Linkage of formate hydrogenlyase with anaerobic respiration in Proteus mirabilis

The linkage between the enzyme system catalysing formate hydrogenlyase and reductases involved in anaerobic respiration in intact cells of anaerobically grown Proteus mirabilis was studied. Reduction of nitrate and fumarate by molecular hydrogen or formate was possible under all growth conditions; reduction of tetrathionate and thiosulphate occurred only in cells harvested at late growth phase from a pH-regulated batch culture and not in cells harvested at early growth phase or in cells grown in pH-auxostat culture. Under all conditions, cells possessed the enzyme tetrathionate reductase. We conclude that linkage between tetrathionate reductase (catalysing also reduction of thiosulphate) and the formate hydrogenlyase chain is dependent on growth conditions. During reduction of high-potential oxidants such as fumarate, tetrathionate (when possible) or the artificial electron acceptor methylene blue by formate, there was no simultaneous H₂ evolution due to the formate hydrogenlyase reaction. H₂ production started only after complete reduction of methylene blue or fumarate, in the case of methylene blue after a lag phase without gas production. In preparations with a low fumarate reduction activity this was accompanied by an acceleration in CO₂ production. During reduction of thiosulphate (a low-potential oxidant) or of tetrathionate in the presence of benzyl viologen (a low-potential mediator) by formate, H₂ was evolved simultaneously. From this we conclude that formate hydrogenlyase is regulated by a factor that responds to the redox state of any electron acceptor couple present such that lyase activity is blocked when the acceptor couple is oxidised to too great an extent.     

Reduction of tetrathionate,trithionate and thiosulphate,and oxidation of sulphide in Proteus mirabilis

The reductase catalyzing the reduction of tetrathionate and thiosulphate in Proteus mirabilis is also concerned with the reduction of trithionate and the oxidation of sulphide. Tetrathionate is reduced to thiosulphate, thiosulphate to sulphite and sulphide, and trithionate is reduced to thiosulphate plus sulphite. The oxidation of sulphide in cell-free extracts proceeds most likely to polysulphanes or to elemental sulphur, depending on the conditions. The kinetics of the reduction of tetrathionate imply a simultaneous interaction of tetrathionate and thiosulphate on the reductase molecule. The reduction of tetrathionate is activated by thiosulphate causing a non-linear progress of this reaction. On the other hand the reduction of thiosulphate is completely blocked until tetrathionate has been depleted. The order of reduction in a mixture of thiosulphate and trithionate is imputed by the enzymatic constants of the reductase for both substrates. Therefore in cell-free extracts thiosulphate is reduced prior to trithionate and afterwards, when thiosulphate has been exhausted, trithionate and the produced thiosulphate are reduced simultaneously. Fast growing cells, however, reduce trithionate first since their intracellular redox potential is insulfficiently low to permit the reduction of any thiosulphate.     

Kinetics of dexamethasone binding to the glucocorticoid receptor of intact erythroid cells from rat fetal liver

The erythroid cells from the rat fetal liver have been shown to possess a receptor for glucocorticoids. In the present work, the characteristics of [3H]dexamethasone binding have been studied on intact cells, in order to minimize receptor degradation, and at 4 degrees C, in order to prevent the activation of the hormone-receptor complex. Dissociation kinetics were those of a first-order reaction and the value of the rate constant of dissociation was similar to the values available in the literature. When studied at low concentrations of the ligand and using short-term incubations, association kinetics were apparently those of a simple bimolecular reaction. But at high ligand concentrations and/or using long-term incubations, association kinetics indicated a more complex reaction. Our results were compatible with the model proposed by Pratt W.B., Kaine J.L. and Pratt V.D. (J. Biol. Chem. 250 (1975) 4584-4591) for cytosolic preparations. This model implies the rapid formation of a transient unstable form of the complex, further converted into a stable form with slower kinetics. Equilibrium dissociation constant of the first (rapid) reaction was 80 microM and the rate constant of 'stabilization' was of the order of 70 X 10(-3) min-1. These values agree with the results of Pratt et al. relative to a cytosolic preparation from rat thymocytes.     

Thiosulfate Oxidation and Tetrathionate Reduction by Intact Cells of Marine Pseudomonad Strain 16B

Levels of thiosulfate-oxidizing enzyme (TSO) and tetrathionate reductase (TTR) were measured in washed cell suspensions of a heterotrophic marine thiosulfate-oxidizing bacterium, strain 16B. TSO activity remained virtually constant in aerobically and anaerobically grown cells and was unaffected by the presence or absence of thiosulfate and tetrathionate in the growth medium. TTR was also present in cells grown aerobically and anaerobically, but its activity was threefold greater in cells cultured in media containing tetrathionate or thiosulfate. Tetrathionate appears to be the inducer of increased TTR activity in both aerobically and anaerobically grown cells. TTR (constitutive or induced) and TSO have different pH and temperature optima. Both TTR activities were unaffected by 10 mM KCN, which reversed oxygen inhibition of tetrathionate reduction. TSO was partially inhibited by 5 μM KCN and completely inhibited by 90 μM KCN. These findings and results of experiments to determine the influence of several inorganic electron donors and acceptors on TSO and TTR activities suggest that constitutive TSO and TTR represent reverse activities of the same enzyme, whereas inducible TTR is a separate enzyme used by strain 16B only for anaerobic respiration of tetrathionate. The bacterium appears well adapted to growth in environments characterized by low oxygen tension, dilute organic carbon concentrations, and the presence of a variety of reduced, inorganic sulfur compounds.     

Relaxation methods for the determination of drug receptor affinities

According to the occupation theory of drug receptor interaction, the response is a functionf of the number of receptors occupied by drug molecules. Considerable controversy exists regarding assumptions about this function. Without knowledge of the nature of the function, it is not possible to determine directly the rate constants, and hence the affinity constant, in the reaction between the receptor and an agonist drug. Instead, indirect determinations involving the use of antagonists have been employed, limiting the determination of affinity to those agents for which specific antagonists exist. The present paper discusses a method for the direct determination of affinity of an agonist drug. It is a “relaxation method,” i.e., the equilibrium is perturbed and the kinetics of the restoration process are studied. Assuming only thatf is non-decreasing and approximately linear over a limited domain of concentrations, it is shown that the change in response obeys first order kinetics, permitting a determination of the rate constants from the time course of the restoration process.     

Kinetics of solvent-free geranyl acetate synthesis by Rhizopus oligosporus NRRL 5905 lipase immobilized on to cross-linked silica

The objective of the present work was to study the kinetics of the solvent-free synthesis of geranyl acetate by a novel lipase (activity 60 U g^?1) made by immobilization of lipase from Rhizopus oligosporous NRRL 5905 on to cross-linked silica gel. Transesterification was performed with vinyl acetate as the acyl donor. Vinyl acetate was used in large excess compared to geraniol, which made the reaction pseudo first order with respect to geraniol and the reaction rate followed Michaelis–Menten kinetics for a single substrate. To obtain the highest yield for geranyl acetate, various relevant physical parameters such as shaking speed, reaction time, enzyme concentration, initial water amount and reaction temperature that influence the activity of lipase were investigated. A maximum molar conversion of 67% was achieved after 48 h of reaction at 30°C, at an enzyme concentration of 25% w/v of reaction mixture. Substrate conversion remained constant for five successive cycles; thereafter the conversion dropped by only 11%. Using a pseudo first-order kinetic model for geranyl acetate synthesis in the absence of organic solvents, apparent K_m and V_max values were evaluated as 60 mM and 141 µmol g^?1 h^?1, respectively.     

The kinetics of in situ hybridization.

The kinetics of the cytological or in situ DNA-RNA hybridization reaction between 125-I HeLa cell 18S and 28S rRNA and the interphase nuclei of chinese hamster cells were studied. The reaction is consistent with the expected first order kinetics, and the value of the rate constant was very similar to the value obtained from analogous filter disc DNA-RNA hybridization experiments. This similarity in the rate constants and the known relationship between the rate constant and the complexity of the RNA hybridized, define conditions to optimize the in situ hybridization reaction.     

Conformational isomerization of lactate dehydrogenase complexes formed by pyruvate and coenzyme analogs]

The kinetics of LDH-catalyzed reduction of pyruvate involving APADH were studied. It was shown that under conditions of a single turnover reaction the first order rate constant is equal to 37+/-4 sec-1. The reaction rate (vo) did not change when a deutero-coenzyme was used. The relationship between vo and pyruvate concentration is hyperbolic. It is concluded that isomerization of the ternary LDH-APADH-pyruvate complex limits the reaction rate. The spectral properties and the kinetics of formation and dissociation of abortive LDH complexes with pyruvate and NAD analogs (APAD and PAAD) were studied. The participation of the carboxamide group of NAD in conformational isomerization of the LDH-NADH-pyruvate and LDH-NAD-pyruvate complexes was studied.     

Studies on Biochemistry of the Thiobacilli

In the oxidation of thiosulfate at pH 4.5 tetrathionate was formed as an intermediate, and the thiosulfate-oxidizing enzyme was active in acidic pH range in contrast to the enzyme of T. thioparus and Thiobacillus X.

Phosphate did not seem to affect the oxidation of thiosulfate but rather affect the conversion of tetrathionate. In the absence of phosphate, tetrathionate, which was produced from thiosulfate oxidation, seemed to accumulate without undergoing further conversion.

Quantitative oxidation of tetrathionate to sulfate was achieved with freshly harvested cells of T. thiooxidans; pH optimum for the oxidation of tetrathionate by the washed cells was 2~3, and the activity fell markedly at pH above 3.5.

Tetrathionate might be enzymatically dismuted to pentathionate and trithionate under anaerobic conditions with crude extracts of T. thiooxidans; pH optimum for the reaction was about 2.7 and the activity fell strikingly at pH 4.7. The formed trithionate might be further hydrolyzed to thiosulfate and sulfate.     

Effect of oxyanions of sulfur onThiobacillus ferrooxidans: Ferrous ion oxidation,oxygen uptake,and cytochrome reduction

Ferrous ion oxidation byThiobacillus ferrooxidans was completely inhibited by 10 mM each of thiosulfate, sulfite, metabisulfite, bisulfite, and tetrathionate. The inhibition was enhanced in a low pH medium (pH 1.5 versus pH 2.5). Oxygen uptake measurements with Fe²⁺ as the electron donor confirmed the toxicity of thiosulfate, but also indicated its dependency on the concentration of Fe²⁺. Cytochrome spectra of intact cells ofT. ferrooxidans showed that metabisulfite, and thiosulfate to a lesser extent, directly reduced electron transport components, in contrast to no direct reduction of cytochromes by tetrathionate and sulfite.     

Mechanism of oxidation of inorganic sulfur compounds by thiosulfate-grown Thiobacillus thiooxidans

Thiobacillus thiooxidans was grown at pH 5 on thiosulfate as an energy source, and the mechanism of oxidation of inorganic sulfur compounds was studied by the effect of inhibitors, stoichiometries of oxygen consumption and sulfur, sulfite, or tetrathionate accumulation, and cytochrome reduction by substrates. Both intact cells and cell-free extracts were used in the study. The results are consistent with the pathway with sulfur and sulfite as the key intermediates. Thiosulfate was oxidized after cleavage to sulfur and sulfite as intermediates at pH 5, the optimal growth pH on thiosulfate, but after initial condensation to tetrathionate at pH 2.3 where the organism failed to grow. N-Ethylmaleimide (NEM) inhibited sulfur oxidation directly and the oxidation of thiosulfate or tetrathionate indirectly. It did not inhibit the sulfite oxidation by cells, but inhibited any reduction of cell cytochromes by sulfur, thiosulfate, tetrathionate, and sulfite. NEM probably binds sulfhydryl groups, which are possibly essential in supplying electrons to initiate sulfur oxidation. 2-Heptyl-4-hydroxy-quinoline N-oxide (HQNO) inhibited the oxidation of sulfite directly and that of sulfur, thiosulfate, and tetrathionate indirectly. Uncouplers, carbonyl cyanide-m-chlorophenylhydrazone (CCCP) and 2,4-dinitrophenol (DNP), inhibited sulfite oxidation by cells, but not the oxidation by extracts, while HQNO inhibited both. It is proposed that HQNO inhibits the oxidation of sulfite at the cytochrome b site both in cells and extracts, but uncouplers inhibit the oxidation in cells only by collapsing the energized state of cells, delta muH+, required either for electron transfer from cytochrome c to b or for sulfite binding.     

Kinetic studies of mold α-galactosidase on PNPG hydrolysis

The kinetic properties of α-galactosidase of Mortierella vinacea were investigated in detail using PNPG (p-nitrophenyl-α-D -galactopyranoside) as a substrate. Consequently, the enzyme was markedly inhibited not only by the substrate, but also by the galactose hydrolized. The initial rate of reaction at sufficiently high substrate concentrations, however, did not fall to zero and did approach a finite value. Galactose behaved as a mixed inhibitor and was neither totally competitive nor totally noncompetitive. A rate equation was obtained from a generalized equation derived from a kinetic model which took both the inhibitions into consideration. The constants used in the equation were appropriately estimated. The calculated rate agreed fairly well with the observed initial rate. Moreover, the PNPG hydrolysis progressing in a batch system was found to be approximately representable by simple first order kinetics in which the rate constant was dependent on the initial substrate concentration.     

Time dependence of activation of muscle AMP-aminohydrolase by substrate and potassium ion

The kinetics of AMP-aminohydrolase, which under steady state conditions shows a typical sigmoid dependence of initial velocities versus substrate concentration, have been examined by rapid mixing methods. Using this technique it was observed that when substrate or substrate plus activator (K(+)) were mixed with enzyme, the rate of appearance of product markedly increased during the first few tenths of a second. The time course of this change in rate was taken to reflect the progress of activation by substrate or by K(+). On the other hand, addition of activator to enzyme prior to mixing with substrate gave process curves for the formation of product consistent with normal Michaelis-Menten behaviour.Under the conditions where the reaction was examined, the enzyme at time zero had less than 10% of the activity of the fully active enzyme. The time course for activation with K(+) followed a first order process with a rate constant of 10.6 sec(-1) at 20 degrees C. A simple mechanism consistent with the data and capable of explaining the sigmoid dependence of initial velocities versus substrate concentrations observed in steady state kinetics was proposed.     

Effect of ionic liquids activation on laccase from Trametes versicolor: Enzymatic stability and activity

The stability and activity of laccase from Trametes versicolor in two water‐soluble ionic liquids (ILs), namely 1‐butyl‐3‐methylimidazolium methyl sulfate, [bmim][MeSO₄] and 1,3‐dimethylimidazolium methyl sulfate, [mmim][MeSO₄], were investigated in this study. Thermal inactivation of laccase was characterized in the presence of these both ILs and as expected first‐order kinetics was followed. Inactivation rate constant (k), half‐life time (t_1/2), and energy of activation (Ea) were determined. Kinetics of 2,2′‐azino‐bis(3‐ethylbenzthiazoline‐6‐sulfonic acid) oxidation by laccase in the presence of these ILs was studied and Michaelis–Menten parameters were calculated. There is no enzymatic inactivation since the maximum reaction rate remained constant for IL concentrations up to 25%, and surprisingly, it was found that laccase was activated for concentrations of 35% of ILs, since the reaction rate increased 1.7 times.     

Effects of hemolysate concentration, ionic strength and cytochrome b5 concentration on the rate of methemoglobin reduction in hemolysates of human erythrocytes

An assay for determining the rate of methemoglobin reduction in hemolysates of human erythrocytes has been developed. The rates obtained by this assay, when corrected for dilution, are comparable to those obtained with intact cells. Increased ionic strength inhibits the reaction, whereas EDTA increases the rate of reduction. The rate with NADPH as electron donor is 65–70% of the rate with NADH. Added cytochrome b₅ stimulates the reaction. The assay has been used to examine erythrocytes from two methemoglobinemic sisters and their asymptomatic mother. Hemolysates of the two patients have both decreased dichlorophenolindophenol reductase activity and decreased ability to reduce methemoglobin. Hemolysates from the heterozygous mother have intermediate dichlorophenolindophenol reductase activity and intermediate methemoglobin reduction ability. The data presented in this paper indicate that the concentrations of cytochrome b₅ and cytochrome b₅ reductase determine the rate of methemoglobin reduction in hemolysates.     

Optimum design of a series of CSTRs performing reversible Michaelis-Menten kinetics: a rigorous mathematical study

The optimum design of a given number of CSTRs in series performing reversible Michaelis-Menten kinetics in the liquid phase assuming constant activity of the enzyme is studied. In this study, the presence of product in the feed stream to the first reactor, as well as the effect of the product intermediate concentrations in the downstream reactors on the reaction rate are investigated. For a given number of N CSTRs required to perform a certain degree of substrate conversion and under steady state operation and constant volumetric flow rate, the reactor optimization problem is posed as a constrained nonlinear programming problem (NLP). The reactor optimization is based on the minimum overall residence time (volume) of N reactors in series. When all the reactors in series operate isothermally, the constrained NLP is solved as an unconstrained NLP. And an analytical expression for the optimum overall residence time is obtained. Also, the necessary and sufficient conditions for the minimum overall residence time of N CSTRs are derived analytically. In the presence of product in the feed stream, the reversible Michaelis-Menten kinetics shows competitive product inhibition. And this is, because of the increase in the apparent rate constant K^' _m that results in a reduction of the overall reaction rate. The optimum total residence time is found to increase as the ratio (‚₀) of product to substrate concentrations in the feed stream increases. The isomerization of glucose to fructose, which follows a reversible Michaelis-Menten kinetics, is chosen as a model for the numerical examples.     

Influence of substrate transport on the activity of immobilized Papaver somniferum cells

Summary Intraparticle diffusion resistance was studied for Papaver somniferum cells immobilized by Ca alginate gel. In callus tissue, these plant cells convert codeinone to codeine. First, the diffusion rates of substrates in the gel were measured, followed by investigation of the consumption rates of the substrates by free cells. The consumption rate of sucrose was zero order in relation to sucrose concentration, whereas that of codeinone was first order in relation to its concentration. The oxygen consumption rate obeyed Michaelis-Menten type kinetics with respect to dissolved oxygen concentration. Combining the reaction rates and diffusion rates allows calculation of the extent of the effect of diffusion limitation on the overall reaction rates. The analysis showed that the effectiveness factor for each substrate was about unity and that the influence of diffusion resistance was negligible. However, the oxygen concentration decreased considerably inside the particle, and this may affect the activity of the plant cell for repeated use over a long time period. Thus, deactivation proceeds due to the oxygen deficit although the temporal reaction rate is not affected.Abbreviations C _c cell concentration (g/l) - C _cod codeinone concentration (g/l) - c _{O 2} dissolved oxygen concentration (g/l) - K _m constant in Eq. (3) (g/l) - K _cod rate constant in Eq. (1) (l/g of cells per second) - k _suc rate constant in Eq. (2) (g sucrose/g of cells per second) - R radius of particles (mm) - r distance from the centre of the particle (mm) - r _cod consumption rate of codeinone (g codeinone/g of cells per second) - r _{O 2} consumption rate of O₂ (g oxygen/g of cells per second) - r _suc consumption rate of sucrose (g sucrose/g of cells per second) - V _m maximum respiration rate (g oxygen/g of cells per second) T. Nozawa is now with the Department of Agricultural Chemistry, University of TokyoT. Isohara is now with the Nippon Steel Corporation     

Kinetics of protein aggregation. Quantitative estimation of the chaperone-like activity in test-systems based on suppression of protein aggregation

The experimental data on the kinetics of irreversible aggregation of proteins caused by exposure to elevated temperatures or the action of denaturing agents (guanidine hydrochloride, urea) have been analyzed. It was shown that the terminal phase of aggregation followed, as a rule, first order kinetics. For the kinetic curves registered by an increase in the apparent absorbance (A) in time (t) the methods of estimation of the corresponding kinetic parameters A _lim and k _I (A _lim is the limiting value of A at t and k _I is the rate constant of the first order) have been proposed. Cases are revealed when the reaction rate constant k _I calculated from the kinetic curve of aggregation of the enzymes coincides with the rate constant for enzyme inactivation. Such a situation is interpreted as a case when the rate of aggregation is limited by the stage of denaturation of the enzyme. A conclusion has been made that, in order to establish the mechanism of protein aggregation, the kinetic investigations of aggregation should be carried out over a wide range of protein concentrations. The refolding experiments after denaturation of proteins by guanidine hydrochloride or urea have been also analyzed. It was shown that aggregation accompanying refolding follows first order kinetics at the final phase of the process. The model of protein refolding explaining such a kinetic regularity has been proposed. When aggregation of protein substrate follows first order kinetics, parameters A _lim and k _I may be used for the quantitative characterization of the chaperone-like activity in the test-systems based on suppression of protein aggregation.     

Comparative Study on Caustic and Enzymatic Cleanings of Stainless Steel Surface Fouled with β-Lactoglobulin

Desorption behavior of β-lactoglobulin (β-Lg), one of the main constituents of fouling deposits in milk processing, from stainless steel surfaces during caustic and enzymatic cleanings was studied by using a glass column packed with stainless steel particles fouled with β-Lg. Both in caustic and enzymatic cleanings, the amount of β-Lg remaining on the stainless steel particles decreased according to first-order kinetics at the initial stage, and gradually reached a constant value. The desorption rate constant at the initial stage and the residual amount of β-Lg after 2 h of cleaning were evaluated as the measures of cleaning efficiency under various conditions. In caustic cleaning, these two values were strongly affected by the concentration of NaOH. The initial desorption rate increased with increasing flow rate of the caustic solution, suggesting a mass transfer effect. In enzymatic cleaning, the maximum desorption rate constant was obtained at around the optimum pH for the enzyme reaction. The temperature dependence of the initial desorption rate constant was stronger in caustic cleaning than in enzymatic cleaning.     

Comparative kinetic studies of the dealkylation reaction and steroid hydroxylations in various oxygen and carbon monoxide:oxygen atmospheres

The time-course kinetics of the cytochrome P-450-catalyzed dealkylations of the exogenous compounds benzphetamine, ethylmorphine, codeine, and 7-ethoxycoumarin were compared to the hydroxylation of the endogenous compound testosterone. Using liver microsomes from phenobarbital-induced rats, the time course of the demethylations of ethylmorphine, codeine, and especially benzphetamine was characterized by a fast initial phase of enzymatic activity and then a steady decline in the rate throughout the remainder of the reaction. In contrast, under the same experimental conditions, both the dealkylation of 7-ethoxycoumarin and the hydroxylation of testosterone showed no initial fast phase of activity and a constant rate of product formation for most of the remainder of the time course. The difference also held for the carbon monoxide inhibition studies in which the degree of inhibition of the demethylation reactions by a variety of CO:O₂ mixtures was time dependent, in contrast to the constant, time-independent degree of CO inhibition of the other two reactions. The kinetics of the demethylation reactions could not be explained by enzyme destruction, back reaction, or product adduct formation and were further confirmed by measurements of the rate of O₂ utilization and NADPH oxidation. The complexity of the demethylation reaction should be taken into consideration in any detailed studies of the monooxygenation reaction system.