Biomedical subjects

O Ristau

Publications and source records attributed to O Ristau.

At least 37 records · Page 2Linked to original sources

PubMed1991

Cytochrome P-450 spin state and leakiness of the monooxygenase pathway.

1. The monooxygenase and oxidase activities of liver microsomes from phenobarbital (PB)-treated rabbits were investigated for their dependence on the high spin shift (delta alpha) of the ferric cytochrome P-450 induced by a series of benzphetamine analogues. 2. The spin shift activity of the substrate determines, via the first electron transfer kinetics, the steady-state level of the reaction intermediate oxycytochrome P-450. Correlation of the amount or oxycytochrome P-450 with delta alpha can be experimentally proved. 3. The spin-state-dependent formation of oxycytochrome P-450 regulates quantitatively the rates of NADPH oxidation and substrate N-demethylation. Both activities correlate with delta alpha. Oxycytochrome P-450 is substrate-stabilized towards decay with the formation of O2- which, upon dismutation, gives rise to H2O2. 4. The ratio of N-demethylase to NADPH oxidase activity (coupling ratio) also increases with the spin shift, delta alpha. Concomitantly, the proportion of NADPH accounted for by H2O2 and H2O formation via two- and four-electron reduction of dioxygen decreases. This indicates that the substrate-induced structural changes in the enzyme active centre which give rise to spin transition may likewise modify the coupling properties. 5. Perfluorinated compounds, which fail to undergo monooxygenation, fall in line with the benzphetamine derivatives with respect to the dependence of NADPH oxidation rate and steady-state oxycytochrome P-450 level on delta alpha. The increased oxidase activity results mostly in H2O formation. 6. The leakiness of the PB-induced monooxygenase pathway in the biotransformation of oxygen in the presence of the benzphetamines and perfluorinated compounds does not result in marked increases in H2O2 formation. Therefore, the increase of NADPH oxidase activity by these substrates does not significantly enhance H2O2-mediated oxygen tissue toxicity.

Animals↗

PubMed1989

Demethylation of tertiary amines by a reconstituted cytochrome P-450 enzyme system: kinetics of oxygen consumption and hydrogen peroxide formation.

Initial reaction rates of oxygen consumption and hydrogen peroxide formation in a cytochrome P-450 catalyzed reaction are practically independent of the nature of tertiary amines that were used as substrates. From the kinetic studies and the substrate conversion results that the amount of water formed in a side reaction is determined by the substrate specificity. Both hydrogen peroxide and water formation lower the efficiency of the monooxygenatic activity of cytochrome P-450.

Amines↗

PubMed1988

Quantitation of interaction between cytochrome P-450scc and adrenodoxin--analysis in the median UV-region by second derivative spectroscopy.

Interaction between the essential protein components of the bovine adrenal mitochondrial enzyme system (cytochrome P-450scc, adrenodoxin and adrenodoxin reductase) were studied in the median UV-region utilizing second derivative difference spectroscopy. Complex formation of cytochrome P-450scc with adrenodoxin induces a signal in the second derivative difference spectrum which can be attributed to tyrosine due to its minimum at 283 nm. Based on this signal cytochrome P-450scc was titrated with adrenodoxin in dependence on different effectors (reductase, phospholipid, cholesterol). The dissociation constants (Kd) of the P-450scc/adrenodoxin complexes derived therefrom revealed an increasing affinity between both components starting from titrations in buffer solution without additional components up to the completely reconstituted system. A high affinity between P-450scc and adrenodoxin corresponds to a high turnover rate of cholesterol. Dissociation constants of the P-450scc/adrenodoxin complex were also derived from spectral changes in the Soret region. But these data do not correlate with the substrate turnover.

Adrenodoxin↗

PubMed1987

Chemical modification of cytochrome P-450 LM4. Identification of functionally linked tyrosine residues.

Cytochrome P-450 LM4 (RH, reduced flavoprotein:oxygen oxidoreductase (RH-hydroxylating), EC 1.14.14.1) from rabbit liver microsomes was chemically modified with tetranitromethane. Nitration of two tyrosine residues inhibits the p-nitrophenetole O-deethylase activity of the enzyme by about 80%. Sequencing the 3-nitrotyrosine-containing peptides after HPLC tryptic peptide mapping reveals that mainly Tyr-243 and Tyr-271 are nitrated, whereas Tyr-71, Tyr-188 and Tyr-365 are modified to a lower extent. Nitration of tyrosine residues affects the complex formation with p-nitrophenetole, alpha-naphthoflavone and metyrapone as indicated by an increased affinity towards p-nitrophenetole and by a decreased affinity for the latter compounds. Furthermore, nitration interferes with the electron transfer from NADPH-cytochrome P-450-reductase to cytochrome P-450 LM4 resulting in a slowed down reduction reaction. The results suggest that Tyr-243 and Tyr-271 of cytochrome P-450 LM4 are functionally involved in the interaction with NADPH-cytochrome P-450 reductase.

Amino Acid Sequence↗

PubMed1985

Spin state control of cytochrome P-450 reduction and catalytic activity in a reconstituted P-450 LM2 system as induced by a series of benzphetamine analogues.

Reconstituted liposomal cytochrome P-450 LM2 was reacted with a series of benzphetamine analogues as substrates. Based on the thermodynamical model of Ristau et al. (Biochim. Biophys. Acta, 536 (1978) 226-234) the free enthalpy of substrate binding to the high spin form of the enzyme was shown to correlate with the total high spin content of the respective enzyme substrate complex. Reduction and substrate N-demethylation rates as well have been evidenced to linearly correlate with the substrate-induced spin shift delta alpha and moreover with the spin content alpha. The data obtained provide further experimental support for the spin state regulation of the reduction and conversion rate of cytochrome P-450 LM2.

Animals↗

PubMed1985

Chemical modification of cytochrome P-450 LM2 with N-acetylimidazole. Evidence for the functional involvement of tyrosyl residues.

Cytochrome P-450 LM2 has been reacted with N-acetylimidazole. About three tyrosyl residues of cytochrome P-450 LM2 are accessible to O-acetylation. The analysis of the spectral dissociation constants, substrate binding kinetics, reduction kinetics, N-demethylase activity and substrate conversion by artificial oxygen-donating agents of differently acetylated enzyme provides evidence for the existence of two groups of accessible tyrosines. One tyrosyl residue is located in the immediate environment of the heme iron and is involved in the binding of type II substrates. This tyrosine is not necessary for N-demethylation. Acetylation of two further tyrosyl residues, however, causes an almost complete inhibition of enzymatic activity. The results strongly suggest tyrosine(s) to be involved in NADPH-cytochrome P-450 reductase dependent N-demethylation.

Acetylation↗

PubMed1985

[Activity of urease in microcapsules].

The activity of urease in microcapsules formed by a symplex membrane from cellulose sulphate and polydimethyldiallyl ammonium chloride is studied and compared with that of free urease in solution. Both free and encapsulated urease exhibits a similar pH optimum. The interaction of urease with cellulose sulphate causes a decrease of the activity down to 35 to 38%. In spite of its lower absolute activity the encapsulated urease shows a higher long time stability than the free urease. The presented immobilization method for urease could be of importance for an active detoxification in uremia.

Capsules↗

PubMed1984

Mechanism of rate control of the NADPH-dependent reduction of cytochrome P-450 by lipids in reconstituted phospholipid vesicles.

The NADPH-supported reduction of cytochrome P-450 LM2 (liver microsomal isozyme 2) in reconstituted phospholipid vesicles in general exhibits two-exponential kinetics. The physiologically relevant rapid partial reaction is favoured in amount with increasing reductase/P-450 ratio. A lipid specificity was observed in that negatively charged lipids favour that process, too. The rate constant increases concomitantly. The data are consistent with the formation of a reactive 1:1 complex the amount of which determines the rate constant. The dissociation constants amount to 0.048 microM for a microsomal lipid extract, 0.051 microM for a 3:1 (w/w) mixture of dioleoylglycerophosphoethanolamine and phosphatidylserine, and 0.47 microM for dioleoylglycerophosphocholine, respectively, in the respective reconstituted systems. At low reductase/P-450 ratio the amount of the rapidly reduced P-450 exceeds the equilibrium concentration of a 1:1 complex. Preformed 1:1 associates, therefore, cannot fit the derived mechanism. Instead, a cluster model based on P-450 association does correspond to the data.

Cytochrome P-450 Enzyme System↗

PubMed1983

Correlations between spin equilibrium shift, reduction rate, and N-demethylation activity in liver microsomal cytochrome P-450 and a series of benzphetamine analogues as substrates.

Cytochrome P-450 forms a thermal ferric spin equilibrium which is significantly shifted by substrate binding. Within a series of benzphetamine analogues the liver microsomal enzyme system exhibits a close correlation of the substrate induced spin equilibrium shift towards the high spin state and both the rate of P-450 reduction, and of substrate turnover, as well. The spin equilibrium regulates the first electron transfer by favoured high spin state reduction and rapid pre-equilibration with respect to the low spin fraction.

Animals↗

PubMed1983

[Immobilization of proteins and cell fragments using a new method of microencapsulation].

Cytochrome c, hemoglobin, urease and liver microsomes were microencapsulated in aqueous solution by use of a new method. The membrane of the microcapsules consist of a symplex, which is formed predominantly by electrostatic interactions between a polymeric polyanion and a polymeric polycation. The membrane is characterized by high mechanical stability and is a barrier for globular substances with molecular weights larger than 12000, but permeable for substances with lower molecular weight. The microcapsules contain the protein or microsomes and additionally the polymeric polyanion in the liquid interior. Structure and function of microencapsulated proteins and microsomes are preserved. The developed method of microencapsulation opens new possibilities for the application of biomacromolecules, particularly for extracorporal detoxification.

Animals↗

PubMed1983

Magnetic properties of ferric hemoproteins and the relation to electronic structure of the prosthetic group.

Recently, it has been shown that the magnetic data of bacterial cytochromes c' and of some model complexes are consistent with a quantum mechanically mixed spin state. This new mixed spin state is to distinguish from thermally mixed pure high-spin and low-spin states. Based on ligand field model, the dependence of the magnetic state of a heme complex on the nature of the axial ligands is discussed.

Chemical Phenomena↗

PubMed1983

Theory of the magnetic properties of ferric hemoproteins: development of a 4-term ligand field model.

A ligand field 4-term model is used to calculate the g-values and the magnetic moments of ferric hemoproteins. The 4-term model allows the common description of the magnetic properties of high spin and low spin hemoproteins with the same approach. The model includes the four energetic lowest cubic terms (6A1, 2T2, 4T1, 4T2) which are mixed by ligand field components of axial and rhombic symmetry and by spin-orbit coupling. Especially the g-values of the high spin cytochrome P-450 LM4 and of the low spin cytochrome P-450 LM2 were simulated and compared with the results of a 3-term model (without 4T2) earlier developed by Ristau [5, 6]. The multiterm model is also suitable to calculate the magnetic properties of spin-mixed ferric hemoproteins with ligand field strengths in the crossover region of ground states.

Animals↗

PubMed1982

Molecular mechanisms of interactions between phospholipids and liver microsomal cytochrome P-450 LM2.

The interactions between cytochrome P-450 LM2, NADPH-dependent P-450 reductase and different phospholipids have been analysed in reconstituted systems utilizing spin equilibrium and the reduction reaction as sensitive probes. The results have been correlated with structural data derived from second derivative spectroscopic measurements. The data obtained indicate that phospholipids with acidic groups are of special importance in shifting the spin equilibrium to the high spin state. The detergent substitution of phospholipids, on the other hand, in the reduction reaction provides evidence that the capability to shift the spin state is not strictly correlated with the functional integrity of the system. The results imply that the phospholipids exert a linker function orienting the proteins within the bilayer into a proper interaction state. This function can be substituted by detergents. The phospholipid effect is specified by their head groups and increases with negative charges.

Animals↗

PubMed1980

[Calculation of the g-factors of hemoproteins with the crystal field theory. Development of a 3-term model].

A 3-term crystal field model is used for the calculation of the magnetic properties of ferric hemoproteins. This model includes the high spin term (6A1), the low spin term (2T2) and the quartet term (4T1). The pertubation matrix between these 3 terms caused by spin orbit coupling and ligand fields of cubic, axial and rhombic symmetry are evaluated. The g-factor of the low spin cytochrome P-450 LM2 from rabbit liver are calculated using this 3-term model and the results are compared with those from the usual 1-term model. Contrary to the 1-term model, no orbital moment correction factor is necessary. That means that the orbital moment correction factor with value > 1 simulates some term interaction, predominantly with the quartet term. The g-factors of the high spin cytochrome P-450 LM4 were also calculated by means of the 3-term model and compared with a spin hamilton treatment. A good agreement between the spin hamilton parameters derived from the experimental g-factors and those calculated by the 3-term model were obtained. For hemoproteins with strongly mixed spin states the 3-term model is also useful, contrary to 1-term models or spin hamiltonians which are completely incorrect. The 3-term model is a minimal model to describe all typical magnetic properties of low spin and high spin ferric hemoproteins with the smallest number of terms.

Animals↗

PubMed1980

[The interaction of methemoglobin and modified poly-(hydroxyethylmethacrylates) studied by spectroscopic methods].

The interaction between methemoglobin (MetHb) and macroporous matrices on the basis of polymethacrylates was investigated by means of optical and e.p.r. spectroscopy. The spectroscopic data show that the adsorption of MetHb to imidazole-containing matrices occurs by complex formation between matrix-bound imidazole and the iron of the prosthetic group, with all 4 polypeptide chains of the MetHb molecule being included in the interaction. The adsorption to hydrophobic side chains containing matrices leads, via the protein-matrix interaction, to considerable disturbances of iron protoporphyrin IX in equilibrium or formed from protein-contacts, which are of general importance with respect to the functional variablity and control, respectively, of iron porphyrins in hemoproteins. In case of matrix containing n-hexyl groups deoxyHb is oxidized by O2 to MetHb, instead of being oxygenated to HbO2. Not all prosthetic groups are able to bind N-3. With the increase in hydrophobicity of the matrix a conformational change is enforced leading in the beta-chains to the direct interaction between iron and sulfur of cysteine (beta-cys 92), as it is proved in all cytochrome P-450 and other model compounds.

Animals↗

PubMed1979

Quantitative analysis of the spin equilibrium of cytochrome P-450 LM2 fraction from rabbit liver microsomes.

The LM2 fraction of cytochrome P-450 from phenobarbital induced rabbit liver microsomes in the presence and in the absence of substrate (benzphetamine) is shown to be a thermal equilibrium of a high spin (S = 5/2) and a low spin (S = 1/2) state each of which exhibiting its individual optical basic spectrum with the Soret maxima at 387 nm and 417 nm for the high spin form and the low spin form, respectively. The equilibrium constants and thermodynamic parameters describing the spin transition and the substrate binding have been evaluated from the temperature and substrate difference spectra. These two interacting equilibria are presented in terms of a thermodynamic model, which provides a quantitative description of the relationship between the substrate binding and the spin equilibrium. From kinetic experiments it is concluded that the spin equilibrium is an electronic one and is not caused by iron ligand dissociation.

Animals↗

PubMed1979

The importance of the spin equilibrium in cytochrome P-450 for the reduction rate of the heme iron.

Based on the existence of a spin equilibrium of P-450 LM a thermodynamic model including the first two elementary steps has been developed. This model has been compared with a modified model of P-450 CAM first presented by SLIGAR [1]. For the quantitative description of the model of P-450 LM seven necessary constants have been calculated. In the model a 5-coordinated ferric heme complex as intermediate and precursor of the reduced state is included. The model is based on experimental data of the spin equilibrium and the binding of benzphetamine to P-450 LM2, on the redox potential of P-450 LM and further on the assumption of a structure of the precursor well adapted to reduction. The dissociation constant (KD) of the 6th heme iron ligand is calculated to be 0.03 meaning that only 3% of the high spin ferric state are 5-coordinated. From the thermodynamic parameters of the model the sequential order of the reaction process can be derived and conclusions can be drawn on possibly reaction controlling steps. Experimentally it is shown that the substrate induced spin shift is correlated with the reduction rate. This has been shown to be valid in dependence on the degree of saturation with only one substrate as well as with different substrates indicating that independent of the individual substrate only the shift of the spin state controls the reduction. From the experiments as well as from the model it can be concluded that P-450 works in the first reaction step in two states (different in structural and functional properties) being in equilibrium with each other. Different affinities of these two states towards the substrate shift the equilibrium. This shift increases the concentration of the reducible intermediate and by this the reduction as second elementary step is regulated.

Animals↗

PubMed1979

Comparison of spectral properties of 3-MC induced cytochrome P-448 from rabbits and rats.

EPR and optical spectral properties of cytochrome P-488 from 3-methylcholanthrene-induced rabbits are compared with those of rats. In the EPR spectra at 77K and in the optical absorption spectra at room temperature a considerable temperature independent high spin content of the rabbit cytochrome is observed which has been estimated to about 55% by titration with n-octylamine. On the other hand the high spin content of the rat cytochrome depends strongly on temperature and amounts to about 6% at 5 degrees C and to about 35% at 34 degrees C. The binding of substrates and ligands to the rabbit cytochrome as well as its reduction by sodium dithionite are slower as compared with the rat cytochrome but also with phenobarbital-induced cytochrome P-450 from rats and rabbits. Contrary to the 3-methylcholanthrene induced cytochrome P-448 from rats, that from rabbits does not bind 3-methylcholanthrene. A particular protein structure establishing the high spin state and an absent binding site for type I substrates is assumed to be responsible for these and other peculiarities of cytochrome P-448 from 3-methylcholanthrene-induced rabbits.

Animals↗