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.     

Separate effects of mercurial compounds on the ionophoric and hydrolytic functions of the (Ca+++Mg++)-ATPase of sarcoplasmic reticulum

Summary We have shown that a Ca⁺⁺-ionophore activity is present in the (Ca⁺⁺+Mg⁺⁺)-ATPase of rabbit skeletal muscle sarcoplasmic reticulum (A.E. Shamoo & D.H. MacLennan, 1974.Proc. Nat. Acad. Sci. USA 71:3522). Methylmercuric chloride inhibited the (Ca⁺⁺+Mg⁺⁺)-ATPase and Ca⁺⁺ transport, but had no effect on the activity of the Ca⁺⁺ ionophore. Mercuric chloride inhibited ATPase, transport and ionophore activity. The ATPase and transport functions were more sensitive to methylmercuric chloride than to mercuric chloride. The two functions were inhibited concomitantly by methylmercuric chloride but slightly lower concentrations of mercuric chloride were required to inhibit Ca⁺⁺ transport than were required to inhibit ATPase. Methylmercuric chloride and mercuric chloride probably inhibited ATPase and Ca⁺⁺ transport by blocking essential-SH groups. However, it appears that there are no essential-SH groups in the Ca⁺⁺ ionophore and that mercuric chloride inhibited the Ca⁺⁺ ionophore activity by competition with Ca⁺⁺ for the ionophoric site. Blockage of Ca⁺⁺ transport by mercuric chloride probably occurs both at sites of essential-SH groups and at sites of ionophoric activity. These data suggest the separate identity of the sites of ATP hydrolysis and of Ca⁺⁺ ionophoric activity.     

Evidence implicating a membrane ATPase in the control of passive permeability of excitable cells

The temperature sensitivity of the ATPase enzyme systems in a muscle microsomal preparation from the crayfish, Astacus pallipes, was studied. Preincubation of the enzyme preparation in the range 33–36°C produced a marked inactivation of the ATPases; the Mg⁺⁺-dependent ATPase was very much more sensitive to this treatment than the Na⁺-K⁺-Mg⁺⁺-dependent ATPase. Thus, the Arrhenius μ for the inactivation of the Mg⁺⁺-dependent ATPase produced by eight minute preincubation is > 100 Kcals. These results are compared with the changes that are observed during the heat death of the whole animal, where exposure to 35°C produces a dramatic change in Na⁺ permeability within five minutes. Arrhenius μ for heat death is also > 100 Kcals and operates over the identical critical temperature range. It is suggested that the Mg⁺⁺-dependent ATPase controls passive permeability in these excitable cells and the results also confirm the view that Mg⁺⁺ and Na⁺-K⁺-Mg⁺⁺ ATPases are separate enzymes.     

Effect of chemical modification of cytoplasmic activator protein on human red cell membrane Ca++ + Mg+ ATPase.

A highly purified cytoplasmic activator protein of human red cell membrane Ca⁺⁺ + Mg⁺⁺ ATPase was prepared by two step purification scheme utilizing Diethylaminoethyl cellulose (DE-52) and sephadex (G-100) column chromatography. This purified protein can elicit a maximum activation of membrane Ca⁺⁺ + Mg⁺⁺ ATPase at low calcium concentrations. The stimulatory effect of this protein can be rendered totally ineffective by chemical modification with N-bromosuccinimide. The results suggest a possible role of methionine oxidation in the regulation of the Ca⁺⁺ + Mg⁺⁺ ATPase activator activity.     

Inhibition of Na+ and K+-stimulated ATPase of rabbit heart sarcolemma after, administration of heparin.

Intravenous heparin administration caused a marked inhibition of Mg⁺⁺-dependent (Na⁺+K⁺)-stimulated ATPase activity of sarcolemmal (SL) membranes prepared from rabbit heart, whereas basal Mg⁺⁺-ATPase was not affected. The inhibition depended on K⁺ concentration and was reversed only in the presence of albumin. Plasma free fatty acid (FFA) concentrations were raised in all animals, after heparin administration. The results obtained support the concept that FFA or other lipids originating in the plasma by the action of lipolytic enzymes released by heparin are involved in the mechanism of inhibition.     

A study of sodium release in the course of ATP hydrolysis by membrane ATPase

Summary With the aid of sodium-sensitive glass electrodes, changes in sodium ion activity were studied in the course of subsequent additions of components required for ATP hydrolysis provided by Na⁺–K⁺-dependent membrane ATPase. Membrane ATPase was obtained from guinea pig kidney cortex. In the presence of ATP, Mg⁺⁺ and Na⁺ in media, the addition of K⁺ caused an increase in Na⁺ activity. The omission of ATP or its substitution by ADP as well as the addition of Ca⁺⁺ to the media eliminated the above-mentioned increase of Na⁺ activity. Quabain did not affect Na⁺ release caused by the addition of K⁺, although it significantly inhibited ATPase activity of the preparation. The data obtained were considered to be a direct indication of ion exchange during the course of membrane ATPase reaction. This ion-exchange stage of the reaction is not inhibited by ouabain. The ratio of sodium ions released per one inorganic phosphate formed in the course of the reaction was found to be much higher than that established for transporting membranes of intact cells. A possible cause of this difference is discussed.     

The inhibition of Na+ and K+ stimulated ATPase activity of rabbit and dog heart sarcolemma by lysophosphatidyl choline.

Lysophosphatidylcholine (LPC) added at 30 μM or 10 μM to sarcolemma (SL) membranes of rabbit or dog causes about 50% inhibition of the Mg⁺⁺ dependent Na⁺ + K⁺ stimulated ATPase activity. Higher concentration (100 μM) of free fatty acid (FFA) is needed to produce the same inhibition. Lysophosphatidyl ethanolamine, phosphorylcholine, phosphorylethanolamine also inhibit the enzyme activity. The inhibition of LPC is competitive for the Na⁺ site of the enzyme and it also affects the K⁺ site. The inhibitory effect of LPC is not additive to that produced by FFA or ouabain, on the contrary at low concentrations of these inhibitors the inhibition of LPC is reversed. The possible effects of increased levels of blood or tissue LPC on heart cell functions related to the Na⁺ + K⁺ ATPase activity are discussed. The possible interference of LPC with cardiac glycoside action is also discussed.     

Histochemische,elektronenmikroskopische und biochemische Untersuchungen über die ATPasen im Vormagenepithel der Ziege

Zusammenfassung Am Epithel der drei Vormägen der Ziege wurden licht- und elektronenmikroskopische Untersuchungen über den Nachweis der Mg⁺⁺-, (Mg⁺⁺-Na⁺-K⁺)- und der Ca⁺⁺-aktivierbaren ATPasen durchgeführt. Bereits an lichtmikroskopischen Präparaten wurde festgestellt, daß sich deutliche Unterschiede in der Enzymaktivität der Mg⁺⁺- und Ca⁺⁺ stimulierbaren ATPasen sowohl für die einzelnen Epithelschichten als auch für die verschiedenen Bereiche der Vormägen nachweisen lassen; die tiefen Epithellagen reagieren stets stärker als die oberflächlichen. Ferner ist die in hohem Maße von der Inkubationszeit abhängige Reaktionsstärke im Epithel des Blättermagens größer als in der Lamina epithelialis des Netzmagens und vor allem in jener des Pansens. Bei dem elektronenmikroskopisch geführten Nachweis der ATPasen zeigt sich, daß die Reaktionsprodukte an der Außenseite der Zellmembranen liegen. Frei von Niederschlägen sind alle Zellmembranabschnitte, die sich an der Bildung der Desmosomen und Halbdesmosomen beteiligen sowie die basalen Abschnitte der Basalzellen.Im Hinblick auf die Verteilung und feinstrukturelle Lokalisation konnten keine Unterschiede zwischen den Mg⁺⁺-, Ca⁺⁺- und (Mg⁺⁺-Na⁺-K⁺)-aktivierbaren ATPasen beobachtet werden.In Anlehnung an die Methode nach Coleman u.a. (1967) wurden Zellmembranen von Pansenepithelzellen isoliert und an diesen biochemische Enzymbestimmungen durchgeführt. Die Ausbeute der gewonnenen Zellmembranen betrug, gemessen anhand der 5-Nukleotidase, dem Leitenzym für Plasmamembranen, gegenüber dem eingesetzten Gesamthomogenat 0,3%. Die mit diesem Verfahren durchgeführten biochemischen Enzymbestimmungen erbrachten den Nachweis, daß in den Plasmamembranen der Pansenepithelzellen neben der Mg⁺⁺- und Ca⁺⁺-aktivierbaren auch eine (Na⁺-K⁺)-abhängige Transport-ATPase vorkommt.

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Histochemical, ultrastructural and biochemical studies of atpases of the goat forestomach epithelium

Summary Light- and electron microscopical studies were carried out to demonstrate ATPases activated by Mg⁺⁺, by (Mg⁺⁺-Na⁺-K⁺), and by Ca⁺⁺. Histological sections showed clear differences of the activity of Mg⁺⁺- and Ca⁺⁺-stimulated ATPases in the light microscope in different layers of the epithelium as well as in different areas of the forestomach. The deeper layers reacted more intensely than the superficial ones. The intensity of the reaction (which depends on incubation time) in the omasal epithelium was stronger than in the lamina epithelialis of the reticulum and much stronger than in the lamina epithelialis of the rumen. In the electron microscope, the reaction products of the ATPase appeared on the outer surface of cell membranes (plasmalemmata). No deposits of the reaction products were observed on those areas of the cell membranes, which are involved in the formation of desmosomes and semidesmosomes. The basal parts of the basal cells were also free from reaction products. As for the distribution and ultrastructural localisation of the deposits, no differences were observed among the ATPase stimulated by Mg⁺⁺, (Mg⁺⁺-Na⁺-K⁺), and Ca⁺⁺.Using the technique of Coleman et al. (1967), the cell membranes of ruminal epithelium were isolated. Biochemical assays of the enzymes were carried out. The quantity of cell membranes obtained was 0.3% of the whole homogenate, when compared with 5-nucleotidase, which is the typical enzyme of plasmalemmata. The biochemical enzyme assays showed that, besides Mg⁺⁺- and Ca⁺⁺-dependent ATPases, a (Na⁺-K⁺)-dependent transport ATPase exists in the cell membranes of ruminal epithelial cells.

Die cytochemischen Ergebnisse wurden auszugsweise auf dem Kongreß der Europäischen Vereinigung der Veterinäranatomen vom 9.–11. September 1968 in Belgrad vorgetragen.     

R 24571: a new powerful inhibitor of red blood cell Ca++-transport ATPase and of calmodulin-regulated functions

Compound R 24571 (1-[bis(p-chlorophenyl)methyl]-3-[2,4-dichloro-β-(2,4-dichlorobenzyloxy)phenethyl]imidazoliniumchloride) is found to be a powerful inhibitor of red blood cell Ca⁺⁺-ATPase as well as Ca⁺⁺ transport into inside-out red blood cell vesicles with an IC₅₀-value of 0.5 and 2 μM, respectively. The inhibitory action of R 24571 is more specific on the calmodulin-dependent fraction of Ca⁺⁺-transport ATPase as compared to the basal Ca⁺⁺-transport ATPase (determined in the absence of calmodulin) and can be antagonized by increasing concentrations of calmodulin in an apparently competitive manner. With respect to other ATPases the action of R 24571 is relatively specific for red blood cell Ca⁺⁺-transport ATPase. Mg⁺⁺-ATPase requires a 40 times higher concentration for halfmaximal inhibition (IC₅₀ = 20 μM) whereas (Na⁺ + K⁺)-transport ATPase is only slightly affected in the investigated concentration range (≤20 μM).     

Effect of mersalyl on mitochondrial Mg++ flux

The mercurial mersalyl has little effect either on rapid Mg⁺⁺ binding by isolated rat liver mitochondria or on the total Mg⁺⁺ content of these organelles measured after 0.75 min of incubation at 20°C. The data do not support the previous suggestion that the increased permeability to K⁺ of mitochondria treated with mersalyl results from release of endogenous Mg⁺⁺. An increased pH-dependence of unidirectional Mg⁺⁺ flux into respiring rat liver mitochondria is suggested to arise indirectly from inhibition by mersalyl of pH shifts associated with exchanges of endogenous phosphate. In addition, mersalyl appears to have a stimulatory effect on Mg⁺⁺ influx. Mersalyl also increases the average rate of unidirectional efflux of endogenous Mg⁺⁺. The stimulatory effects of mersalyl on Mg⁺⁺ flux are similar to, although quantitatively less than, the previously reported effects of mersalyl on mitochondrial K⁺ flux.     

Conformational changes of membrane-bound (Na+−K+)-ATPase as revealed by antibody inhibition

Summary As different structural states of the (Na⁺–K⁺)-ATPase (EC 3.6.1.3) may lead to a changed reactivity to antibodies, the influence of Na⁺, K⁺, Mg⁺⁺, P_i and ATP on the reaction between highly purified (Na⁺–K⁺)-ATPase and antibodies directed against the membrane-bound enzyme was measured. The antigen antibody reaction was registered by measuring the antibody inhibition of (Na⁺–K⁺)-ATPase activity.In themembrane-bound but not in thesolubilized enzyme four different degrees of antibody inhibition were obtained at equilibrium of the antigen antibody reaction if different combinations of Na⁺, K⁺, Mg⁺⁺ and ATP were present during the incubation with the antibodies. Corresponding to the different degrees of inhibition, different rates of enzyme inhibition were measured. (a) The smallest degree of enzyme inhibition was obtained when (i) only Mg⁺⁺, (ii) Mg⁺⁺ and Na⁺ or (iii) Mg⁺⁺ and K⁺ were present during the antigen antibody reaction. (b) The enzyme activity was inhibited more strongly if Na⁺, Mg⁺⁺ and ATP were present together. (c) It was inhibited even more if only (i) Na⁺, (ii) K⁺, (iii) ATP or both (iv) ATP and Na⁺, (v) ATP and K⁺, (vi) ATP and Mg⁺⁺, or if (vii) no ATP and activating ions were present. (d) The highest degree of antibody inhibition was obtained if Mg⁺⁺, ATP and K⁺ were present together.In the presence of Mg⁺⁺ plus ADP and in the presence of Mg⁺⁺ plus the ATP analog adenylyl (--methylene) diphosphonate, Na⁺ and K⁺ did not influence the degree of antibody inhibition as they did in the presence of Mg⁺⁺ plus ATP. It was further found that the degree of antibody inhibition in the presence of Mg⁺⁺, ATP and K⁺ was affected by the sequence in which K⁺ and ATP were added to the enzyme prior to the addition of the antibodies.It is suggested that by antibody inhibition different conformations of the (Na⁺–K⁺)-ATPase could be detected. These conformations may possibly not occur in the solubilized enzyme and therefore do not seem to be necessarily linked to the intermediary steps of the ATP hydrolysis of the enzyme. The structural changes which are induced by Na⁺ and K⁺ in the presence of Mg⁺⁺ plus ATP are proposed to occur during the Na⁺–K⁺ transport.     

Ecto-ATPase

Summary An ecto-adenosine triphosphatase (E.C. 3.6.1.4 ATP-phosphohydrolase) is shown to be localised on the outer surface of varieties of cell membrane. The enzyme is different from the ATPase involved in biological energy transduction and ion transport mechanism. The characteristic of the enzyme lies in having a very broad substrate specificity and is inhibited by EDTA and higher concentration of ATP. The enzyme is dependent on bivalent metal ions, Mg⁺⁺ or Ca⁺⁺ for its optimum activity. The enzyme is highly sensitive to SH-reagents but insensitive to inhibitors of mitochondrial ATPase or Na⁺−K⁺-ATPase. The possible functions of the enzyme in being oriented outside the cell membrane is discussed.     

Use of a concanavalin A polymer to isolate right side-out vesicles of purified plasma membranes from eukariotic cells

We have purified two plasma membrane populations using a Concanavalin A polymer. It was assumed that vesicles retained by the polymer were right side-out, whereas vesicles not retained were inside-out. 5′-nucleotidase and (Na⁺ + K⁺) stimulated Mg⁺⁺ ATPase activities were at least two fold higher in inside-out than in right side-out vesicles, though recovered total activity was about 80 % for both enzymes together. Moreover, Concanavalin A modified 5′-nucleotidase activity of right side-out vesicles according to the dose used.     

Evidence for subclasses of SH groups in (Na++K+)-ATPase.

Changes in the chemical reactivity of the sulfhydryl groups of (Na⁺ + K⁺)-dependent ATPase can be indicative of conformational changes induced by activating ions. Cyanylation of these groups by 5 mM 2-nitro-5-thiobenzoic acid caused a partial inhibition of enzymatic activity. Both this loss and the incorporation of radioactive cyanide from the ¹⁴C-labeled reagent were reduced by inclusion of 50 mM ATP and 150 mM Na⁺ in the incubation. When 10 mM Mg⁺⁺ was added in addition, the inactivation was not different from that produced by cyanylation reagent alone, but the radioactive labeling of protein increased significantly. The data indicate that the sulfhydryl groups of this enzyme exist in two populations, one of which must be free if the enzyme is to function. The other, not essential for enzymatic activity, becomes accessible only when the Na⁺ and Mg⁺⁺-dependent phosphorylation of the enzyme alters its conformation. Inactivation of the enzyme by freezing and thawing increases the incorporation of radioactivity but destroys the responsiveness of labeling to cations and ATP.     

A calorimetric investigation of the heats of binding of Mg ++ to poly A, to poly U, and to their complexes

The heats of binding of Mg⁺⁺ ions to poly A, poly U, and to their complexes, in the presence of Na⁺ ions, have been measurd calorimetrically. In all cases the heat, ΔH(θ), exhibitis a distinct dependence on the extent of binding, θ, and in the cases of poly A and poly U also on the Na⁺ concentration. The values of ΔH(θ) range from +2 to +3 kcal/mole of Mg⁺⁺ bound at θ = 0 to 1.3 kcal/mole at θ = 0.5 except in poly A where at θ = 0 ΔH(θ) = ?2 to ?3 kcal/mole. This is interpreted as being due to a facilitation of base stacking by the binding of Mg⁺⁺. The extent of facilitation is consistent with current estimates of base stacking. A similar effect but of much smaller magnitude is believed to obtain in poly A poly U. An interpretation of the dependence of ΔH(θ) on θ in terms of simple electrostatic interactions, but neglecting solvent effects, was attempted and found to be inadequate.     

Active Uptake of Ca++ and Ca++-Activated Mg++ ATPase in Red Cell Membrane Fragments

Isolated human red blood cell membrane fragments (RBCMF) were found to take up Ca⁺⁺ in the presence of ATP.¹ This ATP-dependent Ca⁺⁺ uptake by RBCMF appears to be the manifestation of an active Ca⁺⁺ transport mechanism in the red cell membrane reported previously (Schatzmann, 1966; Lee and Shin, 1969). The influences of altering experimental conditions on Ca⁺⁺-stimulated Mg⁺⁺ ATPase (Ca⁺⁺ ATPase) and Ca⁺⁺ uptake of RBCMF were studied. It was found that pretreatment of RBCMF at 50°C abolished both Ca⁺⁺ ATPase and Ca⁺⁺ uptake. Pretreatment of RBCMF with phospholipases A and C decreased both Ca⁺⁺ ATPase and Ca⁺⁺ uptake, whereas pretreatment with phospholipase D did not significantly alter either Ca⁺⁺ ATPase or Ca⁺⁺ uptake. Both Ca⁺⁺ ATPase and Ca⁺⁺ uptake had ATP specificity, similar optimum pH's, and optimum incubation temperatures. From these results, it was concluded that Ca⁺⁺ uptake is intimately linked to Ca⁺⁺ ATPase.     

Isolation and Partial Characterization of an Acetylcholine Receptor-Enriched Membrane Fraction from Skeletal Muscle

Abstract

A procedure for purification of the bungarotoxin-binding fraction of sarcolemma from rabbit skeletal muscle is described. Muscle is homogenized in 0.25M sucrose without high salt extraction and membrane fractions separated initially by differential centrifugation procedures. An ultracentrifugation pellet enriched in cell surface and sarcoplasmic reticulum markers is further fractionated on a dextran gradient (density = 1.0 to 1.09). Two fractions are identified as sarcolemma according to high specific activities for lactoperoxidaseiodination, Na⁺, K⁺-ATPase and α-bungarotoxin-binding. No Ca⁺⁺, Mg⁺⁺-ATPase activity is found in these fractions. A third fraction, the dextran gradient pellet, is enriched in Ca⁺⁺, Mg⁺⁺-ATPase activity and lactoperoxidase iodinatable material and characterized by low bungarotoxin binding. This fraction represents a mixture of sarcoplasmic reticulum and transverse tubules with some sarcolemma contamination.     

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.     

Electron microscopic observations on the mitochondrial adenosinetriphosphatase in the rat spinal cord

Summary Nucleosidetriphosphatase activity of the rat spinal cord was studied with a modified Wachstein-Meisel method at ultrastructural level. A short formaldehyde perfusion fixation favoured the preservation of the mitochondrial activity.The reaction product was localized intramitochondrially in the neuropil, while the pericaryonal mitochondria were nonreactive. Similar results were obtained, when ITP was substituted for ATP. No membrane activity was observed by Mg⁺⁺ activation only, but a certain membrane affinity was noticed in the neuropil, when Na⁺ and K⁺ were included.The specificity of the ATPase reaction reported in this paper is further discussed.Abbreviations used ATP adenosinetriphosphate - ATPase adenosinetriphosphatase - IDP inosinediphosphate - ITP inosinetriphosphate - TPP thiaminepyrophosphate - UDP uridinediphosphate     

Effects of calcium ions and quinolinic acid on rat kidney mitochondrial kynurenine aminotransferase

At pH 6.4, rat kidney mitochondrial kynurenine aminotransferase activity is enhanced several-fold by the addition of CaCl₂, apparently because Ca⁺⁺ facilitates the translocation of α-ketoglutarate, one of the substrates, across the mitochondrial inner membrane. Chloride salts or Mg⁺⁺, Mn⁺⁺, Na⁺, K⁺, and NH₄⁺ did not have this effect. At pH 6.8, the enzyme activity was near maximal even without added Ca⁺⁺ but was strongly depressed by either of two calcium chelating agents, quinolinic acid (Q.A.) and ethyleneglycol-bis(β-aminoethyl ether)N,N′-tetraacetic acid (EGTA). These observations support the view that Ca⁺⁺ is involved in regulating kidney mitochondrial translocation of α-ketoglutarate and that the reported interference of polycarboxylate anion translocation by Q.A. $\text{in vivo}$ depends on the ability of that agent to chelate Ca⁺⁺.