzymes of muscle is thought to reflect some perturbation of muscle structure that occurs during heat death.Abbreviations ATP Ademosine 5-Triphosphate - EGTA Ethyleneglycol-bis [-amino-ethyl ether] - N N-tetraacetic acid - Hepes N-2-Hydroxyethylpiperazine-N-2-ethanesulphonic acid - FSR Fragmented sarcoplasmic reticulum - Tris Tris (hydroxymethyl)aminomethane