Intracellular glucose oxidase from Penicillium funiculosum 46.1

A method for isolating extracellular glucose oxidase from the fungus Penicillium funiculosum 46.1, using ultrafiltration membranes, was developed. Two samples of the enzyme with a specific activity of 914-956 IU were obtained. The enzyme exhibited a high catalytic activity at pH above 6.0. The effective rate constant of glucose oxidase inactivation at pH 2.6 and 16 degrees C was 2.74 x 10(-6) s-1. This constant decreased significantly as pH of the medium increased (4.0-10.0). The temperature optimum for glucose oxidase-catalyzed beta-D-glucose oxidation was in the range 30-65 degrees C. At temperatures below 30 degrees C, the activation energy for beta-D-glucose oxidation was 6.42 kcal/mol; at higher temperatures, this parameter was equal to 0.61 kcal/mol. Kinetic parameters of glucose oxidase-catalyzed delta-D-glucose oxidation depended on the initial concentration of the enzyme in the solution. Glucose oxidase also catalyzed the oxidation of 2-deoxy-D-glucose, maltose, and galactose.     

Effect of temperature on Brettanomyces bruxellensis: metabolic and kinetic aspects

The effect of temperatures ranging from 15 to 35 degrees C on a culture of Brettanomyces bruxellensis was investigated in regards to thermodynamics, metabolism, and kinetics. In this temperature range, we observed an increase in growth and production rates. The growth behavior was well represented using the Arrhenius model, and an apparent activation energy of 16.61 kcal/mol was estimated. A stuck fermentation was observed at 35 degrees C as represented by high cell death. The carbon balance established that temperature had no effect on repartition of the glucose consumption between biomass and products. Hence, the same biomass concentration was obtained for all temperatures, except at 35 degrees C. Moreover, using logistic and Luedeking-Piret models, we demonstrated that production rates of ethanol and acetic acid were partially growth associated. Parameters associated with growth (alpha eth and alpha aa) remained constant with changing temperature, whereas, parameters associated with the population (beta eth and beta aa) varied. Optimal values were obtained at 32 degrees C for ethanol and at 25 degrees C for acetic acid.     

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.     

Effect of Decreasing Growth Temperature on Cell Yield of Escherichia coli

Studies of the relationship between yield coefficient and growth rate, as affected by temperature of growth, in Escherichia coli have shown that, over a wide range of temperature, yield is relatively constant until the specific growth rate falls below about 0.2 hr(-1), at which point the yield begins to fall off precipitously. No intermediates of glucose metabolism in a form utilizable at higher temperatures could be found in the medium, and no toxic product was produced which limited growth. At 10 C, 37% of the carbon from glucose-UL-(14)C was assimilated into cellular material, whereas, at 30 C, 53% was assimilated. Cells grown at 10 C contained more carbohydrate than did cells grown at 37 C, and the glycogen-to-protein ratio of cells grown at 10 C was approximately three times higher than that of cells grown at 37 C. Adenosine triphosphatase activities of cells grown at 10 and 35 C were similar. Growth rates on glucose, glycerol, and succinate were quite similar at 10 C, but at 35 C growth was most rapid on glucose and slowest on succinate. The data suggest that the decrease in yield with decrease in temperature is a result of uncoupling of energy production from energy utilization.     

Microaerophilic growth of Wolinella recta ATCC 33238

Abstract The influence of oxygen on growth and fumarate-dependent respiration of Wolinella recta ATCC 33238 was studied in continuous culture. Steady states were obtained with formate-limited cultures grown at a specific growth rate of 0.1 h⁻¹ with different levels of oxygenation. The extent of aeration was regulated by means of a redox control system permitting reproducible cultivation at oxygen levels below the detection limit of conventional lead-silver probes. The ratio of succinate produced to that of formate consumed (Suc/For) decreased from 0.99 in strictly anaerobic cultures to 0.06–0.10 in aerated cultures. The growth yield did not change significantly with increasing redox readings: 4.9–5.2 g cell carbon/mol formate. The ability to use O₂ as the sole electron acceptor was demonstrated in a chemostat culture with formate as electron donor and succinate as carbon source. Washed cells from all chemostat cultures comsumed O₂ with formate as electron donor at a high rate (2.1–3.7 μmol/min per mg protein) and possessed b - and c -type cytochromes and CO-binding pigments. These results clearly indicated the microaerophilic nature of W. recta .     

Comparison of temperature effects on soil respiration and bacterial and fungal growth rates

Temperature is an important factor regulating microbial activity and shaping the soil microbial community. Little is known, however, on how temperature affects the most important groups of the soil microorganisms, the bacteria and the fungi, in situ. We have therefore measured the instantaneous total activity (respiration rate), bacterial activity (growth rate as thymidine incorporation rate) and fungal activity (growth rate as acetate-in-ergosterol incorporation rate) in soil at different temperatures (0-45 degrees C). Two soils were compared: one was an agricultural soil low in organic matter and with high pH, and the other was a forest humus soil with high organic matter content and low pH. Fungal and bacterial growth rates had optimum temperatures around 25-30 degrees C, while at higher temperatures lower values were found. This decrease was more drastic for fungi than for bacteria, resulting in an increase in the ratio of bacterial to fungal growth rate at higher temperatures. A tendency towards the opposite effect was observed at low temperatures, indicating that fungi were more adapted to low-temperature conditions than bacteria. The temperature dependence of all three activities was well modelled by the square root (Ratkowsky) model below the optimum temperature for fungal and bacterial growth. The respiration rate increased over almost the whole temperature range, showing the highest value at around 45 degrees C. Thus, at temperatures above 30 degrees C there was an uncoupling between the instantaneous respiration rate and bacterial and fungal activity. At these high temperatures, the respiration rate closely followed the Arrhenius temperature relationship.     

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.     

Cell growth and catecholase production for Polyporus versicolor in submerged culture.

Cell growth and catecholase production for Polyporus veriscolor (ATCC 12679) were studied in mechanically agitated submerged culture, as functions of temperature. The exponential-phase growth rate exhibited a maximum at 28 degrees C. Over the range of 20 degrees C to approximately 30 degrees C, both cell mass and enzyme yield factors were constant. At higher temperatures (30 to 40 degrees C) cell mass yield factor decreased and enzyme yield factor increased. Specific respiration rate of P. versicolor was determined. Thermal deactivation of catecholase was investigated between 30 and 50 degrees C, and deactivation rates were fit to an Arrhenius rate expression.     

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.     

Fundamental denitrification kinetic studies with Pseudomonas denitrificans

Fundamental kinetic studies on the reduction of nitrate, nitrite, and their mixtures were performed with a strain of Pseudomonas denitrificans (ATCC 13867). Methanol served as the carbon source and was supplied in excess (2:1 mole ratio relative to nitrate and/or nitrite). Nitrate and nitrite served as terminal electron acceptors as well as sources of nitrogen for biomass synthesis. The results were explained under the assumption that respiration is a growth-associated process. It was found that the sequence of complete reduction of nitrate to nitrogen gas is via nitrite and nitrous oxide.It was found that the specific growth rate of the biomass on either nitrate or nitrite follows Andrews inhibitory kinetics and nitrite is more inhibitory than nitrate. It was also found that the culture has severe maintenance requirements which can be described by Herbert's model, i.e., by self-oxidation of portions of the biomass. The specific maintenance rates at 30 degrees C and pH 7.1 were found to be equal to about 28% of the maximum specific growth rate on nitrate and 23% of the maximum specific growth rate on nitrite. Nitrate and nitrite were found to be involved in a cross-inhibitory noncompetitive kinetic interaction. The extent of this interaction is negligible when the presence of nitrite is low but is considerable when nitrite is present at levels above 15 mg/L.Studies on the effect of temperature have shown that the culture cannot grow at temperatures above 40 degrees C. The optimal temperature for nitrate or nitrite reduction was found to be about 38 degrees C. Using an Arrhenius expression to describe the effect of temperature on the specific growth rates, it was found that the activation energy for the use of nitrate by the culture is 8.6 kcal/mol and 7.21 kcal/mol for nitrite. Arrhenius-type expressions were also used in describing the effect of temperature on each of the parameters appearing in the specific growth rate expressions. Studies on the effect of pH at 30 degrees C have shown that the culture reduces nitrate optimally at a pH between 7.4 and 7.6, and nitrite at a pH between 7.2 and 7.3. (c) 1995 John Wiley & Sons, Inc.     

Catalytic efficiency and some structural properties of cold-active protein-tyrosine-phosphatase

A procedure was established for expression and purification of abundant recombinant cold-active protein-tyrosine-phosphatase (RCPTPase), which showed identical enzymatic characteristics to the native enzyme (NCPTPase). The purified RCPTPase showed high catalytic activity at low temperature and maximal activity at 30 degrees C. RCPTPase has a thermodynamic characteristic in that its activation enthalpy was determined to be low, 4.3 kcal/mol, at temperatures below 19.3 degrees C, where the Arrhenius relationship exhibited an inflection point, in comparison with 20.3 kcal/mol above 19.3 degrees C. Also, the thermostability, DeltaG(water), of the catalytic site in the RCPTPase molecule was increased with a decrease in temperature. It was considered that cold-active protein-tyrosine-phosphatase could maintain its catalytic site in a stable conformation for eliciting high catalytic activity with low activation enthalpy at low temperature.     

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.     

Temperature dependence of rat liver mitochondrial respiration with uncoupling of oxidative phosphorylation by fatty acids. Influence of inorganic phosphate

The respiration rate of liver mitochondria in the course of succinate oxidation depends on temperature in the presence of palmitate more strongly than in its absence (in state 4). In the Arrhenius plot, the temperature dependence of the palmitate-induced stimulation of respiration has a bend at 22°C which is characterized by transition of the activation energy from 120 to 60 kJ/mol. However, a similar dependence of respiration in state 4 is linear over the whole temperature range and corresponds to the activation energy of 17 kJ/mol. Phosphate partially inhibits the uncoupling effect of palmitate. This effect of phosphate is increased on decrease in temperature. In the presence of phosphate the temperature dependence in the Arrhenius plot also has a bend at 22°C, and the activation energy increases from 128 to 208 kJ/mol in the range from 13 to 22°C and from 56 to 67 kJ/mol in the range from 22 to 37°C. Mersalyl (10 nmol/mg protein), an inhibitor of the phosphate carrier, similarly to phosphate, suppresses the uncoupling effect of laurate, and the effects of mersalyl and phosphate are not additive. The recoupling effects of phosphate and mersalyl seem to show involvement of the phosphate carrier in the uncoupling effect of fatty acids in liver mitochondria. Possible mechanisms of involvement of the phosphate carrier in the uncoupling effect of fatty acids are discussed.     

Temperature response of oxygen uptake in brain mitochondria from normal and ischemic rats

Mitochondria isolated from rat brains following 30 min of complete (decapitation) ischemia showed a 3-fold increase in free fatty acid content, but no significant decreases in the total fatty acid or phospholipid content.This free fatty acid increase was associated with an altered mitochondrial function: a 50% inhibition of state 3 (+ ADP) respiration and a decrease in the respiratory control ratio from 5.5 to 3 to 24°C. The P:O ratio remained unchanged at 3, and there was no increase in stage 4 respiration. When glutamate and malate supported respiration was determined as a function of temperature in control mitochondria, the resulting Arrhenius plot of the state 3 respiration was biphasic with a transition temperature around 30°C, while ischemic mitochondria exhibited a linear Arrhenius plot with energy of activation (approximately 10 kcal/mol) similar to that of control mitochondria below the transition temperature.The difference in temperature response between control and ischemic mitochondria reflects a change in mitochondrial lipid composition, and is therefore a functional manifestation of the altered cerebral lipid composition commonly observed during ischemia.     

Temperature dependence of growth and membrane-bound activities of Chloroflexus aurantiacus energy metabolism

The temperature dependence of various activities related to the energy metabolism of isolated membranes and whole cells of the thermophilic bacterium Chloroflexus aurantiacus was determined after phototrophic growth at either 40, 50, or 60 degrees C. The data obtained were expressed by use of Arrhenius plots. Maximum activities were determined at about 65 degrees C for succinate 2,4-dichlorophenol-indophenol reductase as well as NADH oxidase and at about 70 degrees C for Mg-ATPase and for light-induced proton extrusion by cells. Activation energies for Mg-ATPase and light-induced proton extrusion were about 40 kJ mol-1 from 30 degrees C to about 50 degrees C and they increased significantly at higher temperatures. Essentially the same dependency was detectable with NADH oxidase, except for an increase in activation energy below 41 degrees C. All of these responses were independent of growth temperature. Succinate-2,4-dichlorophenol-indophenol reductase showed a change in activation energy around 41 degrees C only with cells grown at 60 degrees C. Differences in the responses of cells grown at different temperatures were identified on the basis of changes from sigmoidal to hyperbolic kinetics for light saturation of proton extrusion. Moreover, the thermostability of proton extrusion was maximal when assayed at the corresponding growth temperatures. In any case, thermostability was lowest at the 65 and 68 degrees C assay temperatures. Differential scanning calorimetry with membranes revealed irreversible heat uptake from about 60 to 72 degrees C. The results are discussed in light of the activation energy for the specific growth rate, which is lowest at temperatures from 40 degrees C to the optimum at 60 degrees C.     

Hormones and liver mitochondria: Influence of growth hormone,thyroxine, testosterone,and insulin on thermotropic effects of respiration and fatty acid composition of membranes

The effects of hypophysectomy and subsequent administration of growth hormone, thyroxine, insulin, and testosterone were examined in rat liver for the relationship between the thermotropic effects on State 3 respiration (ADP induced) and fatty acid composition of the phospholipid fraction of intact mitochondria as well as of inner membrane vesicles. The Arrhenius profile for energy-linked (succinate) State 3 respiration of mitochondria from hypophysectomized rats lacked the discontinuity at 23.5 °C seen with mitochondria from normal rats. After injections of the hormones the discontinuity representing the transition temperature from gel to liquid crystalline state of lipids occurred at different temperatures: 18.5 °C for growth hormone, 26.0 °C for thyroxine, 19.5 °C for growth hormone + thyroxine, 27.6 °C for insulin, and 25.3 °C for testosterone. The energy of activation between 37.5 and 23.5 °C was 1.9 times greater for hypophysectomy than for controls. Growth hormone was the most effective in restoring the energy of activation to normal, above as well as below transition temperature. The effect of thyroxine appears to be due to a larger stimulation of the State 4 respiration than that of growth hormone, insulin, or testosterone, especially at higher temperatures. Phospholipids extracted from intact mitochondria or inner membrane vesicles of hypophysectomized rats contained less arachidonic acid (20:4) and more linoleic acid (18:2) than those of normal rats. In addition, the contents of some of the minor fatty acids were also changed. Calculated unsaturation index showed an 18.8 and 14.9% depletion in unsaturation in whole mitochondria and inner membranes, respectively. Among the different hormones used to treat the hypophysectomized rats, growth hormone was the most effective in restoring the transition temperature and fatty acid composition to normal levels and increasing the gain in body weight. Although the other hormones increased total unsaturation index to some extent, some of the individual fatty acids were affected differently. Good correlation exists between the unsaturation index of mitochondrial fatty acids and transition temperature of State 3 respiration. These results strongly suggest a role for the hormones, particularly growth hormone, in the control of mitochondrial membrane fluidity of hypophysectomized rat liver, through fatty acid composition of phospholipids.     

Interaction between succinate dehydrogenase and ubiquinone-binding protein from succinate-ubiquinone reductase

Purified ubiquinone-binding protein in succinate-ubiquinone reductase (QPs) reconstitutes with pure soluble succinate dehydrogenase to form succinate-ubiquinone oxidoreductase upon mixing of the two proteins in phosphate buffer at neutral pH. The maximal reconstitution was found with a weight ratio of succinate dehydrogenase to QPs of about 5, which is fairly close to the calculated value of 6.5, a value obtained by assuming one mole of QPs reacts with one mole of succinate dehydrogenase. Succinate-cytochrome c reductase was reconstituted when succinate dehydrogenase and QPs were added to Complex III or cytochrome b-c₁ III complex (a highly purified ubiquinol-cytochrome c reductase). The reconstituted enzyme possessed kinetic parameters which were identical to those of the native enzyme complex. Interaction between QPs and succinate dehydrogenase resulted in the disappearance of low K_m ferricyanide reductase activity from the latter. Unlike soluble succinate dehydrogenase, the reconstituted enzyme, as well as native succinate-cytochrome c reductase, reduced low concentration ferricyanide only in the presence of excess ubiquinone. The apparent K_m for ubiquinone was 6 μM for reduction of ferricyanide (300 μM) by succinate, which is similar to the K_m when ubiquinone was used as electron acceptor. When 2,6-dichlorophenolindophenol was used as electron acceptor for reconstitution of succinate-ubiquinone reductase very little or no exogeneous ubiquinone was needed to show the maximal activity with QPs made by Method II, indicating that the bound ubiquinone in QPs is enough for enzymatic activity. In addition to restoring the succinate-ubiquinone reductase activity the interaction between QPs and succinate dehydrogenase not only stabilized succinate dehydrogenase but also partially deaggregated QPs. The reconstituted succinate-ubiquinone reductase had a minimal molecular weight of 120000 when the reconstituted system was dispersed in 0.2% Triton X-100. The maximal reconstitution was observed at neutral pH in phosphate buffer, Tris-acetate or Tris-phosphate buffer. Tris-HCl buffer, however, produced a less efficient reconstitution. These results indicate that the interaction between QPs and succinate dehydrogenase may involve some cationic group which has a high affinity for Cl^?. Primary amino groups of QPs are not directly involved in the interaction as the reconstitution showed no significant difference when the amino groups of QPs were alkylated with fluorescamine. The Arrhenius plots of reconstituted succinate-ubiquinone reductase show that the enzyme catalyzes the reaction with an activation energy of 19.7 kcal/mol and 26.6 kcal/mol at temperatures above and below 26°C, respectively. These activation energies are similar to those obtained with native enzyme. The Arrhenius plots of the interaction between QPs and succinate dehydrogenase also have a break point at 26°C. The activation energy for this interaction was calculated to be 11.2 kcal/mol and 6.9 kcal/mol for the temperatures above and below the break-point. The significance of the difference in activation energies between the enzymatic reaction and the reconstitution reaction are further explored in the discussion.     

Effect of polyanions on the refolding of human acidic fibroblast growth factor.

Acidic fibroblast growth factor (aFGF) is unstable at physiological temperatures in the absence of polyanions such as heparin. Therefore, the effect of temperature on the kinetics of refolding of aFGF has been examined in the presence and absence of several polyanions. The protein folds into its native state at temperatures up to 30 degrees C without polyanions with an activation energy of approximately 14 kcal/mol, but does not acquire native structure above this temperature. When heparin, inositol hexasulfate, or sulfate ion are present, aFGF refolds below 30 degrees C with a slightly reduced activation energy (10-11 kcal/mol). In addition, the protein now also renatures between 30 and 50 degrees C with activation energies of 1-2 (heparin), 16 (inositol hexasulfate), and 7 (sulfate) kcal/mol. Trace heavy metals appear to inhibit the refolding process, but a molecular chaperone (bovine 70-kDa heat shock cognate protein) and a peptidylprolyl isomerase (the FK506-binding protein) have no effect. It is concluded that the rate of refolding of aFGF at physiological temperatures is probably controlled by the interaction of a native-like state of the protein with an unknown polyanionic species.     

Effect of Temperature on Respiration of Mitochondria and Shoot Segments from Cold hardened and Nonhardened Wheat and Rye Seedlings

The effect of temperature on respiration of mitochondria and tissue segments from three wheat (Triticum aestivum L.) and one rye (Secale cereale L.) cultivar grown at 2 and 24 C has been examined. Discontinuities in Arrhenius plots of respiratory activity against temperature were observed for mitochondria and tissue segments from seedlings grown at both temperatures. The rates of respiration decreased abruptly below the transition temperatures, resulting in increased energy of activation values for respiration. Transition temperatures were observed from 6 to 10 C during tissue segment respiration, and from 10 to 14 C during respiration by isolated mitochondria. Respiratory control and efficiency of phosphorylation were not affected markedly by either reaction temperature or growth temperature of the seedlings. No correlation was observed between the cold hardiness of the cultivars and the temperature at which structural transitions occurred in the mitochondria. Dry matter content of the seedlings increased markedly during growth at 2 C, but no appreciable changes in the levels of mitochondrial protein were observed. The results support the view that changes other than fatty acid unsaturation are involved in the abrupt change in mitochondrial membrane properties at low temperature.     

Thermodynamic analysis of catalysis by the dihydroorotases from hamster and Bacillus caldolyticus, as compared with the uncatalyzed reaction

Dihydroorotase (DHOase, EC 3.5.2.3) from the extreme thermophile Bacillus caldolyticus has been subcloned, sequenced, expressed, and purified as a monomer. The catalytic properties of this thermophilic DHOase have been compared with another type I enzyme, the DHOase domain from hamster, to investigate how the thermophilic enzyme is adapted to higher temperatures. B. caldolyticus DHOase has higher Vmax and Ks values than hamster DHOase at the same temperature. The thermodynamic parameters for the binding of L-dihydroorotate were determined at 25 degrees C for hamster DHOase (deltaG = -6.9 kcal/mol, deltaH = -11.5 kcal/mol, TdeltaS = -4.6 kcal/mol) and B. caldolyticus DHOase (deltaG = -5.6 kcal/mol, deltaH = -4.2 kcal/mol, TdeltaS = +1.4 kcal/mol). The smaller enthalpy release and positive entropy for thermophilic DHOase are indicative of a weakly interacting Michaelis complex. Hamster DHOase has an enthalpy of activation of 12.3 kcal/mol, similar to the release of enthalpy upon substrate binding, rendering the kcat/Ks value almost temperature independent. B. caldolyticus DHOase shows a decrease in the enthalpy of activation from 12.2 kcal/mol at temperatures from 30 to 50 degrees C to 5.3 kcal/mol for temperatures of 50-70 degrees C. Vibrational energy at higher temperatures may facilitate the transition ES --> ES(double dagger), making kcat/Ks almost temperature independent. The pseudo-first-order rate constant for water attack on L-dihydroorotate, based on experiments at elevated temperature, is 3.2 x 10(-11) s(-1) at 25 degrees C, with deltaH(double dagger) = 24.7 kcal/mol and TdeltaS(double dagger) = -6.9 kcal/mol. Thus, hamster DHOase enhances the rate of substrate hydrolysis by a factor of 1.6 x 10(14), achieving this rate enhancement almost entirely by lowering the enthalpy of activation (delta deltaH(double dagger) = -19.5 kcal/mol). Both the rate enhancement and transition state affinity of hamster DHOase increase steeply with decreasing temperature, consistent with the development of H-bonds and electrostatic interactions in the transition state that were not present in the enzyme-substrate complex in the ground state.