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C Balny

Publications and source records attributed to C Balny.

At least 73 records · Page 4Linked to original sources
PubMed1985

Isolation and partial characterization of a rifampicin induced rabbit liver microsomal cytochrome P-450.

Rifampicin administration to New Zealand male rabbits increased the concentration of an LM3 form of cytochrome P-450 to up to 30% of the microsomal P-450 concentration. This enzyme was purified to electrophoretic homogeneity with a yield of 8% of the original total microsomal P-450 concentration. Isolated as a low spin hemoprotein in its substrate free oxidized form, it displays in its reduced CO-complexed form an absorption maximum at 449 nm. Immunological assays, as well as activity measurements, in particular its stereospecific progesterone hydroxylation in the 6 beta-position, show a relationship between LM3,Rif and LM3c (from untreated rabbits).

Animals↗
PubMed1985

Cryobaroenzymic studies as a tool for investigating activated complexes: creatine kinase.ADP.Mg.nitrate.creatine as a model.

By combining cryoenzymology with baroenzymology (a technique we term "cryobaroenzymology") one can obtain "stop-action" pictures of the intermediates in an enzyme reaction pathway and then observe their structural and energetic features ("motion features"). We illustrate the potential of this approach by considering the formation of a transient state analogue complex of creatine kinase (ATP:creatine N-phosphotransferase, EC 2.7.3.2): enzyme.ADP.nitrate.creatine, where nitrate mimics the transferable gamma-phosphate of ATP. Formation of the analogue complex is accompanied by a conformational change that manifests itself by tryptophan perturbation and thus allows kinetic studies by the stopped-flow method. We studied the formation of the analogue complex under cryoenzymic conditions as a function of pressure and solvent composition. This allowed a detailed description of the structural and energetic features of the activation process of an elementary step in an enzyme pathway.

Adenosine Diphosphate↗
PubMed1984

Inductive and repressive effects of rifampicin on rabbit liver microsomal cytochrome P-450.

New Zealand White rabbits were treated with rifampicin at a dose of 50 mg/kg for 4 days. The total amount of microsomal hepatic cytochrome P-450 was not modified in treated, with respect to control, animals. However, further studies involving SDS-PAGE analysis, monooxygenase activity measurements and radial immunodiffusion assays indicated that rifampicin strongly affects the level of two P-450 isoenzymes. An LM3 form was induced; this form, apparently associated with erythromycine demethylase activity and hydroxylation of progesterone preferentially in position 6 beta, was shown to be immunologically and functionally different from LM3a and LM3b. On the other hand, an LM4 form, typically induced by beta-naphthoflavone, was repressed. The concomitant inductive/repressive effect of rifampicine on two cytochrome P-450 isoenzymes makes this drug a very atypical inducer, at least in the rabbit.

Animals↗
PubMed1984

Kinetics of reduction by substrate or dithionite and heme-heme electron transfer in the multiheme hydroxylamine oxidoreductase.

Hydroxylamine oxidoreductase of Nitrosomonas catalyzes the dehydrogenation of NH2OH. It contains hemes c553, c559 and P460 in the ratio 5:2:1. At equilibrium four or five c hemes are reduced by NH2OH or NH2NH2, respectively. Heme P460 is the site of electron entry into the enzyme; electrons exit via P460 to O2 or H2O2 with rate constants of 30s-1. We report that hydroxylamine oxidoreductase has two categories of electron-accepting sites: (a) heme P460, an H2O2-sensitive site, which is reactive with NH2OH (2.2 hemes c557 and 2 hemes c559 are reduced) or NH2NH2 (3.3 heme c 553 and 2 heme c559 are reduced) and (b) an H2O2-insensitive site(s) which is reactive with H2O2 (approximately 0.15 heme c553 is reduced); hydroquinone, pyrogallol, N-methyl hydroxylamine, pyocyanine, and ascorbate (approximately 0.8 heme c553 is reduced); or Na2S2O4 or EDTA-photoreduction with proflavin, deazalumiflavin or acridine orange and methylviologen (all hemes are reduced). The rate constants at 19 degrees C for reduction by dithionite were: 0.7 heme c553 (7s-1), 4.3 hemes c553 (0.07 s-1), 0.7 heme c559 (0.8s-1), 1.3 hemes c559 (0.1s-1), P460 (0.013s-1). At 2 degrees C the rate constant for 0.8 heme c559 was 1.7s-1. The data indicate that one heme c552 is reduced by dithionite at the same rate as mammalian cytochrome c; other hemes are reduced much more slowly and are possibly inaccessible to the solvent. The rate constants at 2 degrees C for reduction by NH2OH were: 1.8 hemes c553 (30s-1), 0.2 heme c553 (2.4s-1), 1.7 hemes c559 (19s-1), 0.3 heme c559 (1.4s-1). For reduction by NH2NH2 the values were: 2.6 hemes c553 (23s-1), 0.7 heme c553 (1.6s-1), 1.3 hemes c559 (22s-1), 0.7 heme c559 (4.2s-1). Thus reduction by NH2OH at the substrate site was at least an order of magnitude faster than reduction of hydroxylamine oxidoreductase heme by Na2S2O4. Comparison of rates of heme-heme electron transfer on the enzyme during reoxidation by O2 or H2O2, reduction by Na2S2O4 and reduction by NH2OH or NH2NH2 indicates that the enzyme can exist in distinct states which result in different rates of heme-heme electron transfer. Comparison of the rate of substrate reduction of c hemes of hydroxylamine oxidoreductase (HAO) with the turnover of the enzyme in vivo is consistent with the electron path NH2OH----HAO P460----HAO c hemes----biological electron acceptor.

Anaerobiosis↗
PubMed1984

High-pressure stopped-flow spectrometry at low temperatures.

A stopped-flow instrument operating over temperature and pressure ranges of +30 to -20 degrees C and 10(-3) to 2 kbar , respectively, is described. The system has been designed so that it can be easily interfaced with many commercially available spectrophotometers of fast response time, with the aid of quartz fiber optics. The materials used for the construction are inert, metal free and the apparatus has proven to be leak free at temperatures as low as -20 degrees C under a pressure of 2 kbar . The performance of the instrument was tested by measuring the rate of reduction of cytochrome c with sodium dithionite and the 2,6-dichloroindophenol/ascorbate reaction. The dead time of the system has been evaluated to be 20, 50, and congruent to 100 ms in water at 20 degrees C, in 40% ethylene glycol/water, and at 20 degrees C and -15 degrees C, respectively. These values are rather pressure independent up to 2 kbar . Application of the bomb was demonstrated using the cytochrome c peroxidase/ethyl peroxide reaction. This process occurred in two phases and an increase in pressure decreased the rates of reactions indicating two positive volumes of activation (delta V not equal to app (fast) = 9.2 +/- 1.5 ml X mol-1; delta V not equal to app (slow) = 14 +/- 1.5 ml X mol-1, temperature 2 degrees C). The data suggest that the fast reaction could involve a hydrophobic bond, whereas the slow process could be associated with a stereochemical change of the protein. The problem of temperature equilibrium for high-pressure experiments is also discussed.

2,6-Dichloroindophenol↗
PubMed1984

Kinetics of electron transfer between cardiac cytochromes c1 and c.

Highly purified cytochrome c1, which consists of only one heme peptide and does not form a stable c1-c complex (c1-H-c complex), was used in studies of electron transfer between cytochrome c1 and c. Results show that a stable and ionic-strength-sensitive c1-c complex (i.e., the c1-H-c complex) in the forms of the various oxidation states is not required, in contrast to the current belief of the participation of the complex in the electron transfer between cytochromes c1 and c. A minimum mechanism for electron transfer between these two cytochromes is suggested in accord with the experimental results.

Animals↗
PubMed1983

Heme P460 of hydroxylamine oxidoreductase of Nitrosomonas. Reaction with CO and H2O2.

Hydroxylamine oxidoreductase (HAO) of Nitrosomonas catalyzes the dehydrogenation of NH2OH and subsequent addition of oxygen to form nitrite. HAO contains c hemes and the CO-binding heme P460 in a 7:1 ratio; dehydrogenation of NH2OH involves passage of electrons to P460 and then c hemes. We now report that electrons rapidly pass from c hemes of HAO to the P460 center and then to H2O2. This conclusion is supported by (a) inhibition of c heme oxidation with CO and (b) loss of H2O2-oxidizability of ferrous c hemes following specific destruction of heme P460. Reaction of ferrous P460 with H2O2 is rate-limiting. Activation of dioxygen for N-oxidation by ferrous HAO may involve the two-electron reduction of O2 by P460. The reaction of ferrous HAO with H2O2 was studied as it may reveal aspects of the mechanism of activation of dioxygen. Reaction of ferrous heme P460 with CO is slow and with low affinity as compared with other hemoproteins. Values for reaction of CO with enzyme were: k1, 1.1 X 10(-3) M-1 s-1 and Kd, 12 microM.

Carbon Monoxide↗
PubMed1981

Direct evidence for electron transfer from ferrous cytochrome b5 to the oxyferrous intermediate of liver microsomal cytochrome P-450 LM2.

Interaction and electron transfer between highly purified microsomal cytochrome P-450 from phenobarbital-induced rabbits and cytochrome b5 from uninduced rabbits was investigated by difference and stopped-flow spectrophotometry. Formation of a 1:1 complex between ferric P-450 and b5, demonstrated by difference spectrophotometry, was observed only when both cytochromes were incorporated into micelles of phosphatidylcholine. The dissociation constant (Kd) of the complex was decreased from 2.3 microM to 0.4 microM in the presence of 1 mM benzphetamine. The apparent Kd of benzphetamine was reduced from 220 microM to 50 microM upon addition of b5. The influence of ferrous b5 on the autooxidation of the oxyferrous intermediate of P-450 in the presence and in the absence of substrate was investigated by stopped-flow spectrophotometry. Both cytochromes were reduced photochemically, so that experiments could be carried out in the absence of the corresponding reductases and reduced pyridine nucleotides. Kinetic analysis of the data showed that formation of a 1:1 complex between ferrous P-450 and b5 was a prerequisite for electron transfer between the cytochromes. Here again, incorporation of both cytochromes into micelles was absolutely required for this process. Kd was decreased from 7.5 microM to 2.2 microM in the presence of 1 mM benzphetamine. The rate of electron transfer from b5 to oxyferrous P-450, derived from the kinetics of reoxidation of either b5 or P-450, was increased from 2.5 s-1 to about 4 to 7 s-1 in the presence of 1 mM benzphetamine. These results provide the first quantitative data on the electron transfer between the b5 and P-450, whose rate constant is compatible with the observations made on the effect of b5 on hydroxylation reactions catalyzed by the P-450 enzyme system.

Animals↗
PubMed1981

Lack of evidence for a tetrahedral intermediate in the hydrolysis of nitroanilide substrates by serine proteinases. Subzero-temperature stopped-flow experiments.

We have used a stopped-flow apparatus to reinvestigate reports, based on the observation of "burst" kinetics, of an intermediate prior to the acyl-enzyme complex in hydrolysis reactions of anilides catalyzed by trypsin and elastase [M. W. Hunkapiller, M. D. Forgac and J. H. Richards (1976) Biochemistry 15, 5581-5588; D. D. Petkov (1978) Biochim. Biophys. Acta, 523, 538-541; A. L. Fink and P. Meehan (1979) Proc. Natl Acad. Sci. USA, 76, 1566-1569; P. Compton and A. L. Fink (1980) Biochem. Biophys. Res. Commun. 93, 427-431]. We studied the hydrolysis of several anilide substrates by bovine and porcine trypsin and porcine elastase between -30 degrees C and 20 degrees C. In no case did we record true "burst" kinetics. We show that confusion spectral changes can arise from incomplete mixing, thermal gradients, or heterogeneity of the substrate. We conclude that there is no solid spectroscopic evidence at present for the existence of a tetrahedral intermediate in the hydrolysis of amides by serine proteinases. The substrate N-acetyl-L-alanyl-L-prolyl-L-alanine 4-nitroanilide is a mixture of two isomers trans and cis about the L-alanyl-L-propyl peptide bond. It appears that elastase hydrolysis the cis isomer more rapidly than the trans isomer and this could lead to false "burst" kinetics. We describe the construction of the stopped-flow apparatus designed for cryoenzymology used for this work that has novel features and is adaptable to a variety of spectrophotometers. Solutions can be handled under anaerobic conditions. A window allows the drive syringes to be observed or exposed to light for photochemical experiments. The apparatus operates over the temperature range -35 degrees C to + 25 degrees C. The dead time is under 5 ms. A recording system is described that permits one to follow reactions over a wide time scale covering half-time of the order of several milliseconds to hours.

Anilides↗
PubMed1979

Affinity chromatography at sub-zero temperatures. A model study with porcine pancreatic elastase.

A new variety of affinity chromatography of enzymes is described which consists of building up an affinity adsorbent composed of a real substrate. The chromatography is performed at a sub-zero temperature where the turnover of the enzyme is very low or stopped. As a model system Sepharose-bound L-trialanine p-nitroanilide was for used the affinity binding of porcine pancreatic elastase, which was adsorbed to the column in a hypersaline medium at--14 degrees and eluted from the column at the same temperature using 50% (v/v) ethylene glycol. The affinity adsorbent proved to be vary specific as it did not retain trypsin, chymotrypsin and ovalbumin and retained only 20% of cytochrome c.

Animals↗
PubMed1979

Fast photochemical reactions of cytochrome P450 at subzero temperatures.

Several reactions of the cytochrome P450 multi-step cycle have been studied by fast light activation combined with subzero temperatures. A flash device was adapted to an Aminco-Chance DW 2 spectrophotometer equipped for subzero temperature thermostatisation. The first electron can be introduced into the cycle by non specific reducing agents such as reduced flavin mononucleotide (FMNH2) or methylviologen radical (MV.). This first reduction remains a fast process even at subzero temperatures. The oxy-compound Fe2+-O2 can thus be formed either directly from Fe2+ or via the photodissociation of the carboxy-ferro adduct. Fe2+-O2 is stable at subzero temperatures towards spontaneous autoxidation as well as further reduction by FMNH2 or MW.. In addition, the recombination of CO after flash photodissociation of Fe2+-CO was used to study in more details the specific behaviors of the purified microsomal cytochrome.

Bacterial Proteins↗