Characterization of rat heart plasma membrane Ca2+/Mg2+ ATPase

The Ca2+/Mg2+ ATPase of rat heart plasma membrane was activated by millimolar concentrations of Ca2+ or Mg2+; other divalent cations also activated the enzyme but to a lesser extent. Sodium azide at high concentrations inhibited the enzyme by about 20%; oligomycin at high concentrations also inhibited the enzyme slightly. Trifluoperazine at high concentrations was found inhibitory whereas trypsin treatment had no significant influence on the enzyme. The rate of ATP hydrolysis by the Ca2+/Mg2+ ATPase decayed exponentially; the first-order rate constants were 0.14-0.18 min-1 for Ca2+ ATPase activity and 0.15-0.30 min-1 for Mg2+ ATPase at 37 degrees C. The inactivation of the enzyme depended upon the presence of ATP or other high energy nucleotides but was not due to the accumulation of products of ATP hydrolysis. Furthermore, the inactivation of the enzyme was independent of temperature below 37 degrees C. Con A when added into the incubation medium before ATP blocked the ATP-dependent inactivation; this effect was prevented by alpha-methylmannoside. In the presence of low concentrations of detergent, the rate of ATP hydrolysis was reduced while the ATP-dependent inactivation was accelerated markedly. Both Con A and glutaraldehyde decreased the susceptibility of Ca2+/Mg2+ ATPase to the detergent. These results suggest that the Ca2+/Mg2+ ATPase is an intrinsic membrane protein which may be regulated by ATP.     

Effect of n-chloromercuribenzoate and albumin on Mg2+, Ca2+-ATPase activity of plasma membranes of smooth muscle cells

It is shown that Mg2+, Ca2+-ATPase activity of plasma membrane fragments from the rat small intestine myocytes is inhibited by p-chloromercuribenzoate and dithionitrobenzoate (50 and 90%, respectively). The effect of p-chloromercuribenzoate inhibition is removed by serum albumin promoting a rise in the ATPase activity of the plasma membranes and shifting the temperature maximum point up to 32 degrees C (in the norm the maximum is observed at 36 degrees C). According to the data presented, albumin changes the composition and phase state of the lipid surrounding of the membrane enzymes.     

Diamide inhibited (Ca++ + Mg++) and (Mg++) dependent ATPase in erythrocyte membranes: activity at different temperatures.

After inhibition of the monovalent cation dependent ATPase, a (Ca++ + Mg++) and a (Mg++) dependent ATPase activity can be detected. The inhibition due to diamide on the (Mg++) ATPase, assayed in the 12.5 degrees C - 30 degrees C temperature range, is almost complete. On the contrary the diamide induced inhibition of (Ca++ + Mg++) ATPase, in the same temperature range, is not complete and the residual activity increases with temperature. The reported data indicate that the ATPase activity induced by calcium is much less diamide-sensitive and -SH-dependent than that elicited by Mg++ alone.     

Rotational motion and flexibility of Ca2+,Mg2+-dependent adenosine 5'-triphosphatase in sarcoplasmic reticulum membranes

Ca2+,Mg2+-dependent adenosine 5'-triphosphatase (ATPase) in sarcoplasmic reticulum vesicles is labeled with the triplet probe, 5-iodoacetamidoesin. Rotational mobility of the ATPase is investigated by measuring flash-induced transient dichroism of the eosin probe. The absorption anisotropy measured 20 mus after the exciting flash is found to be small at 37 degrees C but increases considerably with decreasing temperature and upon fixation with glutaraldehyde. A purified Ca2+,Mg2+-dependent ATPase preparation partially depleted of membrane lipids exhibits similar properties. The low value of the anisotropy at 37 degrees C is due to the existence of a fast motion which in part is assigned to independent segmental motion of the protein. This internal flexibility of the ATPase may have considerable significance for the functional properties of the enzyme. At times longer than 20 mus, the anisotropy decays with a time constant which varies from approximately 90 mus at 0 degrees C to approximately 40 mus at 37 degrees C. This decay is assigned to rotation of the ATPase about an axis normal to the plane of the membrane. There is some evidence for self-aggregation of the protein at lower temperatures.     

Effect of concanavalin A on the activity of membrane-bound and detergent-solubilized Mg2+ -ATPase

Plasma membranes were isolated after binding liver and hepatoma cells to polylysine-coated polyacrylamide beads, and the effect of concanavalin A on the membrane-bound Mg2+ -ATPase and the Mg2+ -ATPase solubilized by octaethylene glycol monododecyl ether (C12E8) was studied. In the experiment of membrane-bound Mg2+ -ATPase, plasma membranes were pretreated with Concanavalin A and the activity was assayed. Concanavalin A stimulated the activity of both liver and hepatoma enzymes assayed above 20 degrees C. Concanavalin A abolished the negative temperature dependency characteristic of liver plasma membrane Mg2+ -ATPase. On the other hand, Concanavalin A prevented the rapid inactivation due to storage at -20 degrees C, which was characteristic of hepatoma plasma membrane Mg2+ -ATPase. With solubilized Mg2+ -ATPase from liver plasma membranes, the negative temperature dependency was not observed. Concanavalin A, which was added to the assay medium, stimulated the activity of the enzyme solubilized in C12E8 at a high ionic strength. However, Concanavalin A failed to show any effect on the enzyme solubilized in C12E8 at a low ionic strength. With solubilized Mg2+ -ATPase from hepatoma plasma membranes, Concanavalin A could not prevent the inactivation of the enzyme during incubation at -20 degrees C.     

Temperature-induced transitions in the conformation of intermediates in the hydrolytic cycle of myosin.

The conformations of the transitory intermediates of the myosin ATPase occurring during the hydrolytic cycle, enzyme without ligand, enzyme-substrate complex and two different forms of enzyme-product complex, have been characterized in terms of numbers and classes of reactive thiol groups based on incorporation of radioactively labeled alkylation reagent. The techniques employed allowed this to be done under steady-state conditions in the presence of high ligand concentrations on intact myosin from rabbit fast skeletal muscles at low ionic strength where the protein is in the gel state as it is in muscle. The binding of a divalent cation (Mg2+ or Ca2+) nucleotide complex exposes thiol-1 as well as thiol-2 groups. The long-lived ATPase intermediate occurring at temperatures above 10 degrees C adopts the same conformation with Mg2+ and Ca2+ ions. This intermediate does not protect the thiol-1 and thiol-2 groups but exposes a number of thiol-3 groups which seem to be located distant from the active site. The conformation of the intermediate prevailing in the presence of ATP changes with lowering temperature below 10 degrees C and is identical with that found in the presence of ADP at 0 degree C indicating a change in the rate-limiting step of the hydrolytic cycle. In the absence of divalent cations no such temperature-dependent change in conformation was observed. Evaluation of the activation entropies shows that the structure of the long-lived intermediate occurring above 10 degrees C in the presence of Mg2+ ions goes through a transformation from low to high order at around 20 degrees C. In the case of the monovalent-cation-stimulated ATPase a constant activation energy of around 70 kJ/mol, typical of many enzyme reactions, was found over the entire temperature range from 0--35 degrees C.     

Properties of the Na+, K+-stimulated adenosine triphosphatase system associated with the plasma membrane of pig thyroid glands.

The enzymatic properties of plasma membrane-bound Na+, K+-ATPase [EC 3.6.1.3], isolated with high specific activity and in good yield from pig thyroid cells, were examined. The enzyme activity required the presence of both Na+ and K+ at physiological concentrations; it exhibited high sensitivity to K+ and an absolute requirement for Na+. It showed highly specific requirement for Mg2+ and ATP. The apparent Km for ATP was 0.14 mM under the assay conditions. Arrhenius plots had a point of inflection at about 22 degrees C, activation energies being 24.2 kcal/mol at 5-22 degrees C and 19.0 kcal/mol at 22-40 degrees C. In addition to ouabain, the ATPase was strongly inhibited by fluoride and the SH-blocking reagent, PCMB. Iodide and TSH had no appreciable effect on the enzyme activity.     

Plasma membrane Mg(2+)-ATPase of Pachysolen tannophilus: characterization and role in alcohol tolerance.

Following cell fractionation in sucrose density gradients, plasma membrane Mg(2+)-ATPase from Pachysolen tannophilus was studied. The ATPase displayed an apparent Km for ATP of 1.42 mM and was inhibited by high concentrations of Mg2+. The inhibitory effects of ethanol, 1-propanol, 1-butanol, and benzyl alcohol on Mg(2+)-ATPase were evaluated, and the concentration of each alcohol that inhibited ATPase activity by 50% (IC50) was determined. The IC50 decreased as the chain length of the alcohol increased. Moreover, the IC50 for ATPase activity was similar to the IC50 for growth rate, suggesting an association between impaired growth and ATPase inhibition. Almost complete inhibition of ATPase activity occurred at temperatures approaching 60 degrees C, and the optimal temperature was around 44 degrees C for ATPase from both control and ethanol-treated cells. Inclusion of 50 mM MgCl2 or CaCl2 in the medium did not rescue cells from the deleterious effects of ethanol.     

Plasma membrane Mg(2+)-ATPase of Pachysolen tannophilus: characterization and role in alcohol tolerance.

Following cell fractionation in sucrose density gradients, plasma membrane Mg(2+)-ATPase from Pachysolen tannophilus was studied. The ATPase displayed an apparent Km for ATP of 1.42 mM and was inhibited by high concentrations of Mg2+. The inhibitory effects of ethanol, 1-propanol, 1-butanol, and benzyl alcohol on Mg(2+)-ATPase were evaluated, and the concentration of each alcohol that inhibited ATPase activity by 50% (IC50) was determined. The IC50 decreased as the chain length of the alcohol increased. Moreover, the IC50 for ATPase activity was similar to the IC50 for growth rate, suggesting an association between impaired growth and ATPase inhibition. Almost complete inhibition of ATPase activity occurred at temperatures approaching 60 degrees C, and the optimal temperature was around 44 degrees C for ATPase from both control and ethanol-treated cells. Inclusion of 50 mM MgCl2 or CaCl2 in the medium did not rescue cells from the deleterious effects of ethanol.     

Inhibition of gill (Na+ + K+)-ATPase in rainbow trout (Salmo gairdneri) by a purinedisulfide analog of adenosine triphosphate.

The effect of the adenosine triphosphate analog, 6,6'-dithiobis(inosinyl imidodiphosphate), (sIMP-PNP)2, was tested on the ouabain-sensitive (Na+ + K+)-ATPase (ATP phosphohydrolase, EC 3.6.1.3) and the ouabain-insensitive Mg2+ - ATPase in microsomes prepared from gill tissue of sea water-adapted rainbow trout, Salmo gairdneri. The (Na+ + K+)-ATPase was completely inhibited by low concentrations of (sIMP-PNP)2 (6 micrometer) but the Mg2+ - ATPase was unaffected by the inhibitor at concentrations as high as 28 micrometer, supporting the suggestion that the two activities represent separate enzymes. The specificity of inactivation could be demonstrated both at a physiological temperature (13 degrees C) and at 37 degrees C. The rates of inactivation were similar at both temperatures. Inactivation of the (Na+ + K+)-ATPase by (sIMP-PNP)2 was reversed by dithiothreitol, suggesting that the inhibitor forms a mixed disulfide with sulfhydryl groups on the enzyme. The inability of substrate (either ATP or its analog, adenyl-5'-yl imidodiphosphate) to protect against inactivation suggests that (sIMP-PNP)2 is reacting with sulfhydryl groups which are not associated with the active site.     

Alterations in phospholipid-dependent (Na+ +K+)-ATPase activity due to lipid fluidity. Effects of cholesterol and Mg2+.

The (Na+ +K+)-activated, Mg2+-dependent ATPase from rabbit kidney outer medulla was prepared in a partially inactivated, soluble form depleted of endogenous phospholipids, using deoxycholate. This preparation was reactivated 10 to 50-fold by sonicated liposomes of phosphatidylserine, but not by non-sonicated phosphatidylserine liposomes or sonicated phosphatidylcholine liposomes. The reconstituted enzyme resembled native membrane preparations of (Na+ +K+)-ATPase in its pH optimum being around 7.0, showing optimal activity at Mg2+:ATP mol ratios of approximately 1 and a Km value for ATP of 0.4 mM. Arrhenius plots of this reactivated activity at a constant pH of 7.0 and an Mg2+: ATP mol ratio of 1:1 showed a discontinuity (sharp change of slope) at 17 degrees C, with activation energy (Ea) values of 13-15 kcal/mol above this temperature and 30-35 kcal below it. A further discontinuity was also found at 8.0 degrees C and the Ea below this was very high (greater than 100 kcal/mol). Increased Mg2+ concentrations at Mg2+:ATP ratios in excess of 1:1 inhibited the (Na+ +K+)-ATPase activity and also abolished the discontinuities in the Arrhenius plots. The addition of cholesterol to phosphatidylserine at a 1:1 mol ratio partially inhibited (Na+ +K+)-ATPase reactivation. Arrhenius plots under these conditions showed a single discontinuity at 20 degrees C and Ea values of 22 and 68 kcal/mol above and below this temperature respectively. The ouabain-insensitive Mg2+-ATPase normally showed a linear Arrhenius plot with an Ea of 8 kcal/mol. The cholesterol-phosphatidylserine mixed liposomes stimulated the Mg2+-ATPase activity, which now also showed a discontinuity at 20 degrees C with, however, an increased value of 14 kcal/mol above this temperature and 6 kcal/mol below. Kinetic studies showed that cholesterol had no significant effect on the Km values for ATP. Since both cholesterol and Mg2+ are known to alter the effects of temperature on the fluidity of phospholipids, the above results are discussed in this context.     

Temperature-specific inhibition of human red cell Na+/K+ ATPase by 2,450-MHz microwave radiation

The ATPase activity in human red blood cell membranes was investigated in vitro as a function of temperature and exposure to 2,450-MHz continuous wave microwave radiation to confirm and extend a report of Na+ transport inhibition under certain conditions of temperature and exposure. Assays were conducted spectrophotometrically during microwave exposure with a custom-made spectrophotometer-waveguide apparatus. Temperature profiles of total ATPase and Ca+2 ATPase (ouabain-inhibited) activity between 17 and 31 degrees C were graphed as an Arrhenius plot. Each data set was fitted to two straight lines which intersect between 23 and 24 degrees C. The difference between the total and Ca+2 ATPase activities, which represented the Na+/K+ ATPase activity, was also plotted and treated similarly to yield an intersection near 25 degrees C. Exposure of membrane suspensions to electromagnetic radiation, at a dose rate of 6 W/kg and at five temperatures between 23 and 27 degrees C, resulted in an activity change only for the Na+/K+ ATPase at 25 degrees C. The activity decreased by approximately 35% compared to sham-irradiated samples. A possible explanation for the unusual temperature/microwave interaction is proposed.     

A functionally inactive, cold-stabilized form of the Escherichia coli F1Fo ATP synthase

An unusual effect of temperature on the ATPase activity of E. coli F1Fo ATP synthase has been investigated. The rate of ATP hydrolysis by the isolated enzyme, previously kept on ice, showed a lag phase when measured at 15 degrees C, but not at 37 degrees C. A pre-incubation of the enzyme at room temperature for 5 min completely eliminated the lag phase, and resulted in a higher steady-state rate. Similar results were obtained using the isolated enzyme after incorporation into liposomes. The initial rates of ATP-dependent proton translocation, as measured by 9-amino-6-chloro-2-methoxyacridine (ACMA) fluorescence quenching, at 15 degrees C also varied according to the pre-incubation temperature. The relationship between this temperature-dependent pattern of enzyme activity, termed thermohysteresis, and pre-incubation with other agents was examined. Pre-incubation of membrane vesicles with azide and Mg2+, without exogenous ADP, resulted in almost complete inhibition of the initial rate of ATPase when assayed at 10 degrees C, but had little effect at 37 degrees C. Rates of ATP synthesis following this pre-incubation were not affected at any temperature. Azide inhibition of ATP hydrolysis by the isolated enzyme was reduced when an ATP-regenerating system was used. A gradual reactivation of azide-blocked enzyme was slowed down by the presence of phosphate in the reaction medium. The well-known Mg2+ inhibition of ATP hydrolysis was shown to be greatly enhanced at 15 degrees C relative to at 37 degrees C. The results suggest that thermohysteresis is a consequence of an inactive form of the enzyme that is stabilized by the binding of inhibitory Mg-ADP.     

Induction of gel-phase lipid in plasma membrane of chick intestinal cells after coccidial infection.

When chickens are infected with the coccidial parasite Eimeria necatrix, the plasma membrane of intestinal cells harbouring second-generation schizonts becomes refractory to mechanical shearing, hypotonic shock and ultrasonication. Plasma membrane from these infected cells was isolated to high purity as judged by enriched levels of ouabain-sensitive (Na+ + K+)-stimulated Mg2-dependent ATPase activity and sialic acid content, the lack of detectable cytochrome oxidase and glucose-6-phosphatase activities and electron microscopic analysis of the final preparation. Wide-angle X-ray diffraction patterns recorded from the isolated membranes revealed that during the later stages of parasite maturation the host cell plasma membrane acquires increasing proportions of gel-phase lipid. By contrast, purified membrane from isolated parasites is in a liquid-crystalline state. The transition temperature of host cell plasmalemma at 100 h postinfection is 61 degrees C, about 20 degrees C above physiological temperature. By contrast, liposomes of plasma membranes from infected cells undergo a thermal transition at about 28 degrees C. The accumulation of gel-phase lipid in the host cell plasma membrane is not attributable either to an increase in the constituent ratio of saturated to unsaturated fatty acids or to a significant change in the cholesterol to phospholipid ratio. During the late stages of infection, the cells become stainable with trypan blue which suggests that the acquisition of crystalline phase lipid disrupts the permeability of the host cell plasmalemma.     

Cholesterol effect on enzyme activity of the sarcolemmal (Ca2+ + Mg2+)-ATPase from cardiac muscle

The effect of cholesterol incorporation and depletion of the cardiac sarcolemmal sacs on (Ca2+ + Mg2+)-ATPase activity was examined. Cholesterol incorporation to the sarcolemmal sacs was achieved utilizing an in vivo and an in vitro procedure. Cholesterol depleted membranes were obtained in vitro after incubation of the sarcolemmal sacs with inactivated plasma. Arrhenius plots of the (Ca2+ + Mg2+)-ATPase activity showed a triphasic curve when the assays were carried out using a temperature range between 0 and 40 degrees C. The sarcolemmal (Ca2+ + Mg2+)-ATPase activity was shown to be inversely proportional to the cholesterol concentration of the membranes, showing a low ATPase activity with a high cholesterol content and a high ATPase activity when the cholesterol concentration was low. Although the (Ca2+ + Mg2+)-ATPase activity was found to be inhibited in the cholesterol incorporated sarcolemmal sacs, the withdrawal of small amounts of cholesterol from the membranes produced an important stimulatory effect. Changes in (Ca2+ + Mg2+)-ATPase activity due to variation in the membrane cholesterol concentration were shown to be reversible. Our results indicate the possibility of a slow exchange of cholesterol between the tightly bound lipid surrounding the (Ca2+ + Mg2+)-ATPase and the bulk lipid of the sarcolemma.     

Effect of amiodarone on membrane fluidity and Na+/K+ ATPase activity in rat-brain synaptic membranes

In rat-brain synaptic membranes at a fixed temperature (37 degrees C), amiodarone dose-dependently inhibits the Na+/K+ ATPase activity (IC50 approximately equal to 2.10(-5)M) and produces a linear increase in the degree of fluorescence depolarization (P) of 1,6-diphenylhexatriene embedded in the lipid matrix. Amiodarone has no effect on Mg++ ATPase and K+PNPase activity up to 3.10(-4)M. Studies carried out at different temperatures indicate that 10(-5)M amiodarone inhibits the Na+/K+ ATPase and decreases the lipid fluidity at all the temperatures studied (9 - 40 degrees C). The compound significantly displaces the temperature of transition observed around 20 degrees C in both Na+/K+ ATPase activity and lipid fluidity to 24 degrees C with no changes in slopes. The results suggest that part of the selective inhibition of Na+/K+ ATPase activity by amiodarone could be due to the effects of the drug on lipid dynamics.     

Freezing and thawing of myosin with no alteration in ATPase activity

Myosin can be frozen in liquid nitrogen (-70 degrees C) and stored at this temperature for 5 months with no loss in K+, Ca2+, or actin + Mg2+ -stimulated ATPase activities. Furthermore, myosin can be refrozen and thawed in this manner for at least 5 consecutive times with no alteration in ATPase activity.     

Ordering of bulk membrane lipid or protein promotes activity of plasma membrane Mg2+ATPase

Treatment of liver plasma membranes with phospholipase A2 or high doses of concanavalin A enhances the activity of Mg2+ATPase assayed at temperatures greater than 30 degrees C. The effects of the two treatments are not additive. Both the removal of phospholipids and binding of the lectin increase the degree of polarization of fluorescence of the lipid-soluble fluorophores, diphenylhexatriene and beta-parinaric acid, suggesting that decreased lipid fluidity may activate Mg2+-ATPase. In fact modification of lipid fluidity by reconstitution of phospholipase-treated membranes with phosphatidylcholines of defined fatty acid composition or by addition of cis-vaccenic acid showed a strong inverse correlation between Mg2+ATPase activity and lipid fluidity as monitored by fluorescence polarization. However, despite the ability of concanavalin A to nonspecifically order membrane lipid, its effect on Mg2+ATPase is apparently not mediated in this manner because other enzyme-activating lectins such as Ricinus communis agglutinin and wheat germ agglutinin are without effect on lipid fluidity. The facts that lectins of lower valency than tetravalent native concanavalin A such as divalent succinyl concanavalin A are far less effective in activating the enzyme and that paraformaldehyde treatment also activates suggests that cross-linking of membrane proteins is responsible. Hence, the diminution in activity of this membrane enzyme due to the disordering effect of heat in the physiological temperature range can be counteracted by isothermally increasing the order of either membrane lipid or protein.     

Reconstitution of the purified (Na+ + Mg2+)-ATPase from Acholeplasma laidlawii B membranes into lipid vesicles and a characterization of the resulting proteoliposomes

The purified (Na+ + Mg2+)-ATPase from Acholeplasma laidlawii B membranes was reconstituted with dimyristoylphosphatidylcholine using a cholate solubilization and dialysis procedure. The incorporation of this enzyme into the phospholipid bilayer is accompanied by an enhancement of its specific activity and by a restoration of its lipid phase state-dependent properties which were lost during solubilization and purification from native membranes. Moreover, reconstitution of this ATPase with phospholipid also stabilizes it against cold inactivation at low temperatures (approximately equal to 0 degrees C), oxidative degradation at room temperature, and thermal denaturation at elevated temperatures (approximately equal to 55 degrees C). The elution profile from a Sepharose 4B-CL column indicates that all of the ATPase protein is associated with the phospholipid vesicles and that the Stoke's radius of the proteoliposomes formed is smaller than that of the lipid vesicles formed in the absence of any protein. The latter conclusion is supported by sedimentation velocity measurements and by an electron microscopic examination of negatively stained preparations. The electron microscopic studies demonstrate that sealed vesicles are formed only at low protein-to-lipid ratios. These observations indicate that the Acholeplasma laidlawii B (Na+ + Mg2+)-ATPase has been structurally and functionally reconstituted into lipid vesicles and that the proteoliposomes formed are amenable to studies aimed at the clarification of its proposed role as a sodium ion pump.     

Characterization of gill (Na+ + K+)-ATPase in the sea bass (Dicentrarchus labrax L.)

Bass gill microsomal preparations contain both a Na+, K+ and Mg2+-dependent ATPase, which is completely inhibited by 10(-3)M ouabain and 10(-2)M Ca2+, and also a ouabain insensitive ATP-ase activity in the presence of both Mg2+ and Na+. Under the optimal conditions of pH 6.5, 100 mM Na+, 20 mM K+, 5 mM ATP and 5 mM Mg2+, (Na+ + K+)-ATPase activity at 30 degrees C is 15.6 mumole Pi hr/mg protein. Bass gill (Na+ + K+)-ATPase is similar to other (Na+ + K+)-ATPases with respect to the sensitivity to ionic strength, Ca2+ and ouabain and to both Na+/K+ and Mg2+/ATP optimal ratios, while pH optimum is lower than poikilotherm data. The enzyme requires Na+, whereas K+ can be replaced efficiently by NH+4 and poorly by Li+. Both Km and Vm values decrease in the series NH+4 greater than K+ greater than Li+. The break of Arrhenius plot at 17.7 degrees C is close to the adaptation temperature. Activation energies are scarcely different from each other and both lower than those generally reported. The Km for Na+ poorly decreases as the assay temperature lowers. The comparison with literature data aims at distinguishing between distinctive and common features of bass gill (Na+ + K+)-ATPase.