Agonist interactions at hepatic alpha 1- and beta-adrenergic receptors: affinity-state regulation by guanine nucleotides and temperature

We investigated the binding characteristics of agonists to alpha 1- and beta-adrenergic receptors of intact liver cells, broken rat liver cell membranes, and detergent-solubilized preparations under varying experimental conditions, focusing on the different "states" of the receptor for agonists and the regulation of these states by temperature and guanine nucleotides. While only low-affinity binding of agonists to both receptor subtypes was evident in studies performed at 37 degrees C with solubilized preparations, biphasic competition curves for agonists were observed in both intact cells and membrane preparations; the majority of sites were of low affinity. In membrane preparations, the nonhydrolyzable GTP analogue Gpp(NH)p caused a rightward shift of agonist competition curves and a loss of high-affinity binding. These results are consistent with the involvement of guanine nucleotide binding proteins in both alpha 1- and beta-adrenergic transduction pathways. When competition studies were performed at 4 degrees C, receptor sites existed predominantly in the high-affinity configuration, in intact cells and membranes, as well as in soluble preparations. In contrast to the studies conducted at 37 degrees C, no Gpp(NH)p-induced conversion to the lower affinity state could be demonstrated in studies performed with membrane preparations at 4 degrees C. Thus, the high-affinity state of alpha 1- and beta-adrenergic receptors is stabilized at 4 degrees C in intact cells, membranes, and soluble preparations. After incubations had been performed at 37 degrees C, high-affinity binding of agonists could not be restored by subsequent incubation at 4 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)     

Temperature dependence of beta receptor, adenosine receptor, and sodium fluoride stimulated adenylate cyclase from turkey erythrocytes

The individual temperature dependencies of the process which control the activity of turkey erythrocyte adenylate cyclase have been determined. The temperature dependence of the fraction of activable cyclase units experiences a thermal transition at 24 degrees C for all three modes of enzyme activation: l-epinephrine, adenosine, and NaF. This thermal transition probably reflects the phase transition in the inner monolayer of the membrane which influences the behavior of the GTP regulatory unit which is involved in all three modes of enzyme activation. The "rate constant" of enzyme activation by adenosine reflects two thermal transitions, at 24 and at 35 degrees C; the apparent rate constant of cyclase activation by NaF activation experiences a transition only at 24 degrees C whereas the rate constant of the beta-receptor-bound agonist decreases monotonously with no "breaks" on the Arrhenium plot. Following the temperature dependence of the fluorescence intensity of dansylphosphatidylethanolamine embedded in both sides of the membrane and exclusively in the outer monolayer, one can assign the thermal transition of 24 degrees C to the inner monolayer and the other two transitions to the outer monolayer (10 and 35 degrees C). We interpret these results as follows. (a) The monomolecular rate constant characterizing the activation of cyclase by the precoupled adenosine receptor experiences both the transition at 24 and 35 degrees C, indicating that the latter may span the bilayer. (b) The bata receptor activates the cyclase units only in fluid areas since it can diffuse exclusively in the fluid areas of the membrane and is unable to interact with cyclase units in "frozen" areas. the linear dependence of the logarithm of the rate constant on 1/T for the bata receptor reflects the change of membrane fluidity as a function of temperature.     

Calorimetric investigations of the structural stability and interactions of colicin B domains in aqueous solution and in the presence of phospholipid bilayers

The effects of pH and temperature on the stability of interdomain interactions of colicin B have been studied by differential-scanning calorimetry, circular dichroism, and fluorescence spectroscopy. The calorimetric properties were compared with those of the isolated pore-forming fragment. The unfolding profile of the full-length toxin is consistent with two endothermic transitions. Whereas peak A (T(m) = 55 degrees C) most likely corresponds to the receptor/translocation domain, peak B (T(m) = 59 degrees C) is associated with the pore-forming domain. By lowering the pH from 7 to 3.5, the transition temperature of peaks A and B are reduced by 25 and 18 degrees C, respectively, due to proton exchange upon denaturation. The isolated pore-forming fragment unfolds at much higher temperatures (T(m) = 65 degrees C) and is stable throughout a wide pH range, indicating that intramolecular interactions between the different colicin B domains result in a less stable protein conformation. In aqueous solution circular dichroism spectra have been used to estimate the content of helical secondary structure of colicin B ( approximately 40%) or its pore-forming fragment ( approximately 80%). Upon heating, the ellipticities at 222 nm strongly decrease at the transition temperature. In the presence of lipid vesicles the differential-scanning calorimetry profiles of the pore-forming fragment exhibit a low heat of transition multicomponent structure. The heat of transition of membrane-associated colicin B (T(m) = 54 degrees C at pH 3.5) is reduced and its secondary structure is conserved even at intermediate temperatures indicating incomplete unfolding due to strong protein-lipid interactions.     

The phospholipid-dependence of uridine diphosphate glucuronyltransferase. Temperature-dependence of microsomal enzyme activity and thermotropic changes in membrane structure.

Arrhenius plots of the non-latent UDP-glucuronyltransferase (p-nitrophenol acceptor) activity of guinea-pig microsomal membranes prepared with 154 mM-KCl were linear from 5 to 40 degrees C. Arrhenius plots for other microsomal preparations from guinea pig and rat liver that show various degrees of transferase latency, exhibited two linear regions intersecting at a sharp transition point near 20-25 degrees C. This discontinuity was abolished or greatly decreased when transferase latency was removed by treating the membranes with perturbants of phospholipid bilayer strucutre. The fluorescent probe N-phenyl-1-naphthyl-amine detected a thermotropic change in the fluidity of the phospholipid acyl chains of all the microsomal membrane preparations studied, at temperatures close to those of the Arrhenius-plot transitions. It is concluded that the thermotropic change in the structure of the membrane bilayer probably is a 'phase separation' or clustering of phospholipids, which affects a permeability barrier that restricts access of substrate to the transferase molecules.     

Thermotropic phase transitions in the plasma membrane of ram spermatozoa

A steady-state fluorescence polarization technique, using the membrane probe 1,6-diphenyl-1,3,5-hexatriene (DPH), showed that separately detectable transitions occurred in the regions of 17, 26 and 36 degrees C in isolated preparations of ram sperm plasma membrane. An independent technique based on the temperature-related behaviour of calcium- and magnesium-activated ATPase detected a single phase transition in the region of 24 degrees C. Modulation of ATPase by neighbouring lipid composition was inferred from findings that phospholipase A2 caused significant stimulation of the enzyme. Cholesterol-rich liposomes caused an upward shift of the phase-transition temperature from 24 degrees C to 30 degrees C, but the reasons for this are unclear. It is considered that these phase transitions may have profound effects on sperm survival and physiology, both during normal fertilization processes and in response to cryostorage.     

Cooling and freezing damage platelet membrane integrity.

Cytoskeletal rearrangements and a membrane lipid phase transition (liquid crystalline to gel) occur in platelets on cooling from 23 to 4 degrees C. A consequence of these structural alterations is irreversible cellular damage. We investigated whether platelet membrane integrity could be preserved by (a) previously studied combinations of a calcium chelator (EGTA) and microfilament stabilizer (cytochalasin B) with apparent benefit in protecting platelets from cooling injury or (b) agents of known benefit in protecting membranes and proteins from freezing injury. Platelet function and activation before and after freezing or cooling were measured by agglutination with ristocetin, aggregation with thrombin or ADP, platelet-induced clot retraction (PICR), and expression of P-selectin. Platelets were loaded with 10 nM fluorescein diacetate. After freezing or cooling, the preparations were centrifuged and the supernatant was measured for fluorescein. For cooling experiments, fresh platelets were chilled at 4 degrees C for 1 to 21 days with or without the combination of 80 microM EGTA/AM and 2 microM cytochalasin B (EGTA/AM-CytoB) and then warmed rapidly at 37 degrees C. For freezing experiments, 5% dimethyl sulfoxide (Me2SO) or 5 mM glycerol were added to fresh platelets. The preparations were then frozen at -1 degrees C/min to -70 degrees C and then thawed rapidly at 37 degrees C. Platelet membrane integrity, as measured by supernatant levels of fluorescein, correlated inversely with platelet function. Chilling platelets at 4 degrees C with EGTA/AM-CytoB showed a gradual loss of membrane integrity, with maximum loss reached on day 7. The loss of membrane integrity preceded complete loss of function as demonstrated by PICR. In contrast, platelets chilled without these agents had complete loss of membrane integrity and function after 1 day of storage. Freezing platelets in Me2SO resulted in far less release of fluorescein than did freezing with or without other cryoprotectants (P < 0.001). This result correlated with enhanced function as demonstrated by PICR and supports earlier observations that Me2SO protects platelet membranes from freezing injury. Release of fluorescein into the surrounding medium reflected loss of membrane integrity and function in both cooled and frozen platelets. Membrane cytoskeletal rearrangements are linked to membrane changes during storage. These results may be generally applicable to the study of platelet storage.     

Agonist-induced isomerization of the alpha 1-adrenergic receptor: kinetic analysis using broken-cell and solubilized preparations

The affinity of agonists but not antagonists at hepatic membrane alpha 1-adrenergic receptors is temperature dependent; a 100-fold higher affinity is observed at 4 degrees C than at 37 degrees C. The relationship between these two agonist affinity states was investigated by using a strategy that allows the kinetics of this transition to be examined under equilibrium conditions. When competition assays are performed at 37 degrees C for varying intervals and the reaction mixture is then rapidly cooled by freezing, allowed to thaw, and further equilibrated at 4 degrees C, a rapid and progressive decrease (t1/2 of 1-2 min) in agonist affinity occurs, the extent of which is directly related to the incubation time at 37 degrees C. This decrease in agonist affinity is sustained as long as agonist is present but can be reversed by its subsequent removal. In contrast, no change in affinity is seen in identical experiments when antagonists are employed as the competing ligand. High-affinity binding of agonists is also demonstrated in short-term nonequilibrium experiments, indicating that the low-temperature incubations do not induce, but rather stabilize, a receptor conformation of high affinity for agonists. These findings suggest that the predominantly low-affinity binding of agonists to alpha 1-adrenergic receptors demonstrated in equilibrium studies at physiological temperatures may be the result of a ligand-driven decrease in affinity. Since the transition in receptor affinity for agonists occurs not only in broken-cell preparations but also after detergent solubilization of the membrane receptor, it most likely is due to an agonist-induced change in the conformation of the receptor protein per se.(ABSTRACT TRUNCATED AT 250 WORDS)     

Influence of lipid membranes on the conformational transitions of nucleic acids

The conformational transitions of nucleic acids which were enclosed in reverse phase evaporation vesicles (REV) were studied by thermal denaturation with optical recording. Cloned fragments of double-stranded DNA containing 179 base pairs and 187 base pairs, respectively, and polyA.polyU were enclosed in REV with a yield up to every vesicle containing 50 nucleic acid molecules. With the 179 base pairs DNA enclosed in the vesicle from egg lecithin two well resolved helix-coil transitions could be measured; one is very similar in the midpoint-temperature Tm and halfwidth delta T1/2 to the transition of the free nucleic acid, and the other transition occurs stabilized at a 3.5 degrees C higher Tm-value and with a broader delta T1/2, 2.7 degrees C instead of 0.6 degree C. Both transitions are from nucleic acids inside the vesicles. Varying the surface charge of the lipid membrane by adding the negatively charged phosphatidylserine or phosphatidylglycerol, an optimum in the yield of enclosure and a maximum in the increase in Tm (4.5 degrees C) and delta T1/2 (5.5 degrees C instead of 1.0 degrees C) was obtained at 20% phosphatidylserine or phosphatidylglycerol. In vesicles from pure negatively charged lipids no second population of nucleic acids was observed. Qualitatively, similar effects were observed with polyA.polyU. Stabilization and broadening of the second transition is higher for nucleic acids inside vesicles from lipids with unsaturated fatty acids, as dioleoyl-phosphatidylcholine, than with saturated fatty acids, dipalmitoyl-phosphatidylcholine. Stabilization and broadening decrease with increasing ionic strength, whereas the relative contributions of both transitions to the total hypochromicity remain unchanged; the second transition coincides with the first at 90 mM Na+. From the experimental results it was concluded that the interaction of nucleic acids and lipid membranes is mainly of electrostatic nature. The nucleic acids exist inside the vesicles in two populations, one behaving like nucleic acid free in solution and one influenced by the contact with the membrane. All results are in accordance with a model in which the interaction between the nucleic acid and the membrane is in competition with the dipole-dipole interaction inside the membrane surface.     

The effect of acclimation temperature on the activation energies of state III respiration and on the unsaturation of membrane lipids of goldfish mitochondria.

The influence of the acclimation temperature on the thermotropic behaviour of mitochondrial respiration and on the degree of unsaturation of mitochondrial membrane lipids has been studied. The mitochondria were isolated from red muscle, white muscle and liver of goldfish acclimated to 5, 20 and 30 degrees C. ADP-activated succinate oxidation was measured at different temperatures and resulted in non-linear Arrhenius-plots with breaks between 10 and 23 degrees C. As for the break-temperatures, there was found a shift downwards in preparations of decreased acclimation temperatures. This could be caused by a changed composition of membrane lipids and a simultaneous shift of the membrane phase transition temperature. Therefore, the fatty acid composition of all membrane preparations was analyzed. However, no consistent change of the degree of unsaturation due to a changed acclimation temperature could be found.     

Platelet-derived growth factor receptors form a high affinity state in membrane preparations. Kinetics and affinity cross-linking studies

The specific binding of 125I-PDGF (platelet-derived growth factor) to intact fibroblasts becomes relatively nondissociable during incubation at 37 degrees C. To characterize the interaction of PDGF with its receptors under conditions in which there is no receptor internalization, we have studied the binding of 125I-PDGF to membrane preparations derived from mouse 3T3 cells and rat liver. The binding sites had the affinity and specificity characteristics expected of PDGF receptors. At 37 degrees C (but not at 4 degrees C) the specific binding of 125I-PDGF to membranes gradually became nondissociable as assessed by either dilution or by addition of excess unlabeled PDGF. This tight binding was not due to a covalent interaction since the polyanionic compound suramin readily dissociated specifically bound 125I-PDGF. This property of suramin was used to expose rat liver PDGF receptors which were occupied by endogenous PDGF. Affinity cross-linking studies demonstrated that the formation of the nondissociable state of 125I-PDGF binding was associated with the binding of 125I-PDGF to a 160,000-dalton protein and to a 110,000-dalton species. The cross-linked binding sites could be adsorbed to wheat germ agglutinin and to anion exchange resins. The isoelectric point of both cross-linked species determined by two-dimensional gel electrophoresis was approximately 4.7. These data demonstrate that in membrane preparations, PDGF binds to an anionic 160,000-dalton glycoprotein which is likely to be the receptor. A high affinity state of PDGF binding, which is formed rapidly at 37 degrees C, can be dissociated by suramin.     

Quick-freeze differential scanning calorimetry and saturation transfer electron spin resonance: novel techniques for assessing phase transitions in biological membranes

Quick-freeze differential scanning calorimetry (QF-DSC) and saturation transfer-electron spin resonance (ST-ESR) spectroscopy were used to study lipid gel-phase transitions in mature green tomato fruit microsomal membranes. ST-ESR of 12-doxyl methyl stearate labelled membranes proved to be reproducible and provided increased sensitivity to temperature-induced structural changes, allowing the detection of several transitions in isolated membranes (6 degrees C, 21 degrees C, 28 degrees C). QF-DSC led to the assessment of lipid gel phase transitions in isolated microsomal membranes and microsomal membrane lipids by enhancing the transition. A phase transition enthalpy of 114 J/g and an onset temperature of 29.8 degrees C were obtained for whole membranes while with isolated lipids values of 370 J/g and 19.9 degrees C were found.     

Phase transitions in monoglyceride bilayers. A light scattering study.

Thermotropic phase transitions in single planar bilayers of glycerol mono-oleate have been investigated using quasi-elastic light scattering from thermally excited membrane fluctuations. In certain cases both spectroscopic and intensity information were derived from the observations. For solvent-free bilayers transitional changes were observed in several membrane parameters: in tension, viscosity and thickness, in a combination of lipid orientational order parameter and dielectric anisotropy, and in the lateral compression modulus. These changes, particularly those in membrane thickness and in the anisotropy/order combination, were clearly indicative of a chain-melting transition in the lipid molecules. The chain-melting transition temperature was identified as 16.6 +/- 0.03 degrees C (delta T 1/2 = 1.5 degrees C). The other changes tended to cluster around 12.5 and 16.6 degrees C, suggesting that a two-stage transition was involved. Analysis of pretransitional fluctuations in membrane viscosity, based on a Landau approach, suggested that at the transition the membrane was close to a critical point (T = 12.7 degrees C). Less information was accessible for membranes containing n-decane within their structure. In this case, the change in membrane tension was much smaller than in the solvent-free case and the transition was considerably broadened. These effects accord with an increase in 'interactive volume' within the bilayer due to solvent inclusion.     

Fourier transform infrared spectroscopy suggests unfolding of loop structures precedes complete unfolding of pig citrate synthase

Pig citrate synthase (PCS) can be used as a model enzyme to gain some insight into the structural basis of protein thermostability. The thermal unfolding characteristics of the specific secondary structure elements within PCS were monitored in detail by following changes in its amide I band components. The result of our study indicates that PCS undergoes irreversible thermal denaturation. Detailed analysis reveals that the different secondary structures display a multistep transition with a major and a minor transition at different temperatures and a very small initial transition at the same temperature (30 degrees C). A plot of temperature-induced changes in (1)H-(2)H exchange, the decrease in the absorbance of the alpha-helical structures, and the increase in the absorbance of aggregated structures all have in common a multistep transition, the minor one centered at 45 degrees C and the major one around 59 degrees C. In contrast, a band that is tentatively assigned to loop structures displays these same minor and major transitions but at lower temperatures (39 and 52 degrees C, respectively). The transition, which occurs at 39-45 degrees C, is not associated with the appearance of aggregated structures. This transition may reflect a change in the tertiary structure of the protein. However, the final transition, which occurs at a higher temperature (52-59 degrees C), reflects unfolding and aggregation of the polypeptide chains. The Fourier transform infrared (FTIR) analysis suggests that PCS has a thermolabile region that unfolds first, some 7 degrees C below the main unfolding of the protein. We propose that this reflects the unfolding of the highly flexible loop segments, which in turn triggers the unfolding of the predominantly helical core structure of PCS.     

A high melting (105 degrees C) form of chromatin characterizes the potential of cells for mitosis

The nuclei from different types of dividing cultured cells melted as four thermal transitions: I (60 degrees C), II (76 degrees C), III (88 degrees C), and IV (105 degrees C). The fourth transition was the predominant endotherm in all types of cells examined. In the melting profile of nuclei obtained from nondividing density-inhibited fibroblasts, transition IV remained the major endotherm; however, it was lost in nuclei from differentiated myoblasts and nutrient-depleted cells. In these cells, the loss in transition IV was compensated by a concomitant increase in transition III. In the nutrient-deprived cells the decrease in transition IV was followed by a gradual lowering in its melting temperature. The complete loss of transition IV was correlated with loss of cellular capacity to divide.     

Effect of chain unsaturation on the structure and thermotropic properties of galactocerebrosides.

Differential scanning calorimetry (DSC) and x-ray diffraction have been used to study the effect of increasing chain-unsaturation on the structure and properties of the hydrated cerebrosides N-stearoyl, -oleoyl, and -linoleoyl galactosylsphingosine (NSGS, NOGS, and NLnGS, respectively). DSC of hydrated (70 wt% water) NSGS shows an endothermic transition at 85 degrees C (delta H = 18.0 kcal/mol NSGS) and a broad exothermic transition at 40-60 degrees C, the latter being dependent upon the previous cooling rate. X-Ray diffraction patterns recorded at 21, 61, and 86 degrees C provide evidence for interconversions between metastable and stable crystalline NSGS bilayer phases. The properties of the unsaturated-chain cerebrosides are more complex. Hydrated NOGS shows a single endothermic transition at 44.8 degrees C (delta H = 11.5 kcal/mol NOGS). However, incubation of NOGS at 49 degrees C for 24 h results in a second transition at 55.5 degrees C. By cycling NOGS between 0 and 49 degrees C complete conversion into this higher melting phase (delta H = 12.1 kcal/mol NOGS) is achieved. X-ray diffraction confirms a bilayer phase at all temperatures and delineates the conversions between a crystalline phase at 21 degrees C (bilayer period d = 56.5A), a second crystalline phase at 47 degrees C (d = 69.9A), and a liquid crystalline phase at 59 degrees C (d = 52.0A). The more unsaturated NLnGS shows two transitions, a sharp transition at 28 degrees C (delta H = 8.0 kcal/mol NLGS) and a broad, low-enthalpy transition at 42 degrees C (delta H = 0.4 kcal/mol NLGS). Again, incubation between the two transitions leads to a single transition at 44 degrees C (delta H = 9.3 kcal/mol NLGS). X-ray diffraction demonstrates conversions between two crystalline bilayer phases (d = 55.2A and d = 68.4A), and a liquid crystalline bilayer phase (d = 51.8A). Thus, increased unsaturation in the amide-linked fatty acyl chain of cerebrosides results in decreased chain-melting temperatures (NSGS greater than NOGS greater than NLnGS) and has marked effects on their structural properties.     

Phosphatidylcholine Biosynthesis in the Neuroblastoma-Glioma Hybrid Cell Line NG 108-15: Stimulation by Phorbol Esters

Studies were conducted on curaremimetic neurotoxin binding to the nicotinic acetylcholine receptor present on membrane fractions derived from the human medulloblastoma clonal line, TE671. High-affinity binding sites (KD = 2 nM for 1-h incubation at 20 degrees C) and low-affinity binding sites (KD = 40 nM) for 125I-labeled alpha-bungarotoxin are present in equal quantities (60 fmol/mg membrane protein). The kinetically determined dissociation constant for high-affinity binding of toxin is 0.56 nM (k1 = 6.3 X 10(-3) min-1 nM-1; k-1 = 3.5 X 10(-3) min-1) at 20 degrees C. Nicotine, d-tubocurarine, and acetylcholine are among the most effective inhibitors of high-affinity toxin binding. The quantity of toxin binding sites and their affinity for cholinergic agonists is sensitive to reduction, alkylation, and/or oxidation of membrane sulfhydryl residues. High-affinity toxin binding sites that have been subjected to reaction with the sulfhydryl reagent dithiothreitol are irreversibly blocked by the nicotinic receptor affinity reagent bromoacetylcholine. High-affinity toxin binding is inhibited in the presence of either of two polyclonal antisera or a monoclonal antibody raised against nicotinic acetylcholine receptors from fish electric tissue. Taken together, these results indicate that curaremimetic neurotoxin binding sites on membrane fractions of the TE671 cell line share some properties with nicotinic acetylcholine receptors of peripheral origin and with toxin binding sites on other neuronal tissues.     

Relationship of hyperthermia-induced hemolysis of human erythrocytes to the thermal denaturation of membrane proteins

Hemolysis of human erythrocytes as a function of time of exposure to 47.4-54.5 degrees C was measured and correlated to thermal transitions in the membranes of intact erythrocytes as determined by differential scanning calorimetry (DSC). Curves of hemoglobin leakage (a measure of hemolysis) as a function of time have a shoulder region exhibiting no leakage, indicative of the ability to accumulate sublethal damage (i.e., damage not sufficient to cause lysis), followed by a region of leakage approximating pseudo-first-order kinetics. Inverse leakage rates (Do) of 330-21 min were obtained from 47.4-54.5 degrees C, respectively. A relatively high activation energy of 304 +/- 22 kJ/mol was obtained for leakage, eliminating the involvement of metabolic processes but implicating a transition as the rate-limiting step. Membrane protein involvement was suggested by the very low rate (10(-2) of the rate from erythrocytes) and low activation energy (50 +/- 49 kJ/mol) of hemoglobin leakage from liposomes containing no membrane protein. A model was developed that predicts a transition temperature (Tm) for the critical target (rate-limiting step) of 60 degrees C when measured at a scan rate of 1 K/min. DSC scans were obtained from intact erythrocytes and a procedure developed to fit and remove the transition for hemoglobin denaturation which dominated the scan. Three transitions remained (transitions A, B, and C) with Tm values of 50.0, 56.8, and 63.8 degrees C, respectively. These correspond to, but occur at slightly different temperatures than, the A, B, and C transitions of isolated erythrocyte membranes in the same salt solution (Tm = 49.5, 53-58, and 65.5 degrees C, respectively). In addition, the relative enthalpies of the three transitions differ between isolated membranes and erythrocytes, suggestive of membrane alterations occurring during isolation. Thus, all analyses were conducted on DSC scans of intact erythrocytes. The B transition is very broad and probably consists of several transitions. An inflection, which is seen as a distinct peak (transition B3) in fourth-derivative curves, occurs at 60.8 degrees C and correlates well with the predicted Tm of the critical target. Ethanol (2.2%) lowers the Tm of B3 by 4.0-4.5 K, close to the shift of 3.3 K predicted from its effect on hemolysis. Glycerol (10%) has very little effect on both hemolysis and the Tm of B3, but it stabilizes spectrin (delta Tm = 1.5 K) against thermal denaturation.(ABSTRACT TRUNCATED AT 400 WORDS)     

In situ assessment of erythrocyte membrane properties during cold storage

Membrane fluidity and overall protein secondary structure of human erythrocytes were studied in situ using Fourier transform infrared spectroscopy (FTIR). Erythrocyte membranes were found to have weakly cooperative phase transitions at 14 degrees C and at 34 degrees C, which were tentatively assigned to the melting of the inner membrane leaflet and the sphingolipid rich outer leaflet, respectively. Cholesterol depletion by methyl-beta-cyclodextrin (MbetaCD) resulted in a large increase in the cooperativity of these transitions, and led to the appearance of another phospholipid transition at 25 degrees C. Multiple, sharp membrane phase transitions were observed after 5 days cold storage (4 degrees C ), which indicated phase separation of the membrane lipids. Using fluorescence microscopy, it was determined that the lipid probe 1,1'-dioctadecyl-3,3,3',3-tetramethyl-indocarbocyanine perchlorate (dil-C18) remained homogeneously distributed in the erythrocyte membrane during cold storage, suggesting that lipid domains were below the resolution limit of the microscope. Using thin layer chromatography, changes in the membrane lipid composition were detected during cold storage. By contrast, assessment of the amide-II band with FTIR showed that the overall protein secondary structure of haemoglobin was stable during cold storage.     

Phospholipase A2 hydrolysis of membrane phospholipids causes structural alteration of the nicotinic acetylcholine receptor

Thermal perturbation techniques have been used to probe structural alteration of the nicotinic acetylcholine receptor as a function of perturbations of its native membrane environment. Differential scanning calorimetry and a technique involving heat inactivation of the alpha-bungarotoxin-binding sites on the receptor protein reveal that there is a profound destabilization of the acetylcholine receptor structure when receptor-containing membranes are exposed to phospholipase A2. The characteristic calorimetric transition assigned to irreversible denaturation of the receptor protein and the heat inactivation profile of alpha-bungarotoxin-binding sites are shifted to lower temperatures by approx. 7 and 5 C degrees, respectively, upon exposure to phospholipase A2 at a phospholipase/neurotoxin binding site molar ratio of about 1:100. The effects of phospholipase A2 on receptor structure can be (i) reversed by using bovine serum albumin as a scavenger of phospholipase hydrolysis products of membrane phospholipids, and (ii) stimulated by incorporation into the membranes of free, polyunsaturated fatty acids. In particular, linolenic acid (18:3(n-3] causes detectable destabilization of the alpha-bungarotoxin binding sites on the receptor at free fatty acid/receptor molar ratios as low as 10:1. Furthermore, alteration of receptor structure by added phospholipase occurs very rapidly, which is consistent with the observation of rapid in situ phospholipase A2 hydrolysis of membrane phospholipids, particularly highly unsaturated phosphatidylethanolamine and phosphatidylserine. Based on previously published data on the inhibition of acetylcholine receptor cation-gating activity caused by the presence of either phospholipase A2 or free fatty acids (Andreasen T.J. and McNamee M.G. (1980) Biochemistry 19, 4719), we interpret our data as indicative of a correlation between structural and functional alterations of the membrane-bound acetylcholine receptor induced by phospholipase A2 hydrolysis products.     

Evidence for a physiological role for membrane rafts in human platelets.

We have investigated raft formation in human platelets in response to cell activation. Lipid phase separation and domain formation were detected using the fluorescent dye 1,1'-dioctadecyl-3,3,3',3'-tetramethyl-indocarbocyanine perchlorate (diI-C(18)) that preferentially partitions into gel-like lipid domains. We showed that when human platelets are activated by cold and physiological agonists, rafts coalesce into visible aggregates. These events were disrupted by depletion of membrane cholesterol. Using Fourier transform infrared spectroscopy (FTIR), we measured a thermal phase transition at around 30 degrees C in intact platelets, which we have assigned as the liquid-ordered to the liquid-disordered phase transition of rafts. Phase separation of the phospholipid and the sphingomyelin-enriched rafts could be observed as two phase transitions at around 15 and 30 degrees C, respectively. The higher transition, assigned to the rafts, was greatly enhanced with removal of membrane cholesterol. Detergent-resistant membranes (DRMs) were enriched in cholesterol (50%) and sphingomyelin (20%). The multi-functional platelet receptor CD36 selectively partitioned into DRMs, whereas the GPI-linked protein CD55 and the major platelet integrin alpha(IIb)beta(3a) did not, which suggests that the clustering of proteins within rafts is a regulated process dependent on specific lipid protein interactions. We suggest that raft aggregation is a dynamic, reversible physiological event triggered by cell activation.