Comparative study of the lipid composition of rabbit and lobster sarcoplasmic reticulum.

Sarcoplasmic reticulum (SR) membranes isolated from rabbit and lobster muscles have similar phospholipid classes, but they differ in plasmalogen content. The plasmalogenic species are mostly distributed among phosphatidylethanolamines (PE's) and make up about 62% of the total in rabbit SR and about 46% in lobster membranes. Lobster SR phospholipids contain large amounts of polyunsaturated fatty acids which are present in low amounts in rabbit membranes. The total unsaturated fatty acids of phosphatidylcholines (PC's) represent about 53% and 73% of the total fatty chains for rabbit and lobster SR, respectively. The values found for PE's were about 56% and 64%, respectively. Furthermore, lobster membranes contain significant amounts of PC and PE molecular species with unsaturated fatty acids in positions 1 and 2, whereas rabbit SR contain low amounts.     

Modulation of vitronectin receptor binding by membrane lipid composition.

The vitronectin (Vn) receptor belongs to the integrin family of proteins and although its biochemical structure is fully characterized little is known about its binding affinity and specificity. We report here that Vn receptor binding to different matrix proteins is influenced by the surrounding lipid composition of the membrane. Human placenta affinity purified Vn receptor was inserted into liposomes of different composition: (i) phosphatidylcholine (PC); (ii) PC+phosphatidylethanolamine (PE); (iii) PC+PE+phosphatidylserine (PS) + phosphatidylinositol (PI) + cholesterol (chol). The amount of purified material that could be incorporated into the three lipid vesicle preparations was proportional to the efficiency of the vesicle formation that increased from PC (38%) to PC+PE and PC+PE+PS+PI+chol (about 50%) vesicles. Electron microscopy analysis showed that the homogeneity and size of the three liposome preparations were comparable (20-nm diameter) but their binding capacity to a series of substrates differed widely. Vn receptor inserted in PC liposomes bound only Vn, but when it was inserted in PC+PE and PC+PE+PS+PI+chol liposomes it also attached to von Willebrand factor (vWF) and fibronectin (Fn). Vn receptor had higher binding capacity for substrates when it was inserted in PC+PE+PS+PI+chol than PC+PE liposomes. Antibodies to Vn receptor blocked Vn receptor liposome binding to Vn, vWF, and Fn. The intrinsic emission fluorescence spectrum of the Vn receptor reconstituted in PC+PE+PS+PI+chol liposomes was blue-shifted in relation to PC liposomes, suggesting a conformational change of the receptor in the membranes. These data provide direct evidence that the Vn receptor is "promiscuous" and can associate with Vn, vWF and Fn. The nature of the membrane lipid composition surrounding the receptor could thus influence its binding affinity, possibly by changing its conformation or exposure or both.     

A fluorescence decay study of parinaroyl-phosphatidylinositol incorporated into artificial and natural membranes

Phosphoinositide metabolism in the plasma membrane is linked to transmembrane signal transduction. In this study we have investigated some physical properties (e.g. molecular order and dynamics) of phosphatidylinositol (PI) in various membrane preparations by time-resolved fluorescence techniques, using a synthetic PI derivate with a cis-parinaroyl chain on the sn-2 position. Phospholipid vesicles, normal and denervated rat skeletal muscle sarcolemmal membranes, and acetylcholine receptor rich membrances from Torpedo marmorata were investigated both at 4°C and 20 °C. For comparison we have also included 2-parinaroyl-phosphatidylcholine (PC) in this study. The fluorescent lipids were incorporated into the membrane preparations by way of specific phospholipid transfer proteins, to ensure an efficient and non-perturbing insertion of the lipid-probes. In the Torpedo membranes the order parameters measured for the parinaroyl derivatives of both PC and PI were higher than in phospholipid vesicles. For the Torpedo membrane preparations the acyl chain order for the PI was lower than that for PC, whereas the opposite was true for the vesicles. This inversion strongly suggests that PI has different interactions with certain membrane components as compared to PC. This is also suggested by the significantly higher rate of restricted rotation of PI as compared to PC. In contrast to the order parameters, the correlation times were almost identical for both probes and showed little difference between vesicles and the Torpedo membranes. In contrast to Torpedo membranes, the time-dependent fluorescence anisotropy of the two lipid probes in the sarcolemmal membranes showed, after an initial fast decay, a subsequent gradual increase. This phenomenon was satisfactorily analyzed by assuming two populations of probe lipids with distinct lifetimes, rotational correlation times and molecular order. The order parameter of the population with a short lifetime compared with that of phospholipid vesicles, whereas the population with a long lifetime agreed with that of the Torpedo membranes.Abbreviations PI phosphatidylinositol - PC phosphatidylcholine - PA phosphatidic acid - PE phosphatidylethanolamine - PS phosphatidylserine, PnA, cis-parinaric acid: cis,trans,trans,cis-9,11,13,15-octadecatetraenoic acid - 2-PnA-PC 1-acyl, 2-parinaroyl-PC - 2-PnA-PI 1-acyl,2-parinaroyl-PI - DPH diphenylhexatriene - POPOP 1,4-di[2-(5-phenyloxazolyl)]-benzene - NMR nuclear magnetic resonance - ESR electron spin resonance - I parallel fluorescence intensity component - I perpendicular fluorescence intensity component - SET-buffer 0.25 M Sucrose, 1 mM EDTA, 10mM Tris-HCl, pH 7.4     

Temperature-induced transitions of porcine intestinal brush border membranes

Thermotropic transitions of the membrane components in porcine intestinal brush border membranes were studied by means of fluorimetry using a fluorogenic thiol reagent, N-[7-dimethylamino-4-methylcoumarinyl]maleimide (DACM), and a lipophilic fluorescent probe, 1,6-diphenyl-1,3,5-hexatriene (DPH). 1. The reactivity of the sulfhydryl groups of the membrane proteins with DACM was dependent on temperature, with a transition point at about 33°C. A conspicuous transition was also observed in the relation between temperature and the fluorescence intensity of DACM-labeled membranes at 35°C. 2. Temperature dependence profiles of the solubilization of DPH in the membranes and of the fluorescence polarization of DPH-membrane complex suggested that the phase transition of the lipid from gel to liquid-crystalline state occurs over a temperature range of 30 to 35°C. 3. Efficient fluorescence energy transfer was observed from tryptophan residues of the membrane proteins to DPH located in the lipid phase of the membranes, and its efficiency was extremely enhanced, dependent on temperature, above 35°C. The intensity of the tryptophan fluorescence of the membrane proteins decreased with increasing temperature and a discontinuity was observed at about 33°C. Based on these results, it may be concluded that there are co-operative interactions between proteins and lipids in the membranes and that the temperature-induced conformational changes of the membrane proteins are closely related to the dynamics of the hydrocarbon cores of the lipid.     

Strength of Ca(2+) binding to retinal lipid membranes: consequences for lipid organization

There is evidence that membranes of rod outer segment (ROS) disks are a high-affinity Ca(2+) binding site. We were interested to see if the high occurrence of sixfold unsaturated docosahexaenoic acid in ROS lipids influences Ca(2+)-membrane interaction. Ca(2+) binding to polyunsaturated model membranes that mimic the lipid composition of ROS was studied by microelectrophoresis and (2)H NMR. Ca(2+) association constants of polyunsaturated membranes were found to be a factor of approximately 2 smaller than constants of monounsaturated membranes. Furthermore, strength of Ca(2+) binding to monounsaturated membranes increased with the addition of cholesterol, while binding to polyunsaturated lipids was unaffected. The data suggest that the lipid phosphate groups of phosphatidylcholine (PC), phosphatidylethanolamine (PE), and phosphatidylserine (PS) in PC/PE/PS (4:4:1, mol/mol) are primary targets for Ca(2+). Negatively charged serine in PS controls Ca (2+) binding by lowering the electric surface potential and elevating cation concentration at the membrane/water interface. The influence of hydrocarbon chain unsaturation on Ca(2+) binding is secondary compared to membrane PS content. Order parameter analysis of individual lipids in the mixture revealed that Ca(2+) ions did not trigger lateral phase separation of lipid species as long as all lipids remained liquid-crystalline. However, depending on temperature and hydrocarbon chain unsaturation, the lipid with the highest chain melting temperature converted to the gel state, as observed for the monounsaturated phosphatidylethanolamine (PE) in PC/PE/PS (4:4:1, mol/mol) at 25 degrees C.     

Age-dependent effect of oxidative stress on cardiac sarcoplasmic reticulum vesicles

The oxidative stress hypothesis of aging suggests that accumulation of oxidative damage is a key factor of the alterations in physiological function during aging. We studied age-related sensitivity to oxidative modifications of proteins and lipids of cardiac sarcoplasmic reticulum (SR) isolated from 6-, 15- and 26-month-old rats. Oxidative stress was generated in vitro by exposing SR vesicles to 0.1 mmol/l FeSO4/EDTA + 1 mmol/l H2O2 at 37 degrees C for 60 min. In all groups, oxidative stress was associated with decreased membrane surface hydrophobicity, as detected by 1-anilino-8-naphthalenesulfonate as a probe. Structural changes in SR membranes were accompanied by degradation of tryptophan and significant accumulation of protein dityrosines, protein conjugates with lipid peroxidation products, conjugated dienes and thiobarbituric acid reactive substances. The sensitivity to oxidative damage was most pronounced in SR of 26-month-old rat. Our results indicate that aging and oxidative stress are associated with accumulation of oxidatively damaged proteins and lipids and these changes could contribute to cardiovascular injury.     

Effects of different cultivation temperatures on plasma membrane ATPase activity and lipid composition of sugar beet roots.

Sugar beet seedlings (Beta vulgaris L. cv. Monohill) were cultivated for 3 weeks at different root and shoot temperatures and the plasma membranes (PM) from roots were purified by aqueous two-phase partitioning and analyzed for lipid composition and ATPase activities. Lipid analyses, undertaken immediately after PM purification from the roots, showed that a low root zone temperature (10 degrees C) decreased the ratio between the major lipids phosphatidylcholine (PC) and phosphatidylethanolamine (PE). A low temperature in the root environment increased the mol% of PE and decreased the mol% of phosphatidic acid (PA), independent on the shoot growth temperature. A low temperature also decreased the mol% of linoleic acid (18:2) and increased mol% of linolenic acid (18:3) in the analyzed lipid classes, especially in PC and PE. The ratio between acyl chain lipids and protein generally increased in PM from roots grown at 10 degrees C, compared with higher temperature. The changes in lipid composition correlated with changes in ATPase activities, detected as hydrolyses of MgATP. The kinetic parameters, K(m) and V of the PM H(+)ATPase in roots increased at a low cultivation temperature, independent on shoot temperature. Moreover, Arrhenius analyses showed that the transition temperature was independent of both root or shoot growth temperature at 10-24 degrees C, whereas the activation energy of the ATPase was dependent on the growth temperature of the root, and independent on shoot temperature. Thus, acclimation processes can take place in roots, irrespective of the shoot temperature.     

Effect of temperature on fluorescence of labels and probes of various localizations incorporated into sarcoplasmic reticulum samples

Studies were carried out of temperature relationship of dansylchloride, N-3-pyrenylmaleinimide fluorescence, SR membranes, self-luminescence caused by Ca-ATPase tryptophane - provided fluorescence and of pyrene excimerization in membrane preparations of sarcoplasmic reticulum (SR) of rabbit skeletal muscles. Temperature relationship of fluorescence intensity of dansylchloride and N-3-pyrenylmaleimide in Arrhenius coordinates has bends at 15 and 35 degrees. Selffluorescence of protein samples linearly depends on temperature. Temperature relationship of the ratio between the intensities of exsimeric and monomeric forms of pyrene Fa/Fm in Arrhenius coordinates has the bend at 20-22 degrees. Hence only the latter relationship coincides with the shape of Arrhenius graph for enzymatic activity of SR Ca-ATPase.     

Correlation between lipid fluidity and tryptic susceptibility of Ca2+-ATPase in sarcoplasmic reticulum membranes

Lipid fluidity in native and denatured sarcoplasmic reticulum membranes and extracted lipids was monitored between -30 and 30 degrees C using trans-parinaric acid as a fluorescent probe. In addition to a large increase in fluidity between -30 and 0 degree C in each system, a phase change centered near 10 degrees C was observed in the extracted lipids but not in either the native or denatured membranes. A significant change in fluorescence intensity near 15 degrees C was observed in native sarcoplasmic reticulum membranes, however, when trans-parinaric acid was excited by energy transfer from tryptophan residues of the membrane protein. When Ca2+-ATPase was subjected to proteolytic cleavage by trypsin as a function of temperature, a change in susceptibility was detected at about 15-20 degrees C in the native membranes but not in a solubilized preparation. It is proposed that one or more structural changes in the microenvironment of Ca2+-ATPase in the native membrane occur between 15 and 20 degrees C which may be related to the change in apparent activation energy which is observed for this enzyme.     

Phospholipid uptake by Plasmodium knowlesi infected erythrocytes

The uptake of phosphatidylcholine (PC), phosphatidylethanolamine (PE) and phosphatidylserine (PS) in Plasmodium knowlesi infected erythrocytes has been studied. Whereas uptake of phospholipids, in the absence of phospholipid transfer proteins, is negligible in control cells, the infected cells can incorporate considerable amounts of added phospholipids. The uptake is enhanced by the presence of lipid transfer proteins. Doubly labeled [3H]oleate, [14C]choline) PC does not undergo any appreciable remodelling following uptake, which strongly suggests that plasma PC is used as such for the biogenesis of the parasite membranes. Transport of extracellularly offered PS and PE towards the intraerythrocytic parasite and utilization of these lipids by the parasite are confirmed by the observation that these lipids are converted into respectively PE and PC. The extent and rate of these conversions depend on the way the phospholipids are introduced into the infected cells.     

Effects of zinc on lipids of erythrocytes from carp (Cyprinus carpio L.) acclimated to different temperatures

We compared the effect of zinc (0.01, 0.1, 0.5 and 1 mM) at two temperatures (5 and 20 degrees C) on erythrocytes from summer and winter acclimatised carp. An increase in temperature from 5 to 20 degrees C increased the unsaturation index (UI) and relative proportion (UI/SFA) of unsaturated to saturated fatty acids in total lipids of the red cells. At 5 degrees C, the unsaturation index of phosphatidylcholine (PC), phosphatidylserine (PS), phosphatidylethanolamine (PE) and phosphatidylinositol (PI) decreased (30-40%) in the presence of 1 mM zinc. The change in unsaturation of phospholipids in the presence of zinc at 5 degrees C is probably responsible for the alteration in structural integrity of erythrocyte membrane as observed by hemolysis and the decreased thiol group content in the erythrocytes. In light of this result, zinc may be considered an environmental hazard for these fish at low temperatures.     

Time-resolved tryptophan fluorescence anisotropy investigation of bacteriophage M13 coat protein in micelles and mixed bilayers

Coat protein of bacteriophage M13 is examined in micelles and vesicles by time-resolved tryptophan fluorescence and anisotropy decay measurements and circular dichroism experiments. Circular dichroism indicates that the coat protein has alpha-helix (60%) and beta-structure (28%) in 700 mM sodium dodecyl sulfate micelles and predominantly beta-structure (94%) in mixed dimyristoylphosphatidylcholine/dimyristoylphosphatidic acid (80/20 w/w) small unilamellar vesicles. The fluorescence decay at 344 nm of the single tryptophan in the coat protein after excitation at 295 or 300 nm is a triple exponential. In the micelles the anisotropy decay is a double exponential. A short, temperature-independent correlation time of 0.5 +/- 0.2 ns reflects a rapid depolarization process within the coat protein. The overall rotation of the coat protein-detergent complex is observed in the decay as a longer correlation time of 9.8 +/- 0.5 ns (at 20 degrees C) and has a temperature dependence that satisfies the Stokes-Einstein relation. In vesicles at all lipid to protein molar ratios in the range from 20 to 410, the calculated order parameter is constant with a value of 0.7 +/- 0.1 from 10 to 40 degrees C, although the lipids undergo the gel to liquid-crystalline phase transition. The longer correlation time decreases gradually on increasing temperature. This effect probably arises from an increasing segmental mobility within the coat protein. The results are consistent with a model in which the coat protein has a beta-structure and the tryptophan indole rings do not experience the motion of the lipids in the bilayer because of protein-protein aggregation.     

Studies on the temperature effect on bacteriorhodopsin of purple and blue membrane by fluorescence and absorption spectroscopy

Fluorescence and absorption spectra were used to study the temperature effect on theconformation of bacteriorhodopsin (bR) in the blue and purple membranes (termed as bRb and bRprespectively).The maximum emission wavelengths of tryptophan fluorescence in both proteins at roomtemperature are 340 nm,and the fluorescence quantum yield of bRb is about 1.4 fold higher than that of bRp.As temperature increases,the tryptophan fluorescence of bRb decreases,while the tryptophan fluorescenceof bRp increases.The binding study of extrinsic fluorescent probe bis-ANS indicated that the probe can bindonly to bRb,but not to bRp.These results suggest that significant structural difference existed between bRband bRp.It was also found that both kinds of bR are highly thermal stable.The maximum wavelength of theprotein fluorescence emission only shifted from 340 nm to 346 nm at 100℃.More interestingly,as tempera-ture increased,the characteristic absorption peak of bRb at 605 nm decreased and a new absorption peak at380 nm formed.The transition occurred at a narrow temperature range (65℃-70℃).These facts indicatedthat an intermediate can be induced by high temperature.This phenomenon has not been reported before.     

Effects of moderately enhanced levels of ozone on the acyl lipid composition and dynamical properties of plasma membranes isolated from garden pea (Pisum sativum)

Plasma membranes were isolated from leaves of 16-day-old garden pea, Pisum sativum L., that had been grown in the absence or presence of 65 nl l⁻¹ ozone for 4 days prior to membrane isolation. Plasma membranes from ozone-fumigated plants contained significantly more acyl lipids per protein than those from leaves of plants grown in filtered air on a molar/weight ratio. The ratio between the major acyl lipids, phosphatidylethanolamine (PE) and phosphatidylcholine (PC), also increased due to the ozone fumigation, while the fatty acid unsaturation level was unaltered in total plasma membrane acyl lipids, as well as in PC and PE. The amount of free sterols per protein was unaltered, but the percentage of campesterol increased, concomitant with a decrease in stigmasterol. The dynamical properties of the isolated plasma membranes were assessed using Laurdan fluorescence spectroscopy, which monitors water penetration and mobility at the hydrophilic-hydrophobic interface of the membrane. At 0°C, the molecular mobility was slightly lower in plasma membranes from ozone-fumigated plants than in plasma membranes from control plants, possibly reflecting the increased PE/PC, campesterol/stigmasterol and lipid/protein ratios, and suggesting that ozone-fumigated pea plants may be more susceptible to freezing injuries.     

Lipid transfer proteins in the study of artificial and natural membranes

Summary Lipid transfer proteins, differing in their specificity for the transfer of lipids and for the surfaces on which they act, have been purified from various mammalian tissues and subsequently characterized. Several of their properties make them useful research tools. They have been used alone or with other techniques to study the distribution and mobility of phospholipids in artificial vesicles and in natural membranes, and have been used to create asymmetric phospholipid vesicles.Lipid transfer proteins are capable of altering the lipid composition of membranes by introducing new lipids or by depletion of existing lipids. Some of the transfer proteins can effect a net transfer of phospholipids, glycosphingolipids and cholesterol from one structure to another, whereas others appear to act primarily in promoting exchange. Some lipid transfer proteins are capable of introducing spin labeled and fluorescent lipid analogs into the outer surface of membranes. Because lipid transfer proteins do not seem to alter membrane lipid asymmetry or permeability of membranes, they are useful tools for studying the effect of lipid substitution on membrane-mediated transport processes and on various membrane-bound enzyme systems.Abbreviations PA phosphatidic acid - PC phosphatidylcholine - PE phosphatidylethanolamine - PI phosphatidylinositol - PG phosphatidylglycerol - PS phosphatidylserine - DPG diphosphatidylglycerol - SPH sphingomyelin - G_{m t} II³--N-Acetylneuraminosylgangliotetraglycosylceramide - GbOse₄Cer globotetraglycosylceramide Career Investigator of C.O.N.I.C.E.T. (Argentina)Career Investigator of the American Heart Association.     

Lipid changes of goat sperm plasma membrane during epididymal maturation

Highly purified plasma membranes of maturing goat caput-, corpus- and cauda-epididymal spermatozoa were isolated by aqueous two-phase polymer methods and their lipid constituents were analysed. Phospholipid (approx. 75% w/w), neutral lipid (approx. 15% w/w) and glycolipid (approx. 10% w/w) were the major sperm membrane lipids. There was a significant decrease in the total lipids (approx. 25% w/w), phospholipid (approx. 30% w/w) and glycolipid (approx. 80% w/w) contents of sperm membrane during epididymal maturation. On the contrary, the mature cauda-sperm membrane showed greater (approx. 50% w/w) neutral lipid content than that of the immature caput sperm. Phosphatidylcholine (PC), phosphatidylethanolamine (PE) and sphingomyelin were the phospholipids of the sperm membrane, the former two being the major lipids. Both PC and PE fractions consisted of three species--diacyl, alkylacyl and alkenylacyl forms, the last one being the dominant species in both PC and PE. Of all the phospholipids, diacyl PE decreased most strikingly (approx. 65% w/w) during sperm maturation. The neutral lipid fraction contained sterols, wax esters, 1-O-alkyl-2,3-diacylglycerol, triacylglycerol and fatty acids. Sterols represented nearly 75% w/w of the neutral lipids and cholesterol was the major component (approx. 95% w/w) of the sterol fraction. The sperm maturity was associated with marked increase of sterol (approx. 60% w/w) and steryl ester (approx. 200% w/w) and decrease (approx. 50-65% w/w) of the other membrane-bound neutral lipids. The glycolipid was identified as monogalactosyldiacylglycerol. The fatty acid profile of the various membrane lipids underwent marked alteration during the epididymal transit of the male gametes. Cholesterol/phospholipid and saturated/unsaturated fatty acid ratios increased greatly in the maturing sperm membrane. The altered lipid profile of the mature sperm membrane leads to changes in its fluidity that play an important role in determining the structure and functions of the biomembrane.     

Bidirectional transbilayer movement of phospholipid analogs in human red blood cells. Evidence for an ATP-dependent and protein-mediated process.

The transbilayer movement of fluorescent and isotopically labeled analogs of phosphatidylserine (PS), phosphatidylethanolamine (PE), and phosphatidylcholine (PC) from the outer to the inner leaflet (flip) and from the inner to the outer leaflet (flop) of human red blood cells (RBC) was examined. The inward movement of 1-oleoyl-2-(N-4-nitrobenzo-2-oxa-1,3-diazole-aminocaproyl)- (C6-NBD-), 1-oleoyl-2-(N-(3-(3-[125I]iodo-4-hydroxyphenyl)propionyl)aminocaproyl)- (C6-125I-), or 1-oleoyl-2-(N-(3-3-[125I]iodo-4-azido-phenyl)propionyl)aminocaproyl- (C6-125I-N3-) analogs of PC and PE were relatively slow. In contrast, all analogs of PS and PE analogs containing aminododecanoic acid (C12 lipids) were rapidly transported to the cell's inner leaflet. Analysis of 125I-N3 lipids cross-linked to membrane proteins revealed labeling of 32-kDa Rh polypeptides that was dependent on the lipid's capacity to be transported to the inner leaflet but was independent of lipid species. To investigate whether lipids could also be transported from the inner to the outer leaflet, lipid probes residing exclusively in the inner leaflet were monitored for their appearance in the outer leaflet. Lipid movement could not be detected at 0 degrees C. At 37 degrees C, however, approximately 70% of the PC, 40% of the PE, and 15% of the PS redistributed to the cells outer leaflet, thereby attaining their normal asymmetric distribution. Continuous incubation in the presence of bovine serum albumin depleted the cells of the analogs (t1/2 approximately 1.5 h) in a manner that was independent of lipid species. Similar to the inward movement of aminophospholipids, the outward movement of PC, PE, and PS was ATP-dependent and could be blocked by oxidation of membrane sulfhydryls and by the histidine reagent bromophenacyl bromide. Evidence is presented which suggests that the outward movement of lipids is an intrinsic property of the cells unrelated to compensatory mechanisms due to an imbalance in lipid distribution.     

Importance of phosphatidylethanolamine for association of protein kinase C and other cytoplasmic proteins with membranes.

Biological membranes exhibit an asymmetric distribution of phospholipids. Phosphatidylserine (PS) is an acidic phospholipid that is found almost entirely on the interior of the cell where it is important for interaction with many cellular components. A less well understood phenomenon is the asymmetry of the neutral phospholipids, where phosphatidylcholine (PC) is located primarily on exterior membranes while phosphatidylethanolamine (PE) is located primarily on interior membranes. The effect of these neutral phospholipids on protein-phospholipid associations was examined using four cytoplasmic proteins that bind to membranes in a calcium-dependent manner. With membranes containing PS at a charge density characteristic of cytosolic membranes, protein kinase C and three other proteins with molecular masses of 64, 32, and 22 kDa all showed great selectively for membranes containing PE rather than PC as the neutral phospholipid; the calcium requirements for membrane-protein association of the 64- and 32-kDa proteins were about 10-fold lower with membranes containing PE; binding of the 22-kDa protein to membranes required the presence of PE and could not even be detected with membranes containing PC. Variation of the PS/PE ratio showed that membranes containing about 20% PS/60% PE provided optimum conditions for binding and were as effective as membranes composed of 100% PS. Thus, PE, as a phospholipid matrix, eliminated the need for membranes with high charge density and/or reduced the calcium concentrations needed for protein-membrane association. A surprising result was that PKC and the 64- and 32-kDa proteins were capable of binding to neutral membranes composed entirely of PE/PC or PC only. The different phospholipid headgroups altered only the calcium required for membrane-protein association. For example, calcium concentrations at the midpoint for association of the 64-kDa protein with membranes containing PS, PE/PC, or PC occurred at 6, 100, and 20,000 microM, respectively. Thus, biological probes detected major differences in the surface properties of membranes containing PE versus PC, despite the fact that both of these neutral phospholipids are often thought to provide "inert" matrices for the acidic phospholipids. The selectivity for membranes containing PE could be a general phenomenon that is applicable to many cytoplasmic proteins. The present study suggested that the strategic location of PE on the interior of the membranes may be necessary to allow some membrane-protein associations to occur at physiological levels of calcium and PS.     

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.     

Quenching of intrinsic tryptophan fluorescence in membranes of rat pituitary cells.

The GH4C1 strain of hormone-producing rat pituitary cells has specific receptors for the tripeptide thyrotropin-releasing hormone (TRH). Membranes prepared from GH4C1 cells show intrinsic tryptophan fluorescence which was quenched by low concentrations (10--100 nM) of TRH and Ntau-methyl TRH but not by biologically inactive analogs of TRH. Membranes from GH4C1 cells were subjected to thermal denaturation. A conformational transition was noted above 40 degrees C and an irreversible denaturation was observed at 52 degrees C. TRH-induced quenching of intrinsic fluorescence was lost completely in membranes previously incubated for 10 min at 30 degrees C while loss of [3H]-TRH binding was only about 20% at this temperature. Collisional quenching by iodide revealed that about 38% of the tryptophanyl residues in GH4C1 membranes were exposed to solvent. Quenching by TRH occurred with a shift in wavelength maximum from 336 to 342 nm suggesting that few of the tryptophanyl residues quenched by the tripeptide are totally exposed. Membranes prepared from cells preincubated with 20 nM TRH for 48 h, in which TRH receptors were decreased to 30% of control values, showed no quenching of tryptophan fluorescence in response to freshly added TRH. We conclude that the TRH-receptor interaction in GH4C1 cells is associated with a change in membrane conformation that can be measured by differential spectrofluorometry of intrinsic tryptophan fluorescence.