Doi:10.1016/j.bpc.2005.09.007Biophysical Chemistry 119 (2006) 69 – 77 Influence of N-dodecyl-N,N-dimethylamine N-oxide on the activity of sarcoplasmic reticulum Ca2+-transporting ATPase reconstituted into diacylphosphatidylcholine vesicles: Effects of bilayer physical parameters J. Karlovska´ a,*, D. Uhrı´kova´ a, N. Ku*erka a, J. Teixeira b, F. Devı´nsky a, I. Lacko a, P. Balgavy´ a a Faculty of Pharmacy, Comenius University, Odboja´rov 10, 832 32 Bratislava, Slovakia b Laboratoire Le´on Brillouin (CEA-CNRS), CEA Saclay, 91999 Gif-sur-Yvette Cedex, France Received 6 May 2005; received in revised form 31 August 2005; accepted 1 September 2005 Available online 11 October 2005 Sarcoplasmic reticulum Ca-transporting ATPase (EC 220.127.116.11) was isolated from rabbit white muscle, purified and reconstituted into vesicles of synthetic diacylphosphatidylcholines with monounsaturated acyl chains using the cholate dilution method. In fluid bilayers at 37 -C, thespecific activity of ATPase displays a maximum (31.5 T 0.8 IU/mg) for dioleoylphosphatidylcholine (diC18 : 1PC) and decreases progressively forboth shorter and longer acyl chain lengths. Besides the hydrophobic mismatch between protein and lipid bilayer, changes in the bilayer hydrationand lateral interactions detected by small angle neutron scattering (SANS) can contribute to this acyl chain length dependence. When reconstitutedinto dierucoylphosphatidylcholine (diC22 : 1PC), the zwitterionic surfactant N-dodecyl-N,N-dimethylamine N-oxide (C12NO) stimulates theATPase activity from 14.2 T 0.6 to 32.5 T 0.8 IU/mg in the range of molar ratios C12NO : diC22 : 1PC = 0 1.2. In dilauroylphosphatidylcholines(diC12 : 0PC) and diC18 : 1PC, the effect of C12NO is twofold—the ATPase activity is stimulated at low and inhibited at high C12NOconcentrations. In diC18 : 1PC, it is observed an increase of activity induced by C12NO in the range of molar ratios C12NO : diC18 : 1PC 1.3 inbilayers, where the bilayer thickness estimated by SANS decreases by 0.4 T 0.1 nm. In this range, the 31P-NMR chemical shift anisotropy increasesindicating an effect of C12NO on the orientation of the phosphatidylcholine dipole N+ – P accompanied by a variation of the local membranedipole potential. A decrease of the ATPase activity is observed in the range of molar ratios C12NO : diC18 : 1PC = 1.3 2.5, where mixed tubularmicelles are detected by SANS in C12NO + diC18 : 1PC mixtures. It is concluded that besides hydrophobic thickness changes, the changes indipole potential and curvature frustration of the bilayer could contribute as well to C12NO effects on Ca2+-ATPase activity.
D 2005 Elsevier B.V. All rights reserved.
Keywords: N-dodecyl-N,N-dimethylamine N-oxide; Diacylphosphatidylcholine; Ca2+-transporting ATPase; Sarcoplasmic reticulum; Small-angle neutron scattering;31P NMR spectroscopy studied intrinsic membrane proteins because it is a single-chaintransmembrane present at high concentration in the P-type ATPases are fundamental in establishing ion gradi- SR membrane with a well known function. It transports 2 ents by coupling the ATP hydrolysis to ion transport across moles of Ca2+ from the cytoplasm into the reticulum across the biological membranes Among many P-type ATPases SR membrane with concomitant hydrolysis of 1 mol of ATP; known today, Mg2+-dependent Ca2+-ATPase (ATP phosphohy- two or three moles of H+ are counter-transported. The drolase, EC 18.104.22.168, SERCA1) from skeletal muscle sarco- determination of crystal structures of the SR Ca2+-ATPase plasmic reticulum (SR) is structurally and functionally the best with two bound Ca2+ ions in the transmembrane protein region studied member SR Ca2+-ATPase is one of the most and in absence of Ca2+ ions and in presence of the inhibitorthapsigargin provided an opportunity to interpret instructural terms Ca2+-ATPase conformational changes accom-panying the reaction ]. However, to fully elucidate * Corresponding author. Tel.: +421 2 50117289; fax: +421 2 50117100.
E-mail address: [email protected] (J. Karlovska´).
the structure and function of Ca2+-ATPase in the membrane, 0301-4622/$ - see front matter D 2005 Elsevier B.V. All rights reserved.
doi:10.1016/j.bpc.2005.09.007 J. Karlovska´ et al. / Biophysical Chemistry 119 (2006) 69 – 77 and particularly the role of lipid – protein interactions that 2. Material and methods influence ATP hydrolysis and ion transport, it is necessary toreconstitute it into defined synthetic phospholipids. The most successful approach so far involves the use of variousdetergents for Ca2+-ATPase solubilization and reconstitution.
Synthetic 1,2-dilauroylphosphatidylcholine (diC12 : 0PC), Using this approach, it has been found that the Ca2+-ATPase 1,2-dimyristoleoylphosphatidylcholine (diC14 : 1PC), 1,2- activity depends on phase states, hydrocarbon chain lengths, dipalmitoleoylphosphatidylcholine (diC16 : 1PC), 1,2-dioleoyl- structure and charges of polar head groups of annular lipids phosphatidylcholine (diC18 : 1PC), 1,2-dieicosenoylphosphati- surrounding the protein : a) the activity is practically dylcholine (diC20 : 1PC), 1,2-dierucoylphosphatidylcholine zero in the solid-like (gel phase) bilayer, high in the fluid (diC22 : 1PC) and 1,2-dinervonoylphosphatidylcholine (liquid crystalline) bilayer, but the particular value of fluidity in (diC24 : 1PC) were purchased from Avanti Polar Lipids the fluid state has no effect; b) for high activity, a fluid bilayer (Alabaster, USA). Egg yolk phosphatidylcholine (EYPC) was from lipids with zwitterionic head groups is required- charged isolated from fresh hen eggs and purified by a column lipids support low activities; c) lower activity is observed in chromatography according to Singleton et al. with lipids under conditions when they form non-bilayer aggregates modifications detailed in Ref. C12NO was prepared from in isolation; d) the activity in (zwitterionic) diacylphosphati- N,N-dimethyldodecylamine by oxidation with hydrogen per- dylcholines is highest in the fluid bilayer of 1,2-dioleoylpho- oxide and purified as described by Devı´nsky et al. Cholic sphatidylcholine, but lower in fluid bilayers with shorter or acid (Sigma, St. Louis, USA), Hepes (Serva, Heidelberg, longer acyl chains. These results indicate that the ATPase Germany), Tris (Serva, Heidelberg, Germany), sucrose (Slavus, activity is modulated by a delicate interplay of several physical Bratislava, Slovakia), histidine (Sigma, St. Louis, USA), factors—amongst which the most important seem to be dithiothreitol (DTT) (Sigma, St. Louis, USA), phenyl methyl- hydrophobic thickness, hydrogen bonding potential, hydration, sulfonyl fluoride (PMSF) (Sigma, St. Louis, USA), Amberlite surface charge, dipole potential and curvature frustration of the XAD-4 (Sigma, St. Louis, USA), EGTA (Sigma, St. Louis, USA), ATP (Sigma, St. Louis, USA), phosphoenolpyruvate To specify the interplay of these different physical factors (Boehringer, Mannheim, Germany), NADH (Sigma, St. Louis, in the lipid-Ca2+-ATPase protein interactions, the correlation USA) of the best available purity were used. Organic solvents of between Ca2+-ATPase activity and bilayer structural para- the p. a. purity obtained from Mikrochem (Bratislava, Slovakia) meters in presence of various amphiphilic and hydrophobic and water were redistilled before use. Heavy water (99.9% 2H) compounds has been useful. Such studies using cholesterol was from Isotec (Matheson, USA). Potassium cholate was and normal alkanes contributed to finding non-annular lipid prepared by the action of KOH on cholic acid and purified by binding sites in the Ca2+-ATPase In the present crystallization and diethyl ether extraction. The other chemicals study, we correlate effects of the zwitterionic amphiphile N- were from Lachema (Brno, Czech Republic).
dodecyl-N,N-dimethylamine-N-oxide (C12NO) on the activityof purified Ca2+-ATPase reconstituted into synthetic diacyl- phosphatidylcholines with its effects on the structural bilayerproperties. We found in our previous studies that N-alkyl- Pyruvate kinase (EC 22.214.171.124) from rabbit muscle (Boeh- N,N-dimethylamine-N-oxides (CnNOs) stimulate the activity ringer, Mannheim, Germany) and lactate dehydrogenase (EC of purified Ca2+-ATPase at low concentration and inhibit it at 126.96.36.199) from pig heart (Boehringer, Mannheim, Germany) high concentration In the delipidated monomeric were used as obtained. Ca2+-ATPase was isolated from the form, Ca2+-ATPase binds about 240 C12NO molecules that spinal region white muscle of a female rabbit (about 2.5 kg) cover the hydrophobic surface of the protein in the form of a and purified according to the methods outlined by Warren et al.
prolate monolayer ring It was also observed that C12NO th modifications described by Karlovska´ et al. predominantly interacts with the lipid component of Ca2+- The protein concentration was determined by measuring the ATPase membranes C12NO is widely used as a mild absorbance at 280 nm biological detergent for solubilization, purification, reconsti-tution and crystallization of membrane proteins In the 2.3. Ca2+-ATPase reconstitution bilayer, CnNOs penetrate between phospholipids and affectthe fluidity and the thickness of the bilayer. At The purified Ca2+-ATPase reconstitution into phosphatidyl- high concentration, C12NO destabilizes the bilayers and choline vesicles was performed using the cholate dilution converts them into non-bilayer phases  mixed method of Johannsson et al. Briefly, C12NO and micelles present communication, we report phosphatidylcholine were mixed at the needed molar ratio in results of the study of the effects of C12NO on the specific chloroform/methanol in a glass tube; the solvent was evapo- activity of purified Ca2+-ATPase reconstituted into synthetic rated under a stream of gaseous nitrogen and its traces removed diacylphosphatidylcholines and correlate them with the effects by an oil vacuum pump. The dry C12NO + phosphatidylcholine of C12NO on the bilayer thickness, on the conformation of mixture was solubilized by adding 40 Al of the micellar the phosphatidylcholine head group and on the bilayer solution of cholate in buffer (0.025 mol/l cholate, 0.01 mol/ l Hepes/Tris, 0.44 mol/l sucrose, 0.005 mol/l MgATP, pH 8.0).
J. Karlovska´ et al. / Biophysical Chemistry 119 (2006) 69 – 77 The content was sealed under gaseous nitrogen, vortex-mixed, passed through the filters. The final phospholipid concentration sonicated, eventually freezed/thawed until obtaining a trans- was not checked, but it was 1 wt.% in all samples because parent solution of mixed micelles. The purified ATPase (0.125 some amount of lipid could remain in the extruder. The mg) was then added in the volume of 2 – 3 Al; the amount added samples were filled into 1 mm quartz cells (Hellma, Mu¨llheim, was controlled by gravimetry. The content was vortex-mixed Germany), closed and stored at room temperature. As the and incubated depending on the length of acyl chain as follows: reference sample, the same cell containing heavy water without diC12 : 0PC—15 min at room temperature and at 8 – 10 -C for a vesicles was used. The maximum period between the sample further 45 min, diC14 : 1PC diC18 : 1PC—30 min at room preparation and its measurement was 5 h. The neutron temperature and at 8 – 10 -C for a further 30 min, scattering experiments were performed on the PAXE spec- diC20 : 1PC diC24 : 1PC—1 h at room temperature. After trometer located at the end of the G5 cold neutron guide of the this incubation, the samples were diluted by adding 0.4 ml of Orphe´e reactor (Laboratoire Le´on Brillouin, CEA Saclay, buffer (0.02 mol/l Hepes/Tris, 0.1 mol/l KCl, 0.005 mol/ France). The experiments were performed with sample to l MgSO4, pH 7.2 T 0.1). The reconstitution procedure used is detector distances of 1700 and 5000 mm and the neutron described in more detail by Filı´pek et al. wavelength of k = 0.6 nm. The sample temperature was set andcontrolled electronically at 30.0 T 0.1 -C. The acquisition time 2.4. Ca2+-ATPase activity for one sample was 40 min. The normalized SANS intensityI( q) as a function of the scattering vector value q = 4ksin h / k, The ATPase activity was determined at 37 -C using the where 2h is the scattering angle, was obtained as described in coupled assay system as described earlier The Ca2+- detail by Kucˇerka et al.
ATPase was diluted in the assay mixture and after incubation at For the evaluation of I( q) data, we used the recently 37 -C for 15 min, the reaction was started by the addition of a developed strip-function model of the coherent neutron CaCl2 solution to reach the final volume 2.46 ml. The final scattering length density distribution q(z) taken perpendicular- composition of the assay system was 10.06 Ag of Ca2+-ATPase ly to the bilayer surface In this model, the bilayer in per sample (2.46 ml), 0.079 mmol/l phosphatidylcholine, 40 unilamellar vesicles is divided into concentric strips with radii mmol/l Hepes, 0.1 mol/l KCl, 5.1 mmol/l MgSO4, 2.1 mmol/ from the inner bilayer radius R0 to the outer radius R6. The l ATP, 0.53 mmol/l phospoenolpyruvate, 1.1 mmol/l EGTA, methyl, methine and part of methylene groups of diCn : 1PC 0.152 mmol/l NADH, 7.5 IU/ml of PK, 18 IU/ml of LDH, pH acyl chains are located in the region spanning two strips from 7.2. The reaction was followed by measuring the decrease of R2 to R4, the dividing surface at R3 is located at the centre of NADH absorbance at 340 nm, at 37 -C. The specific activity A the bilayer. In the region from R2 to R4, the value of q(z) is (in international units IU per mg of enzyme) was calculated constant. The strips from R0 to R2 and from R4 to R6 contain according to A = DA340Vass / 6.22mprot, where DA340 is the ‘‘dry'' polar diCn : 1PC head groups (including choline, change in NADH absorbance at 340 nm per min, Vass is the phosphate, glycerol and acyl chain carbonyls), some limited final assay volume in ml (2.46 ml), and mpro is the weight of number of water molecules per one diCn : 1PC molecule nW, Ca2+-ATPase in mg in the assay volume. The values of A given and the rest of diCn : 1PC acyl chain methylene groups. The below are the mean values from triplicate experiments. The contribution of the head groups to q(z) is triangular, increasing conditions for optimal pH, temperature, calcium and magne- from R0(R6) to R1(R5) and decreasing then to R2(R4). The sium concentration and enzyme stability were determined for contribution of water molecules to q(z) decreases linearly from the assay system in preliminary experiments. Absorbance R0(R6) to R2(R4). Finally, the contribution of diCn : 1PC acyl measurements were made using the diode-array HP8452A chain methylene groups not located in the strips between R2 spectrophotometer (Hewlett Packard, Palo Alto, USA) and 1 and R4 decreases linearly from R2 to R1 and from R4 to R5. The cm quartz or plastic cuvettes.
fragmental molecular volumes of constituents in R0 to R2 andin R4 to R6 strips are additive and the decrease in volume due to 2.5. Small-angle neutron scattering one constituent is compensated by the increase due to anotherone. The steric bilayer thickness is equal to dS = R6 R0. This The dry diCn : 1PC powder was dispersed in heavy water to model mimics the coherent neutron scattering length density reach the 1 wt.% concentration and closed in a plastic tube. The distribution qsim(z) obtained from the molecular dynamics dispersion was sonicated in a bath sonicator and homogenized (MD) simulations of fluid phosphatidylcholine bilayers more by hand shaking and vortex mixing. From the homogenized closely than other models frequently used in SANS data dispersions, extruded unilamellar vesicles were prepared: The evaluation . The experimental I( q) versus q data were diCn : 1PC dispersions were extruded through two stacked fitted by the function minimization and error analysis program polycarbonate filters (Nucleopore, Plesanton, USA) with pores Minuit (CERN Program Library entry D506), using the vesicle of diameter 50 nm mounted in the LiposoFast Basic extruder structure factor derived from the strip model described above, (Avestin, Canada) fitted with two gas-tight Hamilton syringes convoluted by the Gamma function distribution of vesicle radii (Hamilton, Reno, USA). Each sample was subjected to 25 and by the PAXE spectrometer resolution function. Besides the passes through the filters at room temperature. An odd number experimental I( q) data, the input values were the fragmental of passes were performed to avoid contamination of the sample volumes of different parts of the bilayer (2H2O 0.030104 nm3, by large and oligolamellar vesicles, which might not have dry polar head group 0.319 nm3, acyl methine group 0.0218 J. Karlovska´ et al. / Biophysical Chemistry 119 (2006) 69 – 77 nm3, acyl methylene group 0.0283 nm3, acyl methyl group 0.0522 nm3) taken from the literature and the valuesof their coherent scattering amplitudes calculated by using theknown scattering amplitudes of nuclei During the minimization, the distances of R2 R0 = R6 R4 were con-strained to the value 1.2 nm obtained from MD simulationsThe result of fitting is a pair of d S and nW values; the area AL of one diCn : 1PC molecule at the bilayer—aqueous phase interface is calculated from dS and nW using the knownfragmental volumes of different parts of the bilayer.
2.6. 31P-NMR spectroscopy Changes in the phosphatidylcholine head group conforma- tion were followed using the proton decoupled 31P-NMRspectroscopy. C12NO and EYPC were mixed at the needed Fig. 1. Dependence of the specific Ca2+-ATPase activity A at 37 -C on the molar ratio in chloroform/methanol in a glass tube; the solvent number n of the carbon atoms of the acyl chain of diacylphosphatidylcholine.
was evaporated under a stream of gaseous nitrogen and its For n = 12 the data were obtained with diC12 : 0PC, and for n = 14 – 24 withdiCn : 1PC. The dashed curve is drawn to guide eye.
traces removed by an oil vacuum pump. The dryEYPC + C12NO mixtures were transferred to other glass tubesand evacuated again. Redistilled water was added to these dry the cholate-phosphatidylcholine mixed micelles interact with mixtures at the weight ratio H2O : EYPC = 1 : 1; the amount of the ATPase: most probably 15 min and 0 -C in Ref. water added was controlled by gravimetry. Finally, the tubes resulted in a partial lipid substitution comparing to more were flame sealed, and the content was homogenized by extensive substitution at higher temperatures and longer repeated freezing and thawing and by forth-and-back centrifu- incubation times in Refs. and in the present work gation. Before the measurement, the samples were equilibrated (detailed above in Material and methods). The chain length at room temperature in a dark place. 31P-NMR spectra were dependence of activity is frequently explained by the hydro- recorded on a VXR 300 NMR spectrometer (Varian, USA) at phobic mismatch hypothesis: it is supposed that the thickness 121.4 MHz using the deuterium lock, HF pulse width 45 – 55- of the hydrophobic region of the bilayer must match the length and the interpulse relaxation delay 0.7 – 1.0 s. The spectra were of the hydrophobic part of Ca2+-ATPase to support the recorded using the strong proton inverse gated proton maximum activity; increasing or decreasing of this thickness decoupling. The sample temperature was maintained at 25 -C should cause conformation changes and/or lateral using the gas-flow system of NMR spectrometer. Exponential aggregation of Ca2+-ATPase resulting in a reduced multiplication of free induction decays corresponding to 50 Hz phosphohydrolase activity and Ca2+ transport.
line broadening was applied prior to their Fourier transforma- To relate the changes of the specific activity of Ca2+-ATPase tion. The effective 31P-NMR chemical shift anisotropy, with the physical parameters of the bilayer, we estimated the Dreff, was evaluated as the distance between extremes of steric thickness dS, the surface area AL per lipid at the bilayer- the first derivative spectra and corrected for the Lorentzian aqueous phase interface and the number nW of water molecules linewidth broadening using the spectra simulated by computer.
per lipid located in the bilayer polar region of unilamellar The details of the method were described by Uhrı´kova´ diCn : 1PC vesic2). These parameters have never beenmeasured so extensively, the only relevant work being that of 3. Results and discussion Lewis and Engelman published more than 20 years ago.
They estimated the phosphate – phosphate separation across the 3.1. Effects of the phosphatidylcholine acyl chain length bilayer from the position of the first peak in the Pattersonfunction after inversion of the small-angle X-ray scattering We have found that the specific activity of Ca2+-ATPase (SAXS) of sonicated unilamellar diC18 : 1PC, diC22 : 1PC and reconstituted in the fluid phosphatidylcholine bilayers is diC24 : 1PC vesicles at 20, 24 and 36 -C, respectively.
sensitive to the length of the acyl chain with a maximum at However, their values are underestimated due to systematic diC18 : 1PC while phosphatidylcholines with shorter or longer truncation errors, i.e. inversion of SAXS data in a limited range acyl chains support progressively decreasing activities when of momentum transfer, as noted by Nagle and Tristram-Nagle approaching the chain length extre1). The maximum Furthermore, the SAXS experiments were done at a of activity at diC18 : 1PC was observed earlier by Lee Lee constant reduced temperature above the gel – fluid transition et al. , Caffrey and Feigenson and by Cornea and temperature, while the protein interactions with phosphatidyl- Thomas In the classical paper of Johannsson et al. choline bilayers as a function of n are studied experimentally at the maximum was observed at diC20 : 1PC. This small the same absolute temperature. As expected, we have found in discrepancy could be caused by differences in incubation time our SANS experiments that the bilayer thickness increases with and temperature during the crucial step in reconstitution where the increase of the length n of the acyl chain: a simple linear fit J. Karlovska´ et al. / Biophysical Chemistry 119 (2006) 69 – 77 between acyl chains and the dipolar interactions betweenheadgroups. The main repulsive components include steric interactions, hydration forces, and entropic contributions due to the ordering of acyl chains. The equilibrium area AL is given by the balance of these forces that minimizes the interfacial free energy. At constant temperature, the increase of n increases the van der Waals attraction what will reduce AL. However, the reduction of AL yields a concomitant reduction of gauche conformers in chains, which decreases the chain disorder (i.e.
the entropy) and this may depend on the position of the double bonds in the acyl chains. These two effects which act in opposite directions can cause the observed peculiar dependenceof AL (and nW) in diCn : 1PC bilayers. The value of AL is thus a measure of lateral interactions in the bilayer. Irrespective of theactual mechanisms resulting in the chain length dependences of AL and nW, the results summarized indicate that the bilayer hydration and lateral interactions can influence theactivity of Ca2+-ATPase reconstituted into diCn : 1PC vesicles.
Fig. 2. Dependences of steric thickness dS, surface area AL and number of Changes in lateral interactions yield changes of the bilayer water molecules per lipid nW in bilayers of unilamellar diCn : 1PC vesicles as a lateral pressure profile which may affect the conformation of function of the number n of the carbon atoms of the acyl chain ofdiacylphosphatidylcholine. The dashed lines are drawn to guide eye. Full membrane . Besides the dominant ‘‘hydropho- symbols: measurements at 30 -C, open symbols: extrapolations to 37 -C.
bic mismatch'', this could be another mechanism contributingto the dependence of the reconstituted Ca2+-ATPase activity on (weighted by uncertainties in dS) gives dS = (1.64 T 0.20) + the length of the acyl chain.
(0.17 T 0.01)n in nm. Unexpectedly, the area AL displays amaximum at n = 18. Similarly, the number nW of water 3.2. Effects of N -dodecyl-N,N -dimethylamine-N -oxide molecules located in the polar region of the bilayer depends on n (Since the parameters in were obtained at30 -C and the Ca2+-ATPase activities in were measured The effect of C12NO on the activity of Ca2+-ATPase at 37 -C, we have extrapolated AL and dS in to 37 -C reconstituted into diC22 : 1PC is enormous—the activity correcting for temperature effects. The area AL was corrected increases up to A = 32.5 T 0.8 IU/mg at the molar ratio using the lateral thermal expansivity b = 0.003 K 1 as in C12NO : diC22 : 1PC = 1.21 in sample ). This value is and the thickness of the bilayer using the transversal thermal comparable to A = 31.5 T 0.7 IU/mg observed in diC18 : 1PC, expansivity a = 0.001 K 1 found recently for diC18 : 1PC which is the lipid with the optimum length of the acyl chain, The steric thickness of the bilayer increases with n as i.e. corresponding to the maximum activity ). Assuming dS = (1.66 T 0.20) + (0.17 T 0.01)n in nm at 37 -C, i.e. it remains that all the C12NO molecules are located in the bilayer, one almost unchanged. After temperature corrections, at 37 -C, the calculates a value of the mean hydrocarbon chain length area AL is slightly higher but the maximum is always for n = 18 n = 18.23 carbon atoms in the bilayer at the molar ratio The maxima of AL and nW as a function of n seem surprising. Previous studies reported the decrease of AL with nin bilayers prepared from diacylphosphatidylcholines withsaturated acyl chains (diCn : 0PC) a) in multilamellar vesicles in the solid-like gel state for n = 16 – 18 by small-angle X-raydiffraction (SAXD) b) in multilamellar diCn:0PC vesiclesin the fluid state for n = 12 – 18 by 2H NMR and c) inunilamellar diCn : 0PC vesicles in the fluid state for n = 12 – 18 by SANS The specific dependence of AL (and nW) on n in diCn : 1PC bilayers in comparison to diCn:0PC bilayers can bedue to the presence and position of the double bond in the diCn : 1PC acyl chains (S. J. Marrink, personal communica-tion): the position of the double bonds in diC14 : 1PC,diC16 : 1PC and diC18 : 1PC is 9-cis, in diC20 : 1PC 11-cis, in diC22 : 1PC 13-cis, and in diC24 : 1PC 15-cis. The value of L is the result of attractive and repulsive forces at the aqueous phase-bilayer interface. The main attractive compo- Fig. 3. Dependence of Ca2+-ATPase specific activity A at 37 -C on the molar nents are the hydrophobic interaction, the van der Waals forces ratio C12NO : diC22 : 1PC in the sample.
J. Karlovska´ et al. / Biophysical Chemistry 119 (2006) 69 – 77 C12NO : diC22 : 1PC = 1.21. This value compares well with activity must be excluded. The effect of the surface charge n = 18 in diC18 : 1PC which corresponds to the maximum must be excluded too, because the phosphatidylcholines activity. Therefore, the dominant contribution to mechanisms and C12NO are zwitterionic under experimental conditions responsible for the increase of Ca2+-ATPase activity in the mixed C12NO + diC22 : 1PC bilayer can be ascribed to the The effects of C12NO concentration on the behaviour of the reduction of the hydrophobic mismatch between the protein Ca2+-ATPase activity can be compared with a) those observed and the bilayer.
by Uhrı´kova´ et al. using SANS on the structure of bilayers The evolution of the changes of activity due to C12NO is of diC18 : 1PC vesicles and b) those observed in the present different for the Ca2+-ATPase reconstituted into diC12 : 0PC work by 31P-NMR spectroscopy on the conformation of the and diC18 : 1PC vesicles: the dependence of the activity on the lipid head group in EYPC multilamellar vesicles. For this molar ratio C12NO : PC goes through a maximum. This comparison, the molar ratio C12NO : PC in the lipid phase is behaviour is seen in the dependences of normalized Ca2+- needed because different experiments were performed at ATPase specific activities A / A0 on the molar ratio C12NO : PC different diC18 : 1PC and C12NO concentrations in sam- in the sample ), where A and A0 are the specific activities ples—the effect of the partition equilibrium on results must in presence or not of C12NO, respectively. Several other be thus eliminated. In the following, we calculate this molar amphiphiles stimulate Ca2+-ATPase activity at low concentra- ratio from the data obtained in our laboratory.
tion, e.g. oleic acid, methyl oleate, and oleyl alcohol In the sample, the molecules of C12NO partition between tertiary amine local anestheti, pentobarbital the aqueous phase and the lipid phase; in equilibrium, this nonylphenol hexanol . Ca2+-ATPase is surrounded process is characterized by the molar partition coefficient by a shell (annulus) of about 30 – 32 phospholipid molecules located at the bilayer-protein interface Besides these annular sites, the hydrophobic and amphiphilic molecules can ¼ nC12NO;PC=VPC = nC12NO;W=VW bind to non-annular sites located between transbilayer a-helices or at protein – protein interfaces in Ca2+-ATPase where cC12NO,i and nC12NO,i are molar concentrations and oligomers . It was suggested that the stimulation of numbers of moles of C12NO, respectively, Vi are volumes, and Ca2+-ATPase activity could result from the binding of indices i = W and i = PC denote the aqueous and lipid phases, amphiphilic and hydrophobic molecules to these non-annular respectively. Using simple algebra, one can calculate the molar binding sites (see Froud et , Fernandez-Salguero et al.
ratio C12NO : PC in the lipid phase at any concentration of and Lee for references). While the increase of Ca2+- C12NO (cC12NO) and lipid (cPC) in the sample using the known ATPase activity depicted 4 can be caused by the C12NO molar partition coefficient K, the absolute specific volume of binding to the non-annular binding sites, the cause of the the lipid vPC and the lipid molar weight MPC as subsequent decrease of activity is not clear. Evidently, it cannot C12NO : PC ¼ cC12NO=ðcPC þ 1=vPCMPCKÞ: be the bilayer thickness in case of diC12 : 0PC. Because thelength of the alkyl chain of C12NO is equal to that of The molar partition coefficient K = 630 has been recently diC12 : 0PC acyl chains, the insertion of C12NO into the measured for C12NO in the system consisting of unilamellar diC12 : 0PC bilayer should not induce any significant change of diC18 : 1PC vesicles in the aqueous phase at 37 -C (Karlovska´ the thickness, then the effect of hydrophobic mismatch on the and Balgavy´, in preparation) using methods described in Ref.
. The absolute specific volume of diC18 : 1PC is vPC = 0.9985 ml/g at 30 -C and MPC = 786.12 g/mol. Byusing these data and the thermal volume expansion coefficientc = 0.0008 K 1 found experimentally for diC18 : 1PC obtains the molar ratios C12NO : PC in the lipid phase used inCa2+-ATPase experiments as well as in SANS experiments (). The partition coefficient K = 507 was measured for C12NO in EYPC vesicles in the aqueous phase at room temp. Since the volume of aqueous phase in EYPC samples for NMR spectroscopy was the same as thevolume of EYPC lipid phase, the effect of C12NO partitioningis neglected and the values of C12NO : EYPC molar ratio in the lipid phase are taken to be the same as those in the samples( The SANS parameter r characterizes the geometry of C12NO + diC18 : 1PC aggregates: r = 1 for bilayers, r = 2 for C12NO:PC (mol:mol) rod-like micelles and r = 3 for globular micelles (see Refs.
and references therein). The SANS parameter dg Fig. 4. Dependence of the normalized Ca2+-ATPase specific activity A / A0 at 37 characterizes the thickness of the bilayer; its value follows -C on the molar ratio C12NO : PC in the sample; PC = diC12 : 0PC (?),PC = diC18 : 1PC (0).
the distance between phosphate groups dPP across the bilayer J. Karlovska´ et al. / Biophysical Chemistry 119 (2006) 69 – 77 causes an increase of anisotropy. Secalculated that theN+ – P dipole reorientation is accompanied by variations of the local membrane dipole potential of the order of 105 V/cm and suggested that this could play a regulatory role in the membrane function. Cafiso suggested that changes in membrane dipole potentials could affect the conformation ofthe protein in the membrane. It is possible that the reorientation of the N+ – P dipole of diC18 : 1PC induced by C12NO is (nm)d g another factor that compensates the influence of the decrease ofthe thickness of the bilayer on the activity of Ca2+-ATPase.
The specific activity of Ca2+-ATPase reconstituted into diC18 : 1PC bilayers decreases at molar ratios C12NO : PC > 2where mixed rod-like (cylindrical) C12NO + diC18 : 1PC micelles (r > 1) are formed ). The geometry of the surfactant + phospholipid aggregates consisting of two compo- nents depends on the effective molecular packing parameter . The theory predicts that, depending on the packing parameter d, molecules form spherical micelles (d < 0.33),normal cylindrical micelles (0.33 < d < 0.5), curved bilayers (0.5 < d < 1), flat bilayers (d = 1) or inverted micelles (d > 1). We conclude that the inhibition of Ca2+-ATPase observed in diC18 : 1PC bilayers is most probably caused by C12NO:PC (mol:mol) a severe deformation of the bilayer resulting in the formation of Fig. 5. Dependences of the specific Ca2+-ATPase activity A at 37 normal tubular mixed C12NO + diC18 : 1PC micelles in isola- thickness (SANS parameter dg) of the diC18 : 1PC bilayer (?), SANS tion (d < 0.5). A decrease of the activity of Ca2+-ATPase is parameter r (0), and 31P-NMR chemical shift anisotropy Dreff (r) on the observed when reconstituted into phosphatidylethanolamines molar ratio C12NO : PC in the lipid phase. The dg and r data were recalculated under conditions where the phosphatidylethanolamine in from the original raw data published by Uhrı´kova´ et al. and corrected for isolation forms an inverse hexagonal phase consisting of inverted cylindrical micelles . This situation corre-sponds to an effective molecular packing parameter d > 1. The as well as the thickness dS shows that the inhibition of the activity of Ca2+-ATPase is observed therefore bilayer is stable (r = 1) for molar ratios C12NO : PC < 2 (in for both positive and negative deviations from the optimal bilayers) and that its thickness decreases minimally by bilayer packing (d = 1). These deviations do not necessarily 0.39 T 0.12 nm in this range. Simultaneously, the activity of result in the formation of non-bilayer lipid aggregates in Ca2+-ATPase increases by about 4.4 T 2.0 IU/mg. The lower contact with the protein; instead, there is a change of the lateral bound of the modification of the thickness of the bilayer pressure profile resulting in a change of the protein conforma- induced by C12NO in diC18 : 1PC (0.27 nm) is comparable to tion, thus of the function of the protein, as suggested by Cantor that observed when going from diC18 : 1PC to diC16 : 1PC . Similarly, the change of the thickness of the bilayer (0.24 nm) (which is accompanied by a decrease of the found in isolation does not imply any change in the thickness activity of Ca2+-ATPase equal to 18.1 T 2.0 IU/mg ( of the lipid annulus around the protein, because the bilayer can These findings strongly support the idea that the effect of the deform to match the protein. However, the result could be thickness of the bilayer on the activity of Ca2+-ATPase due to again a change of the bilayer lateral pressure profile.
C12NO has to be compensated by other factors. One of these In conclusion, we have observed that C12NO modulates the factors could be the C12NO binding to non-annular binding activity of Ca2+-ATPase through several mechanisms, depend- sites discussed above. Another feature follows from 31P-NMR ing on the protein lipid environment. Besides the hydrophobic experiments (In the C12NO : PC interval where the mismatch between the lipid bilayer and the protein, the Ca2+-ATPase activity increases, it is observed an increase of conformation of the lipid head group and the deformation of the effective 31P-NMR chemical shift anisotropy, Dreff.
the bilayer could contribute to the optimal protein function.
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Versión Preliminar COMISION de SELECCIÓN y MANTENIMIENTO del DONANTE de ÓRGANOS Página 1 de 1 Versión Preliminar " MUERTE HUMANA" "La muerte es la pérdida de la percepción y justamente por esto, una forma de no ser " EPICUREO Es una tendencia inherente a las ciencias (y en lo que nos atañe a la Ciencia Médica) intentar definir los distintos conceptos, fenómenos, procesos y parámetros que utiliza. Con frecuencia esta tendencia confronta con la imposibilidad de definir conceptos de tipo universal, los cuales por sus características no pueden ser definidos siguiendo la lógica clásica (una definición describe el campo en el que el concepto se halla inserto). No obstante la Ciencia Médica utiliza y aún mensura muchos de estos conceptos (ej. "tiempo", "energía") sin ser posible obtener una definición lógica de los mismos. Esto puede ser aplicado a dos conceptos en el tema que nos atañe: "vida" y "muerte", ambos son tratados, y mensurados sin reparar en la definición de los mismos (ej. tasas de natalidad y mortalidad). A tal respecto, las generalmente declamadas "definiciones", no lo son realmente desde el punto de vista conceptual, sino una enunciación de criterios o condiciones para declarar que tales eventos han ocurrido; podría reservarse el término "definición operativa" a tales efectos
Microsoft word - implant consent.docx
Implant Patient Information and Consent Form 1. I understand that dental implant procedures involve a surgical phase (inserting of implants) and a prosthodontics phase (replacing teeth) and that a separate fee is charged for each phase. 2. I understand that after the surgical phase is completed, the completion of my treatment may range from 4-10 months and that additional procedures may extend completion time.