Effect of reduced temperatures on protein synthesis in mouse L cells.

The rate of incorporation of leucine into protein, the rate of polypeptide elongation and termination, and the relative quantity and size of polysomes were analyzed in mouse L cells grown in suspension culture at various temperatures between 0 degrees C and 36 degrees C. Between 10 degrees C and 36 degrees C protein synthesis exhibited two different apparent activation energies (39 kcal/mole, 10-25 degrees C; 14 kcal/mole, 25-36 degrees C), whereas elongation and termination had only one (16 kcal/mole). Below 36 degrees C, the polysome level and size decreased, reaching a minimum of 30% of the control 36 degrees C values at 10 degrees C; below 10 degrees C the level increased again back to control values at 0 degrees C. The polysome decline was time dependent, requiring about 5 hr to reach the equilibrium value. This decline is completely reversible within 60 min, even in the presence of 4 mug/ml of actinomycin D, and even after 15 hr of incubation at the lower temperature. The results suggest that polypeptide initiation is rate limiting, particularly below 25 degrees C; whereas above this temperature, elongation or perhaps some other process may be limiting. These results are quite different from those obtained for E. coli and rabbit reticulocyte protein synthesis.     

The role of cations and other factors on the apparent energy of activation of (Na + K)-ATPase

Data concerning the temperature dependence of ouabain-sensitive (Na⁺ + K⁺)-activated ATPase have enabled estimates of the apparent activation energies of this process to be obtained. Arrhenius plots show a point of inflection at about 20 °; at higher temperatures the activation energy is about 13.5 kcal/mole while below this temperature the value increases to 28.5 kcal/mole. Storage at −5 ° or reduction in total cation concentration without alteration of the Na⁺:K⁺ ratio causes no significant change in these values, although the specific activity is markedly reduced. Reduction in the sodium concentration alone, however, increases the apparent activation energy at lower temperatures. These results support the hypothesis that two independent processes are involved in ATP hydrolysis, one operating above the critical temperature and one operating below this temperature. Storage, or reduction in the concentrations of both sodium and potassium ions, appears to reduce the number of functional ATPase units, without significantly altering the properties of those which can still hydrolyze ATP. Reduction in the sodium concentration alone, however, may also cause some inhibition of all units. This is more marked at lower temperatures, and may arise from competition by potassium for sodium-binding sites.     

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.     

Catalytic and thermodynamic properties of the urocanate hydratase reaction.

Urocanate hydratase (4-imidazolone-5-propionate hydro-lyase, EC 4.2.1.49) isolated from Pseudomonas putida contains covalently bound alpha-ketobutyrate as its cofactor. In the process of examining the mechanism by which alpha-ketobutyrate serves in this capacity, various thermodynamic parameters and temperature effects on urocanate hydratase activity were determined. As the equilibrium constant at 15 degrees C for imidazooone propionate formation from urocanate is approximately 69, regardless of whether urocanic acid or chemically synthesized imidazolone propionate is used as the initial substrate, it is concluded that the reaction is freely reversible. DeltaG degrees ', deltaH degrees ' and deltaS degrees ' were --2.5 kcal/mole, +5.2 kcal/mole and +26 cal/deg mole, respectively. Measurement of first-order reaction rates at various temperatures, in order to calculate the Arrhenius activation energy, showed a sharp break in the Arrhenius plot at 29 degrees C. Further examination of this phenomenon by determining s20,w values of urocanate hydratase as a function of temperature revealed a dramatic change at 31 degrees C. Since the enzyme in both experiments reverts to its original state when the temperature is lowered back below the transition point, it is proposed that urocanate hydratase undergoes a reversible conformational change or partial dissociation which affects its catalytic properties in the range of 29--31 degrees C.     

Reconstitution and characterization of the adenine nucleotide transporter derived from bovine heart mitochondria.

1. Adenine nucleotide exchange-transport was reconstituted in vesicles prepared from phospholipids and protein fractions derived from bovine heart submitochondrial particles. The transport, which was specific for ATP and ADP was measured either as ADP/ADP, ATP/ATP, or ADP/ATP exchange. The highest specific activity (370 nanomoles of ADP/ADP exchange/min/mg of protein at room temperature) was obtained with a protein fraction prepared by cholate extraction of partly resolved submitochondrial particles followed by ammonium sulfate fractionation. 2. At 200 muM external nucleotide, the exchange reactions were inhibited by low concentrations of bongkrekate, atractyloside, and palmitoyl-CoA, with Ki values of 1.8, 3.0, and 7.5 muM, respectively. The ADP/ADP nucleotide exchange was stimulated about 5-fold by 500 muM MgCl2 or MnCl2(km of 40 muM) and about 3-fold by 500 muM CaCl2(Km of 90 muM). It was optimal between pH 6.0 and 7.0 and decreased rapidly above pH 7.5. Arrhenius plots between 0 degrees and 40 degrees showed a break point at 15 degrees with soybean phospholipids and an activation energy of 29.5 kcal/mole from 0 degrees-15 degrees and 9.0 kcal/mole from 15 degrees-40 degrees. With mitochondrial phospholipids the break point was at 9 degrees and activation energies were 42.4 kcal/mole from 0 degrees-9 degrees and 7.6 kcal/mole from 9 degrees-40 degrees. 3. The phospholipid requirements for adenine nucleotide exchange were similar to those of oxidative phosphorylation. Optimal rates were observed with a phosphatidylethanolamine to phosphatidylcholine ratio of 4:1. Cardiolipin had a slight stimulatory effect. 4. The uptake of ADP into vesicles containing ATP was stimulated by KCl or by KPi as well as by hexafluoracetonylacetone, and uncoupler of oxidative phosphorylation. The uptake of ATP into vesicles containing ADP was inhibited by KCl or by KPi, but was also stimulated by hexafluoracetonylacetone. In both cases valinomycin reversed the effects of KCl, while mersalyl or N-ethylmaleimide prevented the effects of KPi. In contrast, none of these salts nor hexafluoracetonylactone affected the ADP/ADP or ATP/ATP exchange. These findings suggest that in the reconstituted system the ADP/ATP exchange is electrogenic.     

Energies of Activation and Uncoupled Growth in Streptococcus faecalis and Zymomonas mobilis

Growing cultures of Streptococcus faecalis at temperatures above 30 C have activation energies for both rates of growth and glycolysis of 10.3 kcal mole(-1), and a constant growth yield; when growth takes place below this temperature, the growth yield decreases and the activation energy for growth increases to 21.1 kcal mole(-1), but the activation energy for glycolysis is unchanged. The adenosine triphosphate pool in the organisms behaves differently above and below 30 C, suggesting that the energetic coupling between anabolism and catabolism is less effective below 30 C. Washed suspensions of S. faecalis have repressed glycolytic activity and an activation energy for glycolysis of 15.6 kcal mole(-1) over the whole temperature range studied. Growing cultures of Zymomonas mobilis below 33 C have a constant growth yield of 8.3 g (dry weight) of organisms per mole of glucose degraded, and activation energies for both glycolysis and growth of 11.1 kcal mole(-1); above this temperature, the growth yield falls, the activation energy for growth changes to -6.9 kcal mole(-1), but the activation energy for glycolysis is unchanged, so that the coupling between anabolism and catabolism is less effective above 33 C. The findings support the view that energy turnover in these bacteria is not well regulated.     

USE OF ARRHENIUS PLOTS OF Na-K ATPase and ACETYLCHOLINESTERASE AS A TOOL FOR STUDYING CHANGES IN LIPID-PROTEIN INTERACTIONS IN NEURONAL MEMBRANES DURING BRAIN DEVELOPMENT

Abstract— The activities of Na-K ATPase and acetylcholinesterase in the rat brain cortex were measured at different postnatal ages as a function of temperature. It was found that compared to acetylcholinesterase, Na-K ATPase is more strongly affected by the rise in temperature and that this response is further enhanced with age. Arrhenius plots of the data were prepared and the apparent energies of activation were computed for each plot. It was observed that all plots were biphasic except that for Na-K ATPase of the immature (5-day-old) brain which showed no transition temperature, with an apparent energy of activation of 15.5 kcal/mol. The enzyme from the mature brain (25-day-old) showed an average transition temperature of 22.6°C, with average apparent energies of activation of 15.3 and 27.2 kcal/mol above and below the transition temperature respectively. The cortex of 1-day-old rat showed no Na-K ATPase activity. Arrhenius plots of acetylcholinesterase studied at ages 1, 5 and 25 days postnatally all showed transition temperatures which increased from an average of 16.1°C for 1-day-old to 17 and 21.5°C for 5- and 25-day-old animals respectively. The average apparent energies of activation for acetylcholinesterase below the transition temperature changed from 8.3 kcal/mole at day 1 to 8.7 and 7.2 kcal/mol at days 5 and 25, while above the transition temperature they were 4.3, 5.2 and 4.1 at days 1, 5 and 25 respectively. The results are discussed in terms of the differences and changes in the interactions of Na-K ATPase and acetylcholinesterase with membrane lipids during the postnatal phase of brain development.     

Several properties of the Ca-pump of rabbit skeletal muscle sarcoplasmic reticulum in hypercholesteremia]

Sarcoplasmic reticulum (SR) fragments from the skeletal muscles of rabbit with marked atherosclerosis possessed decreased Ca2+-accumulating capacity. Lowering of transport efficiency, namely reduction of the Ca/ATP ratio from 1.9--normal value--to 0.9 during the experiment at 26 degrees C was accompanied by activation of Ca-ATPase and simultaneously of the rate of Ca2+ outflux from the SR. Arrhenius plots of Ca-ATPase temperature dependence characterized under normal conditions by a break at 20--21 degrees C was linearized under hypercholesterolemia. At the same time there was a rise (from 0.03 under normal conditions to 0.15 in atherosclerosis) of cholesterol/protein ratio in the SR membrane preparations. Activation energy for Ca-ATPase crude membranes under normal conditions was equal to 15.6 and 28.7 kcal/mol above and below the break point respectively; this value for Ca-ATPase of membranes with increased cholesterol level was 19 kcal/mol for all the temperatures investigated.     

Regulation of translation in rabbit reticulocytes and mouse L-cells; comparison of the effects of temperature.

Various parameters of protein synthesis were analyzed in rabbit reticulocytes exposed to various temperatures for up to five hours. Between 10 degrees C and 40 degrees C total protein synthesis exhibited two different apparent activation energies (36 kcal/mole, 10-24 degrees C; 22 kcal/mole, 24-40 degrees C), as did protein elongation and release (35 kcal/mole, 10-25 degrees C; 12 kcal/mole, 25-40 degrees C). However, the level of polysomes remained essentially unchanged between 0 degrees C and 42 degrees C which implies that the activation energy for polypeptide initiation is quite similar to that for elongation and is also biphasic. This situation is different from that in cultured mouse L-cells where the polysome level is dependent on temperatures. Nevertheless, reticulocytes and L-cells appear to be similar in their temperature dependence of initiation and in their rate of elongation (5-6 amino acids/second at 36 degrees C.     

Premelting effects in DNA under saltfree conditions

We have studied the electrical conductivity of NaDNA solutions under “saltfree” conditions at temperatures well below the melting point of DNA, using radio-frequency dielectric and noise measurements. A conductivity discontinuity is observed at a temperature well below that at which the usual denaturation processes and trans conformation may commence. The radio-frequency permittivity also exhibits a discontinuity at the same temperature. For the premelting phase, the conductivity versus temperature curves consist of two linear regions with a change in slope occurring at 23°C. This effect is related to the behavior of the ionic sheath covering the DNA macromolecule. The activation energy of the alternative current conductivity as well as that the equivalent noise conductivity results as 3.11 kcal/mole below and 4.08 kcal/mole.     

Fluidity of human erythrocyte membrane and effect of chlorpromazine on fluidity and phase separation of membrane

The fluidity of human erythrocyte membrane, and the effect of chlorpromazine at prelytic and lytic concentrations on the fluidity have been studied by using three kinds of fatty acid spin labels and measuring the temperature dependence of Mg2+-ATPase activity. The Arrhenius plot of the apparent rotational correlation time, tau c, for probes I(12,3) and I(5,10) showed an abrupt discontinuity at about 30 degrees C, and the plot for I(1,14) at 25 degrees C, indicating that a large difference in the fluidity exists between the interior and the outer surface of the lipid bilayer. The portions of the fatty acid chain near the ten carbon bond lengths removed from the bilayer surface became more fluid by chlorpromazine treatment; there was a decrease in the break point to around 26 degrees C following treatment with 0.6 or 1 mM of the drug. Two breaks at 21 and 30 degrees C in the Arrhenius plot of the Mg2+-ATPase activity were observed in normal erythrocyte membrane. The activation energy of the Mg2+-ATPase reaction has the values of 3.0 and 22.1 kcal/mol above the upper break and below the lower break, respectively. The drug exposure induced only a slight shift in the break temperatures, while the treatment significantly enhanced the associated activation energies of the reaction. These results suggest that the boundary phospholipids of the Mg2+-ATPase in the membrane are probably more rigid than the bulk lipids.     

Temperature dependence of ultrasound-induced cell killing: The role of membrane fluidity

Chinese hamster cells in suspension were exposed to 20 kHz ultrasound (US) at 54 W/cm² and various temperatures between 2 and 44 °C. Activation energies were 2.6 and 24 kcal/mole below and above 35 °C, respectively. Procaine, a local anaesthetic drug known to increase membrane fluidity, enhanced cellular inactivation by US above 41 °C, increasing the activation energy to 62 kcal/mole. The inactivation of the bacterium Salmonella typhimurium by US was also dependent on the exposure temperature, with an activation energy of 2.9 kcal/mole between 2 and 44 °C. These data are most simply explained by the hypothesis that membranes are a major target for cellular inactivation by US and that the fluidity of the membranes is important in this respect.     

Temperature-dependent kinetics of the activities of influenza virus

The temperature dependence of membrane interactions between PR8 influenza virus and virus receptor (GD1a)-containing liposomes was studied. For quantitation, the octadecylrhodamine B chloride (R18) membrane marker was incorporated into liposomes at quenched concentrations. Upon interaction with target membranes, the marker gets diluted, and dequenching can be measured in a fluorescence spectrophotometer. Rate constants were calculated from the dequenching curves under low pH conditions, which allow for fusion, and at neutral pH, where no specific fusion occurs. Activation energies were determined from Arrhenius plots. The results were compared with the temperature dependence of other viral activities like infectivity, hemolysis, and fusion with erythrocytes. For the slow reaction at pH 7.4, where only non-specific lipid transfer takes place, the activation energy was about 24 kcal/mole between 15 degrees C and 45 degrees C. For the fast, hemagglutinin (HA)-specific fusion reaction (pH 5.3), a very low activation energy (approximately 7 kcal/mole) was found between 25 degrees C and 37 degrees C, whereas below 25 degrees C it was much higher (approximately 34 kcal/mole). The temperature range with low activation energy coincides with the one for optimal infectivity, hemolysis, and fusion with erythrocytes. Furthermore, it is the same range in which the conformational change of HA takes place, which in the absence of a partner membrane leads to an irreversible inactivation of the fusion protein.     

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.     

The effect of temperature on the activity of the adenylate cyclase system of liver plasma membranes

The rat liver adenylate cyclase system shows a discontinuity in the Arrhenius plots at 20°C in the nonstimulated activity (basal) with activation energies of 16 and 28 Kcal/mole. The discontinuity disappears when the enzyme is stimulated either by glucagon, sodium fluoride, 5′ guanylyl-imidodiphosphate or glucagon plus 5′ guanylyl-imidodiphosphate and the energy of activation was the same with all the compounds tested. If the activator was initially in contact with the membranes at 0°C the energy of activation was similar to that observed below the break (26 Kcal/mole) but it changed to that above the break if the compound contacted the membranes at temperatures above the break (22–24°C). We discuss the possibility of two different conformations of the enzyme; both conformations can be “frozen” by any of the compounds tested, “isolating” the enzyme from any subsequent physical change of the membrane due to temperature.     

Effects of temperature and pH on the water exchange through erythrocyte membranes: Nuclear magnetic resonance studies

Summary The temperature and pH dependence of water exchange has been studied on isolated erythrocytes suspended in isotonic buffered solutions. At pH 7.4 a break in the Arrhenius plot of water exchange time at around 26°C was found. The mean value of the apparent activation energy of the water exchange time at temperatures higher than that of the discontinuity was 5.7 kcal/mole (±0.4); at lower temperatures the values of the apparent activation energy were below 1.4 kcal/mole. The pH dependence of water exchange time of isolated erythrocytes revealed a marked increase of the water exchange time values in the acid range of pH; a much smaller variation of the same parameter occurs between pH 7.0 and 8.0. These finding could be correlated with other processes involving erythrocyte membranes that showed similar pH and temperature dependence and were considered to indicate state transitions in the membranes. It is suggested that the temperature and pH effects on water diffusion indicate that conformational changes and cooperative effects are implicated in the mechanism of this transport process.Institute for Isotopic and Molecular Technology.     

Studies of Photosynthesis Using a Pulsed Laser: I. Temperature Dependence of Cytochrome Oxidation Rate in Chromatium. Evidence for Tunneling

The rate of oxidation of cytochrome following absorption of a short pulse of light from a ruby laser in the photosynthetic bacterium Chromatium has been measured spectrophotometrically. The half-time is about 2 musec at room temperature increasing to 2.3 msec at about 100 degrees K and constant at the latter value to 35 degrees K or below. The temperature dependence above 120 degrees K corresponds to an activation energy of 3.3 kcal/mole; that below 100 degrees K to less than 80 cal/mol: essentially a temperature-independent electron transport reaction. Since the slowness below 100 degrees K indicates the presence of a barrier, the lack of activation energy is taken to mean penetration by quantum-mechanical "tunneling."     

Conformational and thermodynamic properties of apo A-1 of human plasma high density lipoproteins.

Conformational changes of apo A-1, the principal apoprotein of human plasma high density lipoprotein, have been studied by differential scanning calorimetry and ultraviolet difference spectroscopy as a function of temperature, pH, concentration of apoprotein, and urea concentration. Calorimetry shows that apo A-1 (5 to 40 mg/ml, pH 9.2) undergoes a two-state, reversible denaturation (enthalpy = 64 +/- 8.9 kcal/mole), between 43--71 degrees (midpoint temperature, Tm = 54 degrees), associated with a rise in heat capacity (deltaCvd) of 2.4 +/- 0.5 kcal/mole/degrees C. Apo A-1 (0.2 to 0.4 mg/ml, pH 9.2) develops a negative difference spectrum between 42--70 degrees, with Tm = 53 degrees. The enthalpy (deltaH = 59 +/- 5.7 kcal/mole at Tm) and heat capacity change (2.7 +/- 0.9 kcal/mole/degrees C) in the spectroscopic experiments were not significantly different from the calorimetric values. Below pH 9 and above pH 11, the calorimetric Tm and deltaH of denaturation are decreased. In the pH range of reversible denaturation (6.5 to 11.8), delatH and Tm are linearly related, showing that the heat capacity change (ddeltaH/dT) associated with denaturation is independent of Tm. In urea solutions, the calorimetric Tm and deltaH of denaturation are decreased. At 25 degrees, apo A-1 develops a negative difference spectrum between 1.4 and 3 M urea. Fifty per cent of the spectral change occurs in 2.4 M urea, which corresponds to the urea concentration obtained by extrapolation of the calorimetric Tm to 25 degrees. In urea solution of less than 0.75 M there is hyperchromicity at 285 nm (delta epsilon = 264 in 0.75 M urea), indicating strong interaction of aromatic amino acid residues in the native molecule with the solvent. Spectrophotometric titration of apo A-1 shows that 6.6 of the 7 tyrosine groups of apo A-1 titrate at pH less than 11.9, with similar titration curves obtained in aqueous solutions and in 6 M urea. The free energy of stabilization (deltaG) of the native conformation of apo A-1 was estimated, (a) at 37 degrees, using the calorimetric deltaA and deltaCvd, and (b) at 25 degrees, by extrapolation of spectroscopic data to zero urea concentration. The values (deltaG (37 degrees) = 2.4 and deltaG (25 degrees) = 2.7 kcal/mole) are small compared to typical globular proteins, indicating that native apo A-1 has a loosely folded tertiary structure. The low values of deltaG reflect the high degree of exposure of hydrophobic areas in the native protein molecule. The loosely folded conformation of apo A-1 allows extensive binding of lipid, since this can involve both surface hydrophobic sites and hydrophobic areas exposed by a cooperative, low energy unfolding process.     

The kinetics of transferrin endocytosis and iron uptake from transferrin in rabbit reticulocytes

The endocytosis of diferric transferrin and accumulation of its iron by freshly isolated rabbit reticulocytes was studied using 59Fe-125I-transferrin. Internalized transferrin was distinguished from surface-bound transferrin by its resistance to release during treatment with Pronase at 4 degrees C. Endocytosis of diferric transferrin occurs at the same rate as exocytosis of apotransferrin, the rate constants being 0.08 min-1 at 22 degrees C, 0.19 min-1 at 30 degrees C, and 0.45 min-1 at 37 degrees C. At 37 degrees C, the maximum rate of transferrin endocytosis by reticulocytes is approximately 500 molecules/cell/s. The recycling time for transferrin bound to its receptor is about 3 min at this temperature. Neither transferrin nor its receptor is degraded during the intracellular passage. When a steady state has been reached between endocytosis and exocytosis of the ligand, about 90% of the total cell-bound transferrin is internal. Endocytosis of transferrin was found to be negligible below 10 degrees C. From 10 to 39 degrees C, the effect of temperature on the rate of endocytosis is biphasic, the rate increasing sharply above 26 degrees C. Over the temperature range 12-26 degrees C, the apparent activation energy for transferrin endocytosis is 33.0 +/- 2.7 kcal/mol, whereas from 26-39 degrees C the activation energy is considerably lower, at 12.3 +/- 1.6 kcal/mol. Reticulocytes accumulate iron atoms from diferric transferrin at twice the rate at which transferrin molecules are internalized, implying that iron enters the cell while still bound to transferrin. The activation energies for iron accumulation from transferrin are similar to those of endocytosis of transferrin. This study provides further evidence that transferrin-iron enters the cell by receptor-mediated endocytosis and that iron release occurs within the cell.     

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.