Purification and catalytic properties of the chlorophenol 4-monooxygenase from Burkholderia cepacia strain AC1100.

Burkholderia cepacia strain AC1100 can be induced for the degradation of 2,4,5-trichlorophenol (2,4,5-TCP). We have purified the active enzyme 30-fold to apparent homogeneity with a 44% yield by a two-step chromatographic procedure, and showed that it consists of a single type of subunit of 59 kDa based on SDS-PAGE using Coomassie blue and Sypro staining. This enzyme has no bound prosthetic group but requires exogenous addition of FAD and NADH to perform the dioxygen-dependent hydroxylation in the 4-position of 2,4,6-TCP. Studies of the stoichiometry revealed the consumption of 2 mol of NADH plus 1 mol of dioxygen per mol of 2,4,6-TCP with identification of the reaction product as 2,6-dichlorohydroquinone. Steady state kinetic parameters for cofactors and a variety of substrates were determined. Low K(m) values of 1+/-0.1 microM, 32+/-5 microM and 4+/-2 microM were found for FAD, NADH and 2,6-dichlorophenol (2,6-DCP), respectively, under saturating conditions for the two others. In the presence of 2,6-DCP as a substrate, methimazole (MMI) inhibited the enzyme competitively with a K(i)=27 microM. When other polychlorinated substrates were studied, IC(50) values for MMI were found in a range compatible with their apparent affinity. On the basis of aromatic product formation, NADH and O(2) consumption schemes for 2,4,6-TCP and 2,4,5-TCP degradation are discussed. A Blast search revealed that this enzyme has a high sequence identity (60%) with 2,4,6-TCP-4-monooxygenases from Burkholderia pickettii and from Azotobacter sp. strain GP1 which all of them catalyze para hydroxylative dehalogenation.

Purification and structure of the major product obtained by reaction of NADPH and NMNH with the myeloperoxidase/hydrogen peroxide/chloride system.

The first spectrophotometric study of the reaction of the myeloperoxidase/H2O2/Cl- system with NADPH and NMNH showed that the reaction products were not the corresponding oxidized nucleotides and that modifications would take place on the nicotinamide part of the molecule [Auchère, F. & Capeillère-Blandin, C. (1999) Biochem. J. 343, 603-613]. In this report, in order to obtain more precise information on the structural modifications and mechanism of the reaction, we focus on the purification and isolation of products derived from NADPH and NMNH by RP-HPLC. Electrospray ionization mass spectra indicated that the relative height of the peaks reflected that of the natural isotopic abundance of 35Cl and 37Cl, providing evidence that the products derived from NADPH and NMNH were monochlorinated. Moreover, calculated masses revealed the 1 : 1 addition of HOCl to the molecule. Various 1D and 2D NMR experiments provided data for the assignments of the chemical shifts of protons and carbons and the coupling constants of the protons of the chlorinated nucleotides. Further NOESY experiments allowed the characterization of the spatial structure of the chlorinated product and showed that trans HOCl addition occurred at the C5=C6 carbon double bond of the nicotinamide ring, leading to a chlorohydrin.

NADPH as a co-substrate for studies of the chlorinating activity of myeloperoxidase.

The reinvestigation of the kinetics of myeloperoxidase (MPO) activity with the use of NADPH as a probe has allowed us to determine the effects of H(2)O(2), Cl(-) ion and pH on the MPO-dependent production of HOCl. The chlorination rate of NADPH did not depend on NADPH concentration and was entirely related to the rate of production of HOCl by MPO. The overall oxidation of NADPH occurred similarly in the absence of O(2) and was insensitive to scavengers of the superoxide radical anion. Experiments performed on the direct oxidation of NADPH by MPO in the presence and the absence of H(2)O(2) showed that neither the rate nor the stoichiometry of the reaction could interfere in the NADPH oxidation process involved in the steady-state chlorination cycle. The oxidation of NADPH was characterized by a decrease in the A(339) of the reduced nicotinamide with the concomitant appearance of a new chromophore with absorbance maximum at 274 nm, characterized by isosbestic points at 300 and 238 nm. The reaction product did not possess any enzymic properties with dehydrogenases and led to a metabolite other than NADP(+). Its amount accounted for a stoichiometric conversion of H(2)O(2) into HOCl. Analyses of the NADPH reaction allowed the determination of both kinetic (k(cat) and K(m)) and thermodynamic (K(d)) parameters. When the values of kinetic parameters were compared with previously published ones, the main discrepancy was found with data obtained with the chlorination of monochlorodimedon and a better agreement with diethanolchloramine formation or H(2)O(2) consumption. Variations in the extent of NADPH oxidation with Cl(-) concentration enabled us to determine the dissociation constant for the enzyme-Cl(-) complex. In the course of titration studies, the spectral properties of NADPH reacting with either HOCl or the MPO/H(2)O(2)/Cl(-) system were quantitatively similar in terms of stoichiometry and absorbance coefficient and thus led to identical chlorinated products. However, no spectral modification occurred with NADP(+) and adenine nucleotide analogues under the same conditions. A quantitative comparison of difference spectra obtained with NADPH and NMNH indicated that chlorination occurred on the nicotinamide part of the molecule.

Hydroxylation reaction catalyzed by the Burkholderia cepacia AC1100 bacterial strain. Involvement of the chlorophenol-4-monooxygenase.

The Burkholderia cepacia AC1100 strain, known to degrade the herbicide, 2,4,5-Trichlorophenoxyacetic acid (2,4,5-T), is able to metabolize 4-hydroxyarylaldehyde, not only into the corresponding acid, but also into a new hydroquinone, 2,5-dihydroxyarylaldehyde. When incubated with resting AC1100 cells or cell-free extracts, syringaldehyde and 3,5-dimethoxy-4-hydroxybenzaldehyde were converted into such metabolites, identified by comparison of their mass and 1H-NMR spectra with those of authentic chemically synthesized samples. With 5-bromovanillin, only one metabolite was formed, the structure of which was identified as 2, 5-dihydroxy-4-methoxy-6-bromobenzaldehyde through 1H-NMR two-dimensional NOESY experiments. All these products result formally from a para hydroxylation of the phenol followed by the cis migration of the aldehyde. This reaction is the only one to be associated with the 2,4,5-T degradation pathway, as the acid formation was retained when the AC1100 strain had lost its degradation ability. Through competitive experiments with halophenols and methimazole, an alternative substrate of flavin monooxygenase, the chlorophenol-4-monooxygenase was recognized to be the enzyme involved in the hydroxylation of 4-hydroxyarylaldehyde. The purified enzyme, previously reported to catalyze the para hydroxylation or dehalogenating hydroxylation of chlorophenols, also promotes this hydroxylation reaction in the presence of NADH and FAD. The kcat value determined for the best substrate, syringaldehyde, 0. 08 s-1, was about 20% of that obtained for 2,6-dichlorophenol hydroxylation (0.38 s-1).

Oxidation of guaiacol by myeloperoxidase: a two-electron-oxidized guaiacol transient species as a mediator of NADPH oxidation.

The present study was first aimed at a complete steady-state kinetic analysis of the reaction between guaiacol (2-methoxyphenol) and the myeloperoxidase (MPO)/H2O2 system, including a description of the isolation and purification of MPO from human polymorphonuclear neutrophil cells. Secondly, the overall reaction of the oxidation of NADPH, mediated by the reactive intermediates formed from the oxidation of guaiacol in the MPO/H2O2 system, was analysed kinetically. The presence of guaiacol stimulates the oxidation of NADPH by the MPO/H2O2 system in a concentration-dependent manner. Concomitantly, the accumulation of biphenoquinone (BQ), the final steady-state product of guaiacol oxidation, is lowered, and even inhibited completely, at high concentrations of NADPH. Under these conditions, the stoichiometry of NADPH:H2O2 is 1, and the oxidation rate of NADPH approximates to that of the rate of guaiacol oxidation by MPO. The effects of the presence of superoxide dismutase, catalase and of anaerobic conditions on the overall oxidation of NADPH have also been examined, and the data indicated that superoxide formation did not occur. The final product of NADPH oxidation was shown to be enzymically active NADP+, while guaiacol was generated continuously from the reaction between NADPH and oxidized guaiacol product. In contrast, similar experiments performed on the indirect, tyrosine-mediated oxidation of NADPH by MPO showed that a propagation of the free radical chain was occurring, with generation of both O2(-.) and H2O2. BQ, in itself, was able to spontaneously oxidize NADPH, but neither the rate nor the stoichiometry of the reaction could account for the NADPH-oxidation process involved in the steady-state peroxidation cycle. These results provide evidence that the oxidation of NADPH does not involve a free nucleotide radical intermediate, but that this is probably due to a direct electron-transfer reaction between NADPH and a two-electron-oxidized guaiacol intermediate.

Advanced oxidation protein products as novel mediators of inflammation and monocyte activation in chronic renal failure.

We previously demonstrated the presence of advanced oxidation protein products (AOPP), a novel marker of oxidative stress in the plasma of uremic patients receiving maintenance dialysis. The present study in a cohort of 162 uremic patients showed that plasma concentrations of AOPP increased with progression of chronic renal failure and were closely related to advanced glycation end products (AGE)-pentosidine (r = 0.52, p < 0.001), taken as a marker of AGE. In vivo, the relevance of AOPP and AGE-pentosidine in monocyte-mediated inflammatory syndrome associated with uremia was evidenced by close correlations between AOPP or AGE-pentosidine and monocyte activation markers, including neopterin, IL-1R antagonist, TNF-alpha, and TNF soluble receptors (TNF-sR55 and TNF-sR75). To determine the mechanisms by which AOPP and AGE could be directly involved in monocyte activation, AOPP-human serum albumin (HSA) and AGE-HSA were produced in vitro by treating HSA with oxidants or glucose, respectively. Spectroscopic analysis confirmed that AOPP-HSA contains carbonyls and dityrosine. Both AOPP-HSA and AGE-HSA, but not purified dityrosine, were capable of triggering the oxidative burst of human monocytes in cultures. The AOPP-HSA-induced respiratory burst was dependent on the chlorinated nature of the oxidant and on the molar ratio HSA/HOCI. Collectively, these data first demonstrate that AOPP act as a mediator of oxidative stress and monocyte respiratory burst, which points to monocytes as both target and actor in the immune dysregulation associated with chronic uremia.

Sulphoxidation reaction catalysed by myeloperoxidase from human leucocytes.

The oxidation of alkyl aryl sulphides by myeloperoxidase (MPO) at the expense of hydrogen peroxide was investigated under steady-state conditions. The sulphide concentration effect was studied under saturating H2O2 concentrations at pH 5.0 and 20 degreesC. The kinetic constants, kcat and Km, of the different substrates were determined and the values were in the 1-10 s-1 range and around 43+/-26 microM respectively, whatever the sulphide considered. In the case of p-substituted thioanisoles, the oxidation rate was dependent upon the substituent effect. The correlation of log(kcat) with the substituent constants (sigma+ values) (Hammett equation) could be explained by a reaction mechanism involving the enzyme compound II and a sulphenium radical cation. This conclusion was also supported by spectrophotometric analysis of catalytic intermediates of the enzyme, showing the accumulation of compound II. Moreover, chiral HPLC analyses showed that MPO oxidation of alkyl aryl sulphides produced the corresponding (R)-sulphoxides with a low enantioselectivity (4-8%). Chloride ion effects on the MPO-catalysed oxygenation of sulphides were also studied. Chloride acted as a substrate for MPO and as an activator in MPO-catalysed sulphoxidation. Inhibition occurred at chloride concentrations above 120 mM, whereas below 120 mM, chloride increased the reaction rate when using p-tolyl methyl sulphide as the substrate. In the presence of 100 mM chloride the catalytic efficiency (kcat/Km) of MPO increased 3-4-fold, whatever the sulphide considered, but racemic products were obtained. These data have been interpreted in the light of known structural information on the accessibility of the distal haem cavity.

Molecular interpretation of inhibition by excess substrate in flavocytochrome b2: a study with wild-type and Y143F mutant enzymes.

The crystal structure of flavocytochrome b2 (L-lactate dehydrogenase) from Saccharomyces cerevisiae suggests that Tyr143 plays a dual role at the active site: it contributes to substrate binding and, most importantly, makes a hydrogen bond to a heme propionate, which could facilitate communication between the domains. Previous work on the Y143F mutant enzyme provided support for these hypotheses [Miles, C. S., Rouvière-Fourmy, N., Lederer, F., Mathews, F. S., Reid, G. A., Black, M. T., & Chapman, S. K. (1992) Biochem. J. 285, 187-192; Rouvière-Fourmy, N., Capeillère-Blandin, C., & Lederer, F. (1994) Biochemistry 33, 798-806]. In the course of kinetic comparisons between the wild-type (WT) enzyme and the Y143F mutant protein, we observed for the latter signs of inhibition by excess substrate at much lower concentrations than observed for the former. A detailed investigation of the phenomenon has shown that, for the wild-type and Y143F forms, lactate at high concentrations inhibits both cytochrome c and ferricyanide reduction. In these cases, inhibition appears to be a specific effect, since acetate at identical concentrations exerts an inhibitory effect that is markedly weaker than that of lactate. In the pre-steady-state, in the absence of acceptor, flavin and heme reduction are unaffected by high substrate concentrations in the WT enzyme case. For the Y143F mutant, flavin reduction is similarly unaffected, but heme reduction is inhibited to nearly the same extent by high lactate and acetate concentrations. In this case, inhibition can probably be ascribed to ionic strength effects. The combination of stopped-flow and steady-state results suggests that lactate binds with weak affinity at the active site when the flavin is in the semiquinone state, preventing electron transfer to heme b2 and hence to acceptors. This phenomenon is analogous to the inhibition exerted by pyruvate when bound to the enzyme at the semiquinone stage [Tegoni, M., Janot, J. M., & Labeyrie, F. (1990) Eur. J. Biochem. 190, 329-342]. We suggest that the substrate carboxylate and the heme propionate of the mobile heme-binding domain compete for the Tyr143 hydroxyl group, hence for approach to the flavin. In the Y143F mutant enzyme, in which the interdomain interaction is impaired, competition would play in favor of the substrate, resulting in the inhibition at lower lactate concentrations than observed for the wild-type enzyme.

Advanced oxidation protein products as a novel marker of oxidative stress in uremia.

Evidence suggests an imbalance between antioxidant and oxidant-generating systems resulting in oxidative stress in uremic patients. As plasma proteins are critical targets for oxidants, we developed a novel spectrophotometric assay which allows to detect advanced oxidation protein products (AOPP) in uremic plasma. By size-exclusion chromatography AOPP are retrieved in two distinct peaks at 600 and below 80 kDa in uremic plasma, while no such peaks are found in control plasma. Further biochemical characterization revealed that AOPP are carried by oxidized plasma proteins, especially albumin and do not have oxidant properties. AOPP increased in a dose-dependent manner following in vitro exposure of plasma or purified human serum albumin (HSA) to hypochlorous acid. Advanced glycation end products of human serum albumin (AGE-HSA) also increased AOPP levels. In vivo, plasma level of AOPP was the highest in patients on hemodialysis, followed by those on peritoneal dialysis and by undialyzed patients with advanced chronic renal failure. AOPP levels correlated with plasma concentrations of dityrosine and AGE-pentosidine, as indices of oxidant-mediated protein damage, but not with thiobarbituric reactive substances as lipid peroxidation markers. A close correlation was also found between AOPP and neopterin levels, suggesting that AOPP could be part in the monocyte-mediated inflammatory disorders associated with uremia. In conclusion, we propose the measurement of AOPP as a reliable marker to estimate the degree of oxidant-mediated protein damage in uremic patients and to predict the potential efficacy of therapeutic strategies aimed at reducing such an oxidative stress.

Flavocytochrome b2-cytochrome c interactions: the electron transfer reaction revisited.

This review is concerned with the kinetics and mechanism of electron transfer processes which occur intermolecularly between reduced flavocytochrome b2 and cytochrome c molecules within an encounter complex. Analyses are given of previous reports which aimed at describing the formation of stable complexes obtained at low ionic strength in solution and in the crystalline state with a binding stoichiometry of 1 to 1 heme ratio. Relevant data allow to define the respective role of flavin and heme b2 in the electron transfer towards cytochrome c and give a description of the recognition areas on the two redox partners. The paper also refers to a recent computer model of their postulated interactions as based on the three-dimensional structure of the Saccharomyces cerevisiae single molecules. Special emphasis is given to rapid kinetic investigations of the electron transfer reaction between Hansenula anomala flavocytochrome b2 and cytochrome c studied as a function of concentration, ionic strength and temperature. Data showed that reaction rates were modulated by ionic strength, reaching a saturation behaviour at low ionic strength. In the present paper the temperature effects on Kd and kET have been re-examined. Thermodynamic analysis of the dissociation constant points out the importance of hydrophobic interactions in the complex formation. Analysis of the variations of rate constants in terms of semiclassical theory of electron-transfer reaction yields values of 1.12 eV for the reorganization energy and 0.05 cm-1 for the electronic coupling factor. Interpretation of the electronic coupling in terms of through-bond and/or through-space pathways takes into account the hypothetical model proposed for the binary complex. The functional implications of this model in the electron transfer reaction are discussed. Finally the existence of a conformational equilibrium between the initial binding complex and the complex from which electron transfer occurs is considered.

Ontogeny of immunocytochemically demonstrable androgen- and androgen receptor-containing cells in the hypothalamus and adenohypophysis of the chick embryo.

Brain sections of male chick embryos, 6.5-18.5 days of age, were examined immunocytochemically for the presence of androgen- and androgen receptor-containing cells in the hypothalamus and adenohypophyseal pars distalis. Using antibodies (Ab) against both androgens (T-Ab) and the androgen receptor (AR-Ab), single- and double-immunostained cells were located in a total of five nuclei of the anterior-, mid-, and posterior-hypothalamus, as well as in the rostral and caudal lobes of the adenohypophyseal pars distalis. From Days 9.5-12.5, the mean number of androgen-immunostained cells within the hypothalamus and pars distalis increased significantly (P < 0.01), while from Days 12.5-18.5 there was no further statistically significant increase. The results of the present investigation support previous findings which suggest that in the chick embryo the negative feedback loop of the hypothalamo-adenohypophyseal-testicular (HATest) axis is functional by the 13th day of development (Woods et al., 1989a,b). They also agree with the observations of Wilson and Glick (1970) that in the male chick embryo testosterone organizes masculine mating behavior prior to Day 13.0.

Role of tyrosine 143 in lactate dehydrogenation by flavocytochrome b2. Primary kinetic isotope effect studies with a phenylalanine mutant.

Flavocytochrome b2 catalyzes the oxidation of lactate at the expense of cytochrome c. After flavin (FMN) reduction by the substrate, reducing equivalents are transferred one by one to heme b2, and from there on to cytochrome c. The crystal structure of the enzyme is known at 2.4-A resolution, and specific roles in catalysis have been assigned to active side chains. Tyr143 in particular, located at the interface between the flavodehydrogenase moiety and the heme-binding domain, was thought to take part in substrate binding, as well as to orient the heme-binding domain for efficient electron transfer. A first study of the properties of a Tyr143Phe mutant showed that the major effect of the mutation was to decrease the rate of electron transfer from flavin to heme [Miles, C.S., Rouvière-Fourmy, N., Lederer, F., Mathews, F.S., Reid, G.A., Black, M.T., & Chapman, S.K. (1992) Biochem. J. 285, 187-192]. In the present paper, we focus on the effect of the mutation on catalysis of lactate dehydrogenation. We report the deuterium kinetic isotope effects on flavin reduction as measured with stopped-flow methods and on cytochrome c reduction in the steady-state using L-[2-2H]lactate. For the wild-type enzyme, isotope effects on FMN reduction, D(kredF) and D(kredF)/Km), were 7.2 +/- 0.9 and 4.2 +/- 1.3, respectively, and for the Y143F mutant values of 4.4 +/- 0.5 and 3.9 +/- 1.1 were obtained. Calculations, from deuterium isotope effects, of substrate Kd values, combined with knowledge of kcat/Km values, lead to the conclusion that Tyr143 does stabilize the Michaelis complex by hydrogen bonding to a substrate carboxylate, as was postulated; but the mutation does not destabilize the transition state more than the Michaelis complex.(ABSTRACT TRUNCATED AT 250 WORDS)

Molecular characteristics of cytochrome b558 isolated from human granulocytes, monocytes and HL60 and U937 cells differentiated into monocyte/macrophages.

Enriched cytochrome b558 preparations were obtained from human mature monocytes (MN) and retinoic acid plus interferon gamma induced human myeloid leukemia cell lines HL-60 and U937, using an adaptation of the procedure described by A.W. Segal (Nature (1987) 326, 88-91) for purification of cytochrome b558 from human polymorphonuclear leukocytes (PMN). Spectral characteristics of cytochrome b558 were determined and found to be independent of cell type and specific heme b content of the preparation. To increase the sensitivity of the spectral assay, analysis in the gamma band were used and delta epsilon 427-413 was determined to be equal to 158 mM-1 cm-1. An alpha beta type heterodimeric cytochrome b558 was found for PMN and MN by the concordant elution of heme b spectral activity from heparin agarose and the detection of two polypeptide chains by SDS-PAGE. The expression of the lighter polypeptide alpha chain in the various human monocyte-like cell lines was assessed and its identity, as a component of cytochrome b, was confirmed by immunodetection using a rabbit polyclonal antibody reacting with the alpha subunit of PMN cytochrome b558. Immunoblotting studies detected the alpha subunit in monocyto-macrophagic differentiated HL-60 and U 937 cells and mature MN at 22 kDa, but not in uninduced cells which did not express the respiratory burst. Whatever the specific content or the cell origin of the cytochrome b558-enriched preparations, the heme b binding site was shown to be associated with the alpha subunit defined by a constant molecular mass of 22 kDa, as evidenced by the finding of a constant ratio between the silver stained band intensity and the corresponding heme b amount. The heavy polypeptide beta chain from MN cytochrome b was found to have a significantly higher molecular weight than the beta subunit from PMN at 94 +/- 5 kDa instead of 90 +/- 4 kDa. In contrast, in cytochrome b preparations from induced monocyto-macrophagic cells, isolated with a low heme specific content, the variability in the detection of the staining intensity of the beta band either in SDS-PAGE or immunodetection reactivities precludes accurate definition of its molecular mass and estimation of the stoichiometry between the alpha and beta subunits in the differentiated cells. However, wheat-germ agglutinin binding studies indicated the presence of N-glycosylated protein in the range of 85-110 kDa.

Structural modifications of human beta 2 microglobulin treated with oxygen-derived radicals.

Treatment of human beta 2 microglobulin (beta 2m) with defined oxygen-derived species generated by treatment with gamma-radiation was studied. As assessed by SDS/PAGE, the hydroxyl radicals (.OH) caused the disappearance of the protein band at 12 kDa that represents beta 2m, and cross-linked the protein into protein bands stable to both SDS and reducing conditions. However, when .OH was generated under oxygen in equimolar combination with the superoxide anion radical (O2.-), the high-molecular-mass protein products were less represented, and fragmented derivatives were not obviously detectable. Exposure to .OH alone, or to .OH + O2.- in the presence of O2, induced the formation of beta 2m protein derivatives with a more acidic net electrical charge than the parent molecule. In contrast, O2.- alone had virtually no effect on molecular mass or pI. Changes in u.v. fluorescence during .OH attack indicated changes in conformation, as confirmed by c.d. spectrometry. A high concentration of radicals caused the disappearance of the beta-pleated sheet structure and the formation of a random coil structure. Loss of tryptophan and significant production of dityrosine (2,2'-biphenol type) were noted, exhibiting a clear dose-dependence with .OH alone or with .OH + O2.-. The combination of .OH + O2.- induced a pattern of changes similar to that with .OH alone, but more extensive for c.d. and tryptophan oxidation (2 Trp/beta 2m molecule), and more limited for dityrosine formation. Lower levels of these oxidative agents caused the reproducible formation of species at 18 and 25 kDa which were recognized by antibodies against native beta 2m. These findings provide a model for the protein pattern observed in beta 2m amyloidosis described in the literature.

Electron-transfer steps involved in the reactivity of Hansenula anomala flavocytochrome b2 as deduced from deuterium isotope effects and simulation studies.

The L-lactate-flavocytochrome b2-ferricyanide electron-transfer system from the yeast Hansenula anomala was investigated by rapid-reaction techniques. The kinetics of reduction of oxidized flavocytochrome b2 by L-lactate and L-[2H]lactate were biphasic both for flavin and haem prosthetic groups and at all concentrations tested. The first-order rate constants of the rapid and slow phases depended upon substrate concentrations, a saturation behaviour being exhibited. Substitution of the C alpha-H atom by 2H was found to cause appreciable changes in the rate constants for the initial reduction of flavin and haem (phase I), which were respectively about 3-fold and 2-fold less than with L-lactate. In contrast, no significant isotope effect was noted on the apparent reduction rate constants of the slow phase, phase II. Under steady-state conditions an isotope effect of 2.0 was found on the overall electron transfer from L-lactate to ferricyanide. These transient reduction results were discussed in terms of a kinetic model implying intra- and inter-protomer electron exchanges between flavin and haem b2, all of which have been experimentally described. Computer simulations indicate that the reaction scheme provides a reasonable explanation of the fast-reduction phase, phase I (in absence of acceptor). The pseudo-first-order rate constant for oxidation of reduced haem b2 in flavocytochrome b2 increased with increasing ferricyanide concentration in a hyperbolic fashion. The limiting value at infinite ferricyanide concentration, which was attributed to the intramolecular electron-transfer rate from ferroflavocytochrome b2 to the iron of ferricyanide within a complex, was 920 +/- 50 s-1 at pH 7.0 and 5 degrees C. Stopped-flow and rapid-freezing measurements showed haem b2 and flavin to be 90 and 44% oxidized respectively under steady-state conditions in presence of ferricyanide. Simulation studies were carried out to check the participation of the proposed reduction sequence in the overall catalytic reaction together with the role of reduced haem b2 (Hr) and flavin semiquinone (Fsq) as electron donors to ferricyanide. When the rate of the intramolecular electron-transfer exchange between Fsq and ferricyanide was adjusted to 200 s-1, simulated data accounted for molar activities defined under various conditions of L-lactate, [2H]lactate and ferricyanide concentrations. Simulation studies were extended to data obtained using cytochrome c as acceptor and reaction catalysed by Saccharomyces cerevisiae flavocytochrome b2. The differences in reactivity observed for Hr and Fsq with ferricyanide and cytochrome c were discussed in terms of redox potentials, electrostatic interactions, distances and accessibility of the participating groups.

[A new strategy of neonatal screening for cystic fibrosis. The association of immunoreactive trypsin and molecular biology in dried blood]. / Vers une nouvelle stratégie de dépistage néonatal de la mucoviscidose. Association du dosage de la trypsine immunoréactive et de la biologie moléculaire dans le sang séché.

Cystic fibrosis (CF) screening by means of immunoreactive trypsin (IRT) lacks specificity: only 1 out of 12 hypertrypsinemic neonates has cystic fibrosis. We propose here to analyse the KM.19 polymorphic site in the dried blood spots as an additional test in hypertrypsinemic neonates. A blind retrospective study of 114 hypertrypsinemic samples has been performed after polymerase chain reaction. Twenty-seven of 37 CF (74%) were homozygous for allele 2 (2-2) and could have been diagnosed on the 15th day of life. Fifty-five percent of the infants tested were homozygous for allele 1 (1-1), a very rare feature in CF, conferring them a probability of being normal of 99.8%. At the moment, this test could be of great help in the CF screening, even better than the search for the delta F508 mutation for which 45.9% of CF patients are homozygous.

L-Lactate cytochrome c reductase: rapid kinetic studies of electron transfers within the flavocytochrome b2-cytochrome c assembly.

This study is part of a series aimed at the characterization of individual steps of electron transfer taking place between prosthetic flavin, heme b2, heme c within active sites and complexes. After rapid mixing of ferricytochrome c with partially reduced flavocytochrome b2, the reaction is followed at the level of two reactants, cytochrome b2 and cytochrome c. In order to define the proper reactivity of flavosemiquinone, conditions under which this form is highly stabilized (presence of pyruvate) have been chosen. With the help of simulations, it has been possible to characterize a rapid step of electron transfer from cytochrome b2 to cytochrome c within a complex (at approx. 70% saturation) and a slow step k = 5 s-1 assigned to cytochrome b2 reduction by flavosemiquinone within the active site of the pyruvate-liganded enzyme.

Development of cytochrome b558 and oxidative metabolism in human granulocytes, monocytes and during differentiation of HL-60 and U 937 cells.

The development of cytochrome b558 (Cyt b) as determined spectrophotometrically, was investigated in human polymorphonuclear neutrophils (PMN), monocytes (MN) and during differentiation of HL-60 and U 937 cells induced by retinoic acid (RA) alone or in combination with IFN gamma. O2- release in response to a panel of stimulating agents, ie latex particles, opsonised zymosan, PMA, Con A and fMLP, was monitored by lucigenin-amplified chemiluminescence (CL). In parallel the expression of myeloperoxidase (MPO) was investigated and its catalytic activity on H2O2 related to luminol-amplified CL responses. In mature PMN and MN phagocytes, regardless of the stimulating agent, the O2- production is closely related to Cyt b but not to MPO specific contents. In differentiated HL-60 and U 937 cells, the oxidative metabolism increases in parallel with Cyt b specific contents, both being enhanced by the addition of IFN gamma to the RA treatment. However, marked differences in the O2- production intensities are observed depending on the stimulating agent tested and the state of differentiation considered. The PMA-stimulated O2- production is rather low ie 100 and 20 times less in granulocytic HL-60 and monocyto-macrophagic U 937 cells than in PMN and MN respectively. Latex, zymosan and Con A stimulated responses are close to those of MN, in monocyte-macrophagic U 937 cells. In conclusion, these data show that during differentiation; 1), Cyt b plays a critical role in O2- production; 2), the pathways leading to NADPH oxidase activation are diversely modulated following phagocyte differentiation with IFN gamma and/or with RA.