Temperature-dependencies of various catalytic activities of membrane-bound Na+/K+-ATPase from ox brain, ox kidney and shark rectal gland and of C12E8-solubilized shark Na+/K+-ATPase

The temperature dependence of ouabain-sensitive ATPase and phosphatase activities of membrane fragments containing the Na+/K+-ATPase were investigated in tissue from ox kidney, ox brain and from shark rectal glands. The shark enzyme was also tested in solubilized form. Arrhenius plots of the Na+/K+-ATPase activity seem to be linear up to about 20 degrees C, and non-linear above this temperature. The Arrhenius plots of mammalian enzyme (ox brain and kidney) were steeper, especially at temperatures below 20-30 degrees C, than that of shark enzyme. The Na+-ATPase activity showed a weaker temperature-dependence than the Na+/K+-ATPase activity. The phosphatase reactions measured, K+-stimulated, Na+/K+-stimulated and Na+/K+/ATP-stimulated, also showed a weaker temperature-dependence than the overall Na+/K+-ATPase activity. Among the phosphatase reactions, the largest change in slope of the Arrhenius plot was observed with the Na+/K+/ATP)-stimulated phosphatase reaction. The Arrhenius plots of the partial reactions were all non-linear. Solubilization of shark enzyme in C12E8 did not change the curvature of Arrhenius plots of the Na+/K+-ATPase activity or the K+-phosphatase activity. Since solubilization involves a disruption of the membrane and an 80% delipidation, the observed curvature of the Arrhenius plot can not be attributed to a property of the membrane as such.     

The thermal properties of intestinal alkaline phosphatase of three kinds of deep-water fish

1. The thermal properties of intestinal alkaline phosphatase were investigated with three species of deep-water fish in the temperature range of 0-70 C. 2. A relationship was found between the thermal stability of the enzyme and the fish origin. 3. Maximum activity of alkaline phosphatase of the fish that originated in tropical water, namely, Aphanopus carbo and Epigonus telescopus was 60 C, whereas the respective maximum enzyme activity of Etmopterus princeps that originated in the burial zone was 30 C. 4. A breakpoint at 10 degrees C in the Arrhenius plot of emzyme activity in the case of A. carbo and a lack of a break point in the case of E. telescopus and E. princeps, are in accordance with the stenothermic nature of the former and the everythermic nature of the two latter fish species.     

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.     

Enzyme action in polymer and salt solutions. I. Stability of penicillin acylase in poly(ethylene glycol) and potassium phosphate solutions in relation to water activity

The stability of penicillin acylase (penicillin aminohydrolase, EC 3.5.1.11) was studied in poly(ethylene glycol) and potassium phosphate solutions. Enzyme stability measured as the half-life of the enzymatic activity and the transition temperature determined by differential scanning calorimetry, correlated well. The enzyme stability could not be related to the water activity as a measure of solute-solvent interaction. It seems to be related more to the concentration of the solutes and much less to the molecular weight of poly(ethylene glycol). The stabilizing effect of poly(ethylene glycol) is also discussed in terms of poly(ethylene glycol)-protein interactions.     

Yeast fructose-2,6-bisphosphate 6-phosphatase is encoded by PHO8, the gene for nonspecific repressible alkaline phosphatase.

Yeast fructose-2,6-bisphosphate 6-phosphatase has been purified 7000-fold by heat treatment, poly(ethylene glycol) precipitation, ion-exchange chromatography with Q-Sepharose Fast Flow and Mono Q followed by affinity chromatography with concanavalin-A-Sepharose and gel filtration with Superose 12. The purified dimeric enzyme contains 1.5 mol zinc and 1.3 mol copper/mol subunit. It reacts with fructose 2,6-bisphosphate [Fru(2,6)P2] as well as with p-nitrophenyl phosphate (NpP) showing a pH optimum at pH 6-6.5 with Fru(2,6)P2 [Plankert, U., Purwin, C. & Holzer, H. (1988) FEBS Lett. 239, 69-72] and above pH 9.0 with NpP. The following observations suggest that activity with both substrates depends on the same protein. (a) During 7000-fold purification, the ratio of activity with NpP to that with Fru(2,6)P2 remained constant. (b) The time course of inactivation of enzyme activity in dilute solution at 30 degrees C is similar for both substrates. (c) At increasing temperatures, inactivation of enzyme activity measured with both substrates proceeds at nearly identical rates. (d) Activity with both substrates is found preferentially in the vacuoles. (e) Mutants defective in the nonspecific alkaline phosphatase coded by the PHO8 gene are also defective in Fru(2,6)P2 6-phosphatase activity. (f) A proteinase A mutant, defective in processing and activation of nonspecific alkaline phosphatase coded by the PHO8 gene, also fails to activate Fru(2,6)P2 6-phosphatase.     

Effect of solvent, pressure and temperature on reaction rates of the multiheme hydroxylamine oxidoreductase. Evidence for conformational change

Hydroxylamine oxidoreductase (HAO) of the ammonia-oxidizing bacterium Nitrosomonas catalyzes the oxidation: NH2OH + H2O----HNO2 + 2e- + 2 H+. The heme-like chromophore P460 is part of a site which binds substrate, extracts electrons and then passes them to the many c hemes of the enzyme. Reduction of the c hemes by hydroxylamine is biphasic with apparent first-order rate constants k1 and k2. CO binds to ferrous P460 with apparent first-order rate constants, k1,CO. In this work we have measured the binding of CO to ferrous P460 of hydroxylamine oxidoreductase and the reduction by substrate of some of the 24 c hemes of the ferric enzyme. These reactions have been studied in water and 40% ethylene glycol, at temperatures ranging from -15 degrees C to 20.7 degrees C and at hydrostatic pressures ranging over 0.1-80 MPa. From the measurements, thermodynamic parameters delta V+ (activation volume), delta G+, delta H+, and delta S+ have been calculated. CO binding. Binding of CO to ferrous P460 was similar to the binding of CO to ferrous horseradish peroxidase. The change of solvent had only a limited effect on delta V+ (-30 ml.mol-1), delta G+, delta H+ or delta S+ and did not cause an inflection in the Arrhenius plot or downward displacement of the linear relationship between ln k1,CO and P at a critical temperature. Binding was exothermic at high temperatures. The response of the binding of CO to solvent, temperature and pressure suggested that the CO binding site had little access to solvent and was not susceptible to change in protein conformation. Fast phase of reduction of c hemes. Changing the solvent from water to 40% ethylene glycol resulted in a decrease from 90% to 50% in the relative number of c hemes reduced during the fast phase, an increase in activation volume from -3.6 ml.mol-1 to 57 ml.mol-1 and changes in other thermodynamic parameters. The activation volume increased with decreasing temperature. The Arrhenius plot had a downward inflection at about 0 degrees C and, in water or ethylene glycol, the linear dependence of ln k1 on P was displaced downwards as the temperature changed from 3.5 degrees C to -15 degrees C. Slow phase of reduction of c hemes. Changing the solvent from water to 40% ethylene glycol resulted in an increase in the relative number of c hemes reduced during the slow phase from 10% to 50%. The activation volume, which was not measurable in water because of the low absorbance change, was -30 ml.mol-1 in ethylene glycol. The activation volume increased with increasing temperature.(ABSTRACT TRUNCATED AT 400 WORDS)     

Isatin-enzyme interactions. VII. Mechanism of inhibition of rat kidney alkaline phosphatase.

Isatin has been found to inhibit rat kidney alkaline phosphatase (EC 3.1.3.1). The inhibition is dependent on isatin concentration and is of un-competitive type. The hydrolysis of disodium phenyl phosphate by the enzyme at different temperatures (17--37 degrees C) obeys the Arrhenius equation. Energy of activation in the absence and presence of isatin has been found to be 9.84 and 10.24 kCal/mol. The hyperbolic profile of isatin inhibition; the lowering of both Km and Vmax in the presence of isatin, and, small changes in enthalpy, free energy and entropy in the presence of isatin suggest a non-allosteric un-competitive inhibition of the enzyme.     

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

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

Physico-chemical mechanisms of organic acid transport in the apical membrane cells of the proximal kidney tubules in rats. I. Kinetic transport parameters and effect of the lipid phasic state on transport

The kinetics of the transport of 3H-para-aminohippuric acid (PAH) and the influence of the temperature on the initial rate of transport were studied on the vesicles of a purified fraction of the apical membrane isolated from cells of kidney proximal tubules. The PAH transport is accomplished owing to the facilitate diffusion mechanism. The apparent Michaelis constant at 36 degrees C was equal to 7.0 + 1.0 mM, the maximum rate was 15 nmol/min on 1 mg of protein, the inhibition constant for the PAH transport by probenecid being 0.5 mM. At 22 degrees C the apparent Michaelis constant was drastically increased. When the temperature dependence of the initial rate of PAH transport into vesicles was replotted in the form of the Arrhenius plot, there was a turning-point of the line at 28-30 degrees C. The same turning-point is shown on the Arrhenius plot for temperature dependence of alkaline phosphatase activity (a marker enzyme for the apical membrane). The electron paramagnetic resonance spectra analysis of 5-doxylstearate-labeled apical membrane preparation reveals a thermotropic transition near 21-29 degrees C. It is concluded that the function of the carrier and the activity of alkaline phosphatase depend on the phasic state of membrane lipids; the normal function of membrane proteins is possible under the liquid-crystalline state of the lipid bilayer.     

Fructose bisphosphate aldolase from rabbit muscle. A jump in the van't Hoff plot accompanies the onset of half of the sites' reactivity

In 40% ethylene glycol, gamma/2 = 0.11 and pH* 8.2, fructose 1,6-bisphosphate aldolase from rabbit muscle undergoes a transition: above 3 degrees C it displays 4 equivalent dihydroxyacetone phosphate binding sites, below -1 degree C the sites decrease to 2. The dissociation constant of the aldolase-dihydroxyacetone phosphate complex decreases from 10 microM at 3 degrees C to 2.65 microM at -1 degree C, its van't Hoff plot being linear between -1 degree C and -13 degrees C. The rate of the detritiation of the aldolase-(3S)-[3-3H]dihydroxyacetone phosphate complex is strongly influenced by temperature. In 40% ethylene glycol, gamma/2 = 0.01 and pH* 8.2, the apparent rate constant is 7.6 sec-1 at -5 degrees C and 0.012 sec-1 at -24 degrees C. The Arrhenius plot is linear between -5 degrees C and -24 degrees C.     

Enzymatic removal of alkaline phosphatase from renal brush-border membranes. Effect on phosphate transport and on phosphate binding

Brush-border membrane vesicles prepared from rabbit kidney cortex were incubated at 37 degrees C for 30 min with phosphatidylinositol-specific phospholipase C. This maneuver resulted in a release of approx. 85% of the brush-border membrane-linked enzyme alkaline phosphatase as determined by its enzymatic activity. Transport of inorganic [32P]phosphate (100 microM) by the PI-specific phospholipase C-treated brush-border membrane vesicles was measured at 20-22 degrees C in the presence of an inwardly directed 100 mM Na+ gradient. Neither initial uptake rates, as estimated from 10-s uptake values (103.5 +/- 6.8%, n = 7 experiments), nor equilibrium uptake values, measured after 2 h (102 +/- 3.4%) were different from controls (100%). Control and PI-specific phospholipase C-treated brush-border membrane vesicles were extracted with chloroform/methanol to obtain a proteolipid fraction which has been shown to bind Pi with high affinity and specificity (Kessler, R.J., Vaughn, D.A. and Fanestil, D.D. (1982) J. Biol. Chem. 257, 14311-14317). Phosphate binding (at 10 microM Pi) by the extracted proteolipid was measured. No significant difference in binding was observed between the two types of preparations: 31.0 +/- 9.37 in controls and 29.8 +/- 8.3 nmol/mg protein in the proteolipid extracted from PI-specific phospholipase C-treated brush-border membrane vesicles. It appears therefore that alkaline phosphatase activity is essential neither for Pi transport by brush-border membrane vesicles nor for Pi binding by proteolipid extracted from brush-border membrane. These results dissociate alkaline phosphatase activity, but not brush-border membrane vesicle transport of phosphate, from phosphate binding by proteolipid.     

Nanosecond responses of proteins to ultra-high temperature pulses

Observations of fast unfolding events in proteins are typically restricted to <100 degrees C. We use a novel apparatus to heat and cool enzymes within tens of nanoseconds to temperatures well in excess of the boiling point. The nanosecond temperature spikes are too fast to allow water to boil but can affect protein function. Spikes of 174 degrees C for catalase and approximately 290 degrees C for horseradish peroxidase are required to produce irreversible loss of enzyme activity. Similar temperature spikes have no effect when restricted to 100 degrees C or below. These results indicate that the "speed limit" for the thermal unfolding of large proteins is shorter than 10(-8) s. The unfolding rate at high temperature is consistent with extrapolation of low temperature rates over 12 orders of magnitude using the Arrhenius relation.     

Stabilization of dextransucrase from Leuconostoc mesenteroides NRRL B-512F by nonionic detergents, poly(ethylene glycol) and high-molecular-weight dextran

Dextransucrase (sucrose: 1,6-alpha-D-glucan 6-alpha-D-glucosyltransferase, EC 2.4.1.5) (3 IU/ml culture supernatant) was obtained by a modification of the method of Robyt and Walseth (Robyt, J.F. and Walseth, T.F. (1979) Carbohydr. Res. 68, 95-111) from a nitrosoguanidine mutant of Leuconostoc mesenteroides NRRL B-512F selected for high dextransucrase production. Dialyzed, concentrated culture supernatant (crude enzyme) was treated with immobilized dextranase (EC 3.2.1.11) and chromatographed on a column of Bio-Gel A-5m. The resulting, purified enzyme lost activity rapidly at 25 degrees C or on manipulation, as did the crude enzyme when diluted below 1 U/ml. Both enzyme preparations could be stabilized by low levels of high-molecular-weight dextran (2 micrograms/ml), poly(ethylene glycol) (e.g., 10 micrograms/ml PEG 20 000), or nonionic detergents (e.g., 10 micrograms/ml Tween 80). The stabilizing capacity of poly(ethylene glycol) and of dextran increased with molecular weight. Calcium had no stabilizing action in the absence of other additions, but reduced the inactivation that occurred in the presence of 0.5% bovine serum albumin or high concentrations (greater than 0.1%) of Triton X-100. In summary, dextransucrase could be stabilized against activity losses caused by heating or by dilution through the addition of low concentrations of nonionic polymers (dextran, PEG 20000, methyl cellulose) or of nonionic detergents at or slightly below their critical micelle concentrations.     

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.     

Temperature dependency of molecular mobility in preserved seeds

Although cryogenic storage is presumed to provide nearly infinite longevity to cells, the actual timescale for changes in viability has not been addressed theoretically or empirically. Molecular mobility within preserved biological materials provides a first approximation of the rate of deteriorative reactions that ultimately affect shelf-life. Here, temperature effects on molecular mobility in partially dried seeds are calculated from heat capacities, measured using differential scanning calorimetry, and models for relaxation of glasses based on configurational entropy. Based on these analyses, glassy behavior in seeds containing 0.07 g H(2)O/g dm followed strict Vogel-Tamman-Fulcher (VTF) behavior at temperatures above and just below the glass transition temperature (Tg) at 28 degrees C. Temperature dependency of relaxation times followed Arrhenius kinetics as temperatures decreased well below Tg. The transition from VTF to Arrhenius kinetics occurred between approximately 5 and -10 degrees C. Overall, relaxation times calculated for seeds containing 0.07 g H(2)O/g dm decreased by approximately eight orders of magnitude when seeds were cooled from 60 to -60 degrees C, comparable to the magnitude of change in aging kinetics reported for seeds and pollen stored at a similar temperature range. The Kauzmann temperature (T(K)), often considered the point at which molecular mobility of glasses is practically nil, was calculated as -42 degrees C. Calculated relaxation times, temperature coefficients lower than expected from VTF kinetics, and T(K) that is 70 degrees C below Tg suggest there is molecular mobility, albeit limited, at cryogenic temperatures.     

Different responses of tobacco antioxidant enzymes to light and chilling stress

The effect of elevated light treatment (25 degrees C, PPFD 360 mumol m-2 sec-1) or chilling temperatures combined with elevated light (5 degrees C, PPFD 360 mumol m-2 sec-1) on the activity of six antioxidant enzymes, guaiacol peroxidases, and glutathione peroxidase (GPx, EC 1.11.1.9) protein accumulation were studied in tobacco Nicotiana tabacum cv. Petit Havana SR1. Both treatments caused no photooxidative damage, but chilling caused a transient wilting. The light treatment increased the activities of ascorbate peroxidase (APx, EC 1.11.1.11) and guaiacol peroxidases while catalase (EC 1.11.1.6), superoxide dismutase (SOD, EC 1.15.1.1), monodehydroascorbate reductase (MDHAR, EC 1.6.5.4), dehydroascorbate reductase (DHAR, EC 1.8.5.1), and glutathione reductase (EC 1.6.4.2) were unchanged. In contrast, chilling treatment did not increase any of the antioxidant enzyme activities, but decreased catalase and to a lesser extent DHAR activities. Glutathione peroxidase protein levels increased sporadically under light treatment and constantly under chilling. Both chilling and light stress caused induction of glutathione synthesis and accumulation of oxidised glutathione, although the predominant part of the glutathione pool remained in the reduced form. Antioxidant enzymes from the chilling treated plants were measured at both 25 degrees C and 5 degrees C. Measurements at 5 degrees C revealed a 3-fold reduction in catalase activity, compared with that measured at 25 degrees C, indicating that the overall reduction in catalase after four days of chilling was approximately 10-fold. The overall reduction in activity for the other antioxidant enzymes after four days of chilling was 2-fold for GR and APx, 1.5-fold for MDHAR, 3.5-fold for DHAR. The activity of SOD was the same at 25 and 5 degrees C. These results indicate that catalase and DHAR are most strongly affected by the chilling treatment and may be the rate-limiting factor of the antioxidant system at low temperatures.     

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.     

Effects of carbon tetrachloride on isolated rat hepatocytes. Inhibition of protein and lipoprotein secretion.

Both the protein components Kp1 and Kp2 of nitrogenase from Klebsiella pneumoniae were found to be stable in aq. 50% (v/v) ethylene glycol at +30 degrees C or below. At -20 degrees C in this medium their sensitivities to O2 were diminished somewhat. Though purification could be carried out at -20 degrees C, the product had the same specific activity and was obtained in the same yield as when the purification was carried out by standard procedures. This suggests that such procedures yield enzyme undamaged in the course of the purification by O2, thermal denaturation or proteolytic digestion.     

Cryoenzymology of human plasmin catalysis: comparison of cryosolvents and reactions with nitrophenyl ester and anilide substrates

A comparative study of nucleophilic (methanol), aprotic (dimethyl sulfoxide), and protic but non nucleophilic (ethylene glycol, ethylene glycol/dimethylformamide) solvents on the catalytic and structural properties of human plasmin has been made. All four solvent systems are potentially suitable as cryosolvents for plasmin catalysis at subzero temperatures although the solubility of plasmin is limited in the methanol and dimethyl sulfoxide systems. Each cryosolvent system caused minor effects on the catalytic properties of the enzyme, which could be rationalized in terms of the known physical properties of the cosolvent. Solvent systems containing ethylene glycol induce a minor conformational change which increases the catalytic efficiency of plasmin. The cosolvent effects on Km and Ki indicate that electrostatic interactions dominate the binding of both substrates and inhibitors such as benzamidine. A change in slope of the Arrhenius plots for catalysis, reflecting a temperature-induced isomerization, is observed around 0 degree C; the energies of activation being 13 +/- 2 kcal mol-1 at higher temperatures and 19 +/- 2 kcal mol-1 at subzero temperatures, and essentially independent of solvent. Deacylation was shown to be the rate-limiting step in the hydrolysis of specific p-nitrophenyl ester substrates. Previous stopped-flow studies at room temperature provided observations suggesting that a tetrahedral intermediate could be detected in the plasmin-catalyzed hydrolysis of p-nitroanilide substrates. Experiments at subzero temperatures with such substrates failed to reveal any buildup of a tetrahedral intermediate under the experimental conditions.     

Purification and characterization of a novel thermo-alkali-stable catalase from Thermus brockianus

A novel thermo-alkali-stable catalase from Thermus brockianus was purified and characterized. The protein was purified from a T. brockianus cell extract in a three-step procedure that resulted in 65-fold purification to a specific activity of 5300 U/mg. The enzyme consisted of four identical subunits of 42.5 kDa as determined by SDS-PAGE and a total molecular mass measured by gel filtration of 178 kDa. The catalase was active over a temperature range from 30 to 94 degrees C and a pH range from 6 to 10, with optimum activity occurring at 90 degrees C and pH 8. At pH 8, the enzyme was extremely stable at elevated temperatures with half-lives of 330 h at 80 degrees C and 3 h at 90 degrees C. The enzyme also demonstrated excellent stability at 70 degrees C and alkaline pH with measured half-lives of 510 h and 360 h at pHs of 9 and 10, respectively. The enzyme had an unusual pyridine hemochrome spectrum and appears to utilize eight molecules of heme c per tetramer rather than protoheme IX present in the majority of catalases studied to date. The absorption spectrum suggested that the heme iron of the catalase was in a 6-coordinate low spin state rather than the typical 5-coordinate high spin state. A K(m) of 35.5 mM and a V(max) of 20.3 mM/min.mg protein for hydrogen peroxide was measured, and the enzyme was not inhibited by hydrogen peroxide at concentrations up to 450 mM. The enzyme was strongly inhibited by cyanide and the traditional catalase inhibitor 3-amino-1,2,4-triazole. The enzyme also showed no peroxidase activity to peroxidase substrates o-dianisidine and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid), a trait of typical monofunctional catalases. However, unlike traditional monofunctional catalases, the T. brockianus catalase was easily reduced by dithionite, a characteristic of catalase-peroxidases. The above properties indicate that this catalase has potential for applications in industrial bleaching processes to remove residual hydrogen peroxide from process streams.