Interaction of a membrane preparation of Na+, K+-ATPase with a spin-labeled analog of ATP]

Interaction of membrane Na+, K+-ATPase preparation from brain gray matter with spin-labelled ATP analogue, in which free iminoxyl radical is joined as a result of 2'(3')-OH ribose groups acylation, is studied. The rotatory mobility of spin-labelled ATP analogue in Na+,K+-ATPase preparation is found to change in non-linear manner during temperature variation (the break-point on the curve being at 20-23degrees C). It correlates with temperature dependence of Na+,K+-ATPase and temperature dependence of lipid viscosity in the membranes, determined by means of hydrophobic spin probes. Substitution of Mg2+ ions with paramagnetic Mn2+ ions resulted in an intense magnetic dipole-dipole interaction between a spin label and Mn2+ ion, which indicated the formation of triple complex enzyme--spin-labelled ATP--Mn2+.     

Effect of temperature on Ca-ATPase from sarcoplasmic reticulum membranes: ESR studies

Using spin-labeled fatty acid derivatives and maleimide, the effect of temperature on the structural state of various parts of the lipid bilayer of sarcoplasmic reticulum (SR) membranes and the segmental motion of the Ca-ATPase molecule were investigated. The mobility of the spin probes localized in the hydrophobic zone and the outer part of the SR membrane was shown to increase with a rise in temperature from 4 to 44 degrees C, the temperature of 20 degrees C being critical for these changes. In the presence of ATP, critical changes in the spin probe mobility occur at lower temperatures, while in the presence of ATP and Ca2+ they are observed at 20 degrees C for a spin probe localized in the outer part of the SR membrane. The mobility of a spin probe localized in the hydrophobic part of the membrane increases linearly with a rise in temperature. In the absence of ligands, the segmental motion of Ca-ATPase changes linearly within a temperature range of 10-30 degrees C. However, when ATP alone or ATP and Ca2+ are simultaneously added to the incubation mixture, the protein mobility undergoes critical changes at 20 degrees C. The Arrhenius plots for ATPase activity and Ca2+ uptake rate in SR membrane preparations also have a break at 20 degrees C. It is assumed that changes in the structural state of membrane lipids produce conformational changes in the Ca-ATPase molecule; the enzyme seems to be unsensitive to the structural state of the membrane lipid matrix in the absence of the ligands.     

Structural dynamics in F1ATPase during the first reaction cycle of ATP hydrolysis

The velocity of ATP hydrolysis, catalyzed by purified F1ATPase from Micrococcus luteus, was decelerated on decreasing the temperature. At 13 degrees C one reaction cycle is completed after 20 s. Hydrolysis was triggered upon rapid mixing of the enzyme with ATP. During the first reaction cycle, succeeding structural alterations of the F1ATPase were traced by time resolved X-ray scattering. The scattering spectra obtained from consecutive intervals of 1 s, revealed the F1ATPase to pass a conformational state exhibiting an expanded (6%) molecular shape. The expanded state was observed between 45% and 65% of the time required to complete the reaction cycle. This points out a conformational pulsation during ATP hydrolysis.     

Lipid protein interactions in mitochondria. VII. A comparison of the effects of lipid removal and lipid perturbation of the kinetic properties of mitochondrial ATPase.

We investigated the kinetics of mitochondrial ATPase in bovine heart mitochondria and submitochondrial particles upon treatment with phospholipase A2, or upon addition of n-butanol to perturb the lipid protein interactions. The changes observed are the following: (1) Lipid removal or perturbation with butanol is accompanied by loss of ATPase activity with decrease of both V and of the KM for ATP. (2) There are changes of activation energy of ATPase activity at temperatures above the discontinuity normally observed for membrane-bound enzymes in mitochondria. In particular, butanol abolishes the discontinuity, and induces a constant activation energy of about 32 kcal/mol in the range 8--37 degrees C. (3) Butanol modifies the pH dependence of ATPase shifting the pH optimum from around 10 to less alkaline values. The optimum for Mg2+ concentrations is increased by the solvent. (4) Treatment with phospholipase A2 results in a removal of oligomycin-sensitive ATPase, whereas butanol addition prevents oligomycin inhibition of ATPase. (5) In beef heart mitochondria, a spin-labelled analog of the inhibitor, dicyclohexyl carbodiimide, did not show any change in environment upon butanol addition, unlike that found in mitochondria from Saccharomyces cerevisiae.     

Temperature-induced change in the Ca2+-dependent ATPase activity and in the state of the ATPase protein of sarcoplasmic reticulum membrane.

The temperature dependence of the Ca2+-dependent ATPase activity and of the conformational fluctuation of the ATPase molecule has been measured for four kinds of preparations: fragmented sarcoplasmic reticulum, MacLennan's enzyme (purified ATPase preparation), and DOL and egg PC-ATPase (purified ATPase preparations in which lipids are replaced with dioleoyllecithin and egg yolk lecithin, respectively). It has been found that Arrhenius plots of the Ca2+-dependent ATPase activity show a break at about 18 degrees C for all the preparations. Hydrogen--deuterium exchange kinetics of the peptide NH protons were used to measure the conformational fluctuation of the protein molecules. Van't Hoff plots of the conformational fluctuation amplitude of a region near the surface of the ATPase molecule also show a break at about 18 degrees C for all the preparations. It is concluded that the break at around 18 degrees C is not related to a gel-liquid crystalline transition of lipids but to a change in the conformation of the ATPase molecule existing in fluid lipids.     

Differential scanning calorimetry study of local anesthetic effects on F1ATPase and submitochondrial particles

The differential scanning calorimetry trace of F1ATPase, prepared from beef heart submitochondrial particles, has a single sharp endothermic transition at 80.5 +/- 1.0 degrees C and a half-height peak width of 2.0 +/- 0.2 degrees. The transition enthalpy is 19 +/- 2 cal/g of protein. Submitochondrial particles (SMP) have a similar peak at 75.1 +/- 0.5 degrees C with a half-height peak width of 1.8 +/- 0.1 degrees and an enthalpy of 5 +/- 1 cal/g of SMP protein. The SMP transition is provisionally identified as being due to membrane-bound F1ATPase. Tetracaine and dibucaine cause these transitions to shift to lower temperatures; addition of 0.3 mM dibucaine gives peaks at 71.7 and 64.9 degrees C for F1ATPase and SMP, respectively, and 1.0 mM tetracaine gives peaks at 70.0 and 60.5 degrees C for F1ATPase and SMP, respectively. These anesthetic concentrations also give appreciable inhibition of enzyme activity at 25 degrees C. We conclude that the local anesthetics induce conformational alterations in the F1ATPase-protein complex which result both in enzyme inhibition and in the lowering of the thermal denaturation transition temperature.     

Thyroxine: structural transformations in the membranes of rabbit skeletal muscle sarcoplasmic reticulum

The structure of the membranes of sarcoplasmic reticulum fragments (SRF) normally and in thyrotoxicosis was studied by the spin-label and spin-probe methods and by chemifluorescence. The curves of temperature dependence of the regularity parameter show a typical break for the spin probe at 20 degrees C shifted by 4 degrees C to sower temperatures for thyrotoxins. The same shift was observed with temperature dependence for the correlation period of the spin label covalently bound to the thiol groups of Ca2+ dependent ATPase of sarcoplasmic reticulum. The latent period of thyrotoxins was reduced and the chemifluorescence intensity increased. The results obtained suggest the occurrence of considerable changes in the structure of SRF membranes in thyrotoxicosis.     

The temperature dependence of the surface tension of aqueous solutions of plasma proteins.

The surface behavior of aqueous solutions of fibrinogen, transferrin, gamma-globulin and albumin at the liquid-gas interface has been investigated by a modified Wilhelmy technique. The temperature dependence of the surface tension was studied over a temperature range of 20--80 degrees C and a pH range of 2--12. Most pronounced conformational changes of fibrinogen with this technique were found in physiological conditions: 35--45 degrees C and pH 7--8. A conformational change was found for gamma-globulin and transferrin solutions, but at a higher temperature and less pronounced than fibrinogen. Albumin did not undergo conformational transitions to a significant extent.     

Response of the adenosine triphosphatase activity of the soluble latent F1 enzyme from beef heart mitochondria to changes in Mg2+ and H+ concentrations

The coupling factor of oxidative phosphorylation from beef heart mitochondria obtained as a "latent F1," exhibits negligible levels of ATPase activity, contains stoichiometric amounts of the specific F1 inhibitor protein, and is stable to incubation at low temperature (Adolfsen, R., McClung, J.A., and Moudrianakis, E. N. (1975) Biochemistry 14, 1727-1735). Incubation of the latent F1 enzyme at 60 degrees C activates its ATPase activity. We show in this paper that regulation of the interaction of the inhibitor protein with the latent F1 enzyme can be accomplished under more physiological conditions. At 37 degrees C, variations in the proton concentration led to changes in the degree and extent of activation of the enzyme, with maximal activation rates occurring after preincubation at pH 9.6. The energy for the pH 9.6-induced activation process (12.1 kcal/mol) was similar to that reported for the dissociation of the inhibitor protein from the membrane-bound F1 enzyme in energized mitochondria (Gomez-Fernandez, J. C., and Harris, D.A. (1978) Biochem. J. 176, 967-973). The rates of activation were higher in the presence of 5 mM ATP and inhibited by the presence of Mg2+, suggesting the existence of a specific binding site for Mg2+ between the inhibitor subunit and the F1 enzyme. A model is presented in which the activation of the latent F1 enzyme is brought about by a rapid titration of positively charged amino acid residues on the inhibitor subunit, followed by a slow release of a tightly bound Mg2+ atom. This model predicts that the initial event leading to the appearance of ATP synthesis is the deprotonation of the inhibitor subunit and that the onset of ATPase activity in mitochondria is due to sequestering of the available free Mg2+.     

Temperature-induced transitions of function and structure in sarcoplasmic reticulum membranes

A transition in the temperature dependences of Ca²⁺ accumulation and ATPase activity occurs at 20 ° C in Sarcoplasmic reticulum membranes. The transition is characterized by an abrupt change in the activation energies for the cation transport process and the associated enzyme activities. The difference in activation energies below and above 20 °C appears to be due to changes in the entropy of activation rather than in the free energy of activation. Also, the temperature dependences of spectral parameters of lipophilic spin-labeled probes and protein-bound spin labels exhibit different behaviors on either side of this temperature. Above 20 °C the lipid matrix probed by the labels exhibits a large increase in molecular motion and a decrease in the apparent ordering of lipid alkyl chains. In addition, labels covalently bound to enzymic reactive sites indicate that the motion of protein side-chains is sensitive to this transition. The results are consistent with an order-disorder transition involving the lipid alkyl chains of the Sarcoplasmic membrane, and with a model in which molecular motion, Ca²⁺ transport and enzyme activity are limited by local viscosity of hydrophobic regions at temperatures below the transition.Another modification of the Sarcoplasmic reticulum membrane occurs between 37 and 40 °C. It appears that at this temperature the processes governing Ca²⁺ accumulation and ATPase activity are uncoupled, and Ca²⁺ accumulation is inhibited, while ATPase activity and passive Ca²⁺ efflux proceed at rapid rates. Parallel transitions of spectroscopic parameters originating from spin labels, covalently bound to the Sarcoplasmic reticulum ATPase, indicate that the uncoupling is due to a thermally-induced protein conformational change.     

Influence of the bound nucleotide on the molecular dynamics of actin

Rotational dynamics of actin spin-labelled with maleimide probes at the reactive thiol Cys-374 were studied. Replacement of the bound nucleotide by Br8ATP in G-actin and Br8ADP in F-actin causes significant increase of the rotational correlation time of the spin probe, indicating reduced motion in both G and F-actin. The orientation dependence of the electron paramagnetic resonance spectra in oriented F-actin filaments revealed an altered molecular order of the probe when the nucleotide was a Br-substituted one. The bound nucleotide affects the myosin S1 ATPase activation by actin; both Vmax and K(actin) decreased significantly when the bound nucleotide of actin was Br8ADP.     

Spin-labelled AMP - an activator of phosphorylase.

1. A spin-labelled AMP derivative and its diamagnetic analogue activate phosphorylase b in the same way, but do not activate phosphorylase a. 2. The electron-spin-resonance spectra of the spin-labelled AMP derivative bound to phosphorylase b and a have "powderlike" characteristics indicating that the spin label is immobilised on the protein. From changes in the electron-spin-resonance spectrum of spin-labelled AMP as phosphorylase b or a is added, the dissociation constants were calculated. 3. The interactions of spin-labelled AMP and the diamagnetic analogue with phosphorylase b and a have been monitored by observing changes in the spectral properties of fluorescent and spin-label probes covalently attached to the enzyme. 4. The dissociation constants of spin-labelled AMP and phosphorylase b or a are 175 +/- 25 muM and 15 +/- 5 muM respectively. Similar dissociation constants are obtained for the diamagnetic analogue. The effect of these AMP derivatives on the covalently attached probe groups and on phosphorylase activity is compared to the effect of AMP and IMP.     

Lipid ordering and enzymic activities in chromaffin granule membranes.

1. The interaction of a variety of fluorescent probes with the membranes of adrenal medullary chromaffin granules is described. 2. Changes in the motional properties of the bound probes with temperature were investigated and evidence is presented which indicates that ordering of the membrane lipids occurs below 33 degrees C. 3. The ordering is characteristics of the membrane lipids and is retained by sonicated aqueous dispersions of the total lipid extracted from chromaffin granule membranes. 4. The ATPase and NADH:acceptor oxidoreductase activities of the chromaffin granule membrane have discontinuous Arrhenius temperature versus activity relationships with 'transitions' at 33 degrees C. 5. The ATPase has a second transition at 36.5 degrees C. 6. The 33 degrees C 'transition' for the NADH:acceptor oxidoreductase is removed by treatment with the detergent Triton X-100. 7. The correlation between the onset of lipid ordering and the change in activation energy of the membrane-bound enzyme activities is discussed in terms of the co-operative interactions of the different membrane components. The possible role of lipid ordering in exocytosis is discussed.     

Study of intermolecular interactions and self-organization of adenylic nucleotides by the spin label method

A spin labeling method for obtaining thermodynamic parameters of nucleotide association is proposed. The method is based on the dependence of ESR parameters of the spin-labelled derivative on concentration of the nonlabelled compound due to formation of associates involving both spin-labelled and unmodified molecules. It is found that at pH 7.5 the constant of adenylic nucleotide association practically does not depend on the number of phosphate groups and is equal to 9.7 +/- 0.3 M-1 for AMP, ADP and ATP in 0.1 M NaCl at 28 degrees C. In acidic medium the value of the association constant increases by a factor two. Base stacking is shown to make the main contribution to stability of the associates of adenylic nucleotides at neutral pH, whereas upon base protonation the key role is played, apparently, by base-phosphate interaction. It is thought, that an increase in the solvent entropy is essential for stabilisation of the associates, this factor being more important in the case of nucleotide association as compared to the association of nucleosides. A possible role of nucleotide association in the processes of intracellular regulation is discussed.     

Temperature-induced changes of spin-labelled radioactive lipids in isolated guinea pig liver microsomal membranes before and in mitochondrial membranes after their translocation.

Lipids of isolated guinea pig liver microsomal membranes were labelled biosynthetically with isomeric doxyl stearic acid and temperature-induced changes of these membranes were studied by electron spin resonance. A noticeable discontinuity was detected at 10--12 degree C with 12- or 16-doxyl stearic acid containing membrane lipids which was attributed to the spin-labelled lipid--microsomal membrane protein interactions since no such discontinuity was detected in liposomes prepared from total lipid extracts of microsomal membranes. When microsomal membranes containing radioactive isomeric spin-labelled lipids were incubated with unlabelled mitochondria, reisolated mitochondrial membranes contained translocated radioactive isomeric spin-labelled lipids. Temperature-induced changes in these membranes showed no discontinuity with either isomeric doxyl stearic acid derivative, establishing a difference in the environment of translocated lipids in the membrane donor compared with that in the membrane acceptor. Microsomal membranes recovered from translocation experiments showed the same behaviour as the original membranes and exhibited the same discontinuity at 10--12 degree C, establishing that the translocation incubation itself did not alter the spin-labelled lipid interaction within these membranes. Studies of the loss of paramagnetism of spin-labelled lipids in microsomal membranes before and in mitochondrial membranes after their translocation showed a significant difference and suggested that both the outer and the inner mitochondrial membranes might have been involved.     

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.     

Binding of basic pancreatic trypsin inhibitor and related isoinhibitors to leukocytic elastase. Determination of thermodynamic parameters

The effect of temperature, ionic strength and solvation power of mono- and divalent cations on the interaction of BPTI-like inhibitors with human leukocytic elastase has been determined. The binding process is characterized by a non-linear dependence of the equilibrium association constant on 1/T indicating a thermal transition at temperature values ranging between 20 degrees C and 35 degrees C depending on the solvent. The marked dependence of the thermodynamic parameters (delta H degrees, delta S degrees, delta G degrees) and of the transition temperature on the concentration and nature of the cations present in solution seems to indicate that the transition, probably of conformational nature, is related to removal of water molecules upon enzyme/inhibitor complex formation.     

The non-catalytic nucleotide-binding site of mitochondrial ATPase is localised on the alpha-subunit(s) of factor F1

The incubation of isolated factor F1 with the di-aldehyde derivative of ADP (oxADP) which is formed as a result of ADP treatment by periodate, causes the covalent binding of 0.9--1 molecules of the oxADP with a molecule of the enzyme. This modification of factor F1 is not accompanied by any changes in the ATPase activity of the enzyme. The modification of factor F1 is preceded by the reversible binding of oxADP with the enzyme with a Kd of 80 micro M. ADP partly prevents factor F1 from modification by oxADP. The electrophoresis of modified factor F1 in polyacrylamide gel in the presence of sodium dodecyl sulphate showed that oxADP binds with the alpha-subunit(s) of factor F1. When submitochondrial particles are incubated with [3H]oxADP, the main part of the radioactive label may be discovered in the polypeptide with a molecular weight of some 30 000 which is probably the adenine nucleotides' translocase. The isolation of factor F1 from particles preincubated with [3H]oxADP showed that the membrane-bound factor F1 covalently binds 0.2--0.3 mol of oxADP per mol of enzyme. Here again, all the oxADP is bound with the alpha subunit(s) of factor F1. The modification of membrane-bound factor F1 by oxADP is accompanied by the partial inhibition of the particles' ATPase activity. The results obtained testify to the fact that the non-catalytic site of mitochondrial ATP ase located on the alpha-subunit(s) of factor F1 may participate in the mechanism of ATP hydrolysis by membrane-bound ATPase.     

Binding of two spin-labelled derivatives of chlorpromazine to human erythrocytes.

The binding to human intact erythrocytes of two different spin-labelled derivatives of chlorpromazine has been studied. The influence of the positively charged side chain of the drug has been the focus of our attention. The positively charged amphiphilic compound (spin derivative I) is water-soluble up to 80 microM at pH values below 5.9. The apolar analogue (spin derivative II) aggregates in aqueous buffer from the lowest concentration tested. Both spin derivatives undergo a slow reduction inside the erythrocyte. The reduced nitroxides are readily reoxidized by adding a low, non-quenching, concentration of potassium ferricyanide to the intact erythrocytes. The fractions of spin label I and II bound to the erythrocyte membrane or to the erythrocyte-extracted lipids remain constant as a function of the temperature (3-42 degrees C) and as a function of the concentration of the spin label up to 150 microM. E.s.r. spectra of both spin labels show a two-component lineshape when they are bound to intact erythrocytes. Below 35 degrees C for the positively charged spin probe, and below 32 degrees C for the apolar spin probe, the simulation of the lineshape shows that more than 50% of the spectrum originates from a slow-motion component. This slow-motion component is also found in erythrocyte-extracted lipids probed by the positively charged spin label below 25 degrees C. In contrast, no slow-motion component is detected in the range 4-40 degrees C for the apolar spin label in erythrocyte-extracted lipids. In this environment the apolar probe experiences a single fast anisotropic motion with an exponential dependence on 1/temperature. Detailed lineshape simulations take into account the exchange frequency between binding sites where the probe experiences a fast motion and binding sites where it experiences a slow motion. The exchange frequency is strongly temperature-dependent. Characterization of the different motions experienced inside the different locations has been achieved and compared for whole erythrocytes and for the extracted lipids. The biochemical nature of the binding sites (membrane protein/acidic phospholipid) giving rise to the slow-motion component is discussed as a function of the polarity of the spin-labelled drug and as a function of the temperature controlling the fluidity of the lipid bulk and influencing the distribution of the drug inside the membrane.     

The effect of anionic detergents on the ATPase activity of isolated F1 from the thermophilic bacterium PS3

The ATPase activity of F1 isolated from the thermophilic bacterium PS3 is stimulated at 30 degrees C by the anionic detergents cholate or deoxycholate. Maximal activity obtained with these detergents (35 mumol/min X mg) is similar to the activity reported for the optimal temperature, 75 degrees C. The activity is linearly stimulated by the detergents and maximal activity is obtained at the critical micellar concentration of the respective detergent. The results are discussed in relation to the role of subunit interactions of the oligomeric enzyme during catalysis and the mode of interaction between the subunits.