Sarcoplasmic Reticulum Lipids Research Articles

Abstract 539: Lipogenesis Regulates the Response of Cardiac Muscle to Ischemic Stress Through Sarcoplasmic Reticulum Calcium Atpase

Dysfunctional calcium homeostasis is a hallmark of heart failure, a sequela of myocardial infarction (MI). Previous research has shown that mice deficient in cardiac fatty acid synthase (FAS), the rate-limiting enzyme for de novo lipogenesis, have dysfunctional calcium signaling and a predisposition to heart failure. Yet the mechanisms linking endogenous lipid synthesis, myocardial calcium homeostasis, and cardiac function remain poorly understood. A major step in calcium handling is the transport of cytosolic calcium back into the sarcoplasmic reticulum (SR) by sarcoplasmic reticulum calcium ATPase (SERCA), and recent evidence suggests that SR lipid composition can affect SERCA function. To determine whether FAS plays a role in SERCA activity in the heart, we evaluated FASKard ( FAS K nockout in the Myoc ard ium) mice in the setting of experimental myocardial infarction (MI). Deletion of heart FAS caused a decrease in SERCA activity in younger adult (4 months) and aged (12 months) mice when compared to control (Ctrl) mice of the same age. Lipidomic analysis of the SR showed the reduced SERCA activity in FASKard mice was due to an altered SR phospholipid composition, leading to an imbalance of SR phosphatidylcholine to phosphatidylethanolamine phospholipid ratios. This altered ratio of SR phospholipids was due to a decrease in the abundance of specific phosphatidylethanolamine phospholipid species. MI caused a decrease in SERCA activity in both Ctrl and FASKard mice hearts. FASKard mice after MI had impaired survival with concomitantly decreased SERCA activity compared to Ctrl mice after MI. FASKard MI mice also had a perturbed ratio of SR phosphatidylcholine to phosphatidylethanolamine phospholipids compared to Ctrl MI mice, due to a decrease in certain phosphatidylethanolamine phospholipids. Additionally, MI induced FAS expression in the myocardium. These findings suggest that myocardial de novo lipogenesis is critical to the maintenance of the SR phospholipid composition, SR-mediated calcium homeostasis, and blunting the development of heart failure, particularly ischemic cardiomyopathy. Furthermore, FAS regulation of the SR lipid composition represents a novel therapeutic strategy in the treatment of heart failure.

Differential Effects of Temperature and Lipids on the Gating of RyR and SR K+ Channels

The ryanodine receptor (RyR) is the pathway for release of the sarcoplasmic reticulum (SR) Ca2+ required for cardiac and skeletal muscle contraction. The opening of SR K+ channels is thought to support this process. Movements of the RyR and SR K+ channel gates required for channel opening and closing will be dependent on the biophysical properties of the SR membrane which, in turn, will be affected by factors such as temperature and membrane composition. Cardiac RyR channel opening is hypersensitive to temperature changes (Sitsapesan et al., 1991, J Physiol., 434:469-488) although single-channel recordings are invariably performed at room temperature. We have therefore incorporated mammalian cardiac and skeletal RyR and SR K+ channels into bilayers under voltage-clamp conditions to investigate the effects of changing temperature and membrane lipid composition on single-channel behaviour.Changing bilayer lipid composition from phosphatidylethanolamine (PE) only, to a more physiological composition containing a 5:4:1 ratio of PE: phosphatidylserine (PS): phosphatidylcholine (PC) at 23oC, significantly increased the open probability (Po) and single channel conductance of both channels. For example, RyR Po increased from 0.06±0.03 to 0.26±0.08 (n=8; SEM; P<0.05). With bilayer lipid ratio of 5 PE: 4 PS: 1 PC, raising temperature from 10oC to 37oC caused a graded decrease in RyR Po. In contrast, SR K+ channels exhibited non-linear changes in Po with a minimum at approximately 23oC. Our results demonstrate the importance of investigating intracellular ion channel function at physiological temperatures and show that changes in SR membrane lipid composition markedly influence the behaviour of ion channels in the SR.Supported by the British Heart Foundation.

Rats fed linseed oil in microemulsion forms enriches the cardiac sarcoplasmic reticulum lipids with docosahexaenoic acid and lower calcium transport

The effect of feeding rats with whey protein or lipoid microemulsion of n-3 rich linseed oil (LSO), and n-6 rich sunflower oil (SNO) compared to native or microemulsion forms of each oil for 60days on fatty acid composition, Ca2+Mg2+ ATPase, Ca2+ uptake and membrane fluidity of rat cardiac sarcoplasmic reticulum (SR) were assessed. Rats consuming LSO in lipoid microemulsion showed higher levels of ALA (18:3, n-3) conversion to DHA (22:6, n-3) when compared to rats given LSO in native form. Compared to all other dietary treatments, the alterations in the n-6/n-3 PUFA composition of SR of rats given LSO in lipoid microemulsion effected the greatest changes in Ca2+Mg2+ ATPase activity and Ca2+ uptake which effectively reduced Ca2+ transport. Therefore, the LSO in lipoid microemulsion confers similar heart healthy fatty acid compositional and calcium transport changes and potentially anti-arrhythmic effects similar to fish oils.

Activating and Deactivating Roles of Lipid Bilayers on the Ca2+-ATPase/Phospholamban Complex

The physicochemical properties of the lipid bilayer shape the structure and topology of membrane proteins and regulate their biological function. Here, we investigated the functional effects of various lipid bilayer compositions on the sarcoplasmic reticulum (SR) Ca(2+)-ATPase (SERCA) in the presence and absence of its endogenous regulator, phospholamban (PLN). In the cardiac muscle, SERCA hydrolyzes one ATP molecule to translocate two Ca(2+) ions into the SR membrane per enzymatic cycle. Unphosphorylated PLN reduces SERCA's affinity for Ca(2+) and affects the enzymatic turnover. We varied bilayer thickness, headgroup, and fluidity and found that both the maximal velocity (V(max)) of the enzyme and its apparent affinity for Ca(2+) (K(Ca)) are strongly affected. Our results show that (a) SERCA's V(max) has a biphasic dependence on bilayer thickness, reaching maximum activity with 22-carbon lipid chain length, (b) phosphatidylethanolamine (PE) and phosphatidylserine (PS) increase Ca(2+) affinity, and (c) monounsaturated lipids afford higher SERCA V(max) and Ca(2+) affinity than diunsaturated lipids. The presence of PLN removes the activating effect of PE and shifts SERCA's activity profile, with a maximal activity reached in bilayers with 20-carbon lipid chain length. Our results in synthetic lipid systems compare well with those carried out in native SR lipids. Importantly, we found that specific membrane compositions closely reproduce PLN effects (V(max) and K(Ca)) found in living cells, reconciling an ongoing controversy regarding the regulatory role of PLN on SERCA function. Taken with the physiological changes occurring in the SR membrane composition, these studies underscore a possible allosteric role of the lipid bilayers on the SERCA/PLN complex.

Probing ground and excited states of phospholamban in model and native lipid membranes by magic angle spinning NMR spectroscopy

In this paper, we analyzed the ground and excited states of phospholamban (PLN), a membrane protein that regulates sarcoplasmic reticulum calcium ATPase (SERCA), in different membrane mimetic environments. Previously, we proposed that the conformational equilibria of PLN are central to SERCA regulation. Here, we show that these equilibria detected in micelles and bicelles are also present in native sarcoplasmic reticulum lipid membranes as probed by MAS solid-state NMR. Importantly, we found that the kinetics of conformational exchange and the extent of ground and excited states in detergent micelles and lipid bilayers are different, revealing a possible role of the membrane composition on the allosteric regulation of SERCA. Since the extent of excited states is directly correlated to SERCA inhibition, these findings open up the exciting possibility that calcium transport in the heart can be controlled by the lipid bilayer composition. This article is part of a Special Issue entitled: Membrane protein structure and function.

SOME CHARACTERISTICS OF IRON-CATALYZED NADH-DEPENDENT SARCOPLASMIC RETICULAR LIPID OXIDATION IN A SEAWATER CLAM (MERETRIX LUSORIA) AND FRESHWATER CLAM (CORBICULA FLUMINEA)

Characteristics of an iron-catalyzed enzymic lipid oxidation system requiring nicotinamide adenine dinucleotide (NADH) as the reducing agent in the sarcoplasmic reticulum (SR) of clams were investigated. For both the seawater and freshwater clams, the system preferred NADH to NADPH as the reducing agent (P < 0.05). Lipid oxidation activities of the SR were significantly reduced (P < 0.05) when reaction temperature reached 45 and 65C for the freshwater clam and seawater clam, respectively, indicating that denaturation of the enzymes had occurred. When reaction pH ranged from 6.0 to 7.5, acidic pH tended to amplify the lipid peroxidation activity. Under the same experimental conditions, SR lipid oxidation activities in the seawater clam were greater (P < 0.05) than those in the freshwater clam. The fatty acid composition of SR lipids may have been a factor affecting this outcome as the level of polyunsaturated fatty acids of SR lipids in the seawater clam was higher than that in the freshwater clam. PRACTICAL APPLICATIONS The results of this study enhance our knowledge of the lipid oxidation system at the subcellular level of seawater and freshwater clams. Both of these clams are important cultured species in Taiwan and other Asian countries. Clam culturists can manipulate the fatty acid profiles of the clams by altering the dietary lipid sources during the farming stage, while processors may be able to limit oxidation by avoiding pro-oxidative conditions.

Displacement of SERCA from SR Lipid Caveolae-Related Domains by Bcl-2: A Possible Mechanism for SERCA Inactivation

Bcl-2 exerts its anti-apoptotic effect in part through the regulation of Ca2+ homeostasis at the level of the endoplasmic reticulum. Earlier, we demonstrated that a truncated form of Bcl-2, Bcl-2delta21, interacts with and destabilizes the skeletal muscle sarco/endoplasmic reticulum Ca-ATPase (SERCA) [Dremina, E. S., Sharov, V. S., Kumar, K., Zaidi, A., Michaelis, E. K., and Schöneich, C. (2004) Biochem. J. 383, 361-370]. Here we show that (i) the transmembrane (TM) domain of Bcl-2 accelerates SERCA inactivation, (ii) both Bcl-2delta21 and full-length Bcl-2 selectively interact with SERCA1, and (iii) the inactivation of SERCA is accompanied by a translocation of SERCA from caveolae-related domains (CRD) of the sarcoplasmic reticulum (SR). In rat skeletal muscle SR, intact SERCA1 was detected only in the CRD fractions of a sucrose density gradient. Co-incubation of SR with either Bcl-2delta21 or full-length Bcl-2 resulted in both the appearance of Bcl-2delta21 or Bcl-2 in the fractions containing SERCA1 and translocation of SERCA1 from CRD fractions; the latter effect correlated with the loss of the Ca-ATPase activity of the protein.

Temperature Dependence and Thermodynamic Properties of Ca 2+ Sparks in Rat Cardiomyocytes

To elucidate the temperature dependence and underlying thermodynamic determinants of the elementary Ca 2+ release from the sarcoplasmic reticulum, we characterized Ca 2+ sparks originating from ryanodine receptors (RyRs) in rat cardiomyocytes over a wide range of temperature. From 35°C to 10°C, the normalized fluo-3 fluorescence of Ca 2+ sparks decreased monotonically, but the Δ[Ca 2+] i were relatively unchanged due to increased resting [Ca 2+] i. The time-to-peak of Ca 2+ sparks, which represents the RyR Ca 2+ release duration, was prolonged by 37% from 35°C to 10°C. An Arrhenius plot of the data identified a jump of apparent activation energy from 5.2 to 14.6 kJ/mol at 24.8°C, which presumably reflects a transition of sarcoplasmic reticulum lipids. Thermodynamic analysis of the decay kinetics showed that active transport plays little role in early recovery but a significant role in late recovery of local Ca 2+ concentration. These results provided a basis for quantitative interpretation of intracellular Ca 2+ signaling under various thermal conditions. The relative temperature insensitivity above the transitional 25°C led to the notion that Ca 2+ sparks measured at a “warm room” temperature are basically acceptable in elucidating mammalian heart function.

LIPID PEROXIDATION IN SARCOPLASMIC RETICULUM AND MUSCLE OF TILAPIA IS INHIBITED BY DIETARY VITAMIN E SUPPLEMENTATION

Lipid peroxidation was analyzed in muscle and sarcoplasmic reticulum (SR) of hybrid tilapia fed diets containing 0, 100, 200, and 300 IU vitamin E/kg for 8 months. Iron-catalyzed NADH-dependent lipid peroxidation in SR of tilapia fed diet containing no supplemented vitamin E was significantly greater than that of fish fed other diets (P < 0.05). No difference was observed in SR lipid peroxidation between fish fed 200 and 300 IU vitamin E/kg. Thiobarbituric acid reactive substances (TBARS) produced in muscle stored at either 4C or -40C for 7 days and 8 weeks, respectively, increased when storage time increased. When muscle TBARS were plotted against storage time, the lag phases were longer for fish fed high vitamin E (≥ 200 IU/kg) diets than those from fish fed low vitamin E diets.

Characteristics of NADH-dependent lipid peroxidation in sarcoplasmic reticulum of white shrimp, Litopenaeus vannamei, and freshwater prawn, Macrobrachium rosenbergii

The iron-catalyzed NADH-dependent lipid peroxidation system in sarcoplasmic reticulum (SR) of cultured white shrimp, Litopenaeus vannamei and freshwater prawn, Macrobrachium rosenbergii was characterized. Production of thiobarbituric acid reactive substances was used to measure the activity of lipid peroxidation. In both species, the system preferred NADH to NADPH as the reducing agent. Lipid peroxidation activities of SR from both species increased when reaction temperatures increased from 6 to 26 °C. At 66 °C, the reaction was no longer NADH-dependent. Acidic pH amplified the lipid peroxidation activity. Sarcoplasmic reticular lipid peroxidation activity in white shrimp was always greater than in freshwater prawn. Fatty acid composition of SR lipids could be a major factor for this outcome. The proportion of n–3 highly unsaturated fatty acids, such as C20:5 and C22:6, in sarcoplasmic reticular lipids of white shrimp was twice of that in freshwater prawn. The results of this study provide important tools required for anti-oxidative nutrient study at sub-cellular level.

Aromatic indolinic aminoxyls as antioxidants in cardiac sarcoplasmic reticulum lipid and protein oxidation.

The results presented demonstrate the influence of aromatic indolinic aminoxyls: 1,2-dihydro-2-ethyl-2-phenyl-3H-indole-3-phenylimino-1-oxyl (IA-C2) and 1,2-dihydro-2-octadecyl-2-phenyl-3H-indole-3-phenylimino-1-oxyl (IA-C18) on oxidation of lipids and proteins of cardiac sarcoplasmic reticulum membranes. We have used doxorubicin and t-butyl hydroperoxide as agents inducing oxidative stress in isolated rat cardiac sarcoplasmic reticulum membrane system. Carbonyl groups were measured as the end product of membrane protein oxidation, and thiobarbituric acid reactive substances were assessed as a marker of lipid peroxidation. Inhibition of peroxidation of certain membrane components depends on the length of acyl chain. Aminoxyl IA-C2 inhibits the lipid peroxidation process while IA-C18 is an efficient protector against protein oxidation.

Ca2+-ATPase Activity and Lens Lipid Composition in Reconstituted Systems

Lens lipid composition and lipid hydrocarbon chain structure change with age, region and cataract. Since the lens Ca2+-ATPase pump is important to the maintenance of calcium homeostasis and lens clarity, muscle sarcoplasmic reticulum Ca2+-ATPase was reconstituted with bovine lens lipids and dihydrosphingomyelin, the rare and major phospholipid of the human lens. Ca2+-ATPase activity was found to be about 5 times lower when the pump was reconstituted into dihydrosphingomyelin or lens lipids compared to native sarcoplasmic reticulum lipids. The addition of cholesterol to levels ranging from 13–53 mole%, had no affect on reconstituted Ca2+-ATPase activity. Ca2+-ATPase activity correlated with the degree of hydrocarbon chain saturation. The greater levels of saturation are a consequence of the high sphingolipid content in the reconstituted systems. These data support the hypothesis that changes in lens lipid composition or structure could affect Ca2+-ATPase activity in human lenses. Because the mechanisms governing Ca2+-ATPase activity in vivo are much more complex than in these simple reconstituted systems, this study represents an initial step in the elucidation of the relationships of endogenous membrane lipid composition-structure and function.

Lipid composition, membrane structure relationships in lens and muscle sarcoplasmic reticulum membranes.

Membrane lipid composition varies in different tissues and species. Since a defined lipid composition is essential to the function of many membranes, the relationship between membrane lipid composition and structure was determined using infrared and Raman spectroscopy in four membranes containing a calcium pump: rabbit fast and slow twitch muscle sarcoplasmic reticulum and human and bovine lens fiber cell membranes. We found that membrane sphingolipid and phosphatidylcholine content were correlated to a decrease and increase, respectively, in the infrared lipid CH2 symmetric stretching band frequency. We interpret the change in frequency as a change in lipid hydrocarbon chain structural order. This was confirmed by Raman order parameters. The high degree of hydrocarbon chain saturation found in the variable amide chains of sphingolipids is likely to account for this correlation. Lipid phase transition temperature and cooperativity also correlated to sphingolipid and phosphatidylcholine content, and are the forces defining the order in at physiological temperature in the samples studied. Ca(2+)-ATPase caused an increase in the CH2 symmetric stretching frequency in fast twitch muscle sarcoplasmic reticulum (interpreted as an increase in hydrocarbon chain disorder), but had no effect on slow twitch muscle sarcoplasmic reticulum lipid hydrocarbon chain structure. In the natural systems studied, we find that it is the lipid hydrocarbon chain saturation that defines lipid hydrocarbon chain order.

Interaction between Glycogen Phosphorylase and Sarcoplasmic Reticulum Membranes and Its Functional Implications

Skeletal muscle glycogen phosphorylase b binds to sarcoplasmic reticulum (SR) membranes with a dissociation constant of 1.7 +/- 0.6 mg of phosphorylase/ml at 25 degrees C at physiological pH and ionic strength. Raising the temperature to 37 degrees C produced a 2-3-fold decrease in the dissociation constant. The SR membranes could bind up to 1.1 +/- 0.1 mg of glycogen phosphorylase b/mg of SR protein, whereas liposomes prepared with endogenous SR lipids and reconstituted Ca(2+)-ATPase were unable to bind glycogen phosphorylase. Binding of glycogen phosphorylase b to SR membranes is accompanied by inhibition of its activity in the presence of AMP. The Vmax for glycogen phosphorylase b associated with SR membranes is 40 +/- 5% of that for purified glycogen phosphorylase and shows a decreased affinity for its allosteric activators, AMP and IMP. These kinetic effects are also observed with purified glycogen phosphorylase b when starch or alpha-amylose is used as substrate instead of glycogen. Treatment of SR membranes with alpha-amylase produced dissociation of glycogen phosphorylase b from the SR membranes. Thus, linear polysaccharide fragments of glycogen bound to the SR membranes are likely mediating the binding of glycogen phosphorylase b to these membranes.

Irradiation with Ultraviolet Light in the Presence of Vanadate Increases Ca2+ Permeability of the Sarcoplasmic Reticulum Membrane via Ca2+-ATPase

The sarcoplasmic reticulum (SR) of rabbit skeletal muscle was irradiated with ultraviolet light (UV) in the presence of vanadate plus 2 mM EGTA, 10 mM MgCl2, 20% DMSO, and 50 mM PIPES (pH 6.5) at room temperature. In the presence of 100 microM vanadate, the Ca(2+)-uptake activity of SR rapidly decreased and was almost lost in 20 min. The activity was inhibited as a function of vanadate concentration with an apparent Ki of about 20 microM. On the other hand, Ca(2+)-dependent ATP hydrolytic activity as well as phosphoenzyme (EP) formation activity decreased very slowly, and more than 50% of these activities remained 20 min after initiation of the vanadate-UV treatment. Half inhibition of these activities required about 100 microM vanadate. The loss of the relationship between Ca(2+)-uptake and ATPase reaction was found to be mainly caused by an increase in the Ca2+ permeability of the SR membrane, which was raised by increasing the vanadate concentration or UV irradiation time in a manner similar to that observed for the Ca2+ uptake. No rise in Ca2+ permeability occurred in liposomes reconstituted from SR lipid when they were irradiated with UV in the presence of 100 microM vanadate. When the vanadate-UV-treated SR was allowed to react with fluoral-P (4-amino-3-penten-2-one), an indicator of aldehyde, and the membrane proteins were separated by HPLC in the presence of SDS, the fluorescent probe was found to be closely associated with the Ca(2+)-ATPase fraction.(ABSTRACT TRUNCATED AT 250 WORDS)

The active metabolite hydroxytamoxifen of the anticancer drug tamoxifen induces structural changes in membranes

The effects of hydroxytamoxifen (OHTAM) on lipid organization of pure phospholipid liposomes, native sarcoplasmic reticulum (SR) membranes and liposomes of SR lipids were evaluated by intramolecular excimer formation of 1,3-di(1-pyrenyl)propane (Py(3)Py) and by fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene (DPH) and its derivative 3-[p-(6- phenyl)-1,3,5- hexatrienyl]phenylpropionic acid (DPH-PA). OHTAM promotes alterations in the thermotropic profiles of DMPC, DPPC and DSPC. As detected by Py(3)Py and DPH-PA, OHTAM induces an ordering effect in the fluid phase and a fluidizing effect in the temperature range of the cooperative phase transition. In the gel phase, no significant effects are noticed, except for DSPC bilayers, where Py(3)Py and DPH-PA detect a disordering effect. In the hydrophobic region of the above membrane systems probed by DPH, OHTAM induces only a slight fluidizing effect in the range of the phase transition and a small ordering effect in the fluid phase. As detected by all probes, the drug broadens the transition profile of DMPC and shifts the main transition temperature to lower values. However, these effects, and so those observed for the fluid phase, decrease as the fatty acyl chain length increases. Moreover, the drug removes the pre-transitions of DPPC and DSPC bilayers, as probed by Py(3)Py. In fluid SR native membranes and liposomes of SR lipids, OHTAM induces a moderate ordering effect in the outer regions of the lipid bilayer, as monitored by Py(3)Py and by DPH-PA, DPH failing to detect any apparent effect, as observed for the fluid phase of pure phospholipids. Apparently, OHTAM distributes preferentially in the outer region of the lipid bilayer, without significant effect in the bulk lipid organization of the bilayer interior. The changes of OHTAM in the bilayer dynamic properties and the different location across the bilayer thickness relative to its drug promoter (Custódio et al. (1993) Biochim. Biophys. Acta 1150, 123–129) may be involved in the cytostatic activity of tamoxifen.

The anticancer drug tamoxifen induces changes in the physical properties of model and native membranes

The interactions of tamoxifen with lipid bilayers of model and native membranes were investigated by fluorescence polarization of 1,6-diphenyl-1,3,5-hexatriene (DPH) and by intramolecular excimer formation of 1,3-di(1-pyrenyl)propane (Py(3)Py). The effects of TAM on liposomes of DMPC, DPPC and DSPC are temperature dependent. In the fluid phase, TAM reduces dynamics of the upper bilayer region as observed by Py(3)Py and has no effect on the hydrophobic region as detected by DPH. In the gel phase, the effects of TAM evaluated by Py(3)Py are not discernible for DMPC and DPPC bilayers, whereas DSPC bilayers become more fluid. However, DPH detects a strong fluidizing effect of TAM in the hydrophobic region of the above membrane systems, where DPH distributes, as compared with the small effects detected by Py(3)Py. TAM decreases the main phase transition temperature but does not extensively broaden the transition thermotropic profile of pure lipids, except for bilayers of DMPC where TAM induces a significant broadening detected with the two probes. In fluid liposomes of sarcoplasmic reticulum lipids and native membranes, TAM induces an ordering effect, as evidenced by Py(3)Py, failing DPH to detect any apparent effect as observed for the fluid phase of liposomes of pure lipid bilayers. These findings confirm the hydrophobic nature of tamoxifen and suggest that the localization and effects of TAM are modulated by the order and fluidity of the bilayer. These changes in the dynamic properties of lipids and the non-specific interactions with membrane lipids, depending on the order or fluidity of the biomembrane, may be important for the multiple cellular effects and action mechanisms of tamoxifen.

Transitional steps in the solubilization of protein-containing membranes and liposomes by nonionic detergent.

Membrane solubilization by dodecyl maltoside was studied, using Ca(2+)-ATPase membranes and liposome preparations as prototypes of biological membranes. In equilibrium dialysis experiments, transition from saturable incorporation of monomeric detergent into the membrane to cooperative binding already occurred at a free detergent concentration about 50% of the cmc. This transition was discontinuous for unilamellar liposomes of dioleoylphosphatidylcholine, but gradual for Ca(2+)-ATPase membranes and multilayered liposomes of sarcoplasmic reticulum lipid. Equilibrium detergent binding by Ca(2+)-ATPase membranes (expressed on the basis of lipid content) was the same as for detergent binding by multilamellar liposomes of sarcoplasmic reticulum lipid. Equilibration involving cooperative binding was considerably delayed (for many days) if detergent was presented gradually to the membranous preparations in nonmicellar form by diffusion across the dialysis membrane, while equilibration of detergent occurred rapidly if detergent in micellar form was added directly to the membrane preparations. In contrast, equilibration was rapid in both directions if detergent was added at levels below that required to initiate cooperative binding. Detergent interaction resulted in a biphasic decrease in light scattering of Ca(2+)-ATPase membranes. The first of these decreases coincided with the onset of cooperative binding, while the second one was associated with a decreased sedimentability during ultracentrifugation, i.e., with usual criteria of solubilization. The concentration at which this occurred corresponded to the level of free detergent at which lipid, after detergent solubilization, segregated from detergent after gel chromatography.(ABSTRACT TRUNCATED AT 250 WORDS)

On the effect of lysophosphatidylcholine, platelet activating factor and other surfactants on calcium permeability in sarcoplasmic reticulum vesicles

The effect of low concentrations of lysophosphatidylcholine (LPC), platelet-activating factor (PAF) and other surfactants (Triton X-100, C 12E 8, sodium dodecyl sulfate, sodium cholate and sodium deoxycholate) on membrane permeability of native sarcoplasmic reticulum vesicles and sarcoplasmic reticulum lipid vesicles, has been studied. Triton X-100, C 12E 8, sodium dodecyl sulfate, sodium cholate and sodium deoxycholate were all able to permeabilize membranes at concentrations of surfactants below their critical micellar concentration (CMC) in both lipid and native vesicles, being the K 0.5 of calcium release from native vesicles lower than that from lipid vesicles. The values of these K 0.5 were well correlated with the corresponding CMC values for each type of membrane. However, both LPC and PAF behaved in a different way since, although they induced permeabilization of the native vesicles at values of K 0.5 close to their CMC, their K 0.5 values for permeabilizing vesicles, prepared by using lipids extracted from sarcoplasmic reticulum, were much higher than their corresponding CMC

A reliable and rapid procedure to estimate drug partitioning in biomembranes

A direct method using derivative spectrophotometry was developed for determining membrane-water molar partition coefficients (Kp) of the anticancer drugs tamoxifen (TAM) and 4-hydroxytamoxifen (OHTAM). This method explores a shift in the absorption spectra of the drugs when removed from the aqueous phase to a hydrophobic environment. Partition of TAM and OHTAM depends on membrane composition and on drug concentration, temperature and presence of cholesterol. Unlike OHTAM, partition of TAM in DMPC bilayers, liposomes of sarcoplasmic reticulum (SR) lipids and native membranes of SR and mitochondria decreases linearly with drug concentration. Additionally, the partition of these drugs is higher in SR native membranes than in liposomes of SR lipids. The partition also depends on membrane type, being higher in mitochondria than in SR membranes. Maximal partitionings in DMPC are observed at temperatures in the range of the main phase transition. Cholesterol strongly affects the incorporation of drugs and maximal inhibition was observed in DMPC bilayers.