Sarcoplasmic Reticulum Lipids Research Articles

EFFECT OF CYTOSOL ON LIPID PEROXIDATION IN FLOUNDER SARCOPLASMIC RETICULUM

Studies were carried out to determine the effects of cytosol on sarcoplasmic reticulum lipid peroxidation. Upon addition of unfractionated cytosol to the lipid peroxidation system, inhibition was observed. Fractionation revealed that both the low molecular weight (LMW) cytosol ( 6,000–8,000 MW) fractions inhibited lipid peroxidation. It is suggested that either inorganic phosphate compounds or nucleotides may be responsible for the LMW cytosol's inhibitory characteristics. Addition of the HMW cytosol fraction to the system necessitated the presence of additional adenosine 5′-diphosphate (ADP) for optimal activity. ADP bound to the HMW cytosol fraction may be incapable of chelating the iron and preventing its precipitation as the ferric hydroxide.

Calcium and lanthanide binding in the sarcoplasmic reticulum ATPase.

The interactions of calcium and lathanides with the sarcoplasmic reticulum ATPase, and their respective ability to activate the enzyme, were studied by direct measurements of binding with radioactive tracers, functional effects on the ATPase partial reactions, changes in the quantum yield of tryptophanyl residues and a covalently bound fluorescein label (fluorescein 5-isothiocyanate, FITC), and energy transfer between bound lanthanide and fluorescent labels. We find that: (a) Lanthanides displace calcium from specific ATPase sites with diphasic kinetics that are consistent with sequential exchange. (b) Lanthanides in excess of the calcium stoichiometry are mostly bound to sarcoplasmic reticulum lipids and non-ATPase proteins. (c) Both calcium and lanthanides activate the ATPase and allow formation of the phosphorylated intermediate by utilization of ATP; however, hydrolytic cleavage of the intermediate formed in the presence of lanthanides occurs at a slower rate than the intermediate formed in the presence of calcium. (d) In contrast to a calcium-dependent change in the quantum yield of both the tryptophanyl residues (transmembrane region) and the FITC label (extramembranous region), lanthanides induce only a change in the quantum yield of the FITC label. (e) Measurements of energy transfer between bound lanthanide and fluorescent labels detect lanthanide bound midway between the catalytic site in the globular region of the ATPase outside the membrane, and the transmembrane calcium binding domain which is involved in enzyme activation (Clarke, D. M., Loo, T. W., Inesi, G., and MacLennan, D. H. (1989a) Nature 339, 476-478). It is apparent that cation bound in this midway location controls exchange of calcium bound in the transmembrane region. The possibility that the midway location may provide a domain for binding of a second calcium is discussed.

Evidence that lipid lateral phase separation induces functionally significant structural changes in the Ca+2ATPase of the sarcoplasmic reticulum

We have studied lipid lateral phase separation (LPS) in the intact sarcoplasmic reticulum (SR) membrane and in bilayers of isolated SR membrane lipids as a function of temperature, [Mg+2], and degree of hydration. Lipid LPS was observed in both the intact membrane and in the bilayers of isolated SR lipids, and the LPS behavior of both systems was found to be qualitatively similar. Namely, lipid LPS occurs only at relatively low temperature and water content, independently of the [Mg+2], and the upper characteristic temperature (th) for lipid LPS for both the membrane and bilayers of its isolated lipids coincide to within a few degrees. However, at similar temperatures, isolated lipids show more LPS than the lipids in the intact membrane. Lipid LPS in the intact membrane and in bilayers of the isolated lipids is fully reversible, and more extensive for samples partially dehydrated at temperatures below th. Our previous x-ray diffraction studies established the existence of a temperature-induced transition in the profile structure of the sarcoplasmic reticulum Ca+2ATPase which occurs at a temperature corresponding to the [Mg+2]-dependent upper characteristic temperature for lipid LPS in the SR membrane. Furthermore, the functionality of the ATPase, and in particular the lifetime of the first phosphorylated enzyme conformation (E1 approximately P) in the Ca+2 transport cycle, were also found to be linked to the occurrence of this structural transition. The hysterisis observed in lipid LPS behavior as a function of temperature and water content provides a possible explanation for the more efficient transient trapping of the enzyme in the E1 approximately P conformation observed in SR membranes partially dehydrated at temperatures below th. The observation that LPS behavior for the intact SR membrane and bilayers of isolated SR lipids (no protein present) are qualitatively similar strongly suggests that the LPS behavior of the SR membrane lipids is responsible for the observed structural change in the Ca+2ATPase and the resulting significant increase in E1 approximately P lifetime for temperatures below th.

Selective detection of the rotational dynamics of the protein-associated lipid hydrocarbon chains in sarcoplasmic reticulum membranes

We have developed a saturation transfer EPR (ST-EPR) method to measure selectively the rotational dynamics of those lipids that are motionally restricted by integral membrane proteins and have applied this methodology to measure lipid-protein interactions in native sarcoplasmic reticulum (SR) membranes. This analysis involves the measurement of spectral saturation using a series of six stearic acid spin labels that are labeled with a nitroxide at different carbon atom positions. A large amount of spectral saturation is observed for spin labels in native SR membranes, but not for spin labels in dispersions of extracted SR lipids, implying that the motional properties of those lipids interacting with the Ca-ATPase, i.e., the boundary or annular lipid, can be directly measured without the need for spectral subtraction procedures. A comparison of the motional properties of the restricted lipid, measured by ST-EPR, with those measured by digital subtraction of conventional EPR spectra qualitatively agree, for in both cases the Ca-ATPase restricts the rotational mobility of a population of lipids, whose rotational mobility increases as the nitroxide is positioned toward the center of the bilayer. However, the ability of ST-EPR to directly measure the motionally restricted lipid in a model-independent means provides the greater precision necessary to measure small changes in the rotational dynamics of the lipid at the protein-lipid interface, providing a valuable tool in clarifying the relationship between the physical nature of the protein-lipid interface and membrane function.

Changes in the sarcoplasmic reticulum membrane profile induced by enzyme phosphorylation to E1 approximately P at 16 A resolution via time-resolved x-ray diffraction

Time-resolved x-ray diffraction studies of the isolated sarcoplasmic reticulum (SR) membrane have provided the difference electron density profile for the SR membrane for which the Ca2+ ATPase is transiently trapped exclusively in the first phosphorylated intermediate state, E1 approximately P, in absence of detectable enzyme turnover vs. that before ATP-initiated phosphorylation of the enzyme. These diffraction studies, which utilized the flash-photolysis of caged ATP, were performed at temperatures between 0 and -2 degrees C and with a time-resolution of 2-5 s. Analogous time-resolved x-ray diffraction studies of the SR membrane at 7-8 degrees C with a time resolution of 0.2-0.5 s have previously provided the difference electron density profile for the SR membrane for which the Ca2+ ATPase is only predominately in the first phosphorylated intermediate state under conditions of enzyme turnover vs. that before enzyme phosphorylation. The two difference profiles, compared at the same low resolution (approximately 40 A), are qualitatively similar but nevertheless contain some distinctly different features and have therefore been analyzed via a step-function model analysis. This analysis was based on the refined step-function models for the two different electron density profiles obtained independently from x-ray diffraction studies at higher resolution (16-17 A) of the SR membrane before enzyme phosphorylation at 7.5 and -2 degrees C. The step-function model analysis indicated that the low resolution difference profiles derived from both time-resolved x-ray diffraction experiments arise from a net movement of Ca2+ ATPase protein mass from the outer monolayer to the inner monolayer of the SR membrane lipid bilayer. The conserved redistribution of this protein mass is however somewhat different for the two cases, especially at the extravesicular membrane surface containing the Ca2+ATPase "headpiece." However, the conserved redistribution of protein mass within the SR membrane lipid bilayer common to both cases is clearly due to E1~P formation.

Infrared spectroscopic characterization of the structural changes connected with the E1→E2 transition in the Ca2+-ATPase of sarcoplasmic reticulum.

The Ca2+-transporting ATPase (EC 3.6.1.38) of sarcoplasmic reticulum alternates between several conformational states during ATP-dependent Ca2+ transport. The E1 conformation is stabilized by 0.1 mM Ca2+ and the E2 conformation by vanadate in a Ca2+-free medium. Fourier transform infrared spectroscopy reveals significant differences between the two states that indicate differences in the protein secondary structure. The two states and the corresponding spectra can be interconverted reversibly by changing the Ca2+ concentration of the medium. The infrared spectral changes indicate the appearance of a new alpha-helical substructure connected with the E1----E2 conversion accompanied by small changes in beta-turns, while the beta-sheet content remains essentially unchanged. There are also differences between the E1 and E2 states in the C = O stretching vibrations of the ester carbonyl groups of phospholipids in intact sarcoplasmic reticulum that are not observed under identical conditions in isolated sarcoplasmic reticulum lipid dispersions. These observations imply an effect of proteins on the structure of the interfacial regions of the phospholipids that is dependent on the conformational state of the Ca2+-ATPase. The CH2- and CH3-stretching frequencies of the membrane lipids are not affected significantly by the E1----E2 transition. The Fourier transform infrared spectra of sarcoplasmic reticulum vesicles in the presence of 20 mM Ca2+ suggest the stabilization of a protein conformation similar to the E2 state except for differences in the behavior of COO- and phospholipid ester C = O groups that may reflect charge effects of the bound Ca2+.

The secondary structure of calcium pump protein in light sarcoplasmic reticulum and reconstituted in a single lipid component as determined by Raman spectroscopy.

Raman spectra have been measured of the following samples: active calcium pump protein in light sarcoplasmic reticulum (SR) membranes, lipids extracted from light SR membranes, active calcium pump protein reconstituted in dielaidoylphosphatidylcholine (DEPC), and pure DEPC. The spectra of native SR lipids and of pure DEPC are different, and yet when these spectra are subtracted from the spectra of the respective protein-lipid complexes, the resulting amide I spectra of the calcium pump protein are the same, indicating that appropriate criteria have been chosen for subtraction of the spectrum of a lipid. This spectrum has been analyzed for secondary structure with the following results. The SR calcium pump protein contains 51 +/- 5% helix, in agreement with a prediction of secondary structure obtained from an analysis of the sequence, and 21 +/- 4% beta-strand. In addition, the presence of protein broadens and lowers the main melting transition of DEPC.

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.

Temperature dependence of rotational dynamics of protein and lipid in sarcoplasmic reticulum membranes.

We have investigated the relationship between function and molecular dynamics of both the lipid and the Ca-ATPase protein in sarcoplasmic reticulum (SR), using temperature as a means of altering both activity and rotational dynamics. Conventional and saturation-transfer electron paramagnetic resonance (EPR) was used to probe rotational motions of spin-labels attached either to fatty acid hydrocarbon chains or to the Ca-ATPase sulfhydryl groups in SR. EPR studies were also performed on aqueous dispersions of extracted SR lipids, in order to study intrinsic lipid properties independent of the protein. While an Arrhenius plot of the Ca-ATPase activity exhibits a clear change in slope at 20 degrees C, Arrhenius plots of lipid hydrocarbon chain mobility are linear, indicating that an abrupt thermotropic change in the lipid hydrocarbon phase is not responsible for the Arrhenius break in enzymatic activity. The presence of protein was found to decrease the average hydrocarbon chain mobility, but linear Arrhenius plots were observed both in the intact SR and in extracted lipids. Lipid EPR spectra were analyzed by procedures that prevent the production of artifactual breaks in the Arrhenius plots. Similarly, using sample preparations and spectral analysis methods that minimize the temperature-dependent contribution of local probe mobility to the spectra of spin-labeled Ca-ATPase, we find that Arrhenius plots of overall protein rotational mobility also exhibit no change in slope. The activation energy for protein mobility is the same as that of ATPase activity above 20 degrees C; we discuss the possibility that overall protein mobility may be essential to the rate-limiting step above 20 degrees C.

Distribution of a fatty acid spin probe in sarcoplasmic reticulum. Evidence of membrane asymmetry.

The distribution of a lipophilic spin probe, 5-doxyl stearate, between the inner and outer halves of the sarcoplasmic reticulum (SR) bilayer was determined by titration with Ni X EDTA, a spin broadening agent. Titrations were also performed with Fe(CN)3-6 and with the solvated Ni2+ cation. Ni X EDTA titrations reached a clearly defined asymptote at 35% signal reduction. Fe(CN)3-6 and Ni2+ titrations gave biphasic curves but showed 35% of the signal to be readily eliminated at low concentrations. When the Ni2+ cation was used with ionophore, titrations indicated that 96% of the probe is aligned in the bilayer with the spin moiety at either the inner or outer interface. It was concluded that the spin probe distribution between the outer and inner halves of the SR bilayer is 35:65, respectively. Titrations performed on vesicles of purified SR lipids gave a ratio of 60 exposed:40 protected, consistent with the vesicular geometry. In addition the spin probe distribution in SR vesicles did not vary as a function of temperature, salt concentration, or spin probe concentration. On this basis it was concluded that the spin probe distribution gives a reasonable estimation of the volume of fluid lipids available to readily solubilize the probe in each half of the bilayer and that the observed asymmetry in distribution is due to the presence of SR proteins which were eliminated in the pure lipid vesicles. Furthermore, as EDTA is unique in its ability to chelate transition metals, Ca2+ and EGTA can be used in Ni X EDTA titrations without altering the chelation of Ni2+. Known changes in ATPase conformation accompanying Ca2+ and adenyl-5'-yl imidodiphosphate X Mg binding did not affect the spin probe distribution. However, phosphorylation of the enzyme by Pi gave a small, but clearly discernible, protection of spin probe signal. Chemical reduction with ascorbate indicated that this was due to occlusion of a small fraction of spin probes and thus possibly SR lipids.

The separate profile structures of the functional calcium pump protein and the phospholipid bilayer within isolated sarcoplasmic reticulum membranes determined by X-ray and neutron diffraction

The detailed profile structure of the isolated sarcoplasmic reticulum membrane was studied utilizing a combination of X-ray and neutron diffraction. The water and lipid profile structures within the sarcoplasmic reticulum membrane were determined at 28 Å resolution directly by neutron diffraction and selective deuteration of the water and lipid components. The previously determined electron density profile structure of the sarcoplasmic reticulum membrane at 12 Å resolution was subjected to model refinement analysis constrained by the neutron diffraction results, thereby providing unique higher resolution calculated lipid and protein profile structures. It was found that the lipid bilayer profile structure of the isolated sarcoplasmic reticulum membrane is asymmetric, primarily the result of more lipid residing in the inner versus the outer monolayer of the sarcoplasmic reticulum lipid bilayer. The asymmetry in the lipid composition was necessarily coincident with a complimentary asymmetry in the protein mass distribution between the two monolayers in order to preserve the overall cross-sectional area of lipid and protein throughout the lipid bilayer region of the sarcoplasmic reticulum membrane profile structure. Approximately 50% of the mass of the total protein was found to be localized externally to the sarcoplasmic reticulum membrane lipid bilayer protruding from the outer lipid monolayer into the extravesicular medium. The structural features of the protein protrusion appear to be rather variable depending upon the environment of the sarcoplasmic reticulum membrane. This highly asymmetric structural organization of the sarcoplasmic reticulum membrane profile is consistent with its primary function of unidirectional calcium transport.

Phospholipid asymmetry in the isolated sarcoplasmic reticulum membrane

The total phospholipid content and distribution of phospholipid species between the outer and inner monolayers of the isolated sarcoplasmic reticulum membrane was measured by phospholipase A 2 activities and neutron diffraction. Phospholipase measurements showed that specific phospholipid species were asymmetric in their distribution between the outer and inner monolayers of the sarcoplasmic reticulum lipid bilayer; phosphatidylcholine (PC) was distributed 48 52 ± 2% between the outer and inner monolayer of the sarcoplasmic reticulum bilayer, 69% of the phosphatidylethanolamine (PE) resided mainly in the outer monolayer of the bilayer, 85% of the phosphatidylserine (PS) and 88% of the phosphatidylinositol (PI) were localized predominantly in the inner monolayer. The total phospholipid distribution determined by these measurements was 48 52 ± 2% for the outer/inner monolayer of the sarcoplasmic reticulum lipid bilayer. Sarcoplasmic reticulum phospholipids were biosynthetically deuterated and exchanged into isolated vesicles with both a specific lecithin and a general exchange protein. Neutron diffraction measurements directly provided lipid distribution profiles for both PC and the total lipid content in the intact sarcoplasmic reticulum membrane. The outer/inner monolayer distribution for PC was 47 53 ± 1% , in agreement with phospholipase measurements, while that for the total lipid was 46 54 ± 1% , similar to the phospholipase measurements. These neutron diffraction results regarding the sarcoplasmic reticulum membrane bilayer were used in model calculations for decomposing the electron-density profile structure (10 Å resolution) of isolated sarcoplasmic reticulum previously determined by X-ray diffraction into structures for the separate membrane components. These structure studies showed that the protein profile structure within the membrane lipid bilayer was asymmetric, complementary to the asymmetric lipid structure. Thus, the total phospholipid asymmetry obtained by two independent methods was small but consistent with a complementary asymmetric protein structure, and may be related to the highly vectorial functional properties of the calcium pump ATPase protein in the sarcoplasmic reticulum membrane.

Phase transitions in combined rabbit muscle sarcoplasmic reticulum lipids by Raman spectroscopy

Using Raman spectroscopy, we found that the sarcoplasmic reticulum lipids of combined muscles from rabbit leg undergo at least two reversible temperature phase changes, centered at about −15 and 13°C. Below the first transition, the lipid Raman CH st region is characteristic of the hexagonal lamellar gel phase. Above the second transition, the Raman CH stretch region is that of a “melted” lamellar phase, somewhat more rigid than a monophasic lipid system. The composition of the lipids was determined and the possibility of a relation between the major head group types and the phase transitions is discussed. Since SR Ca 2+ATPase activiity is enhanced at about 14–19°C, the Raman studies suggest that ATPase activity is enhanced when the 13°C transition is complete.

Mechanisms of fatty acid effects on sarcoplasmic reticulum. II. Structural changes induced by oleic and palmitic acids.

The interaction of micromolar concentrations of palmitic and oleic acids with the sarcoplasmic reticulum membrane was studied by electron microscopic techniques in an attempt to define their different effects on ATP-induced calcium sequestration in sarcoplasmic reticulum vesicles. Oleic acid had a concentration-dependent effect on the morphology of sarcoplasmic reticulum vesicles, promoting vesicle fusion and eventual solubilization. Palmitic acid did not alter the morphology of sarcoplasmic reticulum, but its probable site(s) of interaction could be determined. In the presence of palmitic acid, large lamellar structures that formed external to sarcoplasmic reticulum vesicles are probably composed of pure palmitic acid and/or palmitic acid/phospholipid mixed "micelles," but internalization of palmitic acid into sarcoplasmic reticulum vesicles was not detected. Palmitic acid reduced the phospholipid content of sarcoplasmic reticulum membranes with a preservation of the average interparticle protein spacing as observed in freeze-fracture electron micrographs. Thus, palmitic acid appears to be incorporated into the sarcoplasmic reticulum lipid bi-layer. Oleic acid inhibition of ATP-induced calcium sequestration by sarcoplasmic reticulum vesicles is probably caused by net permeability changes of the membrane. A structural mechanism for palmitic acid stimulation of ATP-induced calcium sequestration is proposed in light of the probable insertion of palmitic acid into the sarcoplasmic reticulum lipid bilayer.

Perturbation of the structure and function of a membranous Ca2+-ATPase by non-solubilizing concentrations of a non-ionic detergent.

The present study characterizes the effect of octa(ethyleneglycol)-monododecylether (C12E8) on Ca2+-ATPase membranes, prepared from sarcoplasmic reticulum (SR). At low concentrations C12E8 is incorporated into the membrane (less than or equal to 0.2 g/g protein), without any solubilization or appreciable morphological changes of freeze-fracture replica. Binding studies of C12E8 to ATPase membranes and SR lipid liposomes suggest that the major part of the detergent interacts with lipid. Solubilization of ATPase membranes occurs at a free concentration of C12E8 close to the critical micellar concentration (c.m.c); at low temperatures (2 degrees C) phospholipid is extracted somewhat more easily than ATPase. Electron-spin resonance (ESR) spectra of appropriate spin labels, incorporated into ATPase membranes, show that C12E8 strongly increases the fluidity of the lipid phase and the rotational diffusion of ATPase in the membrane. The effect of C12E8 on the ESR spectra is indistinguishable from that produced by a rise in temperature. Incorporation of C12E8 alters the functional properties of Ca2+-ATPase in a characteristic way: V is decreased and the modulatory effect of high ATP concentrations is reduced, in contrast to what occurs by a rise of temperature. The intrinsic fluorescence of the protein is increased, especially in the absence of Ca2+, suggesting that C12E8 modified in particular the E* form (Ca2+-depleted conformation) of the enzyme. Furthermore, stopped-flow data indicate that C12E8 strongly activates the E* to E transition, which may account for the effect of the detergent on ATP modulation during steady-state ATP hydrolysis. It is concluded that C12E8 perturbs ATPase turnover by direct interaction with the enzyme, rather than by an indirect effect exerted via a change in the lipid phase or protein aggregation.

Functional characteristics of reconstituted sarcoplasmic reticulum membranes as a function of the lipid-to-protein ratio

The ATP-induced Ca2+ accumulation efficiency and rates of Ca2+ uptake of the reconstituted sarcoplasmic reticulum (RSR) model membrane system were measured over an extended range of lipid-to-protein (L/P) molar ratios and were compared to those of isolated light sarcoplasmic reticulum (LSR). Highly purified sarcoplasmic reticulum (SR), dissociated in the presence of deoxycholate, was reconstituted for several L/P ratios, according to the same procedure, forming closed membranes vesicles composed of greater than 95% Ca2+ pump protein and SR lipids which were capable of ATP-induced Ca2+ accumulation in the absence of oxalate or other Ca2+ precipitating agents. This suggests that dissociation of SR and reconstitution to form RSR does not significantly affect the ability of the Ca2+ pump protein incorporated into the SR lipid bilayer to establish Ca2+ gradients. Electron micrographs of fixed and stained dispersions of RSR revealed a structural organization of the membrane that was dependent upon the L/P molar ratio. RSR with L/P greater than 88 were composed of closed vesicles whose membranes stained asymmetrically, similar to that observed for LSR. Closed vesicles of RSR with L/P less than 88 were composed of membrane that stained symmetrically. In addition, reconstituted SR preparations with well-defined L/P molar ratios greater than 88 possess a functional behavior similar to that of LSR (in the absence of oxalate, energy efficiencies are 60-70% and apparent initial uptake rates are 80% that of isolated LSR controls); RSR preparations with L/P less than 88 are characterized by significantly depressed values of the energy efficiencies and apparent initial uptake rates especially at low L/P ratios. Thus, we are the first to report a reconstituted SR model membrane system capable of attaining rates of Ca2+ uptake comparable to isolated LSR controls at comparable L/P ratios in the absence of oxalate or other Ca2+ precipitating agents.

Transbilayer distribution of lipids in a population of sarcoplasmic-reticulum vesicles sealed with their cytoplasmic side outwards.

A population of sarcoplasmic-reticulum vesicles, all of which were sealed with their cytoplasmic side outwards, was obtained by density-gradient centrifugation after loading the vesicles with calcium oxalate. The calcium oxalate could subsequently be removed from the vesicles by the reverse action of the calcium-transport system. Measurements of the catalysed exchange of the phosphatidylcholine in the sarcoplasmic-reticulum cytoplasmic monolayer with an exogenous phosphatidylcholine pool suggested that phosphatidylcholine is symmetrically distributed across the sarcoplasmic-reticulum membrane. A similar result was obtained for phosphatidylethanolamine when sarcoplasmic-reticulum lipids were labelled with trinitrobenzenesulphonic acid. Further catalysed lipid-exchange reactions showed that the transverse movement of phosphatidylcholine across the membrane was exceedingly slow (t 1/2 greater than 15 days).

Stereological analysis of freeze-fracture subfractions from skeletal muscle. I. Relative intrinsic protein. II. Relative lipid content and protein-to-lipid ratio

Standard microsomal subfractions from biological tissues are not homogeneous but mixtures of membranes derived from the various cellular organelles. In the case of skeletal muscle, freeze-fracture replicas show both smooth concave faces and concave faces densely populated with 90-A particles. Stereological sampling techniques have been applied to such replicas and the relative surface area of sarcoplasmic reticulum (SR) membrane calculated. Expressions are derived that estimate the relative fraction of SR intrinsic protein and lipid as a function of the relative surface area. Although most of the protein in our subfraction is SR protein, a significant amount of lipid is non-SR lipid. The effect of this on measurements of the protein-to-lipid ratio is discussed.

Effect of phospholipid substitution on the mobility of protein-bound spin labels in sarcoplasmic reticulum.

The influence of phospholipid environment upon the mobility of spin labels covalently bound to the Ca2+-transport ATPase (ATP phosphohydrolase [EC 3.6.1.3]) was studied by electron spin resonance spectroscopy in native and reconstituted sarcoplasmic reticulum membranes. Fragmented sarcoplasmic reticulum of rabbit skeletal muscle was covalently labeled with maleimide spin-labels of different chain length or with 4-(2-iodoacetamido)-2,2,6,6-tetramethylpiperidinooxyl, and the phospholipids were exchanged for dipalmitoylphosphatidylcholine or dioleoylphosphatidylcholine. With short-chain maleimide or iodoacetamide spin labels, the spectrum of the protein-bound label reflected the change in microenvironment caused by replacement of endogenous phospholipids with dipalmitoylphosphatidylcholine as a decrease in mobility. In contrast, after labeling with long-chain maleimide derivatives, there were no noticeable differences in the spectra before and after substitution with dipalmitophatidylcholine. Replacement of endogenous phospholipids with dioleoylphosphatidylcholine did not affect the spectra. The data indicate that increased viscosity in the environment of Ca2+-transport ATPase produced by replacement of sarcoplasmic reticulum lipids with dipalmitoylphosphatidylcholine reduces the mobility of short-chain maleimide spin labels covalently attached to the Ca2+-transport ATPase polypeptide.

Thermotropic transitions in sarcoplasmie reticulum

Summary Functional transitions of nucleoside triphosphatase activity (NTPase) of sarcoplasmic reticulum (SR) occur at about 17 °C independently of the substrate hydrolysed. Changes in the enthalpies (Δ H*) and entropies (Δ S*) of activation occur at the transition temperature (Tt), but the free energies of activation (Δ G*) remain constant over the range of temperatures studied. Phospholipase-A2 from pig pancreas attacks phospholipids of intact SR only at temperatures above 18 °C, but the enzyme is ineffective at all temperatures in liposomes prepared with isolated SR lipids. The rotational parameter of the fluorescent probe, perylene, imbeded in SR membranes showed a transition at about 18 °C not observed in liposomes of SR lipids.