Effects of domain structure on in-plane reactions and interactions

The existence of an in-plane domain structure in biological membranes raises the question of the physiological function, if any, of this structure. One important function may be to enhance or limit the equilibrium poise and rates of in-plane reactions through control by the cell of the percolation properties of the domain system. At low average domain occupancy by reactants or interactants, which must be the case for most biological membrane components, moving the domain system from connection to disconnection has marked effects on the apparent equilibrium poise and the rates of membrane-confined reactions. This conclusion is based on computer modelling of the effects of disconnection/connection of nine types of bimolecular in-plane reactions. Using the phase structure and percolation properties of two-component, two-phase phospholipid bilayers, it is possible to examine experimentally homo- and heterodimeriztion reactions, and enzyme-catalysed reactions in-plane as well as the effects of a transmembrane peptide on these systems. These theoretical and experimental studies suggest that percolation effects may be physiologically important in biological membranes. Whether this is in fact the case remains to be demonstrated.     

Kinetic deuterium isotope effects on deamination and N-hydroxylation of cyclohexylamine by rabbit liver microsomes

Deuterium isotope effects on the kinetic parameters for deamination and N-hydroxylation of cyclohexylamine (CHA) catalyzed by rabbit liver microsomes with NADPH are investigated. Both reactions are inhibited by carbon monoxide and have the characteristics of typical cytochrome P450-dependent monooxygenase reactions. A small and significant deuterium isotope effect operates in the oxidative deamination of CHA. The apparent isotope effects, i.e., VH/VD and (V/K)H/(V/K)D ratios for deamination, are 1.75 and 1.8-2.3, respectively. On the basis of N-hydroxylation, the VH/VD and (V/K)H/(V/K)D ratios are 0.8-0.9. The N-hydroxylation rate of alpha-deuterated CHA (D-CHA) is somewhat higher than that of CHA. The increased increment of hydroxylamine formation seems to coincide with the decreased amount of deamination. Substitution of deuterium in the alpha-position of CHA results in metabolic switching of cytochrome P450 from deamination to N-hydroxylation with low deuterium isotope effects. The data are interpreted in terms of an initial one-electron abstraction from the nitrogen to form an aminium cation radical followed by recombination with iron-bound hydroxyl radical leading to N-hydroxylamine, or followed by alpha-carbon deprotonation to form a neutral carbon radical. The latter can lead to a carbinolamine intermediate for deamination by way of imine or recombination with nascent iron-bound hydroxyl radical. The relative rates of the reactions depend on the alpha-carbon deprotonation rates of amines.     

The effects of thioureylene compounds (goitrogens) on lactoperoxidase activity.

The rates of oxidation of several goitrogens by lactoperoxidase and the rates of inactivation of lactoperoxidase by the same goitrogens have been measured. The influence of iodide on both reactions has also been evaluated. It has been shown by us that iodide acts catalytically in regulating lactoperoxidase activity at pH 8.8. The rate data have been analyzed by a computer program which solves the differential equations for the above mentioned reactions. From this computer analysis we have been able to obtain binding constants of the goitrogens and inactivation rate constants of lactoperoxidase. Iodide was shown to inhibit goitrogenic activity either by increasing the rate of drug oxidation or by reducing the rate of enzyme inactivation, or both, depending on the particular drug. Iodide had little or no effect on the goitrogen-binding constants. We have also shown that the relative rates of enzyme inactivation can be correlated with the potency of the goitrogen as an antithyroid drug.     

Leukotriene metabolism and hypersensitivity in man and mouse

The relative rates of cysteinyl-leukotriene metabolism were analyzed in fresh human and mouse serum. Human serum contained higher gamma-glutamyl-transpeptidase activity than mouse serum, and a higher percentage of the metabolized leukotriene C4 was recovered as leukotriene D4 in the human serum than in the mouse serum. The results suggest that the patterns of metabolism of the cysteinyl-leukotrienes could be an important factor in determining the relative sensitivity of an animal to the development of hypersensitivity reactions.     

Environmental factors in drug metabolism.

Although various kinds of environmental factors may alter the activity of cytochrome P-450 enzymes in liver micromes, their effects on the pharmacokinetics of drugs and other foreign compounds in living animals may not be as great as might be predicted from assays of these enzymes in vitro. Indeed, the effects will depend on the relative importance of excretory and metabolic mechanisms in the elimination of the drug, the relative importance of various metabolic reactions in different tissues, the extraction ratio of the drug by the liver, and in some instances on the route of administration of the drug. Moreover, the effect of the various environmental factors on the pharmacologic and the toxicologic actions of the drug will depend on whether these actions are caused by the parent foreign compounds or by one or more of their metabolites. It may also be important that the environmental factors may alter not only relative activiteis of the cytochrome P-450 in liver microsomes but also the activities of other drug-metabolizing enzymes and that the relative effects of the environmental factors of these enzymes may differ depending on the animal species or the animal strain. Indeed, a given factor may increase the pharmacologic effects of a drug metabolite in one animal species but decrease it in another. For these reasons, it frequently is not possible to predict the effects of environmental factors on drug action in living animals solely from in vitro rates of metabolism of model substrates.     

Association of a protein with membrane vesicles at the collisional limit: studies with blood coagulation factor Va light chain also suggest major differences between small and large unilamellar vesicles

Vesicle size can be a very sensitive modulator of protein-membrane association. In addition, reactions at the collisional limit may be characteristic of many types of protein-membrane or protein-receptor interactions. To probe these effects quantitatively, we analyzed the association of blood clotting factor Va light chain (Va-LC) with phospholipid vesicles of 15-150-nm radius. The number of protein binding sites per vesicle was approximately proportional to vesicle surface area. Association rates approached the collisional limit, and the activation energy for the association reaction was 4.5 +/- 0.5 kcal/mol. In agreement with diffusional theory for this type of interaction at the collisional limit, the observed association rate constant for filling all sites was approximately proportional to the inverse of vesicle radius. This general property has important implications for many systems such as blood coagulation including possible slower association rates and higher Km values for reactions involving whole cells relative to those obtained for phospholipid vesicles. Dissociation rate constants for reactions that are near the collisional limit should also be proportional to the inverse of vesicle size if diffusional parameters are the only factors influencing dissociation. However, Va-LC bound to small unilamellar vesicles (SUVs, less than or equal to 15-nm radius) gave slower dissociation rates than Va-LC bound to large unilamellar vesicles (LUVs, greater than or equal to 35-nm radius). This indicated a change in KI, the intrinsic protein-phospholipid affinity constant for LUVs vs SUVs. The cumulative effect of association and dissociation rates resulted in higher affinity of Va-LC for SUVs than LUVs under equilibrium conditions. The latter was corroborated by competition binding studies. Furthermore, the temperature dependence of both rate constants indicated an entirely entropy-driven binding to LUVs but a largely enthalpy-driven binding to SUVs. Interactions which are largely entropic are thought to be ionic in nature. The differences observed between binding to LUVs and SUVs may reflect thermodynamic differences between these types of phospholipid structures.     

The effect of chlorophyll content on changes of photochemical reactions in intact kidney bean leaves

Abstract. Activation spectra of photochemical reactions were measured by a flash spectrophotometer in leaves having varying chlorophyll contents at different stages of greening. The increase of chlorophyll concentration up to 30 nmol cm^-2 elevated the rates of photochemical reactions at all wavelengths of light used, and was found to be produced by an increase in the amounts of reaction centres. Further accumulation of chlorophyll up to 40 nmol cm^-2 was associated with an increase in light-harvesting chlorophyll, an improved rate of photochemical reactions around 600 nm and at 700 nm, and self-absorption and screening effects where chlorophyll absorbed maximally (400–450 nm and around 680 nm).     

Microwave-enhanced nucleophilic fluorination in the synthesis of fluoropyridyl derivatives of [3,2-c]pyrazolo-corticosteroids, potential glucocorticoid receptor-mediated imaging agents

Fluoropyridyl derivatives of [3,2-c]pyrazolo-corticosteroids have high affinity for the glucocorticoid receptor (GR) and are highly active glucocorticoids. They are thus considered to be excellent candidates for PET imaging of GR containing tissues when labeled with fluorine-18 (t(1/2)=110 min). Previously reported syntheses of these fluorinated glucocorticoids were accomplished by conventional thermal nucleophilic halogen exchange reactions with chloropyridyl precursors. These reactions were found to proceed at rates too slow for feasible application to radiosynthesis using [(18)F]fluoride. We have applied microwave-heating methods to these reactions and found that significant rate enhancements can be realized. Kinetic experiments showed an average relative rate ratio of 3/1 for microwave versus conventional heating and preparative experiments showed an average relative conversion ratio of 4.5/1 during the initial 120 min, a period approximating one half-life of the isotope. The microwave method described was used to prepare previously unreported 2'-(2-fluoro-4-pyridyl)-11beta,17,21-trihydroxy-16alpha-methyl-20-oxo-pregn-4-eno-[3,2-c]-pyrazole, which was evaluated for biological activity.     

Elongation and termination reactions of protein synthesis on maize root tip polyribosomes studied in a homologous cell-free system

We show that the control of gene expression at the level of elongation and termination of protein synthesis can be observed in vitro. Free cytoplasmic polyribosomes were isolated from maize (Zea mays) root tips, and translated in root tip extracts that had been fractionated with ammonium sulfate to contain elongation factors, and be depleted in initiation factors. The root tip extract performs elongation and termination reactions as efficiently as wheat germ extracts. The translation products of the maize system are the same as made in vivo. The dependence of these in vitro elongation and termination reactions on pH was determined. Total protein synthesis in this system exhibits an optimum at pH ~7.5. However, the pH dependence of rates of synthesis of individual proteins is not at all uniform; many polyribosomes become stalled when translated at low pH. These data were compared with the elongation and termination capacity of polyribosomes isolated from oxygenated and hypoxic root tips (tissue having, respectively, high and low cytoplasmic pH values). We observed an inverse relationship between the relative abundance of many specific translatable mRNAs in polyribosomes of hypoxic root tips, and the relative rates of elongation and termination reactions on the different mRNAs at low pH in vitro. These results suggest that changes in intracellular pH in hypoxic root tips can be sensed directly by the translational machinery and thereby selectively modulate gene expression.     

Analysis of ping-pong reaction mechanisms by positional isotope exchange. Application to galactose-1-phosphate uridyltransferase

A new positional isotope exchange method has been developed that can be used for the analysis of enzyme-catalyzed reactions which have ping-pong kinetic mechanisms. The technique can be used to measure the relative rates of ligand dissociation from enzyme-product complexes. Enzyme is incubated with the labeled substrate and an excess of the corresponding unlabeled product. The partitioning of the enzyme-product complex back toward free enzyme is determined from the rate of positional isotope exchange within the original labeled substrate. The partitioning of the enzyme-product complex forward toward free enzyme is determined from the rate of formation of totally unlabeled substrate. It has been shown that the ratio of the two rates provides a lower limit for the release of product from the enzyme-product complex. The technique has been applied to the reaction catalyzed by galactose-1-phosphate uridyltransferase. The lower limit for the release of glucose 1-phosphate from the uridyl-enzyme relative to the maximal velocity of the reverse reaction was determined to be 3.4 +/- 0.5.     

Acid-catalyzed stereoselective heteronucleophilic substitution and racemization of 3-O-methyloxazepam and 3-O-ethyloxazepam

Enantiomers of 3-O-methyloxazepam (MeOX) and 3-O-ethyloxazepam (EtOX) were resolved by chiral stationary phase high-performance liquid chromatography (CSP-HPLC). Reaction kinetics and deuterium isotope effects of acid-catalyzed racemization of enantiomeric MeOX in ethanol and enantiomeric EtOX in methanol were studied by spectropolarimetry. The acid-catalyzed heteronucleophilic substitution reactions of racemic MeOX in ethanol and racemic EtOX in methanol were studied by reversed-phase HPLC. Thermodynamic parameters involved in the reactions were obtained by temperature-dependent reaction rates. The effects of solvent's dielectric constant on the heteronucleophilic substitution reactions were also determined. A nucleophilically solvated and transient C3 carbocation intermediate resulting from an N4-protonated enantiomer, derived from a 1,4-benzodiazcpine either in M (minus) or P (plus) conformation, is proposed to be an intermediate and responsible for the acid-catalyzed stereoselective nucleophilic substitution and the resulting racemization. © 1994 Wiley-Liss, Inc.     

Towards the development of a minimal cell model by generalization of a model of Escherichia coli: use of dimensionless rate parameters.

A model of a minimal cell would be a valuable tool in identifying the organizing principles that relate the static sequence information of the genome to the dynamic functioning of the living cell. Our approach for developing a minimal cell model is to first generalize an existing model of Escherichia coli by expressing reaction rates as ratios to a set of reference parameters. This generalized model is a prototype minimal cell model that will be developed by adding detail to explicitly include each chemical species. We tested the concept of a generalized model by testing the effect of scaling all enzyme-catalyzed reactions in the E. coli model. The scaling has little effect on cellular function for a wide range of kinetic ratios, where the kinetic ratio is defined as the rate of all enzyme-catalyzed reactions in a given model relative to those in the E. coli model.     

Reinforcement omission in concurrent fixed-interval and random-interval schedules

The present experiment examined overall and local effects of omission of reinforcers in a choice situation. Pigeons' key-pecking responses were reinforced under concurrent fixed-interval and random-interval schedules of food presentation. After some weeks of baseline sessions in which the probability of reinforcement was 1.00, approximately 25% of food presentations from the fixed-interval schedule were omitted and replaced by timeout periods. In such omission sessions, the overall relative rates of responding to the fixed-interval schedule became lower than those in the baseline sessions. On the other hand, when relative rates of responding to the fixed-interval schedule in the omission sessions were calculated separately for fixed-interval cycles preceded by timeout periods and those preceded by food presentations, the relative rates in the former type of fixed-interval cycles were higher than those in the latter type for three out of four pigeons. These results mean that relative rates of responding cannot always be regarded as reflecting a relative value of an alternative, and that the overall effect of the omission of fixed-interval reinforcers is not reducible to the local effect of omission.     

Microwave stimulation of an immunological reaction (CEA/anti-CEA) and its use in immunohistochemistry

Summary The effects of microwave stimulation on different aspects of immunohistochemical reactions were studied using Elisa as a model system. This technique allows monitoring of (i) the rates and recoveries of different antigen-antibody reactions, (ii) the formation of avidin/biotin complexes, (iii) the prevention of non-specific activity and (iv) the washing procedures.The binding reactions seemed to follow second-order kinetics, and reaction rates were considerably enhanced by microwave stimulation. The steps were aimed at preventing non-specific reactivity and the washing procedures could all be shortened to seconds. Such rate increases are far too large to be explained solely by the modest increase in temperature. Furthermore, microwave irradiation caused a major reduction in the yield of antigen-antibody complexes. This inhibitory effect can be compensated for by increasing the concentration of antibody. No significant effects of the microwaves on the antigen-antibody complexes were found once the complexes had been formed.The obtained results were then applied to the demonstration of carcinoembryonic antigen in histological material. By employing this method a complete immunohistochemical staining of a hydrated tissue section could be performed within 15 min, excluding the final development of colours.     

Allosteric effects on oxidative and nitrosative reactions of cell-free hemoglobins

A review of the oxidative and nitrosative reactions of cell-free hemoglobin-based oxygen carriers (HBOCs) shows that these reactions are intimately linked and are subject to allosteric control. Cross-linking reactions used to produce HBOCs introduce conformational constraints and result in Hbs with reduced responses to heterotropic and homotropic allosteric effectors. The Nernst plots of heme oxidation of cross-linked HBOCs are shifted to higher potentials relative to unmodified Hb in the absence of allosteric effectors, in accord with their T-state stabilization and right-shifted Hill plots of O(2) binding. They exhibit enhanced rates of autoxidation and nitrite-induced oxidation, features that appear due to their having more solvent-accessible heme pockets. The stability of their NO-Hb derivatives varies as a result of allosteric effects on the extent of formation of pentacoordinate NO-heme geometry by alpha chains and subsequent oxidation of partner beta chains. The physiological implications of these findings on the safety, efficacy and design of second generation HBOCs are discussed in the framework of a reaction scheme showing linkages between Hb-mediated redox reactions. These redox reactions can drive formation of SNO-Hb and other reactive species and are of significance for the use of cell-free Hbs in vivo.     

Determination of relative rate constants for in vitro RNA processing reactions by internal competition

Studies of RNA recognition and catalysis typically involve measurement of rate constants for reactions of individual RNA sequence variants by fitting changes in substrate or product concentration to exponential or linear functions. A complementary approach is determination of relative rate constants by internal competition, which involves quantifying the time-dependent changes in substrate or product ratios in reactions containing multiple substrates. Here, we review approaches for determining relative rate constants by analysis of both substrate and product ratios and illustrate their application using the in vitro processing of precursor transfer RNA (tRNA) by ribonuclease P as a model system. The presence of inactive substrate populations is a common complicating factor in analysis of reactions involving RNA substrates, and approaches for quantitative correction of observed rate constants for these effects are illustrated. These results, together with recent applications in the literature, indicate that internal competition offers an alternate method for analyzing RNA processing kinetics using standard molecular biology methods that directly quantifies substrate specificity and may be extended to a range of applications.     

Substrate steering by electrostatic fields of enzymes: visualization by computer graphics

The rates of reactions catalysed by certain enzymes are increased by electrostatic effects that steer substrate molecules toward the active sites of the enzyme. This phenomenon can be studied by using Brownian dynamics simulation to generate and analyse diffusional trajectories of substrate in the field of an enzyme. The paper demonstrates that computer graphics can be used to show how electrical fields influence the special dependence of substrate flux as calculated by Brownian dynamics. Because of the statistical noise associated with typical simulation runs, filtering techniques are helpful in determining systematic trends in the graphic displays.     

Metabolic network sensitivity analysis

A linear sensitivity analysis of metabolic regulation in nonsteady states is described. This treatment considers the effects of enzymatic and nonenzymatic reactions and spontaneous rapid equilibria. Sensitivity coefficients summarizing the influence of metabolite concentrations on reaction rates and pathway net flux are defined, as are sensitivity coefficients summarizing the effects of enzymes on metabolite concentrations and net flux. The sensitivity analysis is implemented in an easily used set of computer programs. A four-enzyme test model was shown to be resistant to intuitive interpretation. Sensitivity analysis showed a shift of control from the end of the enzymic sequence to the beginning of the sequence with changing metabolic state. The homeostatic behavior of the test system was shown to depend on the nonenzymatic reactions as well as on the enzymes. Under certain conditions metabolic regulation is shared so intimately among enzymes and spontaneous reactions that separation of their effects is impossible.     

Enzyme isoforms may increase phenotypic robustness

Enzyme isoforms are found in many cellular reactions, and can differ in the kind of reaction they catalyze, in their substrate affinity, or in their reaction rates. The evolutionary significance of enzyme isoforms is only partially understood. We used mathematical modeling to investigate the hypothesis that isoforms may be favored by selection because they can increase the phenotypic robustness of the system. We modify a model for circadian clock gene expression in Drosophila to incorporate the presence of isoforms in the phosphorylation pathway of the period gene. We consider the case in which different isoforms catalyze the same reaction but have different affinities for the substrate. Stability is increased if there is dynamic control of the expression of isoforms relative to each other. Thus, we show that controlling isoform proportion can be a powerful mechanism for reducing the effects of variations in the values of system parameters, increasing system robustness.