Characterization of thermotropic structural transitions of the erythrocyte membrane: a biochemical and electron-paramagnetic resonance approach

The relationship between membrane structural properties and functions has been generally inferred from observed thermotropic phenomena. By the use of 16-dinyloxyl stearic acid spin probe we investigated the red blood cell membrane components involved in three characteristic thermotropic structural transitions occurring at 8, 20, and 40 degrees C. The transition at 8 degrees C is removed by chymotrypsin treatment at the cytoplasmic membrane layer. The 20 degrees C phase transition is unmodified after chymotrypsin treatment and occurs at 15 degrees C after complete proteolysis of intramembrane chymotrypsin-insensitive peptides. Liposomes from the total lipid extract of RBC show only one thermotropic transition at 15 degrees C. The 40 degrees C phase transition is absent in vesicles free of skeletal proteins, in vesicles obtained after RBC storage, and in low-ionic-strength resealed ghosts. Transitions at 8 degrees C and 40 degrees C appear to be due to the interactions of cytoplasmic exposed proteins with membrane, whereas the 20 degrees C transition is intrinsic to the lipid component.     

Dual mesomorphic assemblage of chitin normal acylates and rapid enthalpy relaxation of their side chains

Chitin derivatives having normalacyl groups (C(n)H(2n-1)O-; n = 4-20) were synthesized with pyridine, p-toluenesulfonyl chloride, and normal alkanoic acid in an N,N-dimethylacetamide-lithium chloride homogeneous system. The products (C(n)-ACs; degree of acyl substitution, DS = 1.7-1.9) showed an n-dependent thermal transition behavior: no evident transition (n = 4-10), a glass transition (n = 12 and 14), and a pseudo-first-order phase transition (n = 16-20), the latter two occurring usually below room temperature when examined by differential scanning calorimetry. Wide-angle X-ray diffractometry (WAXD) at 20 degrees C displayed a sharp diffraction peak (2theta = 2 degrees -7 degrees ) and a diffuse halo (2theta approximately 20 degrees ) for the respective C(n)-ACs. The former d-spacing (1.5-3.6 nm) increased with an increase in n to yield two stages of mutually different increasing rates, which reflects a systematic n-dependence of the period of a layered structure of the main chains. The molecular assembly of C(n)-ACs exhibited "dual mesomorphy"; nematic ordering for the semirigid carbohydrate trunk and smectic one for the flexible side chains. On the other hand, WAXD profiles of C(n)-ACs (n = 14-18) indicated almost no temperature dependence from -150 to +220 degrees C. Therefore, it was reasonably assumed that the pseudo-first-order transition observed in thermograms of C(n)-ACs (n = 16-20) was due to the enthalpy relaxation of the side-chain assemblage. An insight was provided into the kinetics of the characteristic aging behavior as a liquid-crystalline glass, in comparison with the corresponding data for other noncrystalline macromolecules.     

Inverted hexagonal and cubic phases induced by alpha-tocopherol in fully hydrated dispersions of dilauroylphosphatidylethanolamine

The effect of alpha-tocopherol on the thermotropic phase behaviour and structure of aqueous dispersions of 1,2-di-lauryl-sn-glycero-3-phosphoethanolamine was examined by synchrotron X-ray diffraction. The pure phospholipid exhibited a lamellar gel to liquid-crystal phase transition at 30 degrees C on heating at 3 degrees C min(-1) between 10 degrees C and 90 degrees C. The transition was reversible with a temperature hysteresis of 0.3 degrees C on cooling. At temperatures less than 10 degrees C only lamellar gel phase of the pure phospholipid was seen in co-dispersions of up to 20 mol % alpha-tocopherol. The presence of 2.5 mol % alpha-tocopherol caused the appearance of inverted hexagonal phase at temperatures just below the main phase transition temperature that co-existed with the lamellar gel phase. The intensity of scattering from the hexagonal-II phase increased with increasing proportion of alpha-tocopherol in the mixture and in proportions greater than 10 mol % it persisted at temperatures above the main transition and co-existed with the lamellar liquid-crystal phase of the pure phospholipid. At higher temperatures all co-dispersions containing up to 15 mol % alpha-tocopherol showed the presence of cubic phases. These phases indexed a Pn3m or Pn3 space grouping. When the proportion of alpha-tocopherol was increased to 20 mol % the only non-lamellar phase observed was inverted hexagonal phase. This phase co-existed with lamellar gel and liquid-crystal phases of the pure phospholipid, but was the only phase present at temperatures >60 degrees C. The X-ray diffraction data were used to construct a partial phase diagram of the lipid mixture in excess water between 10 degrees and 90 degrees C and up to 20 mol % alpha-tocopherol in phospholipid.     

Heterologous expression and optimized one-step separation of levansucrase via elastin-like polypeptides tagging system

Elastin-like polypeptides (ELPs) undergo a reversible inverse phase transition upon a change in temperature. This thermally triggered phase transition allows for a simple and rapid means of purifying a fusion protein. Recovery of ELPs-tagged fusion protein was easily achieved by aggregation, triggered either by raising temperature or by adding salt. In this study, levansucrase has been used as a model enzyme in the development of a simple one-step purification method using ELPs. The levansucrase gene cloned from Pseudomonas aurantiaca S-4380 was tagged with various sizes of ELPs to functionally express and optimize the purification of levansucrase. One of two ELPs, ELP[V-20] or ELP[V-40], was fused at the C-terminus of the levansucrase gene. A levansucrase-ELP fusion protein was expressed in Escherichia coli DH5alpha at 37 degrees C for 18 h. The molecular masses of levansucrase-ELP[V-20] and levansucrase-ELP[V-40] were determined as 56 kDa and 65 kDa, respectively. The phase transition of levansucrase-ELP[V-20] occurred at 20 degrees C in 50 mM Tris-Cl (pH 8) buffer with 3 M NaCl added, whereas the phase transition temperature (Tt) of levansucrase-ELP[V-40] was 17 degrees C with 2 M NaCl. Levansucrase was successfully purified using the phase transition characteristics of ELPs, with a recovery yield of higher than 80%, as verified by SDS-PAGE. The specific activity was measured spectrophotometrically to be 173 U/mg and 171 U/mg for levansucrase-ELP[V-20] and levansucrase-ELP[V-40], respectively, implying that the ELP-tagging system provides an efficient one-step separation method for protein purification.     

Phase transitions as a function of osmotic pressure in Saccharomyces cerevisiae whole cells, membrane extracts and phospholipid mixtures

Fourier Transform Infrared spectroscopy (FTIR) was used to determine the phase transition temperature of whole Saccharomyces cerevisiae W303-1 A cells as a function of Aw in binary water-glycerol media. A phase transition occurred at 12 degrees C in water, at 16.5 degrees C at Aw=0.75, and at 19.5 degrees C at Aw=0.65. The temperature ranges over which transition occurred increased with decreasing Aw. A total lipid extract of the plasma membranes isolated from S. cerevisiae cells was also studied, with a phase transition temperature determined at 20 degrees C in pure water and at 27 degrees C in binary water-glycerol solutions for both Aw levels tested. The pure phospholipids dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylethanolamine (DMPE) and three binary mixtures of these phospholipids (percentage molar mixtures of DMPC/DMPE of 90.5/9.5, 74.8/25.2, and 39.7/60.3) were studied. For DMPC, there was no influence of Aw on the phase transition temperature (always 23 degrees C). On the other hand, the phase transition temperature of DMPE increased with decreasing Aw for the three aqueous solutions tested (glycerol, sorbitol and sucrose), from 48 degrees C in water, to 64 degrees C for a solution at Aw=0.67. For the DMPC/DMPE mixtures, transitions were found intermediate between those of the two phospholipids, and a cooperative state was observed between species at the gel and at the fluid phases.     

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).     

Polytetrapeptide of elastin. Temperature-correlated elastomeric force and structure development

High molecular weight polytetrapeptide of elastin, (L.Val1-L.Pro2-Gly3-Gly4)n, was synthesized using activation of the (GGVP) permutation for polymerization. The temperature-dependence of aggregation was characterized as a function of concentration and the circular dichroism spectra were obtained in the 20 degrees to 70 degrees C temperature range. The latter showed an inverse temperature transition centered near 50 degrees C in which polypeptide order increased on raising the temperature. A concentration of 0.6 g of polytetrapeptide in 1 g of water was gamma irradiation cross-linked (20 Mrad) to form an elastomeric matrix. A study of the temperature-dependence of elastomeric force demonstrated a transition toward increased force on raising the temperature with a midpoint of the transition near 50 degrees C. Thus, there is a correlation between increase in intramolecular order and elastomeric force development. These results are compared to previous results on the polypentapeptide of elastin, (VPGVG)n and on an analog, (IPGVG)n, to demonstrate that the temperature of the transition is proportional to the hydrophobicity of the repeating unit. The point is noted that the elastomeric force development correlates better with intramolecular ordering than with intermolecular processes.     

Calorimetric and spectroscopic studies of lipid thermotropic phase behavior in liver inner mitochondrial membranes from a mammalian hibernator

Arrhenius plots of various enzyme and transport systems associated with the liver mitochondrial inner membranes of ground squirrels exhibit changes in slope at temperatures of 20-25 degrees C in nonhibernating but not in hibernating animals. It has been proposed that the Arrhenius breaks observed in nonhibernating animals are the result of a gel to liquid-crystalline phase transition of the mitochondrial membrane lipids, which also occurs at 20-25 degrees C, and that the absence of such breaks in hibernating animals is due to a major depression of this lipid phase transition to temperatures below 4 degrees C. In order to test this hypothesis, we have examined the thermotropic phase behavior of liver inner mitochondrial membranes from hibernating and nonhibernating Richardson's ground squirrels, Spermophilus richardsonii, by differential scanning calorimetry and by 19F nuclear magnetic resonance and fluorescence polarization spectroscopy. Each of these techniques indicates that no lipid phase transition occurs in the membranes of either hibernating or nonhibernating ground squirrels within the physiological temperature range of this animal (4-37 degrees C). Moreover, differential scanning calorimetric measurements indicate that only a small depression of the lipid gel to liquid-crystalline phase transition, which is centered at about -5 degrees C in nonhibernating animals and at about -9 degrees C in hibernators, occurs. We thus conclude that the Arrhenius plot breaks observed in some membrane-associated enzymatic and transport activities of nonhibernating animals are not the result of a lipid phase transition and that a major shift in the gel to liquid-crystalline lipid phase transition temperature is not responsible for seasonal changes in the thermal behavior of these inner mitochondrial membrane proteins.     

Poly(rA).poly(rU) with Ni(2+) ions at different temperatures: infrared absorption and vibrational circular dichroism spectroscopy

Phase transitions were studied of the sodium salt of poly(rA).poly(rU) induced by elevated temperature without Ni(2+) and with Ni(2+) in 0.07 M concentration in D(2)O (approximately 0.4 [Ni]/[P]). The temperature was varied from 20 degrees C to 90 degrees C. The double-stranded conformation of poly(rA).poly(rU) was observed at room temperature (20 degrees C-23 degrees C) with and without Ni(2+) ions. In the absence of Ni(2+) ions, partial double- to triple-strand transition of poly(rA).poly(rU) occurred at 58 degrees C, whereas only single- stranded molecules existed at 70 degrees C. While poly(rU) did not display significant helical structure, poly(rA) still maintained some helicity at this temperature. Ni(2+) ions significantly stabilized the triple-helical structure. The temperature range of the stable triple-helix was between 45 degrees C and 70 degrees C with maximum stability around 53 degrees C. Triple- to single-stranded transition of poly(rA).poly(rU) occurred around 72 degrees C with loss of base stacking in single-stranded molecules. Stacked or aggregated structures of poly(rA) formed around 86 degrees C. Hysteresis took place in the presence of Ni(2+) during the reverse transition from the triple-stranded to the double-stranded form upon cooling. Reverse Hoogsteen type of hydrogen-bonding of the third strand in the triplex was suggested to be the most probable model for the triple-helical structure. VCD spectroscopy demonstrated significant advantages over infrared absorption or the related electronic CD spectroscopy.     

Studies on the anomalous thermotropic behavior of aqueous dispersions of dipalmitoylphosphatidylcholine-cholesterol mixtures

Examination of the thermotropic behavior of aqueous dispersions of dipalmitoylphosphatidylcholine-cholesterol mixtures by high-sensitivity scanning calorimetry has revealed that the phospholipid gel to liquid-crystalline phase transition consists of two components. One, a relatively sharp transition centered at 39.6-40.7 degrees C, exhibits a transition enthalpy change which decreases linearly with increasing cholesterol content, approaching zero at a cholesterol content of about 25 mol %. The other, a broad, lower intensity transition centered at approximately 41.5 degrees C for cholesterol concentrations of 20 mol %, displays an enthalpy change which is maximal at about 20-25 mol % cholesterol and which decreases as the cholesterol content decreases to zero or increases above 25 mol %. The origin of these two transitions is discussed in terms of a separation of these lipid mixtures into cholesterol-rich and cholesterol-poor domains.     

Binding of basic pancreatic trypsin inhibitor and related isoinhibitors to leukocytic elastase. Determination of thermodynamic parameters

The effect of temperature, ionic strength and solvation power of mono- and divalent cations on the interaction of BPTI-like inhibitors with human leukocytic elastase has been determined. The binding process is characterized by a non-linear dependence of the equilibrium association constant on 1/T indicating a thermal transition at temperature values ranging between 20 degrees C and 35 degrees C depending on the solvent. The marked dependence of the thermodynamic parameters (delta H degrees, delta S degrees, delta G degrees) and of the transition temperature on the concentration and nature of the cations present in solution seems to indicate that the transition, probably of conformational nature, is related to removal of water molecules upon enzyme/inhibitor complex formation.     

Charge changes in loop 2 affect the thermal unfolding of the myosin motor domain bound to F-actin

The thermal unfolding of Dictyostelium discoideum myosin head fragments with alterations in the actin-binding surface loop 2 was studied by differential scanning calorimetry. Lengthening of loop 2 without concomitant charge changes led to decreases in the transition temperature of not more than 1.8 degrees C. Insertions with multiple positive or negative charges had a stronger destabilizing effect and led to reductions in the thermal transition temperature of up to 3.7 degrees C. In the presence of nucleotide, most mutants displayed similar or higher transition temperatures than M765. Only constructs M765(11/+6) and M765(20/+12) with long positively charged inserts showed transition temperatures that were more than 2 degrees C below the values measured for M765 in the presence of ADP, ADP-V(i), and ADP-BeF(3). Interaction with F-actin in the presence of ADP shifted the thermal transition of M765 by 6 degrees C, from 49.1 to 55.1 degrees C. The actin-induced increase in thermal stability varied between 1.2 and 9.1 degrees C and showed a strong correlation with the mutant constructs' affinity for actin. Our results show that length and charge changes in loop 2 do not significantly affect nucleotide-induced structural changes in the myosin motor domain, but they affect structural changes that occur when the motor domain is strongly bound to actin and affect the coupling between the actin- and nucleotide-binding sites.     

Effects of temperature on cytochrome oxidase activity in solubilized form and in lipid vesicle systems.

Isolated mammalian cytochrome oxidase gave an Arrhenius plot with a break (Tb) at about 20 degrees C when assayed in a medium containing Emasol. The activation energies above and below 20 degrees C were 9.3 (EH) and 18.9 kcal/mol (EL), respectively. Isolated cytochrome oxidase was also incorporated into vesicles of dipalmitoyl phosphatidylcholine (DPPC, phase transition temperature Tt = 40 degrees C), dimyristoyl phosphatidylcholine (DMPC, Tt = 23 degrees C) and dioleoyl phosphatidylcholine (DOPC, Tt = -22 degrees C). The DPPC system showed a nearly linear Arrhenius plot between 9 and 36 degrees C with E = 22.8 kcal/mol. When cytochrome oxidase was resolubilized from the DPPC vesicles and assayed in solution a biphasic plot was obtained again. Cytochrome oxidase-DOPC was more active than the solubilized enzyme and exhibited a biphasic Arrhenius plot with Tb = 23 degrees C. EH and EL were 6.6 and 15.8 kcal/mol, respectively. The plot for the oxidase-DMPC also showed a break (Tb = 26 degrees C) with EH = 6.6 and EL = 26.6 kcal/mol. These results indicate that the break in the Arrhenius plot reflects primarily a structural transition in the cytochrome oxidase molecule between the "hot" and "cold" conformations, as proposed previously. This transition, as well as the molecular state of cytochrome oxidase, is affected by the physical state of the membrane lipids as reflected by changes in the kinetic properties.     

Thermal transitions in erythrocyte membranes revealed by their permeability to ANS

A number of breaks were recorded on the curve of Arrhenius relationship of the rate constant of the dye 1-anilino-8-naphthalenesulphonate sodium salt (ANS) input into human erythrocytes of 20, 28, 36, 42 and 46 degrees C. Variations in the values of activation energies within the temperature range of 28-36 degrees and 42-46 degrees C obtained in various blood samples allow to consider these temperatures as those at which structural changes of the membranes take place. The values of activation energy of the process for temperature "conformers" of the erythrocyte membrane are 12(10-20 degrees C), 26.5 (20-28 degrees C), 34.2(36-42 degrees C) and 47 kcal/mol (t is greater than 46 degrees C). Within the temperature range of 28-36 degrees and 42-46 degrees C an irreversible decrease of permeability to ANS of the erythrocyte ghost after their incubation for 10 min at increased temperatures were observed. Thus the temperature regions of the change in erythrocyte permeability correspond to those at which the resealing of ghost takes place. The break in Arrhenius graph at 20 degrees C seems to characterize a highly cooperative "point" transition. The lipid nature of the initiator of structural transition within 28-36 degrees C is proved by a sharp increase of the permeability of liposomes prepared from erythrocyte membrane lipids to ANS at 28 degrees C. The nature of the initiators of two other thermal transitions is discussed.     

Structure and metastability of N-lignocerylgalactosylsphingosine (cerebroside) bilayers

Differential scanning calorimetry (DSC) and X-ray diffraction have been used to study hydrated N-lignocerylgalactosylsphingosine (NLGS) bilayers. DSC of fully hydrated NLGS shows an endothermic transition at 69-70 degrees C, immediately followed by an exothermic transition at 72-73 degrees C; further heating shows a high-temperature (Tc = 82 degrees C), high-enthalpy (delta H = 15.3 kcal/mol NLGS) transition. Heating to 75 degrees C, cooling to 20 degrees C and subsequent reheating shows no transitions at 69-73 degrees C; only the high-temperature (82 degrees C), high-enthalpy (15.3 kcal/mol) transition. Two exothermic transitions are observed on cooling; for the upper transition its temperature (about 65 degrees C) and enthalpy (about 6 kcal/mol NLGS) are essentially independent of cooling rate, whereas the lower transition exhibits marked changes in both temperature (30----60 degrees C) and enthalpy (2.2----9.5 kcal/mol NLGS) as the cooling rate decreases from 40 to 0.625 Cdeg/min. On reheating, the enthalpy of the 69-70 degrees C transition is dependent on the previous cooling rate. The DSC data provide clear evidence of conversions between metastable and stable forms. X-ray diffraction data recorded at 26, 75 and 93 degrees C show clearly that NLGS bilayer phases are present at all temperatures. The X-ray diffraction pattern at 75 degrees C shows a bilayer periodicity d = 65.4 A, and a number of sharp reflections in the wide-angle region indicative of a crystalline chain packing mode. This stable bilayer form converts to a liquid-crystal bilayer phase; at 93 degrees C, the bilayer periodicity d = 59.1 A, and a diffuse reflection at 1/4.6 A-1 is observed. The diffraction pattern at 22 degrees C represents a combination of the stable and metastable low-temperature bilayer forms. NLGS exhibits a complex pattern of thermotropic changes related to conversions between metastable (gel), stable (crystalline) and liquid-crystalline bilayer phases. The structure and thermotropic properties of NLGS are compared with those of hydrated N-palmitoylgalactosylsphingosine reported previously (Ruocco, M.J., Atkinson, D., Small, D.M., Skarjune, R.P., Oldfield, E. and Shipley, G.G. (1981) Biochemistry 20, 5957-5966).     

Thermal transitions of red blood cell deformability. Correlation with membrane rheological properties

Red blood cell deformability has been studied by the initial filtration flow rate as a function of temperature. The well-known transition at 49-50 degrees C (probably due to spectrin denaturation) is shown. Another transition is demonstrated around 18 degrees C (the cell becomes stiffer below this temperature range). The erythrocyte membranes prepared by a mild dialysis technique have the same deformability as intact erythrocytes at room temperature; they also show the same low-temperature transition. No such transition has been found for hemoglobin solutions of viscosity 30 g X dl-1. It is interesting to compare these results with those obtained by other methods which measure the properties of natural or artificial lipid membranes and which also demonstrate a thermal transition at 15-20 degrees C. Therefore, the deformability of intact normal erythrocytes seems to depend mainly on the rheological properties of the membrane.     

The thermal transition in crude myelin proteolipid has a lipid rather than protein origin

Myelin proteolipid has been isolated from bovine brain and purified using organic solvents according to conventional procedures. The protein content of the purified sample, or crude proteolipid, contains a minimum of 75% w/w of proteolipid, with DM-20, a proteolipid molecule with an internal deletion of 35 out of 276 amino acid residues, as the only other component. Biochemical analysis has shown the differences in lipid composition between brain white matter, myelin and crude proteolipid preparations. The latter contained practically no cholesterol, while the other two samples had about 22-23% w/w. High-sensitivity differential scanning calorimetry experiments with both crude proteolipid and its extracted pool of lipids have shown similar reversible thermal transitions at 52 degrees C and 48 degrees C. The effect of increasing amounts of cholesterol on the two calorimetric transitions led in both cases to a continuous decrease in the melting temperature and in the transition enthalpy. Parallel Fourier-transform infrared spectroscopy studies of crude proteolipid have detected a reversible, co-operative lipid transition centred at 49 degrees C, with no detectable change in the amide region between 20 degrees C and 60 degrees C. Once more an increase in cholesterol content led to a decrease in the sharpness of this transition. It is concluded that the thermal transition detected in crude proteolipid, which has in the past been attributed to proteolipid thermal denaturation (Mateo et al. 1986), actually corresponds to a thermotropic phase transition of the lipids included in the crude proteolipid sample.     

Thermal stability and conformational changes of transglutaminase from a newly isolated Streptomyces hygroscopicus

Thermal stability and conformational changes of transglutaminase (TGase) from a newly isolated Streptomyces hygroscopicus were investigated in this study. The inactivation kinetics of the microbial transglutaminase (MTGase) was fitted using one-step inactivation model. It was much more stable under 40 degrees C. The half-lives for the MTGase at 50 degrees C and 60 degrees C were only 20 min and 8 min, respectively. Spectroscopic studies of the enzyme suggested conformational transition from ordered secondary structural elements (alpha/beta-protein) to unordered structure during thermal denaturation. Some polyols could improve the thermal stability of the enzyme. Among the polyols examined, the prolonged half-lives of 40 min at 50 degrees C and 20 min at 60 degrees C were gained by adding 10% glycerol. The results of differential scanning calorimetric (DSC) analysis showed a distinct transition peak with a significant greater Tm and DeltaH for the MTGase mixed with polyols in comparison with the control, which indicated that the polyols could maintain the natural structure of the enzyme to some extent. The SDS-PAGE electrophoresis of cross-linked casein confirmed that the stabilizers could protect the MTGase from thermal denaturation.     

Temperature dependence of unitary properties of an ATP-dependent potassium channel in cardiac myocytes.

The temperature dependence of the properties of unitary currents in cultured rat ventricular myocytes has been studied. Currents flowing through an ATP-dependent K+ channel were recorded from inside-out patches with the bath temperature varied from 10 degrees to 30 degrees C. The channel conductance was 56 pS at room temperature (22 degrees C), and the amplitudes of unitary currents and the channel conductance exhibited a relatively weak (Q10 from 1.4 to 1.6) dependence on temperature. The temperature dependence of channel mean open times was biphasic with the low temperature (10-20 degrees C) range showing a relatively stronger temperature dependence (Q10 of 2.3) than the high temperature (20-30 degrees C) range (Q10 of 1.6). The activation energies for the two regions were determined from an Arrhenius plot with the activation energy, corresponding to the lower temperature range, near 16 kcal/mol. Thermodynamic analysis, using transition rate theory, indicated that the formation of a transition state prior to channel closure to be associated with a positive entropy component for the high Q10 region.     

Ultrasonic studies of lipid bilayer. Phase transition in synthetic phosphatidylcholine liposomes

The ultrasonic velocity at 3 MHz and the density in the nonsonicated and sonicated liposomes of dipalmitoylphosphatidylcholine have been measured in the temperature range from 0 degrees C to 55 degrees C. The results indicate that nonsonicated multilamellar vesicles undergo a weak first order transition which is analogous to the nematic-isotropic transition of liquid crystals. A sharp change in the ultrasonic velocity associated with the first order transition disappears when the multilamellar vesicles are sonicated. The bulk modulus of the lipid bilayer calculated from the ultrasonic velocity and the density of sonicated liposomes has a value of 3.0 X 10(10) dyne/cm2 at 20 degrees C, reaches a minimum value of 2.1 X 10(10) dyne/cm2 at its transition temperature and increases slightly to 2.2 X 10(10) dyne/cm2 at 50 degrees C.