Partial characterization of an endogenous factor which modulates the effect of catecholamines on synaptosomal Na+, K+-ATPase

We have previously presented evidence for the existence of a brain soluble factor which mediates the stimulation of synaptosomal ATPases by catecholamines. The stimulation of synaptosomal ATPases by dopamine plus brain soluble fraction was not modified if the soluble fraction was heated for 5 min at 95°C. One day after preparation, the soluble factor inhibited the Na⁺, K⁺-ATPase, but not the Mg²⁺-ATPase activity, and subsequent addition of noradrenaline stimulated the ATPases activities. The inhibitory effect of a 24 h soluble fraction disappeared if the soluble fraction was dialyzed; in this case, noradrenaline did not activate the enzyme activities. Gel filtration in Sephadex G-50 permitted separating a subfraction which inhibited ATPase activity (peak II) from another which stimulated ATPase activity (peak I). Peak I stimulated both Na⁺, K⁺, and Mg²⁺ ATPases. Peak II inhibited only Na⁺, K⁺-ATPase, and when stored acidified, it mediated ATPases stimulation by noradrenaline.Special Issue dedicated to Prof. Eduardo De Robertis.     

Elevation of Different Ion-Specific ATPase Activities by L-Thyroxine (T4) in Different Tissues of Tasar Silkworm, Antheraea mylitta (Lepidoptera: Saturniidae) during Developmental Stages

Tissue-specific age-dependent changes were observed in Na⁺K⁺-, Ca²⁺-, and Mg²⁺-ATPase activities in tropical tasar silkworm, Antheraea mylitta Drury. Maximum enzyme activity was recorded in all the tissues on day 12 (before spinning) in control group of animals. In testis, Na⁺K⁺-, Ca²⁺-, and Mg²⁺-ATPase activities gradually increased from day 2 to day 12 during fifth larval age and level was maintained up to adult eclosion while, in ovary, a marked decline was noted up to day of adult emergence. Further, a significant and sharp rise was found in ATPase activity in silk gland tissue up to day 12 and afterwards a drastic fall was noted on day 15 (end of spinning) during fifth larval age.Administration of T4 to fifth stage larvae (1 hr old) at doses 0.5–2.0 μg/g significantly elevated the Na⁺K⁺-, Ca²⁺-, and Mg²⁺-ATPase activities in larval and pupal gonads in a dose-dependent fashion. But, in moths, the enhancement was very much confined to Na⁺K⁺- and Ca²⁺-ATPase in testes and only Ca²⁺-ATPase in ovaries. Again, in silk glands thyroxine (0.5–2.0 μg/g) caused a significant rise in the all ion-dependent ATPase activities only during the fifth larval stage. Interestingly, higher doses of T4 (4.0 μg/g) caused a significant reduction in Na⁺K⁺-, Ca²⁺- and Mg²⁺-ATPase in all the tissues almost all the days studied so far. However, lower doses of T4 (0.1 and 0.25 μg/g) remained ineffective in altering the different ion-specific ATPase activities. This study suggests, that mammalian thyroxine has a metabolic influence showing biphasic nature of action in tasar silkworm ATPase system.     

Variable ATPase composition of human tumor plasma membranes

Purified plasma membranes from several transplantable human tumors exhibit very high Mg²⁺-dependent ATPase activities. Three types of Mg²⁺-dependent ATPases can be demonstrated: (1) an ouabain sensitive Na⁺, K⁺-ATPase, which is a minor component of the tumor plasma membrane ATPase, (2) a Mg²⁺-activated ATPase, which is a non-specific nucleoside triphosphatase, and (3) an ATPase activity stimulated by Na⁺ (or K⁺) alone. In three human melanomas, only the first two activities are found. In an astrocytoma and an oat cell carcinoma, all three activities are found. In the same two tumors, the plasma membrane Mg²⁺-ATPase is also stimulated by Con A. The relationship of these ATPases are discussed.     

Trifluoperazine inhibition of calmodulin-sensitive Ca2+-ATPase and calmodulin insensitive (Na+ + K+)- and Mg2+-ATPase activities of human and rat red blood cells

Trifluoperazine dihydrochloride-induced inhibition of calmodulin-activated Ca²⁺-ATPase and calmodulin-insensitive (Na⁺ + K⁺)- and Mg²⁺-ATPase activities of rat and human red cell lysates and their isolated membranes was studied. Trifluoperazine inhibited both calmodulin-sensitive and calmodulin-insensitive ATPase activities in these systems. The concentration of trifluoperazine required to produce 50% inhibition of calmodulin-sensitive Ca²⁺-ATPase was found to be slightly lower than that required to produce the same level of inhibition of other ATPase activities. Drug concentrations which inhibited calmodulin-sensitive ATPase completely, produced significant reduction in calmodulin-insensitive ATPases as well. The data presented in this report suggest that trifluoperazine is slightly selective towards calmodulin-sensitive Ca²⁺-ATPase but that it is also capable of inhibiting calmodulin-insensitive (Na⁺ + K⁺)-ATPase and Mg²⁺-ATPase activities of red cells at relatively low concentrations. Thus the action of the drug is not due entirely to its interaction with calmodulin-mediated processes, and trifluoperazine cannot be assumed to be a selective inhibitor of calmodulin interactions under all circumstances.     

Effect of light on ATPases in eyes and brain of the blowfly,Calliphora

Summary The relation between ATPase-activities and stimulation of a compound eye by light was investigated biochemically: (1) The Mg²⁺- and Na⁺K⁺-ATPases of eyes with different amounts of screening pigments were determined in flies reared under identical light conditions. (2) Enzyme activities in eyes were compared after dark adaptation and illumination, respectively. (3) Activity measurements were also taken from the brains of the same animals.—The ATPase activities-measured in vitro under optimal conditions —remained unchanged under illumination of medium intensity. After high level illumination of a pigment-free mutant, Na⁺K⁺-ATPase (but not Mg²⁺-ATPase) activity raised significantly in eyes as well as in brain (Table 3).—In vivo only activity of the membrane-bound Na⁺K⁺-ATPase changes with illumination, but the amount of enzyme protein is large enough that sub-maximal turnover rates occur under usual light conditions. High intensity light causes synthesis of new enzyme protein in pigment-free eyes.This investigation was supported by Deutsche Forschungsgemeinschaft, Sonderforschungsbereich 114 (Bionach)     

Effects of affinity-purified antibodies on the Ca2+ pumping ATPase of erythrocyte membranes

Antibodies raised in rabbits against the purified erythrocyte membrane Ca²⁺ pumping ATPase were affinity-purified using an ATPase-Sepharose column. Addition of a few molecules of the purified antibody per molecule of ATPase was sufficient to inhibit the ATPase activity. Extensively washed ghosts or preincubated pure ATPase sometimes develop an appreciable Mg²⁺-ATPase activity. In such cases, the antibodies inhibited the Mg²⁺-ATPase as well as the Ca²⁺-ATPase. This is consistent with the hypothesis that a portion of the Mg²⁺-ATPase activity of ghosts is derived from the Ca²⁺-ATPase. When nitrophenylphosphatase activity was observed, both Mg²⁺ - and Ca²⁺-stimulated activities were observed. Only the Ca²⁺ activity was inhibited by the antibodies, confirming that this activity is due to the Ca²⁺ pump, and suggesting that the Mg²⁺-nitrophenylphosphatase is due to a separate enzyme. Amounts of antibody comparable to those which inhibited the Ca²⁺-ATPases had no effect on the Na⁺-K⁺-ATPase; 4-fold higher amounts of antibody significantly stimulated the Na⁺-K⁺-ATPase, but this effect of the antibody was not specific: Immunoglobulins from the nonimmune serum also significantly stimulated the Na⁺-K⁺-ATPase.In resealed erythrocyte membranes, antibodies incorporated into the ghosts inactivated the Ca²⁺-ATPase, while antibodies added to the outside had no significant effect.     

Cyclic AMP and calcium modulated ATPase activity in the salivary glands of the lone star tick Amblyomma americanum (L.)

Na⁺,K⁺-activated ATPase activity in tick salivary glands increases during the rapid stage of tick feeding paralleling similar increases in dopamine and cAMP-stimulated fluid secretion. High concentrations of cyclic AMP increase Na⁺,K⁺-ATPase activity in a plasma membrane-enriched fraction from the salivary glands of rapidly feeding ticks. Cyclic AMP-dependent protein kinase inhibitor protein blocks activation of Na⁺,K⁺-ATPase activity at low but not high concentrations of cAMP indicating that both activator and inhibitor modulator phosphoproteins of Na⁺,K⁺-ATPase activity exist in the plasma membrane-enriched fraction.ATPase activity in the plasma membrane-enriched fraction is not measurable in the absence of Mg²⁺, Ca²⁺ and Na⁺. Ca-stimulated nucleotidase activity is highest with ATP serving as the preferred substrate in a series including CTP, UTP, GTP and ADP. Calcium, Mg²⁺ stimulated ATPase activity is activated further by calmodulin and partially inhibited by low concentration of vanadate, trifluoperazine and oligomycin. Results suggest that the plasma membrane-enriched fraction of tick salivary glands contains both Ca²⁺-ATPase activity and oligomycin-sensitive Ca²⁺, Mg²⁺-ATPase activities, the latter likely from a small amount of mitochondria in the partially purified organelle fraction.     

ATPase, peroxidase and lipoxygenase activity during post-harvest deterioration of cassava (Manihot esculenta Crantz) root tubers

Alterations in the activity of ATPases, peroxidases and lipoxygenases were studied during early stages of post-harvest deterioration of cassava (Manihot esculenta Crantz cv. Oyolu) root tubers. The peak activities of Ca²⁺-ATPase, (Ca²⁺+Mg²⁺)-ATPase, Na⁺+K⁺-ATPase, Mg²⁺-ATPase and peroxidases were observed after the first 24 h and thereafter decreased. The activity of lipoxygenase was biphasic, probably depicting two distinct isoforms expressed during deterioration. The results indicate that ATPases and peroxidases have a role in the post-harvest deterioration of cassava tuber, but the participation of lipoxygenases seems unlikely.     

Characteristics of the Mg-ATPase Activity Associated with the Membrane-Bound Maize Coupling Factor

The membrane-bound coupling factor of maize mesophyll thylakoids is a latent ATPase. Mg²⁺-ATPase activity can be induced in the light with either dithiothreitol or low concentrations of trypsin. Maize thylakoids that are activated with light plus trypsin exhibit considerably higher levels of activity in Na₂SO₃-dependent Mg²⁺-ATPase assays compared to thylakoids that are light and dithiothreitol activated (1400 micromoles per milligram of chlorophyll per hour versus 200 micromoles per milligram of chlorophyll per hour). Treatment with light and dithiothreitol or light plus trypsin were also required to demonstrate high levels of octyl glucoside-dependent Mg²⁺-ATPase activity in maize mesophyll thylakoids. Only small differences in octyl glucoside-dependent Mg²⁺-ATPase activity were observed in preparations that were activated in the light with either trypsin or dithiothreitol. Mg²⁺-ATPase activity can also be induced in maize mesophyll chloroplasts by illuminating intact preparations under appropriate conditions. Little or no ATPase activity was observed in the absence of illumination or in the presence of light plus methyl viologen. The active state decayed in the dark with a t_½ of 6 to 7 minutes at room temperature. Based on the effect of the thiol oxidant, o-iodosobenzoate, and the uncoupler, nigericin, on the kinetics of deactivation of ATPase activity in intact maize chloroplasts, it appears that the activation process requires a transmembrane proton gradient and reduction of a key disulfide bridge in the gamma of chloroplast coupling factor one.     

Adenosine triphosphatase localization in the branchial heart of Sepia officinalis L. (Cephalopoda)

Summary Sodium- and potassium-dependent adenosine triphosphatase (Na⁺–K⁺-ATPase) is demonstrated in the branchial heart of Sepia officinalis L. by biochemical, cytochemical and autoradiographical methods. The biochemical data indicate the presence of Na⁺–K⁺-ATPase, shown by potassium and magnesium dependency and inhibition by ouabain. Cytochemically and autoradiographically, the enzyme is localized in the sarcolemma of the muscle cells. The positive reaction of the transparent cells (type I cells) is due to activity of alkaline phosphatases. The dark cells (type II cells) react negatively. In addition to the Na⁺–K⁺-ATPase, a magnesium-activated adenosine triphosphatase (Mg²⁺-ATPase) and a bicarbonate-stimulated ATPase (HCO ₃ ^- -ATPase) are localized in the mitochondria.This study was supported by the Deutsche Forschungsgemeinschaft and is part of the doctoral dissertation     

Subfractionation of rat liver plasma membrane: Uneven distribution of plasma membrane-bound enzymes on the liver cell surface

Plasma membranes were islotaed from rat liver mainly under isotonic conditions. As marker enzymes for the plasma membrane, 5′-nucleotidase and (Na⁺+K⁺)-ATPase were used. The yield of plasma membrane was 0.6–0.9 mg protein per g wet weight of liver. The recovery of 5′-nucleotidase and (Na⁺+K⁺)-ATPase activity was 18 and 48% of the total activity of the whole-liver homogenate, respectively. Judged from the acitvity of glucose-6 phosphatase and succinate dehydrogenase in the plasma membrane, and from the electron microscopic observation of it, the contamination by microsomes and mitochondria was very low. A further homogenization of the plasma membrane yielded two fractions, the light and heavy fractions, in a discontinuous sucrose gradient centrifugation. The light fraction showed higher specific activities of 5′-nucleotidase, alkaline phosphatase, (Na⁺+K⁺)-ATPase and Mg²⁺-ATPase, whereas the heavy one showed a higher specific activity of adenylate cyclase. Ligation of the bile duct for 48 h decreased the specific activities of (Na⁺+K⁺)-ATPase and Mg²⁺-ATPase in the light fraction, whereas it had no significant influence on the activities of these enzymes in the heavy fraction. The specific activity of alkaline phosphatase was elevated in both fractions by the obstruction of the bile flow. Electron microscopy on sections of the plasma membrane subfractions showed that the light fraction consisted of vesicles of various sizes and that the heavy fractions contained membrane sheets and paired membrane strips connected by junctional complexes, as well as vesicles. The origin of these two fractions is discussed and it is suggested that the light fraction was derived from the bile front of the liver cell surface and the heavy one contained the blood front and the lateral surface of it.     

(Na,K)-ATPase activity of embryonic chick heart and skeletal muscles as a function of age

The specific activity of the membrane (Na⁺,K⁺)-ATPase of intact chick embryonic hearts (ventricles) was measured as a function of embryonic age. The membranes were prepared by a NaI extraction method of the 100 000 × g fraction which selectively removes much of the ouabain-insensitive Mg²⁺-ATPase. The specific activity of the myocardial (Na⁺, K⁺)-ATPase increased markedly during embryonic development from mean levels of about 3.0 μmoles P_i per h per mg protein at day 6 to 7.4 at day 16 and 11.0 at day 20 (1 day prior to hatching); the adult level was about the same as that of the 16-day-old chick. The relative activities with respect to that at day 16 (from paired experiments) averaged 43 % (day 6), 56 % (day 9), 73 % (day 13), 140 % (day 20), 115 % (day 23), 126 % (day 30) and 96 % (adult). There was a similar increase in relative activity of the (Na⁺,K⁺)-ATPase from chick skeletal (leg) muscles during development. If the total protein content per unit membrane area and the turnover number remain constant, the data indicate that the surface density of the (Na⁺,K⁺)-ATPase molecules increases during embryonic development; thus, the cation pumping capabilities of the cells should be enhanced if the surface area/volum ratio of the myocardial cells remains unchanged. However, the pumping capabilities of the very young cells must be sufficient to maintain the known high [K⁺]_i and low [Na⁺]_i already present; their internal activities actually change only to a small extent during development. Since there is a known increase in K⁺ permeability during embryonic development, thereby increasing the demand on the cation pump, the observed increase in activity of the (Na⁺,K⁺)-ATPase tends to compensate for this.     

Tissue specificity of dopamine effects on brain ATPases

Dopamine inhibits Mg²⁺,Na⁺,K⁺- and Na⁺,K⁺-ATPase activities but does not modify Mg²⁺-ATPase activity of nerve ending membranes isolated from rat cerebral cortex. In the presence of the soluble fraction of brain, dopamine activates total, Na⁺,K⁺-, and Mg²⁺-ATPases. Dopamine stimulation of nerve ending membrane ATPases is achieved when soluble fractions of brain, kidney, or liver are used. On the other hand, dopamine effects are not observed on kidney or heart ATPase preparations. These results indicate tissue specificity of dopamine effects with respect to the enzyme source; there is no tissue specificity for the requirement of the soluble fraction to achieve stimulation of ATPases by dopamine.     

Effect of Na(3)VO(4) on the P State of Nitella translucens

The capacity of sodium orthovanadate to inhibit the plasmalemma H⁺ ATPase of Nitella translucens internodal cells in vivo was tested. Here we show that 1 millimolar vanadate added externally depolarizes strongly and permanently the membrane potential, both in dark and light, to the Nernst potential for potassium consistent with pump inhibition by vanadate. From the results it is clear that the H⁺ ATPase is always active, under light or dark conditions, in contradiction with the widespread idea of pump inactivation by darkness. The changes in conductance for light, dark, and vanadate-induced conditions are analyzed. The effect of dark on membrane passive permeabilities and on the possibility that some plasmalemma channels could be regulated by a phosphorylation-dephosphorylation process is discussed.     

Effects of delta9-tetrahydrocannabinol on ATPases in mouse brain subcellular fractions.

The effect of (?)-Δ⁹-THC on the activities of Mg²⁺?, Na⁺?K⁺? and Mg²⁺Ca²⁺-ATPases were studied in mouse brain subcellular fractions. $\text{In vitro}$treatment with Δ⁹-THC produced a dose dependent stimulation of Mg²⁺ ATPase in the crude mitochondrial fraction and its subfractions and a dose-related inhibition of this activity in the microsomal fraction. Na⁺-K⁺- and Mg²⁺-Ca²⁺-ATPase activities were inhibited in a dose-related manner in all subcellular fractions studied.     

Effects of morphine tolerance and dependence on Mg++ dependent ATPase activity of synaptic vesicles.

The effect of morphine on ATPase of synaptic plasma membranes (SPM) and synaptic vesicles isolated from the mouse brain was studied. The activity of synaptic vesicle Mg⁺⁺-dependent ATPase from mice rendered morphine tolerant and dependent by pellet implantation was 40% higher than that from placebo implanted mice. However, the activities of Mg⁺⁺-dependent ATPase and Na⁺, K⁺ activated ATPase of SPM of tolerant and nontolerant mice were not significantly different. The activity of synaptic vesicular Mg⁺⁺-dependet ATPase was dependent on the concentration of Mg⁺⁺ but not of Ca⁺⁺; maximum activity was obtained with 2 mM MgCl₂. On the other hand, Mg⁺⁺-dependent ATPase activity of SPM was dependent on both Mg⁺⁺ and Ca⁺⁺, activity being maximum using 2 mM MgCl₂ and 10^?5 M CaCl₂. It is suggested that this stimulation of ATPase activity may alter synaptic transmission and may thus be involved in some aspects of morphine tolerance and dependence.     

Thyroxine influence on different ion-specific adenosine triphosphatase activities in fat body of Bombyx mori during development

The effects of thyroxine on the activity of different ATPases (Na⁺-K⁺, Ca²⁺, and Mg²⁺) in fat body cells of the silkworm, Bombyx mori, were investigated during different developmental stages. In both sexes the maximum enzyme activity was observed in the fat body cells of day 7 last instar larva (the day before spinning). Na⁺-K⁺, Ca²⁺-, and Mg²⁺-ATPase activity in the fat body markedly declined after pupation and continued to decrease in day 1 adults. Injection of thyroxine (T4) at doses of 1.0 and 2.0 μg/g during fifth instar significantly elevated all ATPase activities in the larval, pupal, and adult stages in both sexes. At a dose of 0.5 μg/g, T4 had no effect on day 2 fifth instar larva, although it increased the ATPase activity at the other stages investigated. A higher dose (3.0 μg/g) caused a significant reduction in enzyme activity in all stages with the exception of day 2 fifth instar larva. Thus, the repression of enzyme activity with the higher dose and the elevation of enzyme activity with the lower dose establish the biphasic nature of T4 action on the ATPase system in fat body cells of the silkworm. Arch. Insect Biochem. Physiol. 37:191–196, 1998. © 1998 Wiley-Liss, Inc.     

ATPase ACTIVITY OF NEUROBLASTOMA CELLS IN CULTURE

—Neuroblastoma cells were cultured in the presence of fetal serum. When serum was omitted from the growth medium and also when dibutyryl cyclic AMP or insulin were added without omitting serum, there was a growth of neuron-like processes which was in most cases accompanied by an increase in Na⁺-K⁺ -ATPase activity, whereas Mg²⁺-ATPase activity increased to a lesser extent. The increase in ATPase activities was related to cell density. When the ATPase activities were measured in the presence of 0·25 m -sucrose or 1% deoxycholate the specific activities were decreased. There was no loss of activity during storage at –20°C for several days when 20% glycerol was added to the medium.