On enzymatic clotting processes. II. The colloidal instability of chymosin-treated casein micelles.

The enzymatic clotting of casein micelles dispersed in 0.01 M CaCl₂ was monitored by turbidimetry and electrophoresis. The relation between the duration of the lag phase and the enzyme concentration, (e), can be represented by t = K(e)^?γ, where K is a constant and the exponent γ is found to vary between 0.92 and 1.00. This result is interpreted in terms of a flocculation rate constant increasing with the concentration of the enzyme. It is shown that the colloidal instability of chymosin-treated casein micelles cannot be explained on the basis of the well-known theory of the stability of lyophobic colloids, but that clotting is achieved through short-range interactions. The short-range effects that most probably account for the clotting are: hydrophobic bond formation, Ca-bridgas and electrostatic interactions. Under typica'. experimental conditions (33°C; maximum rate of enzymatic product formation about 1.8 × 10^?10 mol ml^?1 s^?1) the flocculation rate constant of clotting micelles was found to be 5 × 10⁵ mlmol^?1s^?1. Various factors, which could be responsible for this low value, are discussed. In the initial stages of the clotting process the turbidity of the system passes through a shallow minimum, which is ascribed to the cleavage of a macropeptide from K-casein by the clotting enzyme. The condition for the minimum has been derived.     

Effect of temperature on dynamic viscoelasticity during the clotting reaction of fibrin

The dynamic rigidity and loss moduli for fibrinogen-thrombin solution were determined during clotting in the temperature range between 15 and 45 degrees C. The rigidity of fibrin gel decreased with increasing clotting temperature, owing to the dissociation of cross-links. The rate constant of the dissociation of cross-links increased with increasing temperature. The rate constant of the cross-linking reaction increased and then decreased through a maximum with increasing temperature. It is explained by assuming that denaturation of fibrin occurs at high temperature. The irreversible denaturation becomes appreciable at high ionic strength. The activation energy and the enthalpy change for the cross-linking reaction of fibrin is about 35 and 15 kcal/mol, respectively. The enthalpy change for the reversible denaturation is about 46 kcal/mol.     

Kinetics of internalization and degradation of asialo-glycoproteins in isolated rat hepatocytes

The rate constants for internalization of surface-bound asialo-orosomucoid by hepatocytes were 0.040 min-1 at 20 degrees C, 0.18 min-1 at 30 degrees C and 0.28 min-1 at 40 degrees C. At 40 degrees C, internalization accounted for most of the increase in cell-associated radioactivity. The activation energy over the temperature range 20 to 40 degrees C was 68 +/- 7 (S.D.) kJ/mol. At 10 degrees C, most of the cell-associated asialo-orosomucoid was bound to the cell surface in a reaction which followed ordinary chemical kinetics. Pre-incubation of hepatocytes with a large concentration of unlabelled asialo-orosomucoid did not influence the uptake of subsequently added 125I-asialofetuin; neither was degradation of 125I-asialo-fetuin affected in this experiment. The fractional rate of degradation (the fraction of cell-associated asialo-fetuin which was degraded per unit time) was constant over a twelve-fold range of intracellular asialo-fetuin concentrations. Increasing the temperature from 20 to 30 degrees C produced approximately a ten-fold increase in the rate of degradation of either asialo-fetuin or asialo-orosomucoid. The average activation energies of degradation over the range 20 to 40 degrees C were 125 kJ/mol for asialo-fetuin and 149 kJ/mol for asialo-orosomucoid; however, the Arrhenius plots were not straight lines over this temperature range.     

Temperature response of photosynthesis and internal conductance to CO2: results from two independent approaches

The internal conductance to CO(2) transfer from intercellular spaces to chloroplasts poses a major limitation to photosynthesis, but few studies have investigated its temperature response. The aim of this study was to determine the temperature response of photosynthesis and internal conductance between 10 degrees C and 35 degrees C in seedlings of a deciduous forest tree species, Quercus canariensis. Internal conductance was estimated via simultaneous measurements of gas exchange and chlorophyll fluorescence ("variable J method"). Two of the required parameters, the intercellular photocompensation point (C(i)*) and rate of mitochondrial respiration in the light (R(d)), were estimated by the Laisk method. These were used to calculate the chloroplastic photocompensation point (Gamma*) in a simultaneous equation with g(i). An independent estimate of internal conductance was obtained by a novel curve-fitting method based on the curvature of the initial Rubisco-limited portion of an A/C(i) curve. The temperature responses of the rate of Rubisco carboxylation (V(cmax)) and the RuBP limited rate of electron transport (J(max)) were determined from chloroplastic CO(2) concentrations. The rate of net photosynthesis peaked at 24 degrees C. C(i)* was similar to reports for other species with a C(i)* of 39 micromol mol(-1) at 25 degrees C and an activation energy of 34 kJ mol(-1). Gamma* was very similar to the published temperature response for Spinacia oleracea from 20 degrees C to 35 degrees C, but was slightly greater at 10 degrees C and 15 degrees C. J(max) peaked at 30 degrees C, whereas V(cmax) did not reach a maximum between 10 degrees C and 35 degrees C. Activation energies were 49 kJ mol(-1) for V(cmax) and 100 kJ mol(-1) for J(max). Both methods showed that internal conductance doubled from 10 degrees C to 20 degrees C, and then was nearly temperature-independent from 20 degrees C to 35 degrees C. Hence, the temperature response of internal conductance could not be fitted to an Arrhenius function. The best fit to estimated g(i) was obtained with a three-parameter log normal function (R(2)=0.98), with a maximum g(i) of 0.19 mol m(-2) s(-1) at 29 degrees C.     

Thermal inactivation and reactivation of an enzyme in vivo. Pantothenate hydrolase of Pseudomonas fluorescens

Thermal inactivation and reactivation of pantothenate hydrolase were studied in whole cells of Pseudomonas fluorescens. The enzyme is susceptible to thermal inactivation in whole cells at 37-40 degrees C, and is reactivated when the temperature is lowered again. Chloramphenicol does not prevent reactivation. The activation energy of enzyme inactivation in vivo is about 540kJ/mol. This activation energy is 220kJ/mol in vitro, but it is increased to 550-630kJ/mol by several metabolites, such as succinate, glyoxylate and oxalate. Generally, good carbon sources, causing rapid growth, protect the enzyme from thermal inactivation in vivo, and enable reactivation to occur at a fast rate. The enzyme is also inactivated below 35 degrees C, showing an activation energy of about 35kJ/mol. Good carbon sources prevent this inactivation as well, and cause slight reactivation. Glycine, although not utilized for growth, protects the enzyme well from this inactivation but not from inactivation at 37-40 degrees C, and prevents reactivation totally. From the activation energies of inactivation and the effects of the various carbon sources, it appears possible that changes in the concentrations of intracellular metabolites may be responsible for the changes in inactivation and reactivation.     

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."     

Temperature dependence of mammalian muscle contractions and ATPase activities

Isolated rat and mouse extensor digitorum longus (EDL) and soleus muscles were studied under isometric and isotonic conditions at temperatures from approximately 8 degrees -38 degrees C. The rate constant for the exponential rise of tension during an isometric tetanus had a Q10 of approximately 2.5 for all muscles (corresponding to an enthalpy of activation, delta H = 66 kJ/mol, if the rate was determined by a single chemical reaction). The half-contraction time, contraction time, and maximum rate of rise for tension in an isometric twitch and the maximum shortening velocity in an isotonic contraction all had a similar temperature dependence (i.e., delta H approximately 66 kJ/mol). The Mg++ ATPase rates of myofibrils prepared from rat EDL and soleus muscles had a steeper temperature dependence (delta H = 130 kJ/mol), but absolute rates at 20 degrees C were lower than the rate of rise of tension. This suggests that the Mg++ ATPase cycle rate is not limiting for force generation. A substantial fraction of cross-bridges may exist in a resting state that converts to the force-producing state at a rate faster than required to complete the cycle and repopulate the resting state. The temperature dependence for the rate constant of the exponential decay of tension during an isometric twitch or short tetanus (and the half-fall time of a twitch) had a break point at approximately 20 degrees C, with apparent enthalpy values of delta H = 117 kJ/mol below 20 degrees C and delta H = 70 kJ/mol above 20 degrees C. The break point and the values of delta H at high and low temperatures agree closely with published values for the delta H of the sarcoplasmic reticulum (SR) Ca++ ATPase. Thus, the temperature dependence for the relaxation rate of a twitch or a short tetanus is consistent with that for the reabsorption rate of Ca++ into the SR.     

The bactericidal activity of a methyl and propyl parabens combination: isothermal and non-isothermal studies.

The effect of temperature on the kill rate of Escherichia coli by methyl and propyl parabens was studied. The kill kinetics was first order. It was shown that the Arrhenius equation provided a good model for describing the relationship between the first order rate constant and the temperature. The activation energy was found to be 274 kJ/mol for exponential phase cells and 168 kJ/mol for stationary phase cells. Exponential phase cells were much more susceptible to the lethal effects of the parabens than were the stationary phase cells. For example, at 34 degrees C stationary phase cells, in chemically defined media, had a kill rate constant of 0.072/h while the corresponding value for exponential phase cells was 0.238/h. In water the rate of kill for exponential phase cells was even faster giving a rate constant of 5.25/h at 34 degrees C. Non-isothermal kinetic testing was not found to be useful for modelling bacterial kill kinetics because we could not achieve the precision required in bacterial enumeration.     

Hydrolysis of inulin: a kinetic study of the reaction catalyzed by an inulinase from Aspergillus ficuum

A kinetic study of the hydrolysis of inulin was performed by using as catalyst a commercial inulinase from Aspergillus ficuum. The reaction was studied carrying out initial rate as well as time course measurements. Both inulinase and invertase activities of the enzyme were taken into account, and the corresponding kinetic parameters were determined in the temperature range 30-50 degrees C. The activation energies of the turnover constant for inulinase and invertase activities were found to be similar (56-57 kJ . mol(-1)). The ratio S/I of invertase to inulinase activity was 1.6 regardless of temperature. The thermal degradation of the enzyme was also investigated up to 70 degrees C, and an activation energy of 350-370 kJ . mol(-1) was evaluated.     

Temperature dependence and acclimatory response of amylase in the High Arctic springtail Onychiurus arcticus (Tullberg) compared with the temperate species Protaphorura armata (Tullberg)

Amylolytic activity was measured in whole body homogenates of High Arctic (Onychiurus arcticus) and temperate (Protaphorura armata) springtails (Collembola: Onychiuridae) in the temperature range 5-55 degrees C. A pH of ca. 8 was optimum for amylolytic activity in both species. A higher weight-specific amylolytic activity was observed in P. armata. In O. arcticus, amylolytic activity depended on thermal acclimation, which increased during 2 and 9 weeks of cold acclimation (5 degrees C) and decreased over 7 weeks of warming (15 degrees C) of animals that were previously acclimated to cold for 2 weeks. In cold-acclimated O. arcticus, a slower decrease of amylolytic activity occurred with lowering of temperature in the range 5-20 degrees C in comparison with warm-acclimated specimens and P. armata, which resulted in higher activity at 5 degrees C. The activation energy calculated from an Arrhenius plot for P. armata was 68.7 kJ.mol(-1). In O. arcticus it was between 30.2 and 61.5 kJ.mol(-1), being lower in cold-acclimated samples. The temperature optimum for amylolytic activity was higher in the temperate species (40 degrees C), whilst in O. arcticus it depended on the acclimation regime: it rose to 35 degrees C after warm acclimation and decreased to 20 degrees C after cold adaptation. The total soluble protein content of body tissues of O. arcticus also increased during cold acclimation. These differences between the two species suggest that amylolytic activity is an indicator of cold adaptation in the High Arctic O. arcticus.     

High-pressure effect on the equilibrium and kinetics of cyanide binding to chloroperoxidase

The kinetics of cyanide binding to chloroperoxidase were studied using a high-pressure stopped-flow technique at 25 degrees C and pH 4.7 in a pressure range from 1 to 1000 bar. The activation volume change for the association reaction is delta V not equal to + = -2.5 +/- 0.5 ml/mol. The total reaction volume change, determined from the pressure dependence of the equilibrium constant, is delta V degrees = -17.8 +/- 1.3 ml/mol. The effect of temperature was studied at 1 bar yielding delta H not equal to + = 29 +/- 1 kJ/mol, delta S not equal to + = -58 +/- 4 J/mol per K. Equilibrium studies give delta H degrees = -41 +/- 3 kJ/mol and delta S degrees = -59 +/- 10 J/mol per K. Possible contributions to the binding process are discussed: changes in spin state, bond formation and conformation changes in the protein. An activation volume analog of the Hammond postulate is considered.     

Studies of glutamate dehydrogenase. Regulation of glutamate dehydrogenase from Candida utilis by a pH and temperature-dependent conformational transition.

Glutamate dehydrogenase from Candida utilis undergoes a reversible conformational transition between an active and an inactive state at low pH AND low temperature. This conformational transition can also be followed by fluorescence measurements. The temperature-dependent equilibrium between the active and the inactive state is characterized by a transition temperature of 10.7 degrees C and a delta H value of 148 kcal/mol (620 kJ/mol). The temperature dependence of the enzymic activity above 15 degrees C yields an activation energy of 15 kcal/mol (63 kJ/mol), a larger value than that for the beef liver enzyme (9 kcal/mol; 38 kJ/mol). In contrast to the yeast enzyme the Arrhenius plot is linear and, therefore, the beef liver enzyme is not transformed into an inactive conformation at low temperatures. Sedimentation analysis shows that the inactivation of the Candida utilis enzyme is not caused by change in the quaternary structure. The pH dependence of the conformational transition at low pH measured by fluorescence change is characterized by a pK value of 7.01 for the enzyme in the absence and of 6.89 for the enzyme in the presence of 2-oxoglutarate with a Hill coefficient of 3.4 in both cases. Similar results are found when the pH dependence of the enzymic activity is analyzed. With the beef liver enzyme the same pK value is obtained but with a Hill coefficient of 1 indicating cooperativity only in the case of the Candida utilis enzyme. The best fit of the pH dependence of the rate constants of the fluorescence changes was obtained with pK values of 7.45 and 6.45 for the active and the inactive state respectively. In this model the lowest time constant which is obtained at the pH of the equilibrium was found to be 0.05 s-1. Preincubation experiments with the substrate 2-oxoglutarate but not with the coenzyme shift the equilibrium to the active conformation. The coenzyme obviously reduces the rate constant of the conformational transition. The sedimentation coefficient (SO20, w) and the molecular weight were found to be 11.0 S and 276 000, respectively. The enzyme molecule is built up by six polypeptide chains each having a molecular weight of 47 000.     

Effect of temperature on cuticular transpiration of isolated cuticular membranes and leaf discs

Cuticular transpiration was measured in the temperature range between 10 degrees C and 55 degrees C using tritiated water and five species (Vinca major L., Prunus laurocerasus L., Forsythia intermedia L., Citrus aurantium L., and Hedera helix L.). Cuticular water permeabilities measured with isolated cuticular membranes were not different from cuticular water permeabilities measured with leaf discs. Depending on the species cuticular water permeabilities increased by factors between 12 (V. major) to 264 (H. helix) when temperature was increased from 10 degrees C to 55 degrees C. Arrhenius plots (lnP versus 1/T) of all investigated species were characterized by phase transitions occurring in the temperature range of 30-39 degrees C. Activation energies for water permeability across plant cuticles below and above the midpoint of phase transition were calculated from Arrhenius plots. Depending on the species they varied between 26 (F. intermedia) to 61 kJ mol(-1) (H. helix) below the phase transition and from 67 (V. major) to 122 kJ mol(-1) (F. intermedia) above the phase transition. Since the occurrence of phase transitions always lead to significantly increased rates of cuticular transpiration it is argued that temperatures higher than 35 degrees C caused structural defects to the transport-limiting barrier of the plant cuticles of all species investigated.     

Kinetics of ganglioside transfer between liposomal and synaptosomal membranes

The transfer of ganglioside GM1 from micelles to membranes and between different membrane populations has been examined by using a pyrene fatty acid derivative of the ganglioside. The transfer of gangliosides from micelles to membranes depends on the physical state as well as the molecular composition of the acceptor vesicles. At 30 degrees C, the transfer of micellar gangliosides to dipalmitoylphosphatidylcholine (DPPC) large unilameller vesicles (Tm = 41.3 degrees C) is characterized by a rate constant of 0.01 min-1; at 48 degrees C, however, the rate constant is 0.11 min-1. Below the phase transition temperature, the activation energy is 25 kcal/mol whereas above the phase transition it is 17 kcal/mol. Similar experiments performed with synaptic plasma membranes yielded a rate constant of 0.05 min-1 at 37 degrees C. The rate of transfer of ganglioside molecules, asymmetrically located on the outer layer of donor vesicles, to acceptor vesicles lacking ganglioside depends on the physical state of both the donor and acceptor vesicles. For the transfer of ganglioside from DPPC (donor) vesicles to dimyristoylphosphatidylcholine (DMPC) (acceptor) vesicles, the rates were essentially zero at 15 degrees C in which both vesicle populations were in the gel phase, 0.008 min-1 at 30 degrees C in which DPPC is in the gel phase and DMPC is in the fluid phase, and 0.031 min-1 at 48 degrees C in which both vesicle populations are in the fluid phase. The transfer of ganglioside from DPPC vesicles to synaptic plasma membranes was also dependent on the physical state of the donor vesicles and showed an inflection point at the phase transition temperature of DPPC.(ABSTRACT TRUNCATED AT 250 WORDS)     

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 and structural characterization of manganese(II)-loaded methionyl aminopeptidases

Manganese(II) activation of the methionyl aminopeptidases from Escherichia coli (EcMetAP-I) and the hyperthermophilic archaeon Pyrococcus furiosus (PfMetAP-II) was investigated. Maximum catalytic activity for both enzymes was obtained with 1 equiv of Mn(II), and the dissociation constants (K(d)) for the first metal binding site were found to be 6 +/- 0.5 and 1 +/- 0.5 microM for EcMetAP-I and PfMetAP-II, respectively. These K(d) values were verified by isothermal titration calorimetry (ITC) and found to be 3.0 +/- 0.2 and 1.4 +/- 0.2 microM for EcMetAP-I and PfMetAP-II, respectively. The hydrolysis of MGMM was measured in triplicate between 25 and 85 degrees C at eight substrate concentrations ranging from 2 to 20 mM for PfMetAP-II. Both specific activity and K(m) values increased with increasing temperature. An Arrhenius plot was constructed from the kcat values and was found to be linear over the temperature range 25-85 degrees C. The activation energy for the Mn(II)-loaded PfMetAP-II hydrolysis of MGMM was found to be 25.7 kJ/mol while the remaining thermodynamic parameters calculated at 25 degrees C are DeltaG+ = 50.1 kJ/mol, DeltaH+ = 23.2 kJ/mol, and DeltaS++ = -90.2 J x mol(-1) x K(-1).     

Thermodynamics of the water permeability of plant cuticles: characterization of the polar pathway

The water permeability of cuticles isolated from the leaves of 14 plant species was measured at temperatures from 10 degrees C to 55 degrees C at 5 K intervals. Permeances increased slightly with temperatures < or =35 degrees C and drastically in the higher temperature range. The data were analysed according to the Arrhenius formalism which led to distinct plots for the lower and higher temperature range, respectively. Activation energies of permeation for the lower temperature range were estimated to amount to 15.2-52.5 kJ mol(-1), at higher temperature activation energies ranged from 52.2-117.3 kJ mol(-1). This thermodynamics approach is used for further elucidating the pathway taken by water across the plant cuticle. Based on the results of this study it is hypothesized that the diffusion of water occurs along polysaccharide strands crossing the cuticle and that the transport properties of these polar pathways change with temperature.     

Flow cytometric analysis of the uptake of Hoechst 33342 dye by human lymphocytes

Human peripheral blood lymphocytes have been shown to resist staining with the DNA binding fluorochrome Hoechst 33342 by the cellular membrane. The rate of uptake of the dye is strongly temperature-dependent with minimal uptake rate below 16 degrees C. The activation energy of dye transport was found to be 135 kJ/mol above 20 degrees C and about 20 kJ/mol below 16 degrees C. Metabolic inhibitors accelerated, instead of inhibiting, the transport of the dye. Dead cells have been shown to stain promptly in contrast with the gradually staining viable cells. The uptake process in the vital staining conditions is suggested to involve a carrier mediated mechanism. Application of Hoechst 33342 as a fluorescent indicator of viability is proposed.     

Temperature dependence of force, velocity, and processivity of single kinesin molecules

Using the bead assay in optical microscopy equipped with optical tweezers, we have examined the effect of temperature on the gliding velocity, force, and processivity of single kinesin molecules interacting with a microtubule between 15 and 35 degrees C. The gliding velocity increased with the Arrhenius activation energy of 50 kJ/mol, consistent with the temperature dependence of the microtubule-dependent ATPase activity. Also, the average run length, i.e., a measure of processivity of kinesin, increased on increasing temperature. On the other hand, the generated force was independent of temperature, 7.34 +/- 0.33 pN (average +/- S.D., n = 70). The gliding velocities decreased almost linearly with an increase in force irrespective of temperature, implying that the efficiency of mechano-chemical energy conversion is maintained constant in this temperature range. Thus, we suggest that the force generation is attributable to the temperature-insensitive nucleotide-binding state(s) and/or conformational change(s) of kinesin-microtubule complex, whereas the gliding velocity is determined by the ATPase rate.