Thermally induced changes in the structure and activity of yeast hexokinase B

Yeast hexokinase has been poorly characterized in regard with its stability. In the present study, various spectroscopic techniques were employed to investigate thermal stability of the monomeric form of yeast hexokinase B (YHB). The enzyme underwent a conformational transition with a T(m) of about 41.9 degrees C. The structural transition proved to be significantly reversible below 55 degrees C and irreversible at higher temperatures. Thermoinactivation studies revealed that enzymatic activity diminished significantly at high temperatures, with greater loss of activity observed above 55 degrees C. Release of ammonia upon deamidation of YHB obeyed a similar temperature-dependence pattern. Dynamic light scattering and size exclusion-HPLC indicated formation of stable aggregates. Taking various findings on the influence of osmolytes and chaperone-like agents on YHB thermal denaturation together, it is proposed that the purely conformational transition of YHB is reversible, and irreversibility is due to aggregation, as a major cause. Deamidation of a critical Asn or Gln residue(s) may also play an important role.     

Thermal unfolding and aggregation of human complement protein C9: a differential scanning calorimetry study

The thermotropic behavior of purified human complement protein C9 was investigated by high-sensitivity differential scanning calorimetry. When dissolved in physiological buffers (pH 7.2, 150 mM NaCl), C9 underwent three endothermic transitions with transition temperatures (Tm) centered at about 32, 48, and 53 degrees C, respectively, and one exothermic transition above 64 degrees C that correlated with protein aggregation. The associated calorimetric enthalpies of the three endothermic transitions were about 45, 60, and 161 kcal/mol with cooperative ratios (delta Hcal/delta HvH) close to unity. The total calorimetric enthalphy for the unfolding process was in the range of 260-280 kcal/mol under all conditions. The exothermic aggregation temperature was strongly pH dependent, changing from 60 degrees C at pH 6.6 to 81.4 degrees C at pH 8.0, whereas none of the three endothermic transitions was significantly affected by pH changes. They were, however, sensitive to addition of calcium ions; most affected was Tm1 which shifted from 32 to 35.8 degrees C in the presence of 3 mM calcium, i.e., the normal blood concentration. Kosmotropic ions stabilized the protein by shifting the endothermic transitions to slightly higher temperatures whereas inclusion of chaotropic ions (such as choline), removal of bound calcium by addition of EDTA, or proteolysis with thrombin lowered the transition temperatures. Previous studies had indicated the formation of at least three different forms of C9 during membrane insertion or during heat polymerization, and it is suggested that the three endothermic transitions reflect the formation of such C9 conformers. Choline, which is present at high concentrations on the surface of biological membranes, and calcium ions have the ability to shift the transition temperatures of the first two transitions to be either close to or below body temperature. Thus, it is very likely that C9 is present in vivo in a partially unfolded state when bound to a membrane surface, and we propose that this facilitates membrane insertion and refolding of the protein into an amphiphilic conformation.     

4℃预处理对人中性粒细胞膜电流和极性的影响

对急性分离的人中性粒细胞采用4℃预处理是进行膜片钳实验前经常采取的步骤,但这一步骤对电生理记录结果有何影响尚无文献报道。本实验探讨这一步骤对电生理记录过程和实验结果的影响。结果显示,4℃预处理可以显著提高细胞的封接率,有利于对中性粒细胞进行电生理记录;封接率提高的原因与4℃预处理降低细胞的极性活动有关,但记录到的电压依赖性钾通道全细胞电流和大电导Ca^2＋依赖性K^＋单通道电流动力学没有显著的变化。这些结果表明,4℃预处理可能影响到细胞膜上与极性有关的脂膜变化,但对细胞膜上蛋白的功能影响较少。     

Mechanisms for the spontaneous formation of covalently linked polymers of the terminal membranolytic complement protein (C9)

Purified human C9 spontaneously polymerizes upon prolonged incubation at 37 degrees C, and a fraction of these C9 polymers becomes resistant to dissociation by sodium dodecyl sulfate (SDS) and reducing agents. We examined possible mechanisms for this spontaneous covalent linking of C9. The following results are consistent with the conclusion that the formation of the covalently linked C9 polymer involves disulfide linking. 1) In addition to the SDS/dithiothreitol (DTT)-resistant C9 polymer (Mr = 950,000), disulfide-linked C9 dimers and trimers were formed upon incubation of C9 at 37 degrees C for 64 h. 2) The C9 polymer formed upon incubation at 37 degrees C for 64 h was resistant to dissociation by 6 M guanidine hydrochloride, 20 mM DTT but was dissociated by 6 M guanidine thiocyanate alone, yielding disulfide-linked C9 oligomers. 3) The formation of the SDS/DTT-resistant C9 polymer was completely inhibited by 1 mM iodoacetamide and 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB), while DTNB enhanced the formation of disulfide-linked C9 oligomers. 4) A significant amount of free sulfhydryl group was detected in the polymerized C9 samples with various SH-specific reagents, though native C9 reacted with none of these reagents. In addition, inhibition by 1 mM iodoacetamide of C9 disulfide linking inhibited the self-association of C9 as analyzed by gel filtration on TSK-G4000 SW, whereas enhancement by 1mM DTNB of C9 disulfide linking enhanced C9 self-association. Thus, these results indicate that C9 disulfide linking that occurs upon C9 polymerization is an intrinsic property of C9 which is of importance in the formation of the stable C9 polymer structure.     

Effects of hyperthermia on the membrane potential and Na+ transport of V79 fibroblasts

The effects of hyperthermia (41-43 degrees C) on the membrane potential (calculated from the transmembrane distribution of [3H]tetraphenylphosphonium) and Na+ transport of Chinese hamster V79 fibroblasts were studied. At 41 degrees C, hyperthermia induced a membrane hyperpolarization of log phase cells (5 to 26 mV) that was reversible upon returning to 37 degrees C. The hyperpolarization was inhibited 50% by 1 mM ouabain or 0.25 mM amiloride, an inhibitor of Na+:H+ exchange. Shifting temperature to 41 degrees C increased ouabain-sensitive Rb+ uptake indicating activation of the electrogenic Na+ pump. At 43 degrees C for 60 min, the membrane potential of log phase cells depolarized (20-35 mV). Parallel studies demonstrated enhanced Na+ uptake at 41 degrees C only in the presence of ouabain. At 43 degrees C, Na+ uptake was increased relative to controls with or without ouabain present. At both 41 and 43 degrees C, 0.25 mM amiloride inhibited heat-stimulated Na+ uptake. Na+ efflux was enhanced at 41 degrees C in a process inhibited by ouabain. Thus, one consequence of heat treatment at 41 degrees C is activation of Na+:H+ exchange with the resultant increase in cytosolic [Na+] activating the electrogenic Na+ pump. At temperatures greater than or equal to 43 degrees C, the Na+ pump is inhibited.     

The kinetics of formation and structure of the low-temperature phase of 1-stearoyl-lysophosphatidylcholine

A combination of differential scanning calorimetry (DSC) and X-ray diffraction have been used to study the kinetics of formation and the structure of the low-temperature phase of 1-stearoyl-lysophosphatidylcholine (18:0-lysoPC). For water contents greater than 40 weight %, DSC shows a sharp endothermic transition at 27 degrees C (delta H = 6.75 kcal/mol) corresponding to a low-temperature phase----micelle transition. This sharp transition is not reversible, but is regenerated in a time and temperature-dependent manner. For example, with incubation at 0 degrees C the maximum transition enthalpy (delta H = 6.75 kcal/mol) is generated in about 45 min after an initial slow nucleation process of approx. 20 min. The kinetics of formation of the low-temperature phase is accelerated at lower temperatures and may be related to the disruption of 18:0-lysoPC micelles by ice crystal formation. X-ray diffraction patterns of 18:0-lysoPC recorded at 10 degrees C over the hydration range 20-80% are characteristic of a lamellar gel phase with tilted hydrocarbon chains with the bilayer repeat distance increasing from 47.6 A at 20% hydration to a maximum of 59.4 A at 39% hydration. At this maximum hydration, approx. 19 molecules of water are bound per molecule of 18:0-lysoPC. Electron density profiles show a phosphate-phosphate distance of 30 A, indicating an interdigitated lamellar gel phase for 18:0-lysoPC at all hydration values. The angle of chain tilt is calculated to be between 20 and 30 degrees. For water contents greater than 40%, this interdigitated lamellar phase converts to the micellar phase at 27 degrees C in a kinetically fast process, while the reverse (micelle----interdigitated bilayer) transition is a kinetically slower process (see also Wu, W. and Huang, C. (1983) Biochemistry 22, 5068-5073).     

Bacterial killing and inhibition of inner membrane activity by C5b-9 complexes as a function of the sequential addition of C9 to C5b-8 sites

The assembly of the C5b-9 complex on the outer membrane of C-sensitive cells of Escherichia coli results in a rapid inhibition of inner membrane function and ultimately a loss of cell viability. Cells bearing C5b-8 sites suffer no deleterious effects; however, the addition of C9 results in a rapid inhibition of inner membrane function and cell death. An attempt was made to examine the relationship between the toxic effects of the C5b-9 complex and the number of C9 molecules per C5b-8 site. Cells bearing C5b-8 sites were exposed to excess C9 at 0 degrees C and washed three times at 4 degrees C. The number of C9 molecules bound to each cell was equivalent to the number of C5b-8 sites present on each cell, and no additional C9 molecules could be bound when the cells were maintained at 4 degrees C. These cells were then incubated at 37 degrees C for 3 min and returned to 0 degrees C, a technique which exposed additional C9-binding sites equivalent to the number of C9 molecules previously bound to the cells. This technique was repeated and demonstrated that the sequential build-up of a C5b-9 site with two C9 molecules per C5b-8 site was capable of inhibiting both inner membrane function (respiration and amino acid transport) and cell viability. Three C9 molecules per complex had effects that approached the inhibitory effects of complexes formed in the presence of excess C9.     

Moderate resolution profile structure of the sarcoplasmic reticulum membrane under low temperature conditions for the transient trapping of E1 approximately P.

The calcium uptake reaction kinetics of isolated sarcoplasmic reticulum (SR) vesicles have previously been shown to be at least biphasic over a range of temperatures (26 to 10 degrees C) with a fast phase identified with the formation of E1 approximately P and calcium occlusion and a slow phase with Ca2+ translocation across the membrane and turnover of the Ca2+ ATPase ensemble. At "low" temperatures, namely 0 degrees C or lower, E1 approximately P formation is slowed and E1 approximately P is transiently trapped for at least several seconds, as indicated by the absence of the slow phase for 6 s or more. We now report that a reversible, temperature-induced structural transition occurs at about 2-3 degrees C for the isolated SR membrane. We have investigated the nature of this structural transition utilizing meridional and equatorial x-ray diffraction studies of the oriented SR membrane multilayers in the range of temperatures between 7.5 and -2 degrees C. The phase meridional (lamellar) diffraction has provided the profile structure for the SR membrane at the highest vs. lowest temperature at the same moderate resolution of 16-17 A while the equatorial diffraction has provided information on the average lipid chain packing in the SR membrane plane in the two cases. To identify the contribution of each membrane component in producing the differences between the profile structures at 7.5 and -2 degrees C, step-function models have been fitted to the moderate resolution electron density profiles. Lipid lateral phase separation may be responsible for inducing the structural change in the Ca2+ ATPase, thereby resulting in the slowing of E1 approximately P formation and the transient trapping of E1 approximately P at the "lower" temperatures.     

Conformational states of annexin VI in solution induced by acidic pH

Acidic pH-induced folding of annexin (Anx)VI in solution was investigated in order to study the mechanism of formation of ion channels by the protein in membranes. Using 2-(p-toluidino)naphthalene-6-sulfonic acid as a hydrophobic probe, it was demonstrated that AnxVI exerts a large change in hydrophobicity at acidic pH. Moreover, circular dichroism spectra indicated that the native state of AnxVI changes at acidic pH towards a state characterized by a significant loss of alpha-helix content and appearance of new beta-structures. These changes are reversible upon an increase of pH. It is postulated that the structural folding of AnxVI could explain how a soluble protein may undergo transition into a molecule able to penetrate the membrane hydrophobic region. The physiological significance of these observations is discussed.     

Modification of membrane lipid: physical properties in relation to fatty acid structure.

Differential scanning calorimetry (DSC) and electron spin resonance (ESR) measurements were made to characterize how modifications in the fatty acid composition of Escherichia coli affected the thermotropic phase transition(s) of the membrane lipd. When the fatty acid composition contained between 20 and 60% saturated fatty acids, the DSC curves for isolated phospholipids and cytoplasmic membranes showed a broad (15-25 degree C) gel to liquid-crystalline phase transition, the position of which depended on the particular fatty acid composition. Utilizing multiple lipid mutants, enrichment of the membrane phospholipids with a single long-chain cis-monoenoic fatty acid in excess of that possible in a fatty acid levels less than 20% and gradually replaced the broad peak as the cis-monoenoic fatty acid content increased. These results were obtained with phospholipids, cytoplasmic membranes, and whole cells. With these same phopholipids, plots of 2,2,6,6-tetramethylpiperidinyl-1-oxy partitioning and ESR order parameters vs. 1/T revealed discontinuities at temperatures 40-60 degrees C above the calorimetrica-ly measured gel to liquid-crystalline phase transitions. Moreover, when the membrane phospholipids were enriched with certain combinations of cis-monenoic fatty acids (e.g., cis-delta 9-16:1 plus cis-delta 11-18:1) the DSC curve showed a broad gel to liquid crystalline phase change below 0 degrees C but the ESR studies revealed no discontinuities at temperatures above those of the gel to liquid-crystalline transition. These results demonstrated that enrichment of the membrane lipids with molecules in which both fatty acyl chains are identical cis-monoenoic residues led to a distinct type of liquid-crystalline phase. Furthermore, a general conclusion from this study is that Escherichia coli normally maintains a heterogeneous mixture of lipid molecules and, by so doing, prevents strong lipid-lipid associations that lead to the formation of lipid domains in the membrane.     

Reversible unfolding of beta-sheets in membranes: a calorimetric study

The hexapeptide acetyl-Trp-Leu(5) (AcWL(5)) has the remarkable ability to assemble reversibly and spontaneously into beta-sheets on lipid membranes as a result of monomer partitioning followed by cooperative assembly. This system provides a unique opportunity to study the thermodynamics of protein folding in membranes, which we have done using isothermal titration calorimetry (ITC) and differential scanning calorimetry (DSC). The results, which may represent the first example of reversible thermal unfolding of peptides in membranes, help to define the contribution of hydrogen bonding to the extreme thermal stability of membrane proteins. ITC revealed that the enthalpy change for partitioning of monomeric, unstructured AcWL(5) from water into membranes was zero within experimental error over the temperature range of 5 degrees C to 75 degrees C. DSC showed that the beta-sheet aggregates underwent a reversible, endothermic, and very asymmetric thermal transition with a concentration-dependent transition temperature (T(m)) in the range of 60 degrees C to 80 degrees C. A numerical model of nucleation and growth-dependent assembly of oligomeric beta-sheets, proposed earlier to describe beta-sheet formation in membranes, recreated remarkably well the unusual shape and concentration-dependence of the transition peaks. The enthalpy for thermal unfolding of AcWL(5) beta-sheets in the membrane was found to be about 8(+/-1)kcal mol(-1), or about 1.3(+/-0.2)kcal mol(-1) per residue.     

Laminin polymerization in vitro. Evidence for a two-step assembly with domain specificity

Laminin, a major structural glycoprotein of basement membranes, has been found to self-associate in vitro into large polymers. The formation of these complexes can be followed by the development of turbidity upon incubation in neutral phosphate buffer at 21-35 degrees C and is seen to be time-, concentration-, and temperature-dependent. The process is thermally reversible at 4 degrees C and the protein can be cycled between a dispersed and an aggregated state by alternating between 4 and 35 degrees C. Following incubation at 35 degrees C much of the monomeric laminin, which sediments at 11.4 S, is now seen to sediment at greater than 25 S. Both by turbidometric and sedimentation analysis, an apparent critical concentration for assembly of about 0.1 mg/ml (10(-7) M) is observed and is interpreted as evidence for a nucleation-propagation polymerization mechanism. The relative paucity of intermediates seen in a size-distribution analysis lends further support for this model. On platinum replicas obtained by rotary shadowing analysis, mostly free monomers are seen in the cold while after incubation at 35 degrees C, large multimeric aggregates with smaller amounts of oligomers are observed. The interaction between individual molecules appears to be specific because the dimers, trimers, and smaller oligomers are only associated at the terminal globular domains of the laminin molecules. In addition, removal of the globular domains of laminin with pepsin, which yields fragment P1, abolishes self-association. A divalent cation dependency for polymerization can be demonstrated and incubation in the presence of EDTA stops the polymerization at an oligomeric intermediate step. Hence overall laminin self-assembly can be divided into at least two steps: an initial temperature-dependent, divalent cation independent step followed by a divalent cation-dependent step.     

The intracellular chloride ion channel protein CLIC1 undergoes a redox-controlled structural transition

Most proteins adopt a well defined three-dimensional structure; however, it is increasingly recognized that some proteins can exist with at least two stable conformations. Recently, a class of intracellular chloride ion channel proteins (CLICs) has been shown to exist in both soluble and integral membrane forms. The structure of the soluble form of CLIC1 is typical of a soluble glutathione S-transferase superfamily protein but contains a glutaredoxin-like active site. In this study we show that on oxidation CLIC1 undergoes a reversible transition from a monomeric to a non-covalent dimeric state due to the formation of an intramolecular disulfide bond (Cys-24-Cys-59). We have determined the crystal structure of this oxidized state and show that a major structural transition has occurred, exposing a large hydrophobic surface, which forms the dimer interface. The oxidized CLIC1 dimer maintains its ability to form chloride ion channels in artificial bilayers and vesicles, whereas a reducing environment prevents the formation of ion channels by CLIC1. Mutational studies show that both Cys-24 and Cys-59 are required for channel activity.     

Temperature-dependent changes in the swimming behaviour of Tetrahymena pyriformis-NT1 and their interrelationships with electrophysiology and the state of membrane lipids

The swimming velocity and the amplitude of the helical swimming path of T. pyriformis-NT1 cells grown at 20 degrees C (Tg 20 degrees C) and 38 degrees C (Tg 38 degrees C) were monitored between 0 and 40 degrees C in the presence and absence of electric fields. Within physiological limits the swimming velocity increased and the amplitude decreased as temperature was raised. The temperature profiles of these properties were not linear, and showed discontinuities at different temperatures for the different cultures. The break points in Arrhenius plots of the resting potential, regenerative spike magnitude, repolarization time, swimming velocity and swimming amplitude are tabulated and compared. The initial breakpoints upon cooling were clustered about the breakpoints in fluorescence polarization of D.P.H. in extracted phospholipids, and around the transition temperatures estimated from the literature for the pellicular membrane of these cells. The average of the initial breakpoints on cooling was 22.9 degrees C for Tg 38 degrees C cells and 13.7 degrees C for Tg 20 degrees C cells, a shift of 9.2 degrees C. Unlike Paramecium there is no depolarizing receptor potential in Tetrahymena upon warming. It is suggested that this may be the basis of a behavioural difference between Tetrahymena and Paramecium--namely that in Tetrahymena maximum swimming velocity occurs above growth temperature whereas in Paramecium the two points coincide. Swimming velocity and resting potential were correlated with membrane fluidity within physiological limits, but for other parameters the relationship with fluidity was more complex.(ABSTRACT TRUNCATED AT 250 WORDS)     

Temperature sensitivity of the erythrocyte membrane potential as determined by cyanine dye fluorescence

We have used the cyanine dye fluorescence technique to measure the membrane potential of human erythrocytes as a function of temperature. With erythrocytes starved of glucose, there is an abrupt decrease in membrane potential centered at 38 degrees which is reversible up to 41 degrees, and irreversible at higher temperatures. With erythrocytes supplemented with glucose, the thermally induced transition is centered at 41 degrees and is reversible up to the highest temperature measured, 45 degrees. These results extend previous spectroscopic studies with erythrocyte membranes which demonstrated a thermally induced transition in protein tertiary or quaternary structure that is irreversible above 42 degrees.     

Cooperative phenomena in the membrane potential of parathyroid cells induced by divalent cations

Membrane potentials of mouse parathyroid cells were measured by means of the intracellular microelectrode method. The membrane potential in external Krebs solution containing 2.5 mM of Ca++ was -23.6 +/- 0.4 mV (mean +/- standard error of mean). The low concentration of Ca++ (1.0 mM) caused hyperpolarization of the membrane potential to -61.7 +/- 0.8 mV. The membrane potential was proportional to the logarithm of the concentration of K ion in the solution of low Ca ion. The concentration of external Na+, C1- and HPO4-- had no effect on the membrane potential. The sigmoidal transition of membrane potentials was induced by the change of Ca ion concentration in the range from 2.5 to 1.0 mM. The change of the membrane potentials in low Ca ion is originated from increase in potassium permeability of the cell membrane. The similar sigmoidal changes of the membrane potentials were observed in the solution containing 4 to 3 mM of Sr ion. The Mg and Ba ion showed smaller effect on the membrane potential. The Goldman equation was extended to divalent ions. Appling the extended membrane potential equation, ratios of the permeability coefficients were obtained as follows: PK/PCa = 0.067 for 2.5 mM Ca++, 0.33 for 1.0 mM Ca++; PK/PSr = 0.08 for 4 mM Sr++ and 0.4 for 3 mM Sr++; PK/PMg = 0.5; PK/PBa = 0.67 for all range of concentration. The Hill constants of Sr ion and Ca ion were 20; the relationship between Sr ion and Ca ion was competitive. The Hill constants of Mg and Ba ion were 1 each. The Hill constant of Ca ion was depend of the temperature; nmax = 20 at 36 degrees C, n = 9 at 27 degrees C, n = 2 at 22 degrees C. The enthalpy of Ca-binding reaction was obtained from the Van't Hoff plot as 0.58 kcal. The activation energies of the K+ permeability increase were obtained from the Arrhenius plots as 3.3 kcal and 4 kcal. The difference, 0.7 kcal, corresponds to the enthalpy change of this reaction, of which value is close to that of the Ca-binding reaction.     

Reversible surface aggregation in pore formation by pardaxin.

The mechanism of leakage induced by surface active peptides is not yet fully understood. To gain insight into the molecular events underlying this process, the leakage induced by the peptide pardaxin from phosphatidylcholine/ phosphatidylserine/cholesterol large unilamellar vesicles was studied by monitoring the rate and extent of dye release and by theoretical modeling. The leakage occurred by an all-or-none mechanism: vesicles either leaked or retained all of their contents. We further developed a mathematical model that includes the assumption that certain peptides become incorporated into the vesicle bilayer and aggregate to form a pore. The current experimental results can be explained by the model only if the surface aggregation of the peptide is reversible. Considering this reversibility, the model can explain the final extents of calcein leakage for lipid/peptide ratios of > 2000:1 to 25:1 by assuming that only a fraction of the bound peptide forms pores consisting of M = 6 +/- 3 peptides. Interestingly, less leakage occurred at 43 degrees C, than at 30 degrees C, although peptide partitioning into the bilayer was enhanced upon elevation of the temperature. We deduced that the increased leakage at 30 degrees C was due to an increase in the extent of reversible surface aggregation at the lower temperature. Experiments employing fluorescein-labeled pardaxin demonstrated reversible aggregation of the peptide in suspension and within the membrane, and exchange of the peptide between liposomes. In summary, our experimental and theoretical results support reversible surface aggregation as the mechanism of pore formation by pardaxin.     

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.     

Effect of temperature on the formation and inactivation of syringomycin E pores in human red blood cells and bimolecular lipid membranes

The effects of temperature on the formation and inactivation of syringomycin E (SRE) pores were investigated with human red blood cells (RBCs) and lipid bilayer membranes (BLMs). SRE enhanced the RBC membrane permeability of 86Rb and monomeric hemoglobin in a temperature dependent manner. The kinetics of 86Rb and hemoglobin effluxes were measured at different temperatures and pore formation was found to be only slightly affected, while inactivation was strongly influenced by temperature. At 37 degrees C, SRE pore inactivation began 15 min after and at 20 degrees C, 40 min after SRE addition. At 6 degrees C, below the phase transition temperature of the major lipid components of the RBC membrane, no inactivation occurred for as long as 90 min. With BLMs, SRE induced a large current that remained stable at 14 degrees C, but at 23 degrees C it decreased over time while the single channel conductance and dwell time did not change. The results show that the temperature dependent inactivation of SRE pores is due to a decrease in the number of open pores.     

Thermohaemolysis of human erythrocytes in isotonic NaCl/sucrose media during transient heating

1. 1.|The hyperhermia induced haemolysis of cells and resealed ghosts suspended in isotonic NaCl/sucrose media was studied upon transient heating.

2. 2.|At 61.5°C a process of temperature accelerated disturbance of membrane permeability barrier was initiated, wich was sensed by the consequent volume changes. Concomitantly with this process the thermohaemolysis appeared as a threshold colloid-osmotic lysis.

3. 3.|The initial temperature of this successive barrier disturbance was decreased linearly by ethanol. At 18% ethanol this barrier disturbance took place at 39°C while spectrin was denaturated at about 45°C. Apparently, the spectrin denaturation was not sufficient, nor was involved in, the initiation of this membrane disturbance.

4. 4.|The membrane of cells made ion permeable in the presence of 18% ethanol by heating to 39°C contained irreversible pores with a radius of about 0.45 nm.

5. 5.|This suggests a conformational change of a protein(s) in their formation, but not spectrin nor the anion channel.

6. 6.|Using specific amino reagents it was ascertained, that a superficial NH₃⁺ group dissociable at neutral pH impeded this thermo-induced pore formation.

7. 7.|Consistent results show that this formation of membrane pores initiated at 61.5°C may be included in the still unknown mechanism of thermohaemolysis.