New parameters controlling the effect of temperature on enzyme activity.

Arising from careful measurements of the thermal behaviour of enzymes, a new model, the Equilibrium Model, has been developed to explain more fully the effects of temperature on enzymes. The model describes the effect of temperature on enzyme activity in terms of a rapidly reversible active-inactive (but not denatured) transition, revealing an additional and reversible mechanism for enzyme activity loss in addition to irreversible thermal inactivation at high temperatures. Two new thermal parameters, T(eq) and DeltaH(eq), describe the active-inactive transition, and enable a complete description of the effect of temperature on enzyme activity. We describe here the Model and its fit to experimental data, methods for the determination of the Equilibrium Model parameters, and the implications of the Model for the environmental adaptation and evolution of enzymes, and for biotechnology.

Characterisation of hydrogel gel swelling by molecular exclusion.

A facile method for the characterization of hydrogel swelling is described which is based on the determination of changes in the liquid phase concentration of an excluded tracer as gel swells in a constant volume system. The utility of this approach is demonstrated with two responsive hydrogel preparations, one where swelling is influenced by system pH, the other by changes in specific solute concentration.

NAD-sensitive hydrogel for the release of macromolecules.

A hydrogel membrane containing immobilized ligands and receptors was synthesized and investigated for the controlled diffusion of test proteins (cytochrome C and hemoglobin). Both Cibacron blue (ligand) and lysozyme (receptor) were covalently linked to dextran molecules that were subsequently crosslinked to form a gel. The resulting stable hydrogels contained both covalent and affinity crosslinks such that their intrinsic porosities were sensitive to competitive displacers of the affinity interaction between lysozyme and Cibacron blue. Transport experiments in a twin chamber diffusion cell showed that as NAD was added to the donor side, the dissociation of the binding sites between the Cibacron blue and the lysozyme led to an increase in protein diffusion through the hydrogel. The results showed that addition of NAD caused a saturable concentration-dependent increase in the transport of both cytochrome C and hemoglobin. This effect was shown to be both specific and reversible.

A reversible hydrogel membrane for controlling the delivery of macromolecules.

Glucose-sensitive hydrogel membranes have been synthesized and characterized for their rate-of-delivery of macromolecules. The mechanism for changing this rate is based on variable displacement of the affinity interaction between dextran and concanavalin A (con A). Our main objective was to characterize the diffusion of model proteins (insulin, lysozyme, and BSA) through the membrane, in response to changes in environmental glucose concentrations. Membranes were constructed from crosslinked dextrans to which con A was coupled via a spacer arm. Changes in the porosity of the resulting hydrogel in the presence of glucose led to changes in the diffusion rate observed for a range of proteins. Gels of specified thickness were cast around to nylon gauze support (pore size, 0.1 mm) to improve mechanical strength. Diffusion of proteins through the gel membrane was determined using a twin-chamber diffusion cell with the concentrations being continuously monitored using a UV-spectrophotometer. Changes in the transport properties of the membranes in response to glucose were explored and it was found that, while 0.1M D-glucose caused a substantial, but saturateable, increase in the rates of diffusion of both insulin and lysozyme, controls using glycerol or L-glucose (0.1M) had no significant effect. Sequential addition and removal of external glucose in a stepwise manner showed that permeability changes were reversible. As expected, diffusion rates were inversely proportional to membrane thickness. A maximum increase in permeability was observed at pH 7.4 and at 37 degrees C. The results demonstrate that this hydrogel membrane functions as a smart material allowing control of solute delivery in response to specific changes in its external environment.

Suicide inactivation of xanthine oxidoreductase during reduction of inorganic nitrite to nitric oxide.

Xanthine oxidoreductase (XOR) is progressively inactivated while catalysing the reduction of inorganic nitrite to NO by xanthine. Inactivation results from conversion of the enzyme into its desulpho-form. The rate of inactivation increases with nitrite concentration. Similar behaviour was shown when NADH replaced xanthine as reducing substrate. A kinetic model is proposed incorporating a 'suicide' inactivation involving an enzyme-substrate (product) complex, rather than inactivation by free NO. The model provides a good fit to progress curves of the reaction of xanthine or NADH with nitrite in the presence of the oxidase or dehydrogenase forms of the enzyme. Inorganic nitrate, like nitrite, was shown to be reduced at the molybdenum site of XOR. With xanthine as reducing substrate, nitrite was produced in essentially a 1:1 stoichiometric ratio with respect to urate. Unlike the case of nitrite, the enzyme was not significantly inactivated, implying that inactivation during nitrite reduction depends on the presence of nascent NO in its enzyme complex.

Effect of contact time and inhibitor concentration on the affinity mediated adsorption of cells to surfaces.

Cell detachment by shear stress under conditions of laminar flow was used to investigate the effect of incubation time and soluble binding competitors on affinity mediated cell/surface interactions. Fractional attachment between yeast and a Concanavalin A (Con A) coated surface was studied as a function of adhesion time prior to exposure to shear in a parallel plate flow chamber. Two, four and sixteen hours adhesion times gave rise to significantly different fractional attachment profiles, with four hours giving greater cell retention.The effect of dextran as a competitive displacer of pre-attached cells was also examined using a number of exposure regimes. While the presence of dextran in the displacement buffer led to higher fractional displacement of pre-attached cells, this effect was magnified if an equilibration period between dextran solution and pre-attached cells was allowed before detachment was attempted. The decline in fractional attachment increased with incubation time up to 30 min, with longer periods resulting in a smaller effect. Pre-incubation of the Con A surface with dextran prior to the introduction of cells led to a 60% reduction in attachment.Attempts to determine critical shear values were complicated by the presence of a tightly bound cell fraction of approximately 15% that was not removed at the highest shear values used.

Reduction of organic nitrates catalysed by xanthine oxidoreductase under anaerobic conditions.

Xanthine oxidoreductase catalyses the anaerobic reduction of glyceryl trinitrate (GTN), isosorbide dinitrate and isosorbide mononitrate to inorganic nitrite using xanthine or NADH as reducing substrates. Reduction rates are much faster with xanthine as reducing substrate than with NADH. In the presence of xanthine, urate is produced in essentially 1:1 stoichiometric ratio with inorganic nitrite, further reduction of which is relatively slow. Organic nitrates were shown to interact with the FAD site of the enzyme. In the course of reduction of GTN, xanthine oxidoreductase was progressively inactivated by conversion to its desulpho form. It is proposed that xanthine oxidoreductase is one of several flavoenzymes that catalyse the conversion of organic nitrate to inorganic nitrite in vivo. Evidence for its further involvement in reduction of the resulting nitrite to nitric oxide is discussed.

The temperature optima of enzymes: a new perspective on an old phenomenon.

Careful analysis of the dependence of enzyme activity on assay temperature has revealed that some enzymes might have real temperature optima in which the decrease in catalytic rate at temperatures above the optimum is not primarily a result of irreversible thermal inactivation. The 'equilibrium model' has been formulated to describe genuine temperature optima, and to suggest a simple experimental method by which to distinguish these cases from those in which enzyme instability is the major determinant of temperature optima.

The inhibition of flavoproteins by phenoxaiodonium, a new iodonium analogue.

Iodonium compounds, especially diphenylene iodonium and iodonium diphenyl are used extensively as inhibitors of NADH-ubiquinone reductase and NADPH oxidase activity. Here, the use of a new iodonium compound, phenoxaiodonium is reported. The IC(50) of neutrophil superoxide production, measured using the superoxide dismutase inhibitable rate of cytochrome c reduction, was approximately 0.75 microM, while 50% inhibition of mitochondrial respiration, measured by the rate of oxygen uptake using a Clark type oxygen electrode, was at approximately 20 microM. The inhibition of oxidation of xanthine to urate by xanthine oxidase was also studied, giving a K(i) of 0.2 microM. Inhibition of nitric oxidase synthase (NOS: from rat brain) by 0.2 microM phenoxaiodonium was equivalent to 1 mM N(G)-nitro-L-arginine methyl ester HCl (L-NAME), that is total abolition of activity. We conclude that phenoxaiodonium is an extremely good inhibitor of flavo-enzymes, but like diphenylene iodonium and iodonium diphenyl, will be of limited use as a pharmacological tool for the elucidation of the involvement of such enzymes in specific cellular functions.

A new route to peroxynitrite: a role for xanthine oxidoreductase.

Peroxynitrite, a potent oxidising, nitrating and hydroxylating agent, results from the reaction of nitric oxide with superoxide. We show that peroxynitrite can be produced by the action of a single enzyme, xanthine oxidoreductase (XOR), in the presence of inorganic nitrite, molecular oxygen and a reducing agent, such as pterin. The effects of oxygen concentration on peroxynitrite production have been examined. The physiologically predominant dehydrogenase form of the enzyme is more effective than the oxidase form under aerobic conditions. It is proposed that XOR-derived peroxynitrite fulfils a bactericidal role in milk and in the digestive tract.

Complementation of a glucose transporter mutant of Schizosaccharomyces pombe by a novel Trypanosoma brucei gene.

The African trypanosome Trypanosoma brucei has a digenetic life cycle that involves the insect vector and the mammalian host. This is underscored by biochemical switches in its nutritional requirements. In the insect vector, the parasite relies on amino acid catabolism, but in the mammalian host, it derives its energy exclusively from blood glucose. Glucose transport is facilitated, and constitutes the rate-limiting step in ATP synthesis. Here, we report the cloning of a novel glucose transporter-related gene by heterologous screening of a lambdaEMBL4 genomic library of T. brucei EATRO 164 using a rat liver glucose transporter cDNA clone. Genomic analysis shows that the gene is present as a single copy within the parasite genome. The gene encodes a protein with an estimated molecular mass of 55.9 kDa, which shares only segmental homology with members of the glucose transporter superfamily. Several potential post-translational modification sites including phosphorylation, N-glycosylation, and cotranslational myristoylation sites also punctuate the sequence. It is distinguished from classical transporter proteins by the absence of putative hydrophobic membrane-spanning domains. However, this protein was capable of complementing Schizosaccharomyces pombe glucose transporter mutants. The rescued phenotype conferred the ability of the cells to grow on a broad range of sugars, both monosaccharides and disaccharides. The kinetics of glucose uptake reflected those in T. brucei. In addition to complementation in yeast, we also showed that the gene enhanced glucose uptake in cultured mammalian cells.

Reduction of organic nitrites to nitric oxide catalyzed by xanthine oxidase: possible role in metabolism of nitrovasodilators.

Xanthine oxidase (XO) was shown to catalyze the reduction of isoamyl and isobutyl nitrites to nitric oxide (NO) in the presence of xanthine under anaerobic conditions. NO was produced at a stoichiometric ratio of 2:1 versus urate generation, steady-state analysis of which showed Michaelis-Menten kinetics with xanthine as varied substrate and substrate inhibition with varied organic nitrite. Under the conditions of NO generation from isoamyl nitrite, XO was progressively inactivated by a mechanism involving conversion of Mo=S to Mo=O, yielding "desulfo" enzyme. It is proposed that XO is involved in the metabolism of organic nitrites to NO in vivo and that the observed inactivation serves to explain the phenomenon of tolerance.

Reduction of nitrite to nitric oxide catalyzed by xanthine oxidoreductase.

Xanthine oxidase (XO) was shown to catalyze the reduction of nitrite to nitric oxide (NO), under anaerobic conditions, in the presence of either NADH or xanthine as reducing substrate. NO production was directly demonstrated by ozone chemiluminescence and showed stoichiometry of approximately 2:1 versus NADH depletion. With xanthine as reducing substrate, the kinetics of NO production were complicated by enzyme inactivation, resulting from NO-induced conversion of XO to its relatively inactive desulfo-form. Steady-state kinetic parameters were determined spectrophotometrically for urate production and NADH oxidation catalyzed by XO and xanthine dehydrogenase in the presence of nitrite under anaerobic conditions. pH optima for anaerobic NO production catalyzed by XO in the presence of nitrite were 7.0 for NADH and

The kinetics of affinity-mediated cell-surface attachment.

Data and a semi-empirical model are presented that describe the affinity interaction of yeast cells with a Concanavalin A derivatised surface. The model uses 3 parameters to describe the time course of cell attachment from a flowing suspension of yeast cells, over a range of flow rates, and gives an effective global fit to the data obtained. Further modifications allow the effects of a soluble competitor (glucose) on binding to be quantified in terms of a saturation effect, and an effective global fit is obtained. A comparison was made between the relationship between steady-state attached fraction and applied shear with similar data reported earlier (Ming, F. et al, 1998) for the detachment of pre-adsorbed cells. This shows that there is an order of magnitude difference between the forces required to effect complete detachment in the two systems, and that the nature of the relationship between shear and attached fraction is profoundly different. The magnitude of this time-dependent stabilization might be explained in terms of a progressive reorientation of cell relative to the surface such that the number of bonds is maximized.

Xanthine oxidoreductase catalyses the reduction of nitrates and nitrite to nitric oxide under hypoxic conditions.

Xanthine oxidoreductase (XOR) catalyses the reduction of the therapeutic organic nitrate, nitroglycerin (glyceryl trinitrate, GTN), as well as inorganic nitrate and nitrite, to nitric oxide (NO) under hypoxic conditions in the presence of NADH. Generation of nitric oxide is not detectable under normoxic conditions and is inhibited by the molybdenum site-specific inhibitors, oxypurinol and (-)BOF 4272. These enzymic reactions provide a mechanism for generation of NO under hypoxic conditions where nitric oxide synthase does not function, suggesting a vasodilatory role in ischaemia.

Prospects for antiparasitic drugs. The case of Trypanosoma brucei, the causative agent of African sleeping sickness.

Glycolysis in the bloodstream form of Trypanosoma brucei provides a convenient context for studying the prospects for using enzyme inhibitors as antiparasitic drugs. As the recently developed model of this system (Bakker, B. M., Michels, P. A. M., Opperdoes, F. R., and Westerhoff, H. V. (1997) J. Biol. Chem. 272, 3207-3215) contains 20 enzyme-catalyzed reactions or transport steps, there are apparently numerous potential targets for drugs. However, as most flux control resides in the glucose-transport step, this is the only step for which inhibition can be expected to produce large effects on flux, and in the computer model such effects prove to be surprisingly small (although larger than those obtained by inhibiting any other step). It follows that there is little prospect of killing trypanosomes by depressing their glycolysis to a level incapable of sustaining life. The alternative is to use inhibition to increase the concentration of a metabolite sufficiently to interfere with the viability of the organism. For this purpose, only uncompetitive inhibition of pyruvate export proves effective in the model; in all other cases studied, the effects on metabolite concentrations are little more than trivial. This observation can be explained by the fact that nearly all of the metabolite concentrations in the system are held within relatively narrow ranges by stoichiometric constraints.

NADH oxidase activity of human xanthine oxidoreductase--generation of superoxide anion.

Human xanthine oxidase was purified from breast milk. The dehydrogenase form of the enzyme, which predominates in most mammalian tissues, catalyses the oxidation of NADH by oxygen, generating superoxide anion significantly faster than does the oxidase form. The corresponding forms of bovine enzyme behave very similarly. The steady-state kinetics of NADH oxidation and superoxide production, including inhibition by NAD, by the dehydrogenase forms of both enzymes, are analysed in terms of a model involving two-stage recycling of oxidised enzyme. Established inhibitors of xanthine oxidoreductases (allopurinol oxypurinol, amflutizole and BOF 4272), which block all other reducing substrates, were ineffective in the case of NADH. Diphenyleneiodonium, on the other hand, was a powerful inhibitor of NADH oxidation. The potential involvement of reactive oxygen species arising from NADH oxidation by xanthine oxidoreductase in ischaemia-reperfusion injury and other disease states, as well as in normal signal transduction, is discusssed.

Ferrocenoyl derivatives of alamethicin: redox-sensitive ion channels.

The synthesis and single-channel characterization of two redox-active C-terminal derivatives of alamethicin are herein described. The reduced [Fe(II)] forms of ferrocenoyl-alamethicin (Fc-ALM) and 1'-carboxyferrocenoyl-alamethicin (cFc-ALM) are shown to form voltage-dependent ion channels at cis positive potentials in planar lipid bilayers (PLB) with conductance properties similar to those of alamethicin. In situ oxidation of Fc-ALM [to Fe(III)] in the PLB apparatus causes a time-dependent elimination of channel openings, which can be restored by an increase in the transbilayer potential. In contrast, oxidation of cFc-ALM leads to the formation of shorter-lived channels. Pretreatment of the ferrocenoyl peptides with oxidizing agent alters their single-channel properties in a qualitatively similar manner, establishing that the changes in channel properties in the presence of oxidizing agents are due specifically to ferrocenoyl oxidation. We suggest that the redox sensitivity of these ferrocene-containing ion channels may be governed by a combination of the following factors: (1) changes in hydrophobicity; (2) alteration of peptide molecular dipole; and (3) alterations in tendencies toward self-association. However, oxidation induced changes in peptide conformation cannot be ruled out. Our results provide evidence that it is possible to engineer channel-forming peptides that respond to specific changes in the chemical environment.