The role of conformational changes in the functioning of brain Na,K-ATPase]

In the experiments with enzyme preparations of Na,K-ATPase from normal brain tissue (NBT) and tumorous brain tissue (TBT) the following data were established: 1) the cooperativity of Na,K-ATPase with Na+ from NBT is temperature-dependent, the Hill coefficient (nH) at 37, 27.0-30.5 and 20-22 degrees C being 1.80 +/- 0.07, 1.30 +/- 0.09 and 1.10 +/- 0.08, respectively; the cooperativity of Na+ with Na,K-ATPase from TBT was absent; 2) the cooperativity for ouabain (nH-1.30 +/- 0.05) was revealed only in the case of Na-pump from TBT; 3) the protective effect of ATP against the inhibitory action of pCMB is temperature-dependent and differs significantly in enzyme preparations from NBT and TBT; 4) the parameters of the temperature inactivation of enzyme preparations at 45-52 degrees C, especially the change of entropy (delta S*) were different in the case of NBT and TBT; 5) a peptide fraction isolated from sheep brain differently inhibited the Na,K-ATPase from NBT and TBT. In conclusion, these data demonstrate that there are significant differences in functioning of Na,K-ATPase from NBT and TBT, and that besides lipid-protein interactions the local domenic conformational changes in the enzyme molecule may play a definite role in these differences.     

Temperature-dependencies of various catalytic activities of membrane-bound Na+/K+-ATPase from ox brain, ox kidney and shark rectal gland and of C12E8-solubilized shark Na+/K+-ATPase

The temperature dependence of ouabain-sensitive ATPase and phosphatase activities of membrane fragments containing the Na+/K+-ATPase were investigated in tissue from ox kidney, ox brain and from shark rectal glands. The shark enzyme was also tested in solubilized form. Arrhenius plots of the Na+/K+-ATPase activity seem to be linear up to about 20 degrees C, and non-linear above this temperature. The Arrhenius plots of mammalian enzyme (ox brain and kidney) were steeper, especially at temperatures below 20-30 degrees C, than that of shark enzyme. The Na+-ATPase activity showed a weaker temperature-dependence than the Na+/K+-ATPase activity. The phosphatase reactions measured, K+-stimulated, Na+/K+-stimulated and Na+/K+/ATP-stimulated, also showed a weaker temperature-dependence than the overall Na+/K+-ATPase activity. Among the phosphatase reactions, the largest change in slope of the Arrhenius plot was observed with the Na+/K+/ATP)-stimulated phosphatase reaction. The Arrhenius plots of the partial reactions were all non-linear. Solubilization of shark enzyme in C12E8 did not change the curvature of Arrhenius plots of the Na+/K+-ATPase activity or the K+-phosphatase activity. Since solubilization involves a disruption of the membrane and an 80% delipidation, the observed curvature of the Arrhenius plot can not be attributed to a property of the membrane as such.     

Effect of digitonin on the activity of Na,K-ATPase from bovine brain

The kinetic properties of intact and digitonin-treated Na,K-ATPase from bovine brain were studied. The temperature dependence curve for the rate of ATP hydrolysis under optimal conditions (upsilon 0) in the Arrhenius plots shows a break at 19-20 degrees. The temperature dependence curves for Km' and Km" have breaks at the same temperatures, while the Arrhenius plot for V is linear. The value of the Hill coefficient (nH) for ATP at 37 degrees is variable depending on ATP concentration, i. e. it is less than 1 at ATP concentrations below 50 mkM and is increased up to 3.2 at higher concentrations of the substrate. At high ATP concentrations the value of nH depends on temperature, falling down to 2.1 at 23 degrees and then down to 1 within the temperature range of 21-19 degrees. A further decrease in temperature does not significantly affect the nH value. Digitonin irreversibly inhibits Na, K-ATPase. ATP hydrolysis is more sensitive to the effect of the detergent than is nNPP hydrolysis, i. e. after complete inhibition of the ATPase about 40% of the phosphatase activity are retained. Treatment of Na,K-ATPase by digitonin results in elimination of the breaks in the Arrhenius plots for upsilon 0, Km' and Km", whereas the temperature dependence plot of V remains linear. Simultaneously digitonin eliminates the positive cooperativity of the enzyme for ATP. It is assumed that Na, K-ATPase from bovine brain is an oligomer of the (alpha beta) 4 type. Digitonin changes the type of interaction between the protomers within the oligomeric complex by changing the lipid environment of the enzyme or the type of protein -- lipid interactions.     

Characteristics of (Na+ + K+)-ATPase in the gills of bass

A partial characterization of bass gill (Na+ + K+-ATPase is reported in the present paper. Microsomal preparation from gill homogenate showed optimal (Na+ + K+)-ATPase activity at pH 6,5 in the presence of 100 mM Na+, 20mM K+ and 5mM Mg2+. Under these conditions maximal activity was shown at 45 degrees C, even if an increased lability of the enzyme was shown at temperature greater than 30 degrees C. A complete inhibition of the enzyme occurred in the presence of 1 mM ouabain. The break in the Arrhenius plot occurred approximatively at the temperature of adaptation of these fish (18 degrees C). The energies of activation above and below the break were scarcely different from each other and lower than those reported in other Poikilotherms. Furthermore similar values of Km for Na+ were evidenced at 18 degrees C and 30 degrees C. The whole of data are discussed in comparison with other teleost gill (Na+ + K+)-ATPase reports and related to the physiological role of the enzyme in osmoregulation.     

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.     

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.     

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.     

Variation of scallop sarcoplasmic reticulum Ca2+-ATPase activity with temperature

Methods for preparing native scallop sarcoplasmic reticulum vesicles, largely purified membranous scallop sarcoplasmic reticulum Ca2+-ATPase, and nonionic detergent-solubilized sarcoplasmic reticulum Ca2+-ATPase are described. The effect of a range of polyoxyethylene-based detergents on the solubilized Ca2+-ATPase was tested. Decaethylene glycol dodecyl ether (C12E10) supported the highest levels of activity, although C12E8 and C12E9 were more routinely used. Arrhenius plots of Ca2+-ATPase activity, where the assays were carried out with the same pH at all temperatures (7.4), showed a region of nonlinearity at 10 degrees C. A very similar plot was obtained when no compensation was made for pH variation with temperature. Both the break in the Arrhenius plot and the activation energies for the scallop sarcoplasmic reticulum above and below the break were very similar to those found for lobster sarcoplasmic reticulum (Madeira, V. M. C., Antunes-Madeira, M. C., and Carvalho, A. R. (1974) Biochem. Biophys. Res. Commun. 65, 997-1003). The Arrhenius plot of the scallop Ca2+-ATPase in C12E8 no longer showed the nonlinearity at 10-12 degrees C seen with the native sarcoplasmic reticulum, but instead a break now appeared at 20-21 degrees C. This is close to the Arrhenius break temperature of rabbit Ca2+-ATPase in C12E8 and of a perturbation in C12E8 (Dean, W. L. (1982) Biophys. J. 37, 56-57).     

Partial purification and properties of (Na+ + K+)-ATPase from Potamon potamios.

1. The tissue distribution of the (Na+ + K+)-ATPase in the freshwater/land crab Potamon Potamios was studied. 2. Gills were found to display the highest total activity in the whole animal (47%) but the highest specific activity was detected in the heart (15.15 mumol Pi/mg protein/min). 3. All other organs tested were found to have low enzyme activity. 4. The freshwater/land crab ATPase enzyme was inhibited by ouabain with a Ki of 0.5 mM.Km values for ATP, Mg2+ and K+ were 1.4, 4.0 and 1.2 mM respectively. The enzyme also showed a break in the Arrhenius plot at 23 degrees C. 5. A purification method of microsomal ATPase is described involving ultracentrifugation and electrofocusing.     

Kinetics of Na-ATPase activity by the Na,K pump. Interactions of the phosphorylated intermediates with Na+, Tris+, and K+

To determine the biochemical events of Na+ transport, we studied the interactions of Na+, Tris+, and K+ with the phosphorylated intermediates of Na,K-ATPase from ox brain. The enzyme was phosphorylated by incubation at 0 degrees C with 1 mM Mg2+, 25 microM [32P]ATP, and 20-600 mM Na+ with or without Tris+, and the dephosphorylation kinetics of [32P]EP were studied after addition of (1) 1 mM ATP, (2) 2.5 mM ADP, (3) 1 mM ATP plus 20 mM K+, and (4) 2.5 mM ADP plus Na+ up to 600 mM. In dephosphorylation types 2-4, the curves were bi- or multiphasic. "ADP-sensitive EP" and "K+-sensitive EP" were determined by extrapolation of the slow phase of the curves to the ordinate and their sum was always larger than Etotal. These results required a minimal model consisting of three consecutive EP pools, A, B, and C, where A was ADP sensitive and both B and C were K+ sensitive. At high [Na+], B was converted rapidly to A (type 4 experiment). The seven rate coefficients were dependent on [Na+], [Tris+], and [K+], and to explain this we developed a comprehensive model for cation interaction with EP. The model has the following features: A, B, and C are equilibrium mixtures of EP forms; EP in A has two to three Na ions bound at high-affinity (internal) sites, pool B has three, and pool C has two to three low-affinity (external) sites. The putative high-affinity outside Na+ site may be on E2P in pool C. The A leads to B conversion is blocked by K+ (and Tris+). We conclude that pool A can be an intermediate only in the Na-ATPase reaction and not in the normal operation of the Na,K pump.     

On the temperature- and salt-dependent conformation change in human erythrocyte pyruvate kinase

The influence of temperature, K+, Mg2+ and fructose 1,6-bisphosphate on human red cell pyruvate kinase was investigated. Kinetic measurements between 4 degrees C and 43 degrees C revealed a remarkable influence of the temperature on the allosteric behaviour of the enzyme. Below a transition region between 15 degrees C and 20 degrees C (as obtained from an Arrhenius plot) the enzyme shows non-cooperative behaviour, as can be deduced from Michaelis-Menten, Hill and Scatchard plots. At temperatures above 20 degrees C cooperativity increases with rising temperature. This effect becomes even more pronounced at higher temperatures upon addition of increasing amounts of K+ and Mg2+ accompanied by a slight decrease of the reaction velocity. Fructose 1,6-bisphosphate, however, abolishes cooperativity at every temperature and salt concentration measured. Difficulties which arise in evaluating the correct values of V, Km and the Hill coefficient nH with cooperative systems are met by using a computer program of Wieker, Johannes and Hess, especially designed for the determination of kinetic parameters obtained from sigmoidal steady-state kinetics.     

Effects of temperature on cytochrome oxidase activity in solubilized form and in lipid vesicle systems.

Isolated mammalian cytochrome oxidase gave an Arrhenius plot with a break (Tb) at about 20 degrees C when assayed in a medium containing Emasol. The activation energies above and below 20 degrees C were 9.3 (EH) and 18.9 kcal/mol (EL), respectively. Isolated cytochrome oxidase was also incorporated into vesicles of dipalmitoyl phosphatidylcholine (DPPC, phase transition temperature Tt = 40 degrees C), dimyristoyl phosphatidylcholine (DMPC, Tt = 23 degrees C) and dioleoyl phosphatidylcholine (DOPC, Tt = -22 degrees C). The DPPC system showed a nearly linear Arrhenius plot between 9 and 36 degrees C with E = 22.8 kcal/mol. When cytochrome oxidase was resolubilized from the DPPC vesicles and assayed in solution a biphasic plot was obtained again. Cytochrome oxidase-DOPC was more active than the solubilized enzyme and exhibited a biphasic Arrhenius plot with Tb = 23 degrees C. EH and EL were 6.6 and 15.8 kcal/mol, respectively. The plot for the oxidase-DMPC also showed a break (Tb = 26 degrees C) with EH = 6.6 and EL = 26.6 kcal/mol. These results indicate that the break in the Arrhenius plot reflects primarily a structural transition in the cytochrome oxidase molecule between the "hot" and "cold" conformations, as proposed previously. This transition, as well as the molecular state of cytochrome oxidase, is affected by the physical state of the membrane lipids as reflected by changes in the kinetic properties.     

The effect of hypothermia on kinetic characteristics of the rat brain synaptic membrane Na,K-ATPase

The effect of profound hypothermia (acute or prolonged) on Km for ATP, Vm and strophanthine K affinity to Na,K-ATPase in the rat brain synaptosomal membranes was investigated. The temperature dependence of Na,K-ATPase activity in temperature range 5-40 degrees C was also studied. Hypothermia decreases Km and Vm, and increases affinity of strophanthine K to the enzyme. There are two linear sections in Arrhenius plots ofNa,K-ATPase activity. Hypothermia does not change position of the break point in Arrhenius plots. The mechanisms and biological significance of the changes revealed are discussed.     

Temperature and Ca(2+)-dependence of the sarcoplasmic reticulum Ca(2+)-ATPase in haddock,salmon, rainbow trout and zebra cichlid

Temperature dependence of Ca(2+)-ATPase from the sarcoplasmic reticulum (SR) in rabbit muscle has been widely studied, and it is generally accepted that a break point in Arrhenius plot exist at approximately 20 degrees C. Whether the break point arises as a result of temperature dependent changes in the enzyme or its membrane lipid environment is still a matter of discussion. In this study we compared the temperature dependence and Ca(2+)-dependence of SR Ca(2+)-ATPase in haddock (Melanogrammus aeglefinus), salmon (Salmo salar), rainbow trout (Oncorhynchus mykiss) and zebra cichlid (Cichlasoma nigrofasciatum). The Arrhenius plot of zebra cichlid showed a break point at 20 degrees C, and the haddock Arrhenius plot was non-linear with pronounced changes in slope in the temperature area, 6-14 degrees C. In Arrhenius plot from both salmon and rainbow trout a plateau exists with an almost constant SR Ca(2+)-ATPase activity. The temperature range of the plateau was 14-21 and 18-25 degrees C in salmon and rainbow trout, respectively. Ca(2+)-dependence in the four different fish species investigated was very similar with half maximal activation (K(0.5)) between 0.2 and 0.6 micro M and half maximal inhibition (I(0.5)) between 60 and 250 micro M. Results indicated that interaction between SR Ca(2+)-ATPase and its lipid environment may play an important role for the different Arrhenius plot of the different types of fish species investigated.     

Purification and characterization of (Na+ + K+)-ATPase from toad kidney.

This report describes the partial purification and the characteristics of (Na+ + K+)-ATPase (ATP phosphohydrolase, EC 3.6.1.3) from an amphibian source. Toad kidney microsomes were solubilized with sodium deoxycholate and further purified by sodium dodecyl sulphate treatment and sucrose gradient centrifugation, according to the methods described by Lane et al. [(1973) J. Biol. Chem. 248, 7197--7200], J?rgensen [(1974) Biochim. Biophys. Acta 356, 36--52] and Hayashi et al. [(1977) Biochim. Biophys. Acta 482, 185--196]. (Na+ + K+)-ATPase preparations with specific activities up to 1000 mumol Pi/mg protein per h were obtained. Mg2+-ATPase only accounted for about 2% of the total ATPase activity. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis revealed three major protein bands with molecular weights of 116 000, 62 000 and 26 000. The 116 000 dalton protein was phosphorylated by [gamma-32P]ATP in the presence of sodium but not in the presence of potassium. The 62 000 dalton component stained for glycoproteins. The Km for ATP was 0.40 mM, for Na+ 12.29 mM and for K+ 1.14 mM. The Ki for ouabain was 35 micron. Temperature activation curves showed two activity peaks at 37 degrees C and at 50 degrees C. The break in the Arrhenius plot of activity versus temperature appeared at 15 degrees C.     

Effect of ethanol on synaptosomal (Na+ + K+)-ATPase in control and ethanol-dependent rats

Rats were made dependent upon ethanol by feeding them liquid diets containing ethanol. Synaptosomal plasma membranes (SPM) were isolated from cerebral cortex and midbrain regions of isocaloric-fed control and ethanol-dependent rats. No major alcohol-induced alteration in in vitro (Na+ + K+)-ATPase activity was found in SPM of either brain area. At 37 degrees C, ethanol (0.10 to 0.98 M) added to incubations caused a dose-dependent inhibition of (Na+ + K+)-ATPase activity. The degree of inhibition found was independent of the diet administered or whether ethanol was present in the diet. At temperatures between 14 and 22 degrees C, 0.48 M ethanol caused a temperature-dependent decrease in activity. Arrhenius plots for SPM (Na+ + K+)-ATPase showed that in control and ethanol-dependent rats fed the Lieber de Carli diet, 0.48 M ethanol did not alter the transition temperature of this enzyme. Activation energies both above and below the transition temperature were decreased by the addition of ethanol to incubations. These results indicate that (Na+ + K+)-ATPase, a membrane-bound enzyme that is sensitive to its lipid environment and to the presence of ethanol, is not altered by the chronic administration of ethanol to rats.     

Comparison of sarcoplasmic reticulum Ca2+-adenosine triphosphatase from vitamin E-deficient dystrophic rabbit skeletal muscle with iron-ascorbate-treated and untreated enzyme

Sarcoplasmic reticulum Ca2+-ATPase from rabbit skeletal muscle has an Arrhenius curve of enzyme activity with a discontinuity at about 20 degrees C. Preparations treated with FeSO4 and ascorbic acid and from a vitamin E-deficient dystrophic rabbit have 22% of the normal activity and a linear Arrhenius curve (Promkhatkaew, D., Komaratat, P., & Wilairat, P. (1985) Biochem. Int. 10, 937-943). All three preparations were cross-linked to the same extent by dimethyl suberimidate and copper-phenanthroline reagent at temperatures above and below the temperature of the Arrhenius discontinuity. Both iron-ascorbate-treated Ca2+-ATPase and that from a vitamin E-deficient animal had 50% of the normal sulfhydryl content, but the disulfide and free amino contents were unaltered. These observations suggest that loss of sulfhydryl groups through lipid peroxidation, both in vivo and in vitro, resulted in reduction of Ca2+-ATPase activity and loss of the break in the Arrhenius plot. Changes in Ca2+-ATPase polypeptide aggregational state could not account for the discontinuity in the Arrhenius curve as revealed by the similar extent of cross-linking of the three enzyme preparations at temperatures above and below the temperature of the Arrhenius discontinuity.     

Difference in phospholipid dependence between two isozymes of brain (Na+ + K+)-ATPase

The effect of phospholipase C on two isozymes (alpha (+) and alpha forms) of rat brain (Na+ + K+)-ATPase and the temperature-dependence of their activities were investigated. Phospholipase C from Clostridium welchii inhibited the activities of the enzymes treated with and without pyrithiamin or N-ethylmaleimide, a preferential inhibitor of the alpha (+) form, but the extent of the inhibition was higher in the control enzyme than in the treated enzymes. The treatment of the (Na+ + K+)-ATPase with phospholipase C altered a ratio between high- and low-affinity components for ouabain inhibition. It also caused the similar change in a ratio between the alpha (+) and alpha forms of Na+-stimulated phosphorylation from [gamma-32P]ATP. These findings indicate that the alpha (+) form of rat brain (Na+ + K+)-ATPase is more sensitive to phospholipase C than the alpha form. Analysis of Arrhenius plots of the activities of the control and pyrithiamin-treated enzymes showed that there was a difference between the two enzymes in a break point. We suggest that two isozymes of rat brain (Na+ + K+)-ATPase differ in the interaction with phospholipids or in the lipid-environment.     

Difference between neuronal and nonneuronal (Na+ + K+)-ATPases in their conformational equilibrium

Several experiments were carried out to study the difference between two isozymes (alpha(+) and alpha) of (Na+ + K+)-ATPase in the conformational equilibrium. Rat brain (Na+ + K+)-ATPase was much more thermolabile than the kidney enzyme. Both enzymes were protected from heat inactivation not only by Na+ and K+, but also by choline in varying degrees, though there was a difference between the two enzymes in the protection by the ligands. The brain enzyme was partially protected from N-ethylmaleimide (NEM) inactivation by both Na+ and K+, but the effects of the ligands on NEM inactivation of the kidney enzyme were more complex. Though ligands differentially affected the thermostability and NEM sensitivity of the two enzymes, the effects were not simply related to the conformational states. The sensitivity of phosphoenzyme (EP) formed in the presence of ATP, Na+, and Mg2+ to ADP or K+ and K+-p-nitrophenyl phosphatase (pNPPase) was then studied as a probe of the differences in the conformational equilibrium between the two isozymes. The EP of the brain enzyme was partially sensitive to ADP, while those of the heart and kidney enzymes were not. At physiological Na+ concentrations the percentages of E1P formed by the brain and kidney enzymes were determined to be about 40-50 and 10-20% of the total EP, respectively. The hydrolytic activity of pNPP in the presence of Li+, a selective activator at catalytic sites of the reaction, was much higher in the kidney enzyme than in the brain enzyme. The inhibition of K+-stimulated pNPPase by ATP and Na+ was greater in the latter enzyme than in the former. These results suggest that neuronal and nonneuronal (Na+ + K+)-ATPases differ in their conformational equilibrium: the E1 or E1P may be more stable in the alpha(+) than in the alpha during the turnover, and conversely the E2 or E2P may be more stable in the latter than in the former.     

A saturation transfer electron spin resonance study on the break in the Arrhenius plot for the rotational motion of Ca2+-dependent adenosine triphosphatase molecules in purified and lipid-replaced preparations of rabbit skeletal muscle sarcoplasmic reticulum

By means of saturation transfer electron spin resonance spectroscopy the rotational motion of spin-labeled Ca2+-dependent ATPase molecules has been investigated for three kinds of preparations of rabbit skeletal muscle sarcoplasmic reticulum: MacLennan's enzyme (purified ATPase preparation), DOPC- and egg PC-ATPase (purified ATPase preparations in which endogenous lipids are replaced with dioleoyl and egg yolk phosphatidylcholine, respectively). The rotational mobility of the enzyme in these preparations is somewhat lower than that in the intact membrane, probably due to the reduced amount of lipids. For all the preparations, however, the Arrhenius plot for rotational mobility showed a break at about 18 degrees C, the same temperature at which a break in the Arrhenius plot for Ca2+-ATPase activity occurs. This result provides further evidence that the break in the Arrhenius plot is not related to a lipid phase transition but to a change in the physical state of the Ca2+-ATPase molecule existing in fluid lipids.