Differential scanning calorimetry and Fourier transform infrared analysis of lipid-protein interactions involving the nicotinic acetylcholine receptor

Lipid-protein interactions were studied using Torpedo californica acetylcholine receptor (AChR) as a model system by reconstituting purified AChR into dielaidoylphosphatidylcholine (DEPC, 18:1 trans-9,10) membranes. The structural and thermodynamic behavior of lipids in the vicinity of the protein were studied by differential scanning calorimetry and Fourier transform infrared spectroscopy. The effects of AChR on the thermodynamic parameters associated with lipid phase transitions were to reduce the enthalpy change, lower the transition temperature and reduce the cooperative behavior of the lipid molecules. A stoichiometry of approx. 95 lipids per AChR molecule was found by simulating the decrease in enthalpy in terms of a simple model in which a fixed number of lipid molecules are prevented from undergoing a cooperative phase transition. In parallel, the vibrational spectra of pure DEPC and AChR reconstituted in DEPC membranes at various lipid to protein ratios were examined. Profiles of the 3000-2800 cm-1 C-H stretching region and 1350-950 cm-1 characteristic of the headgroup region of the lipid exhibit little sensitivity to protein/lipid ratio reflecting weak interaction of AChR with DEPC. The lipid carbonyl on the other hand appear to be increasingly hydrogen bonded in the presence of AChR. The results provide new information about the size and physical state of the motionally restricted lipid environment that surrounds the acetylcholine receptor. The results are discussed in the context of lipid-mediated alterations in acetylcholine receptor function.     

Unusual partitioning behavior of CaATPase in dipalmitoylphosphatidylethanolamine/dielaidoylphosphatidylcholine++ + mixtures.

CaATPase from rabbit sarcoplasmic reticulum has been isolated, purified, stripped of its native lipids, and reconstituted into binary lipid mixtures of dielaidoylphosphatidylcholine (DEPC) and dipalmitoylphosphatidylethanolamine (DPPE) or acyl-chain perdeuterated DPPE (DPPE-d62). The partitioning properties of the protein were determined from differential scanning calorimetry (DSC) and Fourier transform infrared (FT-IR) spectroscopy. Acyl-chain perdeuteration allows the separate determination of the order and melting characteristics of each lipid species with FT-IR. The binary lipid mixture has been shown to be phase separated in the gel state (Brauner, J. W., and R. Mendelsohn, 1986, Biochim. Biophys. Acta, 861:16-24). The solid phases present at low temperatures correspond to a pure DEPC phase and a mixed phase of DEPC/DPPE-d62. Insertion of protein at 37 degrees C leads to a domain of relatively protein-free DPPE-d62 and a phase containing both lipids plus CaATPase. We suggest that CaATPase selects a fixed composition (60% DEPC, 40% DPPE-d62) for its immediate environment. The composition of the lipid in the immediate vicinity of protein is largely independent of the initial DEPC/DPPE-d62 ratios in the reconstitution protocol. The relevance of these results to observations of discrete domains in native membranes is discussed.     

Comparable levels of Ca-ATPase inhibition by phospholamban in slow-twitch skeletal and cardiac sarcoplasmic reticulum

Alterations in expression levels of phospholamban (PLB) relative to the sarcoplasmic reticulum (SR) Ca-ATPase have been suggested to underlie defects of calcium regulation in the failing heart and other cardiac pathologies. To understand how variation in PLB expression relative to that of the Ca-ATPase can modulate calcium transport, we have investigated the inhibition of the Ca-ATPase by PLB in native SR membranes from slow-twitch skeletal and cardiac muscle and in reconstituted proteoliposomes. Quantitative immunoblotting in combination with affinity-purified protein standards was used to measure protein concentrations of PLB and of the Ca-ATPase. Functional inhibition of the Ca-ATPase was determined from both the calcium concentrations for half-maximal activation (Ca(1/2)) and the shift in the calcium concentrations following release of PLB inhibition (i.e., (Delta)Ca(1/2)) by incubation with monoclonal antibodies against PLB, which are equivalent to phosphorylation of PLB by cAMP-dependent protein kinase. We report that equivalent levels of PLB inhibition and antibody-induced activation ((Delta)Ca(1/2) = 0.25 +/- 0.02 microM) are observed in SR membranes from slow-twitch skeletal and cardiac muscle, where molar stoichiometries of PLB expressed per Ca-ATPase vary, respectively, from 0.9 +/- 0.1 to 4.1 +/- 0.8. Similar levels of inhibition to those observed in isolated SR vesicles were observed using reconstituted proteoliposomes following co-reconstitution of affinity-purified Ca-ATPase with PLB. These results indicate that total expression levels of one PLB per Ca-ATPase result in full inhibition of the Ca-ATPase and, based on the measured K(D) (140 +/- 30 microM), suggests one PLB complexed with two Ca-ATPase molecules is sufficient for full inhibition of activity. Therefore, the excess PLB expressed in the heart over that required for inhibition suggests a capability for graded responses of the Ca-ATPase activity to endogenous kinases and phosphatases that modulate the level of phosphorylation necessary to relieve inhibition of the Ca-ATPase by PLB.     

Secondary structure of calsequestrin in solutions and in crystals as determined by Raman spectroscopy

Calsequestrin has been precipitated with calcium into five different crystal forms: cruciform twins, flat rectangles, thin needles, bipyramids, rectangular prisms, and a sixth precrystalline form, spheres. Raman spectra of the spheres and the cruciform twins are the same. The Raman spectrum of a physiological concentration (10%) of calsequestrin in calcium-free solution is the same as the spectrum of calcium precipitated calsequestrin in the amide I region, and in the C-C stretching region, but these spectra are different in the amide III region. The Raman spectrum of unfolded calsequestrin in 5 M guanidine hydrochloride is quite different from the other spectra, but it is not similar to the spectra of other unfolded proteins. Estimates of secondary structure from the amide I region indicate that calsequestrin in calcium-free solution and calcium-precipitated forms has 40 +/- 5% helix, 30 +/- 4% beta-strand, and 18 +/- 2% reverse turn. Secondary structure estimates calculated from the amide III region are not significantly different. They indicate 41 +/- 5% helix and 36 +/- 6% beta-strand for the precipitated forms, and 32 +/- 5% helix and 39 +/- 6% beta-strand for solutions. Calsequestrin unfolded in 5 M guanidine hydrochloride at 100 mg/ml gives 24 +/- 5% helix and 48 +/- 6% beta-strand.     

Effects of melittin on lipid-protein interactions in sarcoplasmic reticulum membranes.

To investigate the physical mechanism by which melittin inhibits Ca-adenosine triphosphatase (ATPase) activity in sarcoplasmic reticulum (SR) membranes, we have used electron paramagnetic resonance spectroscopy to probe the effect of melittin on lipid-protein interactions in SR. Previous studies have shown that melittin substantially restricts the rotational mobility of the Ca-ATPase but only slightly decreases the average lipid hydrocarbon chain fluidity in SR. Therefore, in the present study, we ask whether melittin has a preferential effect on Ca-ATPase boundary lipids, i.e., the annular shell of motionally restricted lipid that surrounds the protein. Paramagnetic derivatives of stearic acid and phosphatidylcholine, spin-labeled at C-14, were incorporated into SR membranes. The electronic paramagnetic resonance spectra of these probes contained two components, corresponding to motionally restricted and motionally fluid lipids, that were analyzed by spectral subtraction. The addition of increasing amounts of melittin, to the level of 10 mol melittin/mol Ca-ATPase, progressively increased the fraction of restricted lipids and increased the hyperfine splitting of both components in the composite spectra, indicating that melittin decreases the hydrocarbon chain rotational mobility for both the fluid and restricted populations of lipids. No further effects were observed above a level of 10 mol melittin/mol Ca-ATPase. In the spectra from control and melittin-containing samples, the fraction of restricted lipids decreased significantly with increasing temperature. The effect of melittin was similar to that of decreased temperature, i.e., each spectrum obtained in the presence of melittin (10:1) was nearly identical to the spectrum obtained without melittin at a temperature approximately 5 degrees C lower. The results suggest that the principal effect of melittin on SR membranes is to induce protein aggregation and this in turn, augmented by direct binding of melittin to the lipid, is responsible for the observed decreases in lipid mobility. Protein aggregation is concluded to be the main cause of inactivation of the Ca-ATPase by melittin, with possible modulation also by the decrease in mobility of the boundary layer lipids.     

Comparison of the profile structures of isolated and reconstituted sarcoplasmic reticulum membranes

The profile structures of functional reconstituted sarcoplasmic reticulum (RSR) membranes were investigated as a function of the lipid/protein (L/P) ratio via x-ray diffraction studies of hydrated oriented multilayers of these membranes to a resolution of 10-15 A, and neutron diffraction studies on these multilayers to lower resolutions. Our results at this stage of investigation indicate that reconstitution of SR with variable amounts of Ca2+ pump protein for L/P ratios greater than 88 results in closed membraneous vesicles in which the Ca2+ pump protein is distributed asymmetrically in the membrane profile; a majority of the protein density is contained primarily in the extravesicular half of the membrane profile whereas a relatively lesser portion of the protein spans the hydrocarbon core of the RSR membranes. These RSR membranes are functionally similar and resemble isolated light sarcoplasmic reticulum in both profile structure and function at a comparable L/P ratio. Reconstitution with greater amounts of Ca2+ pump protein (e. g. L/P approximately 50-60) resulted in substantially less functional membranes with a dramatically thicker profile structure.     

The secondary structure of turkey gizzard myosin light chain kinase and the nature of its interaction with calmodulin

The enzymatic activities of native myosin light chain kinases are subject to modification by interaction with Ca2(+)-calmodulin (CaM). The interaction between myosin light chain kinase isolated from turkey gizzard (tgMLCK) and calmodulin isolated from bovine testes (CaMbt) and wheat germ (CaMwg) has been examined by means of the intrinsic tryptophan fluorescence of tgMLCK and the fluorescence of extrinsic fluorescent labels located at Cys-27 and Tyr-139 of CaMwg and Tyr-99 of CaMbt. Static and dynamic fluorescence measurements provide evidence for the involvement of the former two sites in the zone of contact with lesser involvement of the site marked by the probe at Tyr-99. Complex formation protected the primary cleavage site in CaMbt (Lys-77) from proteolysis by trypsin. These results are consistent with involvement of the N- and C-terminal lobes of CaM in stabilization of the complex with tgMLCK, but cannot rule out participation of the connecting strand in the interaction. CD measurements extending to 175 nm, obtained using synchroton radiation, indicate the following secondary structure content for tgMLCK: 17 +/- 2% alpha-helix, 22 +/- 3% antiparallel beta-sheet, 3 +/- 1% parallel beta-sheet, 24 +/- 2% beta-turns, and 34 +/- 2% random coil. Similar measurements of the CD spectra of CaMbt and of the 1:1::CaMbt:tgMLCK complex presently indicate that neither protein undergoes major secondary structure rearrangement during their interaction, although subtle changes in the CD spectrum of tgMLCK appear to be correlated with the interaction with CaM.     

Unsaturated aminophospholipids are preferentially retained by the fast skeletal muscle CaATPase during detergent solubilization. Evidence for a specific association between aminophospholipids and the calcium pump protein.

When fast twitch skeletal muscle vesicles (SR) and purified calcium pump protein are stripped with the nonionic detergent C12E8 (octaethylene glycol dodecyl ether), not all the membrane phospholipids are removed from the calcium pump protein. Maximal extraction produces a remnant of 6-8 mol of phospholipid/mole of calcium ATPase (CaATPase). In contrast to native SR and the prestripped purified CaATPase, the remaining phospholipid is markedly enriched in phosphatidylethanolamine (PE) and phosphatidylserine (PS) in both preparations; the remaining lipid is also enriched in phospholipid that is predominantly unsaturated. In addition, virtually all of the associated PE is plasmalogenic (96% as opposed to 63% in the native SR). The amino-specific cross-linking reagent DFDNB (1,5-difluoro-2,4-dinitrobenzene sulfonic acid) and the amino binding reagent TNBS (2,4,6-trinitrobenzene sulfonic acid) were utilized to identify the monolayer of the native preparation where these phospholipids reside, and to determine which phospholipids are closely associated with the calcium pump protein following detergent treatment. These studies demonstrate that PE and PS are closely associated with the pump protein, PE residing almost exclusively in the outer monolayer of SR, while PS resides in the inner monolayer. Nonspecific phospholipid exchange protein was shown to be capable of exchanging phospholipids from donor vesicles into those phospholipids associated with the CaATPase; stripping of lipid-exchanged vesicles with C12E8 exhibited the same specificity with regard to head-group species (i.e., PE is markedly enriched in the extracted protein associated fraction). The results suggest that specific protein-lipid interactions exist, favoring the association of plasmalogenic aminophospholipids with the calcium pump protein.     

Photosynthetic energy conversion under extreme conditions--II: the significance of lipids under light limited growth in Antarctic sea ice diatoms

Low photosynthetic active radiation is a strong determinant in the development and growth of sea ice algae. The algae appear to have universal mechanisms to overcome light limitation. One important process, which is induced under light limitation, is the desaturation of chloroplast membrane lipids. In order to discover whether this process is universally valid in sea ice diatoms, we investigated three species coexisting in chemostats illuminated with 15 and 2 micromol photons m(-2) s(-1) at -1 degrees C. Growth under 2 micromol photons m(-2) s(-1) caused a 50% increase in monogalactosyldiacylglycerols (MGDG) thylakoid membrane related 20:5 n-3 fatty acids. This fatty acid supports the fluidity of the thylakoid membrane and therefore the velocity of electron flow, which is indicated by increasing rate constants for the electron transport between Q(A) (first stable electron acceptor) and bound Q(B) (second stable electron acceptor) (11.16 +/- 1.34 to 23.24 +/- 1.35 relative units). Two micromol photons m(-2) s(-1) furthermore resulted in higher amounts of non-lipid bilayer forming MGDG in relation to other bilayer forming lipids, especially digalactosydiacylglycerol (DGDG). The ratio of MGDG:DGDG increased from 3.4 +/- 0.3 to 5.7 +/- 0.3. The existence of bilayer thylakoid membranes with high proportions of non. bilayer forming lipids is only possible when sufficient thylakoid pigment-protein complexes are present. If more thylakoid pigment-protein complexes are present in membranes, as found under extreme light limitation, less bilayer forming lipids such as DGDG are required to stabilize the bilayer structure. Differences in protein contents between both light intensities were not found. Consequently pigment contents which nearly doubled under 2 micromol photons m(-2) s(-1) must be responsible in balancing the potential stability loss resulting from an increase in MGDG:DGDG ratio.     

脂肪族类两亲物对肌浆网膜蛋白结构的影响

本文观察和比较了六种C_(18)脂肪族类两亲物(fatty amphiphile,FA),包括硬脂酸(stearic acid)、硬脂胺(stearyl amine)、硬脂醇(stearyl alcohol)、油酸(oleic acid)、油胺(oleylamine)和油醇(oleyl alcohol),对肌浆网(sarcoplasmic reticulum,SR)钙泵蛋白结构的影响。当FA∶SR(μmol∶mg)的比例为2.67∶1—21.33∶1时,除油醇(oleyl alcohol)外,其余五种FA引起天然兔骨骼肌肌浆网蛋白内源荧光强度降低。随FA∶SR比例升高,降低幅度加大。五种FA的最大降低幅度在10—32％之间。其中带电荷FA比不带电荷FA的作用强,但均未见峰位位移。当FA∶SR的比例为2.67∶1时,除硬脂醇(stearyl alcohol)外,其余五种FA使N-(3-芘)-马来酰胺(N-(3-pyrene)maleimide,N-(3-p)-M)修饰的SR蛋白巯基荧光强度分别上升9％,40％,150％,193％和5％,但也未见峰位位移。ATP可减弱胺类FA引起的SR蛋白内源荧光降低和巯基荧光升高的幅度。油酸、油胺和硬脂胺既抑制SRCa~(2+),Mg~(2+)-ATPase活力和SR钙蓄积,又使SR钙泵蛋白的巯基修饰荧光显著上升。提示C_(18)脂肪族类两亲物使SR功能受损与其引起钙泵蛋白构象的显著变化有关。     

Endogenous phosphorylation of sarcoplasmic reticulum fragments of rabbit fast skeletal muscles

The purified membrane fragments of sarcoplasmic reticulum (SR) of rabbit fast skeletal muscles were found to incorporate 32P from[gamma-32P]ATP in endogenous membrane substrates and in histone H1. The existence of membrane-bound protein kinase of SR was demonstrated by steady state binding of [3H]-cAMP to the SR membranes. The constant of [3H]cAMP binding to the membranes is 2.5 +/- 0.003 x 10(6) M-1, the number of binding sites is 6.1 +/- 0.8 pmol per 1 mg of protein. The endogenous phosphorylation of SR components was inhibited by cAMP and cGMP at concentrations of 10(-7)-10(-6) and depended on Mg2+ and Ca2+. The thermostable protein inhibitor of cAMP-dependent protein kinase inhibited the endogenous phosphorylation of SR membranes by 30-40%. The protein phosphoproduct of SR membranes revealed the properties of a phosphoester. The membrane-bound protein kinase was active towards the exogenous substrate--histone H1. Phosphorylation in the presence of histones was independent of cyclic nucleotides, Mg2+ and Ca2+. Fractionation of 32P-labelled solubilized membranes in polyacrylamide gel in the presence of Na-SDS showed that the radioactivity is bound to protein zones with molecular weights of 95 000 and 6000.     

SR compartment calcium and cell apoptosis in SERCA overexpression

The relationship between SR Ca2+ ATPase (SERCA) activities, cell calcium level, SR calcium store and cell cycle events is not clearly understood. We studied SERCA overexpression in Cos cells using an adenovirus vector. Twofold increases in SERCA mRNA and in protein were correlated with a 2.3-fold and a 1.6-fold paralleled increase in SR calcium pump activity (R = 0.97 and R = 0.99 respectively). Dose-related apoptotic cell death was associated with SERCA overexpression (R = 0.92). When serum was reduced to 4%, cell apoptosis further increased from 20.7 +/- 4.8% to 47.5 +/- 12.9% (M+/-SD; P<0.05; n=3). Flow cytometry identified cell cycle arrest at the G2/M phase. The interleukin-1 converting enzyme (ICE) inhibitor z-VAD-fmk reduced apoptosis for low-, medium- and high-expressing constructs, whereas the CPP-32 inhibitor z-DEVD-fmk had no effect. Flow cytometry using Fluo-3 and Fura-Red revealed a 1.5-fold higher basal calcium and a 10-fold SR calcium overload. ICE inhibitor z-VAD-fmk did not alter calcium loading. An epitope-tagged SERCA mutant, which has no intrinsic Ca2+-pump activities, had a much smaller effect on the SR calcium. These findings suggest that SERCA2A overexpression has an intrinsic role in altering cell-cycle progression, augmenting cellular and SR calcium loading, and precipitating ICE protease-mediated apoptosis; this represents as a novel model for primary SR calcium overload and associated cell apoptosis.     

Differential molecular dynamics and transmembrane fluidity gradients in canine myocardial sarcolemma and sarcoplasmic reticulum.

The molecular dynamics of highly purified preparations of canine myocardial sarcolemma (SL) and sarcoplasmic reticulum (SR) were quantified by electron spin resonance spectroscopy (ESR). Canine myocardial SL and SR have substantially different motional regimes in their membrane interiors as demonstrated by alterations in the relative peak height ratios, peak widths and peak splittings in ESR spectra of 16-doxylstearate incorporated into SL and SR. Quantification of the apparent order parameters (S) of 16-doxylstearate in SL and SR by analyses of ESR spectra demonstrated that the interior of the SL membrane was substantially more immobilized than the interior of the SR membrane (e.g. S = 0.168 +/- 0.002 for SL and S = 0.128 +/- 0.003 for SR). In contrast, only modest differences in membrane dynamics near the hydrophobic-hydrophilic interface were present in SL and SR as ascertained by ESR spectra of the probe 5-doxylstearate incorporated into these membranes. Myocardial sarcolemma contained heretofore unsuspected amounts of cholesterol (1.4 +/- 0.1 mumol cholesterol/mg protein) while sarcoplasmic reticulum contained only small amounts of cholesterol (0.17 +/- 0.06 mumol cholesterol/mg protein). Model systems employing binary mixtures of plasmenylcholine/cholesterol and phosphatidylcholine/cholesterol demonstrated that the observed alterations in molecular dynamics were due, in large part, to the differential cholesterol content in these two subcellular membrane compartments. Taken together, these results demonstrate that these two functionally distinct myocardial subcellular membranes have markedly disparate molecular dynamics and transmembrane fluidity gradients which may facilitate their performance of specific functional roles during excitation-contraction coupling in myocardium.     

Structure in solution of a four-helix lipid binding protein.

Because of the low solubility of lipids in water, intercellular and intracellular pathways of lipid transfer are necessary, e.g., for membrane formation. The mechanism by which lipids in vivo are transported from their site of biogenesis (endoplasmatic reticulum and the chloroplasts) to their place of action is unknown. Several small plant proteins with the ability to mediate transfer of radiolabeled phospholipids in vitro from liposomal donor membranes to mitochondrial and chloroplast acceptor membranes have been isolated, and a protein with this ability, the nonspecific lipid transfer protein (nsLTP) isolated from barley seeds (bLTP), has been studied here. The structure and the protein lipid interactions of lipid transfer proteins are relevant for the understanding of their function, and here we present the three-dimensional structure in solution of bLTP as determined by NMR spectroscopy. The 1H NMR spectrum of the 91-residue protein was assigned for more than 97% of the protein 1H atoms, and the structure was calculated on the basis of 813 distance restraints from 1H-1H nuclear Overhauser effects, four disulfide bond restraints, from dihedral angle restraints for 66 phi-angles, 61 chi 1 angles, and 2 chi 2 angles, and from 31 sets of hydrogen bond restraints. The solution structure of bLTP consists of four well-defined alpha-helices A-D (A, Cys 3-Gly 19; B, Gly 25-Ala 38; C, Arg 44-Gly 57; D, Leu 63-Cys 73), separated by three short loops that are less well defined and concluded by a well defined C-terminal peptide segment with no observable regular secondary structure. For the 17 structures that are used to represent the solution structure of bLTP, the RMS deviation to an average structure is 0.63 A +/- 0.04 A for backbone atoms and 0.93 A +/- 0.06 A for all heavy atoms. The secondary structure elements and their locations in the sequence resemble those of nsLTP from two other plant species, wheat and maize, whose structures were previously determined (Gincel E et al, 1995, Eur J Biochem 226:413-422; Shin DH et al, 1995, Structure 3:189-199). In bLTP, the residues analogous to those in maize nsLTP that constitute the palmitate binding site are forming a similar hydrophobic cavity and a potential acyl group binding site. Analysis of the solution structure of bLTP and bLTP in complex with a ligand might provide information on the conformational changes in the protein upon ligand binding and subsequently provide information on the mode of ligand uptake and release. In this work, we hope to establish a foundation for further work of determining the solution structure of bLTP in complex with palmitoyl coenzyme A, which is a suitable ligand, and subsequently to outline the mode of ligand binding.     

Effect of short-chain primary alcohols on fluidity and activity of sarcoplasmic reticulum membranes

Intramolecular excimer formation with the fluorescent probe 1,3-di(1-pyrenyl)propane, differential scanning calorimetry, and X-ray diffraction were used to assess the effect of ethanol, 1-butanol, and 1-hexanol on the bilayer organization in model membranes, sarcoplasmic reticulum (SR) lipids and native SR membranes. These alcohols have fluidizing effects on membranes and lower the main transition temperature of dimyristoylphosphatidylcholine (DMPC), but only 1-hexanol alters the cooperativity of the phase transition and significantly increases the thickness of DMPC bilayers. The interaction of the three alcohols with the SR Ca2+ pump was also investigated. Hydrolysis of ATP and coupled Ca2+ uptake are differently sensitive to the three alcohols. Whereas ethanol and 1-butanol inhibited the Ca2+ uptake, 1-hexanol stimulated it. Nevertheless, the energetic efficiency of the pump (Ca2+/ATP) is not significantly affected by ethanol or 1-hexanol, but uncoupling was observed with 1-butanol at high concentrations. The different effects of alcohols on the activity of SR membranes rule out an unitary mechanism of action on the basis of fluidity changes induced in the lipid bilayer. Depending on the chain length, the alcohols interact with the SR membranes in different domains, perturbing differently the Ca2+-pump activity.     

A comparison of differential scanning calorimetric and Fourier transform infrared spectroscopic determination of mixing behavior in binary phospholipid systems

Fourier transform infrared (FT-IR) spectroscopy and differential scanning calorimetry (DSC) have been used to elucidate the phase behavior of two binary lipid mixtures, acyl chain perdeuterated 1,2-dipalmitoylphosphatidylethanolamine (DPPE-d62)/1,2-dielaidoylphosphatidylcholine (DEPC) and acyl chain perdeuterated 1,2-dipalmitoylphosphatidylcholine (DPPC-d62)/1,2-dimyristoylphosphatidylethanolamine (DMPE). The former shows gel state immiscibility over most of the composition range. The FT-IR data indicate that one of the solid phases is essentially pure DEPC, while the other solid phase contains both lipids. The DPPC-d62/DMPE pair are miscible over the entire composition range. The use of deuterated lipids as one component in the mixture permits the melting characteristics of each component to be separately determined in the FT-IR experiment. The FT-IR data are used to assign the endotherms observed in the DSC to particular molecular components. For the DPPE-d62/DEPC system, two endotherms are observed at compositions between 10 and 67 mol% DPPE-d62. The lower transition is assigned to the DEPC component, while the higher event contains contributions to the enthalpy from both lipids in the mixture. The midpoint of the DEPC melting occurs substantially below that for DPPE-d62. For the miscible pair, each of the lipids melt over approximately the same temperature range. The complementary and consistent nature of the information available from FT-IR and from DSC is demonstrated from the current work.     

Decavanadate is responsible for vanadate-induced two-dimensional crystals in sarcoplasmic reticulum

Two-dimensional protein crystals of the calcium pump protein of sarcoplasmic reticulum (SR) from fast skeletal muscle were induced using Na₃VO₃ as first described by Dux and Martonosi. These crystals exhibit repeat rows 11 nm apart which contain discrete units with 7 nm repeats. Four different methods of sample preparation for electron microscopy, i.e., negative staining, freezedrying, freeze-fracturing, and thin-sectioning electron microscopy, each give complimentary repeat units. The SR-membrane crystals exhibit surface structure by the freeze-drying technique and row-like structures on the normally smooth outer face of normal SR. The formation of the membrane crystals is dependent on the pH and concentration of the vanadate. Only conditions favoring the presence of decavanadate yield crystals. At low concentrations and neutral pH, decavanadate is unstable and with time converts to smaller oligomers and the monomer. The presence of membrane crystals was correlated with the life span of the decavanadate. Membrane crystals were obtained in the SR membrane from fast twitch muscle from light and heavy SR, referable to longitudinal and terminal cisternae as well as from reconstituted SR. Canine cardiac SR did not crystallize under these conditions.Abbreviations Tris (tris[hydroxymethyl])aminomethane - TES (N-tris[hydroxymethyl]methyl-2-aminoethanesulfonic acid), 2-(2-hydroxy-1-bis[hydroxymethyl]ethyl)aminoethanesulfonic acid - SR sarcoplasmic reticulum - CPP calcium pump protein Dedicated to the memory of Prof. David E. Green, friend, mentor, and colleague.     

Ordered arrays of Ca2+-ATPase on the cytoplasmic surface of isolated sarcoplasmic reticulum.

Isolated sarcoplasmic reticulum (SR) vesicles with polymerized calcium pump protein were freeze-dried and rotary shadowed following uranyl acetate stabilization. This technique allows direct observation of a single side of the vesicle without requiring optical filtering. The heads of individual ATPase molecules, projecting above the cytoplasmic surface, are clearly resolved in the replicas. Ca ATPase molecules form extensive arrays in vanadate-treated, rabbit SR vesicles and in gently isolated, native SR vesicles from scallop. Gentle isolation results in limited areas of orderly structure in native SR isolated from vertebrate muscles. Special attention is given to the effect of various shadow thicknesses on the appearance of the heads. This information is essential to the interpretation of images in the accompanying paper (Franzini-Armstrong, C., and D.J. Ferguson, 1985, Biophys. J., 48:607-615).     

Incorporation of synthetic phospholipids into the membranes of the sarcoplasmic reticulum from the rabbit muscle

The hydrophobic spin label used in ESR showed that the iminoxyl radical rotation in the native membrane of sarcoplasmatic reticulum (SR) occurred much faster than in the membranes, modified by a synthetic lipid. Such effect was observed throughout the whole temperature range (7-40 degrees). Experimental technique for the modification of the SR membrane and the lipid by ultrasonic treatment has been developed. Synthetic lipids without ultrasonic treatment did not inhibit the activity of Ca2+-ATPase. The change in both the enzyme activity and its ability to transport the Ca2+ ions through the membrane vesicules was observed after the phospholipids incorporation into the SR membrane. The investigation of the temperature dependence (in Arrhenius coordinates) of native and modified by lecithin Ca2+-ATPase after ultrasonic treatment and also of a "pure enzyme" showed the presence of two sharp breaks at 20 degrees and 40-42 degrees. It was shown tha the break of an Arrhenius anamorphosis was caused by a lipid environment of ATPase, "melting" of a phospholipid bilayer. The break at 20-22 degrees was observed in all cases and even after the incorporation of all the lipids into the SR membrane. This phenomenon can be explained by the distortion of the protein-lipid interaction, affecting the conformation mobility of protein and the geometry of its catalytically active center.