Determination of the kinetic constants of inhibited acetyl cholinesterase

A method for the determination of kinetic constants of inhibited acetylcholinesterase, in the presence of substrate, is proposed. Theoretical, experimental and data treatment aspects are described for a suitable analysis of inhibitors acting with one or two steps. Limitations of other methods recently reported are pointed out.     

Biosynthesis and functions of eicosanoids generated by the coelomocytes of the starfish, Asterias rubens

Eicosanoids are a group of oxygenated fatty acid derivatives formed from C20 polyunsaturated fatty acids, including arachidonic and eicosapentaenoic acids. The potential of the coelomocytes of the starfish, Asterias rubens, to generate eicosanoids through the cyclooxygenase (COX) and lipoxygenase (LOX) pathways was investigated using reverse-phase high performance liquid chromatography, enzyme immunoassay and gas chromatography–mass spectrometry. The principal LOX product was identified as 8-hydroxyeicosatetraenoic acid (8-HETE) with 8-hydroxyeicosapentaenoic acid (8-HEPE) synthesised at significantly lower levels. No classical prostaglandins (PG), such as PGE₂ or PGD₂, were found to be generated by ionophore-challenged coelomocytes. Incubation of coelomocytes with lipopolysaccharides from either Escherichia coli or Salmonella abortus failed to induce an increase in generation of LOX products and the presence of 8-HETE (0–25 μM) had no significant effect on the in vitro phagocytic activity of Asterias coelomocytes. Neither indomethacin (a COX inhibitor) or esculetin (a LOX inhibitor) had any effect on the clearance of the bacterium, Vibrio splendidus, from the coelomic cavity of starfish suggesting that products of these enzymes are not involved in such coelomocyte responses to foreign particles.     

Nematode neuropeptides: Localization, isolation and functions

Historically, peptidergic substances (in the form of neurosecretions) were linked to moulting in nematodes. More recently, there has been a renewal of interest in nematode neurobiology, initially triggered by studies demonstrating the localization of peptide immunoreactivities to the nervous system. Here, David Brownlee, Ian Fairweather, Lindy Holden-Dye and Robert Walker will review progress on the isolation of nematode neuropeptides and efforts to unravel their physiological actions and inactivation mechanisms. Future avenues for research are suggested and the potential exploitation of peptidergic pathways in future therapeutic strategies highlighted.     

Localization of cholinesterase in Pseudomonas aeruginosa strain K

The inducible cholinesterase of Pseudomonas aeruginosa strain K (ATCC 25102) degraded propionylcholine, acetylthiocholine, acetylcholine and acetyl-beta-methylcholine at a high rate and butyrylcholine and succinylcholine at very low rates. The localization of the enzyme in the periplasmic space was indicated by a similar rate of acetylcholine degradation by intact cells or their extracts, by release of cholinesterase together with alkaline phosphatase into the culture medium during cell growth in a low phosphate-containing medium, by liberation of cholinesterase and alkaline phosphatase during lysozyme-induced conversion of cells to spheroplasts and by freezing and thawing. Threatment of cells with diazo-7-amino-1,3-naphthalenedisulphonic acid, which inactivates surface-located enzymes, abolished most of the cholinesterase and 5'-nucleotidase activities.     

Localization of cholinesterase activity in house-fly thoraces: Inhibition of cholinesterase with organophosphate compounds

Cholinesterase (ChE) activity was localized by a histochemical method in thoraces of non-treated, and tepp- and dicrotophos-(Bidrin^®)-treated houseflies. The findings indicate a good correlation between intoxication symptoms (knock-down) and inhibition of ChE activity in the cell body region rather than the neuropile or synaptic region of the compound ganglion. Results of histochemical studies agree well with quantitative pH-stat assays which show wide differences in levels of inhibition of total ChE activity in tepp- and dicrotophos-treated flies at knock-down.In contrast to 2 ChE's in mammalian brain, histochemical evidence is presented to support the conclusion of previous workers that a single ChE in houseflies hydrolyzes both acetylcholine and butyrylcholine.

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Zusammenfassung Die Cholinesterase (ChE)-Aktivität im Thorax von unbehandelten und mit Tepp-(Tetraäthylpyrophosphat) und Dicrotophos- ( Isomer von Dimethylphosphateester mit (E)-3-hydroxy-N, N-dimethylcrotoamide) (Bidrin) behandelten Stubenfliegen wurde mit einer histochemischen Methode lokalisiert. Die Ergebnisse deuten eine gute Korrelation zwischen Vergiftungserscheinungen (knock-down) und Hemmungen der ChE-Aktivität in der Region der Zellkörper an, aber nicht im Nerven bündel oder in der synaptischen Region des zusammengesetzten Ganglions. Resultate dieser histochemischen Versuche stimmen ziemlich gut überein mit den quantitativen pH-stat-Proben. Diese Proben zeigten einen weiten Unterschied in den Hemmungsgraden der totalen ChE-Aktivität von Tepp-und Dicrotophosbehandelten Fliegen während der Vergiftung.In Bestätigung der Schlußfolgerung früherer Bearbeiter beweist die histochemische Untersuchung, daß im Gegensatz zu zwei Cholinesterasen im Säugetiergehirn in der Stubenfliege eine einzige Cholinesterase Acetylcholin und Butyrylcholin hydrolisiert.

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This investigation was supported by National Institutes of Health Research Grant No. 5 ROI ES00253. Journal paper no. 743, University of Georgia College of Agriculture Experiment Stations, College Station, Athens.     

Localization and dynamics of Cdc2-cyclin B during meiotic reinitiation in starfish oocytes

The Cdc2-cyclin B kinase has a central role in regulating the onset of M phase. In starfish oocytes, Cdc2-cyclin B begins to be activated approximately 10 min after application of maturation hormone, followed by accumulation in the nucleus then nuclear envelope breakdown. By immunofluorescence and by expressing a green fluorescent (GFP) chimera of cyclin B, we find that cyclin B is present in aggregates in the cytoplasm of immature oocytes. The aggregates disperse at approximately 10 min, suggesting that the dispersal is closely related to the activation of the kinase. Using cyclin B-GFP, the dispersion begins from the region containing the centrosomes. Extractability of Cdc2-cyclin B changes with similar kinetics during maturation. Active Cdc25 phosphatase released Cdc2-cyclin B from the detergent-insoluble fraction independently of its phosphatase activity. Live cell imaging also showed that Cdc2-cyclin B begins to accumulate in the nucleus before changes in nuclear pore permeability, consistent with Cdc2-cyclin B-induced disassembly of the pores.     

A kinetic model of the cytochrome bf complex. Evaluation of kinetic parameters

A kinetic model of the cytochrome bf complex was developed on the assumption that the Q-cycle operates. The bf complex was considered as a membrane enzyme catalyzing the electron transfer from plastoquinol to plastocyanine, which is coupled with proton translocation from the chloroplast stroma to the thylakoid lumen. The dependence of the electron transfer rates on the value of the transmembrane electric potential was taken into account. The model was applied to describe the experimental data on the flash-induced turnover of cytochromes b, plastocyanine, and the kinetics of proton deposition in the thylakoid lumen. The estimation of model parameters was performed.     

Fumarase: viscosity dependence of the kinetic parameters

Fumarase catalyzes the reversible, stereospecific hydration of fumarate to form L-malate. We have determined the viscosity dependence of V/K and V in both the forward and the reverse directions at pH 6.9 in the absence and presence of several viscosogenic reagents. V/K for fumarate hydration decreases with increasing concentrations of glycerol and sucrose, but is unaffected by increasing concentrations of the polymeric viscosogen polyethyleneglycol (av MW, 10,000 da). V/K for malate dehydration similarly decreases with increasing concentrations of both glycerol and sucrose, but is unaffected by increasing concentrations of polyethylene glycol. Equilibrium constants, calculated from the ratio of V/K values for malate dehydration and fumarate hydration at various concentrations of glycerol, closely match the experimentally determined equilibrium constants at the same concentrations of glycerol. Both experimental and calculated equilibrium constants decrease with increasing concentrations of viscosogens. V/K for the dehydration of (-)-tartrate, a poor substrate, is unaffected by increasing concentrations of glycerol. Analysis of the microviscosity dependence of malate dehydration and fumarate hydration suggests that both substrates bind at diffusion-limited rates. The viscosity dependence of substrate and product dissociation steps may also contribute to the viscosity dependence of V/K values for both substrates. The viscosity dependence of the maximal velocities argues that product dissociation steps are rate-limiting and diffusion controlled.     

Quantifying the kinetic parameters of prion replication.

The mechanism of protein-only prion replication is controversial. A detailed mathematical model of prion replication by nucleated polymerisation is developed, and its parameters are estimated from published data. PrP-res decay is around two orders of magnitude slower than PrP-sen decay, a plausible ratio of two parameters estimated from very different experiments. By varying the polymer breakage rate, we reveal that systems of short polymers grow the fastest. Drugs which break polymers could therefore accelerate disease progression. Growth in PrP-res seems slower than growth in infectious titre. This can be explained either by a novel hypothesis concerning inoculum clearance from a newly infected brain, or by the faster growth of compartments containing smaller polymers. The existence of compartments can also explain why prion growth sometimes reaches a plateau. Published kinetic data are all compatible with our mathematical model, so the nucleated polymerisation hypothesis cannot be ruled out on dynamic grounds.     

Measurement of the kinetic parameters mediating protease-serpin inhibition

Serine protease inhibition by proteins of the serpin family is a unique and complex process involving physical, chemical, and conformational changes. After encounter with the reactive site of inhibitor, the protease is conformationally trapped as a covalent complex resembling the acyl-protease intermediate of catalysis. The stability of the trap is not permanent and may vary for different proteases. In addition, the trapping mechanism is not 100% efficient and a fraction of the serpin may be consumed like a substrate before inactivation is complete. Characterization of protease-serpin inhibition therefore requires the measurement of three parameters: the apparent second order rate constant of inhibition (k(inh)), the stoichiometry of inhibition (SI), and the rate of complex breakdown (k(brkdn)). The basic kinetic methods to establish these parameters are described.     

Mycobacterium tuberculosis mycothione reductase: pH dependence of the kinetic parameters and kinetic isotope effects

The recent identification of the enzyme in Mycobacterium tuberculosis that catalyzes the NADPH-dependent reduction of the unique low molecular weight disulfide mycothione, mycothione reductase, has led us to examine the mechanism of catalysis in greater detail. The pH dependence of the kinetic parameters V and V/K for NADPH, NADH, and an active analogue of mycothione disulfide, des-myo-inositol mycothione disulfide, has been determined. An analysis of the pH profiles has allowed the tentative assignment of catalytically significant residues crucial to the mechanism of disulfide reduction, namely, the His444-Glu449 ion pair and Cys39. Solvent kinetic isotope effects were observed on V and V/K(DIMSSM), yielding values of 1.7 +/- 0.2 and 1.4 +/- 0.2, respectively, but not on V/K(NADPH). Proton inventory studies (V versus mole fraction of D(2)O) were linear, indicative of a single proton transfer in a solvent isotopically sensitive step. Steady-state primary deuterium kinetic isotope effects on V have been determined using NADPH and NADH, yielding values of 1.27 +/- 0.03 and 1.66 +/- 0.14, respectively. The pre-steady-state primary deuterium kinetic isotope effect on enzyme reduction has values of 1.82 +/- 0.04 and 1.59 +/- 0.06 for NADPH and NADH, respectively. The steady-state primary deuterium kinetic isotope effect using NADH coincide with that obtained under single turnover conditions, suggesting the complete expression of the intrinsic primary kinetic isotope effect. Rapid reaction studies on the reductive half-reaction using NADPH and NADH yielded maximal rates of 129 +/- 2 and 20 +/- 1 s(-1), respectively, while similar studies of the oxidation of the two-electron reduced enzyme by mycothiol disulfide yielded a maximum rate of 190 +/- 10 s(-1). These data suggest a unique flavoprotein disulfide mechanism in which the rate of the oxidative half-reaction is slightly faster than the rate of the reductive half-reaction.     

Identification of isoenzymes in cholinesterase preparations using kinetic data of organophosphate inhibition

Commercial preparations of acetylcholinesterase (EC 3.1.1.7) and of cholinesterase (EC 3.1.1.8) were characterized by organophosphate inhibition. Cholinesterase activities were inhibited by varying organophosphate concentration and time of inhibition. Bimolecular rate constants were determined by plotting log activity vs inhibitor concentration or inhibition time. Inhibition of acetylcholinesterase from bovine erythrocytes by diethyl p-nitrophenyl phosphate (Paraoxon), diisopropylphosphorofluoridate (DFP), and N,N′-diisopropylphosphorodiamidic fluoride (Mipafox) in semilogarithmic plots showed a linear decay of activity. Inhibition of acetylcholinesterase from electric eel (Electrophorus electricus) and of cholinesterases from horse serum and from human serum did not show linear characteristics, indicating the presence of more than one single enzyme in these preparations. The corresponding inhibition curves were resolved by subtraction of exponential functions. In each case two different activity components were identified and characterized in respect to partial activity, substrate specificity, and reactivity with organophosphorous compounds. The suitability of the method for application on crude homogenates is discussed.     

Localization and possible functions of Drosophila septins.

The septins are a family of homologous proteins that were originally identified in Saccharomyces cerevisiae, where they are associated with the "neck filaments" and are involved in cytokinesis and other aspects of the organization of the cell surface. We report here the identification of Sep1, a Drosophila melanogaster septin, based on its homology to the yeast septins. The predicted Sep1 amino acid sequence is 35-42% identical to the known S. cerevisiae septins; 52% identical to Pnut, a second D. melanogaster septin; and 53-73% identical to the known mammalian septins. Sep1-specific antibodies have been used to characterize its expression and localization. The protein is concentrated at the leading edge of the cleavage furrows of dividing cells and cellularizing embryos, suggesting a role in furrow formation. Other aspects of Sep1 localization suggest roles not directly related to cytokinesis. For example, Sep1 exhibits orderly, cell-cycle-coordinated rearrangements within the cortex of syncytial blastoderm embryos and in the cells of post-gastrulation embryos; Sep1 is also concentrated at the leading edge of the epithelium during dorsal closure in the embryo, in the neurons of the embryonic nervous system, and at the baso-lateral surfaces of ovarian follicle cells. The distribution of Sep1 typically overlaps, but is distinct from, that of actin. Both immunolocalization and biochemical experiments show that Sep1 is intimately associated with Pnut, suggesting that the Drosophila septins, like those in yeast, function as part of a complex.     

Localization and possible functions of phospholipase D isozymes

The activation of PLD is believed to play an important role in the regulation of cell function and cell fate by extracellular signal molecules. Multiple PLD activities have been characterized in mammalian cells and, more recently, several PLD genes have been cloned. Current evidence indicates that diverse PLD activities are localized in most, if not all, cellular organelles, where they are likely to subserve different functions in signal transduction, membrane vesicle trafficking and cytoskeletal dynamics.     

Estimation of yield, maintenance, and product formation kinetic parameters in anaerobic fermentations

In many anaerobic fermentation processes, high energy bonds in adenosine triphosphate (ATP) are produced when available electrons are converted from organic substrate into extracellular organic products such as ethanol. The true growth yield and maintenance parameters are directly related to the product formation kinetic parameters for these anaerobic processes. Methods are presented which allow all of the experimental measurements to be used simultaneously to estimate these parameters. Results are presented for several different anaerobic fermentations.     

Histochemical investigations of Rotifera Bdelloidea. I. Localization of cholinesterase activity

Summary Cholinesterase activity has been investigated in Rotifera Bdelloidea (Philodina roseola, Philodina tubercolata, Rotaria rotatoria and other unidentified species) by histochemical methods andin vivo observations. Parallel histological studies have been carried out. The enzyme specificity was tested by employing different substrates and inhibitors. The effectsin vivo of tubocurarin, bungarotoxin and acetylcholine were also observed. Acetylcholinesterase activity is localized in the nervous and muscular tissues, in sensory organs and in all the ciliated cells. Secretory cells (subcerebral, salivary and pedal glands) and gonad cells (nuclei of the syncytial vitellarium and follicular layer, oocytes and eggs) show both acetyl-and butyrylcholinesterase activities. The effectsin vivo of cholinesterase inhibitors, as well as those of tubocurarin, bungarotoxin and acetylcholine, are consistent with the histochemical results, indicating a cholinergic system of transmission and acetylcholinesterase, as well as butyrylcholinesterase, activity.This paper is dedicated to my master, the late Professor António Minganti.