A calorimetric study of the thermotropic behavior of pure sphingomyelin diastereomers

The phase-transition properties of sphingomyelins were investigated in detail with totally synthetic, chemically and stereochemically pure (2S,3R)-(N-stearoylsphingosyl)-1-phosphocholine (D-erythro-C18-SPM) (1) and the corresponding 2S,3S isomer (L-threo-C18-SPM) (2). Heating scans of an unsonicated dispersion of 1 right after hydration showed a main transition (I) at 44.7 degrees C (delta H = 6.8 kcal/mol). Upon incubation at 20-25 degrees C a second transition (II) appeared at 36.0 degrees C (delta H = 5.7 kcal/mol). The two gel phases were designated as G alpha and G beta phases, respectively. The G beta phase was also metastable and relaxed to a third gel phase (G gamma) upon incubation below 10 degrees C. Conversion of the G gamma phase to the liquid-crystalline phase occurred via two new endotherms at 33.4 degrees C (2.6 kcal/mol) (III) and 43.6 degrees C (8.0 kcal/mol) (IV) as well as a main transition at 44.7 degrees C (9.5 kcal/mol). Possible interpretations have been proposed to account for the observed phase transitions. The L-threo isomer 2 showed similar thermotropic behavior to dipalmitoylphosphatidylcholine (DPPC): a "main transition" at 44.2 degrees C (6.0 kcal/mol), a "pretransition" at 43.1 degrees C (1.8 kcal/mol), and upon incubation at 7 degrees C for 2 weeks, a very broad "subtransition" at ca. 35 degrees C. The results are substantially different from previous studies of sphingomyelins using mixtures of stereoisomers. Mixing of 1 with 2, 1 with DPPC, and 2 with DPPC removed the metastability of the gel phase and resulted in a single transition.     

Temperature dependence of D-glucose transport in reconstituted liposomes

Sodium-dependent D-glucose uptake into proteoliposomes reconstituted from dimyristoylphosphatidylcholine (DMPC) and hog kidney brush border membrane extract is strongly affected by temperature and the physical state of the membranes. This dependence is defined by a nonlinear Arrhenius plot with a break point at 23 degrees C, a temperature not significantly different from the phase transition temperature of the pure lipid (24 degrees C). The transport process is characterized by different activation energies: 35.1 kcal/mol below and 5.5 kcal/mol above the transition temperature. The shift in the break point for the D-glucose transport activity from 15 degrees C, in the brush border membranes, to 23 degrees C in the reconstituted system leads us to conclude that the lipids surrounding the sodium/D-glucose cotransport system can exchange readily with the bulk lipid used for reconstitution. The results thus provide no evidence for the presence of an annulus of specific lipids surrounding the transport system.     

Temperature dependence of the energy-linked monosaccharide transport across the cell membrane of Rhodotorula gracilis.

The temperature dependence of the active monosaccharide transport across the cell membrane of the yeast Rhodotorula gracilis has been studied between 0 and 55 degrees C with D-xylose as the transported substrate: (i) Between 0 and 10 degrees C there is virtually no transport. (ii) The initial velocity of transport increases exponentially from 15 to 30 degrees C (deltaE equal to 32 plus or minus 2 kcal/mol). (iii) At 30 degrees C a sharp "break" occurs in the Arrhenius plot and with increasing temperature the transport becomes inactivated, with a positive slope of the corresponding straight line ("deltaE equal to minus 15 kcal/mol"). (iv) In the temperature range of 50-55 degrees C, both the transport and the metabolic activity cease. In order to account for the abrupt changes of the membrane permeability, we attempted to ascribe them to phase transitions in the membrane structure: the first one, between 10 and 15 degrees C, to the crystalline: liquid-crystalline phase change; the second one, around 30 degrees C, to a change from highly ordered (low entropy) to less ordered (high entropy) membrane structure. Whereas the former phase transition is reversible, the latter appears to be irreversible. Arrhenius plots of the cell respiration exhibit a "break" at 30 degrees C, as well. However, at higher temperatures there is no thermal inactivation of the respiratory activity. The importance of a proper organization of the cell membrane constituents for the efficient transport function is discussed.     

Phase transitions in yeast mitochondrial membranes. The effect of temperature on the energies of activation of the respiratory enzymes of Saccharomyces cerevisiae.

The effect of temperature on the activation energies of mitochondrial enzymes of the yeast Saccharomyces cerevisiae was examined. Non-linear Arrhenius plots with discontinuities in the temperature range 14-19 degrees C and 19-22 degrees C were observed for the respiratory enzymes and mitochondrial ATPase (adenosine triphosphatase) respectively. A straight-line Arrhenius plot was observed for the matrix enzyme, malate dehydrogenase. The activation energies of the enzymes associated with succinate oxidation, namely, succinate oxidase, succinate dehydrogenase and succinate-cytochrome c oxidoreductase, were in the range 60-85kJ/mol above the transition temperature and 90-160kJ/mol below the transition temperature. In contrast, the corresponding enzymes associated with NADH oxidation showed significantly lower activation energies, 20-35kJ/mol above and 40-85kJ/mol below the transition temperature. The discontinuities in the Arrhenius plots were still observed after sonication, treatment with non-ionic detergents or freezing and thawing of the mitochondrial membranes. Discontinuities for cytochrome c oxidase activity were only observed in freshly isolated mitochondria, and no distinct breaks were observed after storage at -20 degrees C. Mitochondrial ATPase activity still showed discontinuities after sonication and freezing and thawing, but a linear plot was observed after treatment with non-ionic detergents. The results indicate that the various enzymes of the respiratory chain are located in a similar lipid macroenvironment within the mitochondrial membrane.     

Kinetic characteristics of soluble and brush border alkaline phosphatase and sucrase activities in developing rat intestine: effect of hormones

Suckling rat intestine contains 35 and 65% of the cytosolic and membrane-bound alkaline phosphatase (AP) activities. The corresponding values for sucrase were 20 and 80% respectively. The amount of the soluble enzymes was reduced to 7-11% in adult rat intestine. Administration of cortisone, thyroxine or insulin to suckling animals induced adult type distribution of the enzymes. There were apparent differences in kinetic characteristics of soluble and brush border enzymes, but the kinetic properties of the normally developed and hormone-induced AP and sucrase were essentially similar. This suggested identical nature of these enzymes under these conditions. A biphasic Arrhenius plot was obtained for AP in weaned and hormone injected pups with a break point around 18 degrees C, while the soluble enzyme yielded a monophasic curve (Ea = 8-11 kcal/mole). Arrhenius plot for sucrase was monophasic in the suckling, hormone-injected and adult rat intestine (Ea = 8.3-15.1 kcal/mole). Membrane-bound enzymes were generally labile, while soluble enzyme activities were stable to heat treatment (sucrase at 50 degrees C and AP at 60 degrees C) in various experimental groups.     

Events leading to the opening and closing of the exocytotic fusion pore have markedly different temperature dependencies. Kinetic analysis of single fusion events in patch-clamped mouse mast cells.

The earliest event in exocytosis is the formation of a fusion pore, an aqueous channel that connects the lumen of a secretory granule with the extracellular space. We can observe the formation of individual fusion pores and their subsequent dilation or closure by measuring the changes in the admittance of patch-clamped mast cells during GTP gamma S-stimulated exocytotic fusion. To investigate the molecular structure of the fusion pore, we have studied the temperature dependency of the rate constants for fusion pore formation and closure. An Arrhenius plot of the rate of fusion pore formation shows a simple linear relationship with an apparent activation energy of 23 kcal/mol. In contrast, the Arrhenius plot of the rate of closure of the fusion pore is discontinuous, with the break at approximately 13 degrees C. Above the break point, the rate of closure has a weak temperature dependence (7 kcal/mol), whereas below 13 degrees C the rate of closure is temperature independent. This type of temperature dependency is characteristic of events that depend on diffusion in a lipid phase that undergoes a fluid-solid phase transition. We propose that the formation of the fusion pore is regulated by the conformational change of a molecular structure with a high activation energy, whereas the closure of the fusion pore is regulated by lipids that become phase separated at 13 degrees C.     

Effect of 25-hydroxy-cholesterol on the uptake of corticosterone in isolated rat liver cells

Isolated rat liver cells are able to take up corticosterone. The Arrhenius plot of the uptake shows a biphasic course with a change in the slope around 25 degrees C. 25-OH-cholesterol is also taken up; this phenomenon reaches a maximum at 10-15 minutes. After preincubating liver cells at 37 degrees C in the presence of this sterol the phase transition is shifted to a higher temperature (32 degrees C) as shown in the Arrhenius plot of the corticosterone uptake. At the same time the uptake of corticosterone is diminished. This cannot readily be explained by direct competition. The mechanism might involve an inhibition of an active uptake mechanism caused by a change in the plasma membrane.     

Thermally induced changes in reconstituted and membranous cytochrome c oxidase.

The Arrhenius plots of electron transport activity in cytochrome c oxidase reconstituted with well-defined phospholipids have been shown to display a change in slope at 20--25 degrees C regardless of the chemical nature of the incorporated lipid. In native membranous cytochrome c oxidase, the discontinuity in Arrhenius activity plot occurred at 16--18 degrees C. These temperature breaks were found to correlate with changes in spin-label mobilities but not with the bulk lipid transition observed by differential scanning calorimetry. Temperature-dependent reciprocal equilibrium between the immobilized and fluid pools is demonstrated. It is suggested that the changes in kinetic and spin-label spectral characteristics in cytochrome c oxidase membranes are related very likely to a lipid-protein interaction prompted by a thermally induced change in the physical state of the lipids that does not involve a gel to liquid crystalline transition.     

Properties of ganglioside GM1 in phosphatidylcholine bilayer membranes.

Gangliosides have been shown to function as cell surface receptors, as well as participating in cell growth, differentiation, and transformation. In spite of their multiple biological functions, relatively little is known about their structure and physical properties in membrane systems. The thermotropic and structural properties of ganglioside GM1 alone and in a binary system with 1,2-dipalmitoyl phosphatidylcholine (DPPC) have been investigated by differential scanning calorimetry (DSC) and x-ray diffraction. By DSC hydrated GM1 undergoes a broad endothermic transition TM = 26 degrees C (delta H = 1.7 kcal/mol GM1). X-ray diffraction below (-2 degrees C) and above (51 degrees C) this transition indicates a micellar structure with changes occurring only in the wide angle region of the diffraction pattern (relatively sharp reflection at 1/4.12 A-1 at -2 degrees C; more diffuse reflection at 1/4.41 A-1 at 51 degrees C). In hydrated binary mixtures with DPPC, incorporation of GM1 (0-30 mol%; zone 1) decreases the enthalpy of the DPPC pretransition at low molar compositions while increasing the TM of both the pre- and main transitions (limiting values, 39 and 44 degrees C, respectively). X-ray diffraction studies indicate the presence of a single bilayer gel phase in zone 1 that can undergo chain melting to an L alpha bilayer phase. A detailed hydration study of GM1 (5.7 mol %)/DPPC indicated a conversion of the DPPC bilayer gel phase to an infinite swelling system in zone 1 due to the presence of the negatively charged sialic acid moiety of GM1. At 30-61 mol % GM1 (zone 2), two calorimetric transitions are observed at 44 and 47 degrees C, suggesting the presence of two phases. The lower transition reflects the bilayer gel --> L alpha transition (zone 1), whereas the upper transition appears to be a consequence of the formation of a nonbilayer, micellar or hexagonal phase, although the structure of this phase has not been defined by x-ray diffraction. At > 61 mol % GM1 (zone 3) the calorimetric and phase behavior is dominated by the micelle-forming properties of GM1; the presence of mixed GM1/DPPC micellar phases is predicted.     

Calorimetry of apolipoprotein-A1 binding to phosphatidylcholine-triolein-cholesterol emulsions.

The thermotropic properties of triolein-rich, low-cholesterol dipalmitoyl phosphatidylcholine (DPPC) emulsion particles with well-defined chemical compositions (approximately 88% triolein, 1% cholesterol, 11% diacyl phosphatidylcholine) and particle size distributions (mean diameter, approximately 1000-1100 A) were studied in the absence and presence of apolipoprotein-A1 by a combination of differential scanning and titration calorimetry. The results are compared to egg yolk PC emulsions of similar composition and size. Isothermal titration calorimetry at 30 degrees C was used to saturate the emulsion surface with apo-A1 and rapidly quantitate the binding constants (affinity Ka = 11.1 +/- 3.5 x 10(6) M-1 and capacity N = 1.0 +/- 0.09 apo-A1 per 1000 DPPC) and heats of binding (enthalpy H = -940 +/- 35 kcal mol-1 apo-A1 or -0.92 +/- 0.12 kcal mol-1 DPPC). The entropy of association is -3070 cal deg-1 mol-1 protein or -3 cal deg-1 mol-1 DPPC. Without protein on the surface, the differential scanning calorimetry heating curve of the emulsion showed three endothermic transitions at 24.3 degrees C, 33.0 degrees C, and 40.0 degrees C with a combined enthalpy of 1.53 +/- 0.2 kcal mol-1 DPPC. With apo-A1 on the surface, the heating curve showed the three transitions more clearly, in particular, the second transition became more prominent by significant increases in both the calorimetric and Van't Hoff enthalpies. The combined enthalpy was 2.70 +/- 0.12 kcal mol-1 DPPC and remained constant upon repeated heating and cooling. Indicating that the newly formed DPPC emulsion-Apo-A1 complex is thermally reversible during calorimetry. Thus there is an increase in delta H of 1.17 kcal mol-1 DPPC after apo-A1 is bound, which is roughly balanced by the heat released during binding (-0.92 kcal) of apo-A1. The melting entropy increase, +3.8 cal deg-1 mol-1 DPPC of the three transitions after apo-A1 binds, also roughly balances the entropy (-3 cal deg-1 mol-1 DPPC) of association of apo-A1. These changes indicate that apo-A1 increases the amount of ordered gel-like phase on the surface of DPPC emulsions when added at 30 degrees C. From the stoichiometry of the emulsions we calculate that the mean area of DPPC at the triolein/DPPC interface is 54.5 A2 at 41 degrees C and 54.2 A2 at 30 degrees C. The binding of apo-A1 at 30 degrees C to the emulsion reduces the surface area per DPPC molecule from 54.2 A2 to 50.8 A2. At 30 degrees apo-A1 binds with high affinity and low capacity to the surface of DPPC emulsions and increases the packing density of the lipid domain to which it binds. Apo-A1 was also titrated onto DPPC emulsions at 45 degrees C. This temperature is above the gel liquid crystal transition. No heat was released or adsorbed. Furthermore, egg yolk phosphatidylcholine emulsions of nearly identical composition were also titrated at 30 degrees C with apo-A1 and were euthermic. Association constants were previously measured using a classical centrifugation assay and were used to calculate the entropy of apo-A1 binding (+28 cal deg-1 mol-1 apo-A1). This value indicates that apo-A1 binding to a fluid surface like egg yolk phosphatidylcholine or probably DPPC at 45 degrees C is hydrophobic and is consistent with hydrocarbon lipid or protein moities coming together and excluding water. Thus the binding of apo-A1 to partly crystalline surfaces is entropically negative and increases the order of the already partly ordered phases, whereas binding to liquid surfaces is mainly an entropically driven hydrophobic process.     

A jump in an Arrhenius plot can be the consequence of a phase transition. The binding of ATP to myosin subfragment 1

The temperature dependence of the kinetics of the binding of ATP to myosin subfragment-1 was studied by an ATP chase technique in a rapid-flow-quench apparatus: (formula; see text) A temperature range of 30 degrees C to -15 degrees C was obtained with ethylene glycol as antifreeze. The Arrhenius plot of k2 is discontinuous with a jump at 12 degrees C. Above the jump delta H+ = 9.5 kcal/mol, below delta H+ = 28.5 kcal/mol. Few such Arrhenius plots are recorded in the literature but they are predicted from theory. Thus, we explain our results as a phase change of the subfragment 1-ATP system at 12 degrees C. This is in agreement with certain structural studies.     

Alterations in phospholipid-dependent (Na+ +K+)-ATPase activity due to lipid fluidity. Effects of cholesterol and Mg2+.

The (Na+ +K+)-activated, Mg2+-dependent ATPase from rabbit kidney outer medulla was prepared in a partially inactivated, soluble form depleted of endogenous phospholipids, using deoxycholate. This preparation was reactivated 10 to 50-fold by sonicated liposomes of phosphatidylserine, but not by non-sonicated phosphatidylserine liposomes or sonicated phosphatidylcholine liposomes. The reconstituted enzyme resembled native membrane preparations of (Na+ +K+)-ATPase in its pH optimum being around 7.0, showing optimal activity at Mg2+:ATP mol ratios of approximately 1 and a Km value for ATP of 0.4 mM. Arrhenius plots of this reactivated activity at a constant pH of 7.0 and an Mg2+: ATP mol ratio of 1:1 showed a discontinuity (sharp change of slope) at 17 degrees C, with activation energy (Ea) values of 13-15 kcal/mol above this temperature and 30-35 kcal below it. A further discontinuity was also found at 8.0 degrees C and the Ea below this was very high (greater than 100 kcal/mol). Increased Mg2+ concentrations at Mg2+:ATP ratios in excess of 1:1 inhibited the (Na+ +K+)-ATPase activity and also abolished the discontinuities in the Arrhenius plots. The addition of cholesterol to phosphatidylserine at a 1:1 mol ratio partially inhibited (Na+ +K+)-ATPase reactivation. Arrhenius plots under these conditions showed a single discontinuity at 20 degrees C and Ea values of 22 and 68 kcal/mol above and below this temperature respectively. The ouabain-insensitive Mg2+-ATPase normally showed a linear Arrhenius plot with an Ea of 8 kcal/mol. The cholesterol-phosphatidylserine mixed liposomes stimulated the Mg2+-ATPase activity, which now also showed a discontinuity at 20 degrees C with, however, an increased value of 14 kcal/mol above this temperature and 6 kcal/mol below. Kinetic studies showed that cholesterol had no significant effect on the Km values for ATP. Since both cholesterol and Mg2+ are known to alter the effects of temperature on the fluidity of phospholipids, the above results are discussed in this context.     

A saturation transfer electron spin resonance study on the break in the Arrhenius plot for the rotational motion of Ca2+-dependent adenosine triphosphatase molecules in purified and lipid-replaced preparations of rabbit skeletal muscle sarcoplasmic reticulum

By means of saturation transfer electron spin resonance spectroscopy the rotational motion of spin-labeled Ca2+-dependent ATPase molecules has been investigated for three kinds of preparations of rabbit skeletal muscle sarcoplasmic reticulum: MacLennan's enzyme (purified ATPase preparation), DOPC- and egg PC-ATPase (purified ATPase preparations in which endogenous lipids are replaced with dioleoyl and egg yolk phosphatidylcholine, respectively). The rotational mobility of the enzyme in these preparations is somewhat lower than that in the intact membrane, probably due to the reduced amount of lipids. For all the preparations, however, the Arrhenius plot for rotational mobility showed a break at about 18 degrees C, the same temperature at which a break in the Arrhenius plot for Ca2+-ATPase activity occurs. This result provides further evidence that the break in the Arrhenius plot is not related to a lipid phase transition but to a change in the physical state of the Ca2+-ATPase molecule existing in fluid lipids.     

Temperature effects on cholinesterases from rat brain capillaries

The activities of acetylcholinesterase (ACHE) and butyrylcholinesterase (BuChE) in rat brain capillaries were measured as a function of temperature. Arrhenius plots of the data revealed that AChE exhibits a biphasic Arrhenius plot with a distinct break (transition temperature) at about 15.2 kcal/mol. In contrast, BuChE did not show evidence of discontinuity. BuChE showed an activation energy higher than that of AChE in the physiological range of temperature. These data suggest a lack of lipid-protein interaction in the case of BuChE. Although the possibility exists that BuChE is weakly anchored to the membranes, our results indicate that BuChE is not bound, at least significantly, to cellular membranes in brain capillaries as is ACHE.     

Protein and lipid structural transitions in cytochrome c oxidase-dimyristoylphosphatidylcholine reconstitutions

The thermotropic behavior of the mitochondrial enzyme cytochrome c oxidase (EC 1.9.3.1) reconstituted in dimyristoylphosphatidylcholine (DMPC) vesicles has been studied by using high-sensitivity differential scanning calorimetry and fluorescence spectroscopy. The incorporation of cytochrome c oxidase into the phospholipid bilayer perturbs the thermodynamic parameters associated with the lipid phase transition in a manner analogous to other integral membrane proteins: it reduces the enthalpy change, lowers the transition temperature, and reduces the cooperative behavior of the phospholipid molecules. Analysis of the dependence of the enthalpy change on the protein:lipid molar ratio indicates that cytochrome c oxidase prevents 99 +/- 5 lipid molecules from participating in the main gel-liquid-crystalline transition. These phospholipid molecules presumably remain in the same physical state below and above the transition temperature of the bulk lipid, thus providing a more or less constant microenvironment to the protein molecule. The effect of the phospholipid bilayer matrix on the thermodynamic stability of the cytochrome c oxidase complex was examined by high-sensitivity differential scanning calorimetry. Detergent (Tween 80)-solubilized cytochrome c oxidase undergoes a complex, irreversible thermal denaturation process centered at 56 degrees C and characterized by an enthalpy change of 550 +/- 50 kcal/mol of enzyme complex. Reconstitution of the cytochrome c oxidase complex into DMPC vesicles shifts the transition temperature upward to 63 degrees C, indicating that the phospholipid bilayer moiety stabilizes the native conformation of the enzyme. The lipid bilayer environment contributes approximately 10 kcal/mol to the free energy of stabilization of the enzyme complex. The thermal unfolding of cytochrome c oxidase is not a two-state process.(ABSTRACT TRUNCATED AT 250 WORDS)     

Temperature dependence of d-glucose transport in reconstituted liposomes

Sodium-dependent d-glucose uptake into proteoliposomes reconstituted from dimyristoylphosphatidylcholine (DMPC) and hog kidney brush border membrane extract is strongly affected by temperature and the physical state of the membranes. This dependence is defined by a nonlinear Arrhenius plot with a break point at 23°C, a temperature not significantly different from the phase transition temperature of the pure lipid (24°C). The transport process is characterized by different activation energies: 35.1 kcal/mol below and 5.5 kcal/mol above the transition temperature. The shift in the break point for the d-glucose transport activity from 15°C, in the brush border membranes, to 23°C in the reconstituted system leads us to conclude that the lipids surrounding the sodium/d-glucose cotransport system can exchange readily with the bulk lipid used for reconstitution. The results thus provide no evidence for the presence of an annulus of specific lipids surrounding the transport system.     

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.     

Functional interactions of lipids and proteins in rat intestinal microvillus membranes.

Interactions of lipids and proteins in isolated rat intestinal microvillus membranes were examined by studying the temperature dependence of enzyme activities and of D-glucose transport in relation to the membrane lipid thermotropic transition observed by fluorescence polarization (26 +/- 2 degrees C) and differential scanning calorimetry (23--39 degrees C). Two groups of activities were defined. Enzymes of the first group, comprising lactase, maltase, sucrase, leucine aminopeptidase, and gamma-glutamyl transpeptidase, all yielded a single slope on the Arrhenius plot in the range 10--40 degrees C and did not appear to experience functionally the effects of the lipid thermotropic transition. Each activity of the second group, comprising calcium- and magnesium-dependent adenosine triphosphatases, p-nitrophenylphosphatase, and D-glucose transport, showed a change in the slope of the Arrhenius plot in the range 25--30 degrees C, corresponding to the lower region of the lipid transition. The terms "extrinsic" and "intrinsic" activities could be applied to these groups. Delipidation of the particulate p-nitrophenylphosphatase removed the discontinuity in the Arrhenius plot. Subsequent relipidation with a variety of lipids restored a break point, but the temperature corresponded to the original discontinuity (25--29 degrees C) rather than to the phase transition temperature of the exogenous lipid added.     

Membrane lipids can modulate guanylate cyclase activity of rat intestinal microvillus membranes

Studies of the temperature dependence (10-40 degrees C) of guanylate cyclase in rat intestinal microbillus membranes reveal a change in energy of activation (slope of the Arrhenius plot) at 30 +/- 1 degree C. The break point temperature corresponds to the lipid thermotropic transition in these membranes previously characterized by differential scanning calorimetry (range: 23-39 degrees C; peak temperature, 31 degrees C). The break point temperature for guanylate cyclase also corresponds to that of a number of other microbillus membrane enzymes and of D-glucose transport. These activities are defined as "intrinsic" membrane activities by this operational criterion. Treatment with the nonionic detergent Lubrol WX increased the guanylate cyclase activity 4- to 8-fold and removed the discontinuity in the Arrhenius plot.     

Temperature dependence of anion transport in the human red blood cell

Arrhenius plots of chloride and bromide transport yield two regions with different activation energies (Ea). Below 15 or 25 degrees C (for Cl- and Br-, respectively), Ea is about 32.5 kcal/mol; above these temperatures, about 22.5 kcal/mol (Brahm, J. (1977) J. Gen. Physiol. 70, 283-306). For the temperature dependence of SO4(2-) transport up to 37 degrees C, no such break could be observed. We were able to show that the temperature coefficient for the rate of SO4(2-) transport is higher than that for the rate of denaturation of the band 3 protein (as measured by NMR) or the destruction of the permeability barrier in the red cell membrane. It was possible, therefore, to extend the range of flux measurements up to 60 degrees C and to show that, even for the slowly permeating SO4(2-) in the Arrhenius plot, there appears a break, which is located somewhere between 30 and 37 degrees C and where Ea changes from 32.5 to 24.1 kcal/mol. At the break, the turnover number is approx. 6.9 ions/band 3 per s. Using 35Cl- -NMR (Falke, Pace and Chan (1984) J. Biol. Chem. 259, 6472-6480), we also determined the temperature dependence of Cl- -binding. We found no significant change over the entire range from 0 to 57 degrees C, regardless of whether the measurements were performed in the absence or presence of competing SO4(2-). We conclude that the enthalpy changes associated with Cl- - or SO4(2-)-binding are negligible as compared to the Ea values observed. It was possible, therefore, to calculate the thermodynamic parameters defined by transition-state theory for the transition of the anion-loaded transport protein to the activated state for Cl-, Br- and SO4(2-) below and above the temperatures at which the breaks in the Arrhenius plots are seen. We found in both regions a high positive activation entropy, resulting in a low free enthalpy of activation. Thus the internal energy required for carrying the complex between anion and transport protein over the rate-limiting energy barrier is largely compensated for by an increase of randomness in the protein and/or its aqueous environment.