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K Ruckpaul

Publications and source records attributed to K Ruckpaul.

At least 19 recordsLinked to original sources
PubMed1979

Enzymatic activities of matrix-bound components of the liver microsomal cytochrome P-450 system.

Cytochrome P-450 and NADPH-cytochrome P-450 reductase were covalently attached to Sepharose 4B in different ways in order to find out factors which are important for the organization of the individual components to a catalytically active system. Both proteins can be bound individually, simultaneously, and successively to the matrix retaining N-demethylase activity after reconstitution with the complementary essential components. The activity of the system with immobilized components depends on the individual component, the sequence of fixation if both proteins are bound, the degree of purification, and the level of disintegration (detergent-treated samples). The functional importance of the lipid component is beyond doubt, but its specific role needs further investigations. At present it is difficult to differentiate between the influence of chemical modification on the properties of the proteins and the disturbed interactions within the system as the main reason for the decrease in the activity after immobilization. The cluster-like (aggregated) arrangement of the cytochrome P-450 system is necessary for an optimal activity.

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↗
PubMed1979

Comparison of microsomal and solubilized monooxygenases from rat and rabbit by proton magnetic relaxation.

The paper presents results of a comparative study of the haem environment, by proton magnetic relaxation, in P-450 and P-448 monooxygenases from rat and rabbit, induced by phenobarbital and 3-methylcholanthrene, in both species. It was established that the method yields information on the accessibility of the haem iron for solvent molecules (protons), both in microsomes and in solubilized samples of various degrees of purification, i.e. association. The state of micelles in the solutions does not alter the haem iron accessibility. A slight difference was found for the microsomes suspended in a phosphate vs. pyrophosphate buffer, but this is without any consequence with regard to the species and form differences. The correlation time for the highly purified LM2 fraction of rabbit P-450 could not be determined more precisely than before for a sample of lower purity, because the relaxation rates are frequency independent. The correlation time for the rat P-448 monooxygenase was determined by dispersion measurements to be (4.1 +/- 0.4) x 10(-11) s. It was found that the PMRx behaviours of rabbit and rat monooxygenases are more alike in microsomes than in the partially purified solubilized form. The solubilization produces a pronounced alteration of the PMRx temperature dependence only for the rat 3-MC induced monooxygenase P-448. For the P-450 form the haem iron becomes less accessible on solubilization, both for the rabbit and the rat liver monooxygenases, whereas in case of rat liver P-448 the accessibility is considerably enhanced on solubilization. There is a substantial structural specificity of the haem environments from the two animal species, the one from rat being tighter. The reduced, NO-bound rabbit liver monooxygenase was studied also, but the results are not yet conclusive, except the fact that the unpaired spin from NO is thoroughly shielded from the solvent compared with the haem iron from the original sample. The following series of increased haem-iron accessibility emerges from the PMRx studies known so far: rat (P-448) less than rabbit (P-448) less than rat (P-450) less than rabbit (P-450) in microsomes, and rabbit (P-448, with 3-MC bound?) less than Pseudomonas putida (P-450) rat less than (P-448), less than rat (P-450) less than rabbit (P-450) from solubilized samples. For the latter, it appears that increased enzymic specificity goes along with a closing of the haem cleft.

Animals↗
PubMed1979

Stereochemical properties of the binding site of liver microsomal cytochrome P-450 as studied by substrate analogous spin labels.

For the characterization of the substrate binding site optical and EPR measurements with spin labelled substrates on solubilized and pure cytochrome P-450 were performed. Analogously to the unlabelled derivatives spin labelled n-alkylamines and isocyanides with different chain lengths are type II substrates. The Ks-values evaluated from optical (P-450 = 1.98 . 10(-6) M) and ESR (P-450 = 1.98 . 10(-4) M) measurements are very similar indicating no concentration dependences. Contrary to the unlabelled n-alkylamines the spin labelled compounds show an affinity almost independent of the chain lengths. The SL-substrates with a short distance between the functional group and the NO-group bound to P-450 induce pronounced changes of the ligand field of the heme iron and a large broadening of the signal of the immobilized nitroxide indicating intensive interactions between the unpaired electron of the nitroxide group and the paramagnetic heme iron. Elongation of the alkyl chains results in spectra of the Fe3+ complexes with only slight modification and a remained unbroadened signal of the immobilized nitroxide. The binding of the substrate through their functional groups together with a 1:1 stoichiometry of the P-450 SL-IC-complex give evidence for the same binding site in the near vicinity of the heme iron.

Animals↗
PubMed1979

Kinetics of elementary steps in the cytochrome P-450 reaction sequence. III. NADPH reduction of cytochrome P-450LM at different integrational levels.

The aerobic NADPH reduction of cytochrome P-450LM has been investigated on microsomes, as well as on the solubilized enzyme system in the associated, disintegrated, and reconstituted state, respectively. P-450 exhibits biphasic reduction kinetics of about 70/30% phase distribution and rate constants differing 10-fold. The partial reactions are due to organizational asymmetries, the cytochrome being either incorporated into P-450/reductase associates (cluster) or localized outside (randomly distributed, homoassociated, weakly cluster-associated). Triton N-101 disintegrates the different associate structures, consequently followed by the disappearance of the rapid reaction phase. The enzyme system can be reconstituted; at microsomal stoichiometry the respective standard parameters are approached, depending on the composition and structural organization of the phospholipid. The reorganization without any membrane matrix is obviously thermodynamically determined.

Animals↗
PubMed1979

Kinetics of elementary steps in the cytochrome P-450 reaction sequence. IV. Mechanism of the NADPH reduction reaction of cytochrome P-450LM.

The aerobic NADPH reduction of the Triton N-101 disintegrated cytochrome P-450LM system has been studied. At this organization level--the components being dispersed in solution--a first-order monophasic reaction is exhibited. Neither the complex formation of cytochrome and reductase, respectively, nor preceding diffusion is rate limiting. The initial rate follows the ratio reductase/P-450 (mole/mole) thus indicating a Michaelis-Menten type enzyme mechanism. A model treatment of the reaction fits the systems behaviour as a whole. A multiparameter equilibrium state and different specified time function equations were developed for the determination of individual step rate constants and other system parameters as well.

Aerobiosis↗
PubMed1979

NADPH reduction of cytochrome P-450 at different integrational levels of the enzyme system.

The complex monooxygenatic enzyme exhibits different functional behaviour at different integrational levels, thus indicating distinct organizational states. The aerobic NADPH reduction of microsomes, solubilized and reconstituted systems follows a biphasic kinetics, the two phases are attributed to associated state (cluster) and random cytochrome P-450 reduction. States of different cytochrome P-450/reductase ratio (associates) could not be differentiated in rate. Detergents (Triton N-101, cholate) are capable of disintegrating the system, at last only monophasic slow reduction is observed. The hydroxylation activity follows the respective reduction behaviour. Sedimentation analysis proves the distinct structural states. Reconstitution of the system can be achieved by means of detergent dilution as well as by combining the constituents. The activity of the reconstituted system depends on the composition of the phospholipids as well as on its organizational state. The reassociation of the solubilized enzyme system at nearly microsomal components stoichiometry (Triton N-101 dilution) proves to be thermodynamically governed leading to self-organization of the system without matrix prerequisite. Individual step rate constants of the reduction reaction and other system parameters are accessible by means of a model treatment of the disintegrated system. Further application to mixed kinetics systems is in progress.

Aerobiosis↗
PubMed1979

Infrared spectral studies of carbon monoxide complexes of microsomal cytochromes P-450 and P-448.

The characteristic infrared spectral parameters of the carbonyl stretching vibration (the maximum position nu CO, the band width at half height delta nu 1/2 and the apparent molar extinction coefficient epsilon M) have been determined for the CO complexes of microsomal cytochromes P-450 (PB-induced) and P-448 (3-MC-induced) from rabbits and rats. The cytochromes P-450 and P-448 from the same species as well as the cytochromes prepared by the same inducer but from the two different species yielded nu CO band parameters different from each other and from the bacterial cytochrome P-450cam [1]. These differences are discussed in connection with (1) the presence of different protein moities (multiple forms) of cytochromes, (2) changes in the order of the nearest neighbour environment (accessibility) of the CO binding site and (3) the presence of (endogenous bound substrate. The heme-carbonyl of the microsomal cytochromes senses subtle changes in the nearest heme environment by changing the solvent from H2O to D2O.

Animals↗
PubMed1979

Spectral properties of nonequilibrium states in cytochrome P-450 formed by reduction at subzero temperatures.

Nonequilibrium conformational states in cytochrome P-450 in the presence and absence of substrates formed by reduction at subzero temperatures with hydrates electrons were obtained and characterized by their absorption spectra. Different absorption spectra between the relaxed (298 K) and the non-relaxed enzyme forms (77 K) indicate conformational changes proceeding in the relaxed form after reduction of the heme iron which lead to altered interactions between the active centre and its environment in the protein. The two maxima of the nonequilibrium form of cytochrome P-450 without substrate in the visible absorption spectrum (alpha-band, beta-band) and the ratio of their intensities indicate the low-spin character of the heme iron. These spectral properties give evidence for a reduced cytochrome P-450 with two heme-linked axial ligands.

Animals↗
PubMed1978

Quantitative analysis of the spin equilibrium of cytochrome P-450 LM-2 fraction from rabbit liver microsomes.

The LM-2 fraction of cytochrome P-450 from rabbits in the presence and in the absence of substrate (benzphetamine) is shown to be a thermal mixture of a high spin (S = 5/2) and a low spin (S = 1/2) form 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, respectively. These two interacting equilibria are presented in terms of a thermodynamic model, which provides a clear quantitative description of the properties of the cytochrome P-450 substrate system. From the thermodynamic model also the cause of the substrate difference spectra can be explained. The importance of the spin shift in the presence of substrate with respect to the reduction rate is discussed.

Animals↗
PubMed1978

Kinetics of elementary steps in the cytochrome P-450 reaction sequence. II. Temperature dependence and species differences in substrate binding reaction.

Species dependencies and the temperature function of substrate binding reaction have been studied. The solubilized P-450 preparations from rat and rabbit, respectively, exhibit similar substrate binding characteristics with respect to rate constants and substrate specificity. The rabbit P-450 is more sensitive to preparational disintegration, this holds especially for aniline. In the Arrhenius plots a normal temperature dependence without breaks is observed. Iron ligands are bound with relatively low activation energies and negative entropies. The parameters increase for benzphetamine (type 1) and further for aniline (type 2), the latter substrate being entropically favoured too.

Animals↗