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K Ruckpaul

Publications and source records attributed to K Ruckpaul.

At least 91 records · Page 5Linked to original sources
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↗
PubMed1977

Substrate binding to solubilized cytochrome P-450 from rabbits at different temperatures.

The binding affinities of selected type I- and type II-substrates to partially purified cytochrome p-450 from rabbit liver microsomes were studied and found to differ from those of rats. The temperature dependence of the apparent binding constants qualitatively exhibited the same characteristics compared with that of rats. For type I-substrates endothermic and for type II-substrates exothermic reaction characteristics were observed. Taking into account the partition coefficients of the substrates so far investigated it is obvious that type I substrates with increasing hydrophobicity are bound more strongly while type II-substrates show a more complicated behvaiour. This may due to the fact that other types of binding are included besides the hydrophobic interactions.

Animals↗
PubMed1977

[Interactions between the components of the cytochrome P-450-monoxygenase system from liver microsomes. III. N-demethylase activity of cytochrome P-450 relative to the degree of its integration].

Preceding experiments on the immobilization of the multi-component P-450 (LM) system, in which hte order of the first-immobilized component had been varied, gave evidence that the enzymatic activity depends on the degree of association. The present paper deals with the relationship between the degree of association and the demethylase activity. With rising concentrations of the detergent (Triton N-101) up to 0.2%, ultracentrifugation experiments have shown a steady decrease of the sedimentation coefficient from 20-22 down to 4.5 [S]. The demethylase activity (substrate benzphetamine) of the immobilized P-450 declines on reconstitution with solubilized reductase with raising degree of monomerization: 61% (0.02% Triton N-101) and 23% (0.2% Triton N-101) as compared with untreated fixed P-450 equals 100%. The demethylase activity can be increased by raising the reductase concentration in the test preparation.

Animals↗
PubMed1976

Haem accessibility in cytochrome P-450 from rabbit liver. A proton magnetic relaxation study by stereochemical probes.

Cytochrome P-450 was solubilized from phenobarbital induced rabbit liver and purified by affinity chromatography. The longitudinal proton magnetic relaxation rates of this ferric, low-spin sample (as confirmed by ESR) in 20% glycerol aqueous solution are very large compared with low-spin methaemoglobin and myoglobin derivatives. Similarly high rates were measured in a deuterated solution using the aliphatic protons of glycerol as stereochemical markers, which strongly suggests that the haem iron in cytochrome P-450 is much more accessible to the solvent than in harmoglobin or myoglobin. Type I substate (Spasman) produced small but significant increases in NMR rates both in the H2O and in the 2H2O solution, while binding of aniline (Type II substrate) doubled the rates.

Binding Sites↗
PubMed1976

Circular dichroism of partially purified cytochrome P-450 from rabbit liver microsomes.

The heme related circular dichroic bands of solubilized cytochrome P-450 from rabbit liver and some of its complexes with type I- and type II-substrayes and heme iron ligands were measured in the Soret region. All P-450 dersorption. The positions of the dichroic bands together with their ellipticities vary with ligand substitution and the oxidative state of the iron. The results are compared with CD-date of other hemoproteins and discussed with respect to stereochemical properties of the heme pocket.

Animals↗
PubMed1976

Kinetics of elementary steps in the cytochrome P-450 reaction sequence. I. Substrate binding to cytochrome P-450 LM.

The substrate binding step in the reaction sequence of the cytochrome P-450 enzyme system (rat liver microsomes) has been investigated. The type I/II substrate classification kinetically holds too. The rate constants are in the 10(3) to 10(5) (M-1 sec-1) range, the type I compounds are preferably bound by about one order of magnitude. The rate constants of the binding process to the reduced cytochrome are considerably decreased. The results favour the ordered reaction mechanism.

Animals↗
PubMed1976

Differentiation between type I and type II substrate binding to cytochrome P-450 by variation of temperature.

The binding affinities of type I- and type II-substrates to cytochrome P-450 solubilized from phenobarbital induced rat liver microsomes in dependence on the temperature have been determined. Both classes of substrates have been found to exhibit different temperature behaviour. The tendency of type I-substrates (benzphetamine and hexobarbital) to form complexes increases with increasing temperature; type II-substrates show the inverse tendency. From the van't Hoff plot the binding enthalpy was calculated and discussed in connection with the entropy and free enthalpy values. These data ascertain the suggestion of different binding sites for both classes of substrates.

Aniline Compounds↗
PubMed1976

An infrared study of the carbon monoxide complexes of cytochrome P-450 and cytochrome P-420.

The infrared stretch vibrations (upsilonCO) of the CO-complexes of cytochrome P-450 and cytochrome P-420 have been determined from infrared difference spectra. The CO-complexes exhibit IR-bands at 1949 cm-1 and 1966 cm-1 with half widths of approximately 17 cm-1 and approximately 20 cm-1 respectively. These results are compared with the CO-stretch frequencies of other haemoproteins and discussed with respect to specific interactions of the CO-ligand with the protein moiety and to the ligand trans to CO of the cytochromes.

Binding Sites↗
PubMed1975

Evidence for the existence of a low spin complex in acidic methemoglobin: its structure and formation.

By means of electron spin resonance and magneto-optical rotation, specific low spin complexes in acidic methemoglobin are obtained. The formation of these complexes is explained by a specific stereochemical arrangement of the distal histidine in the absence of allosteric effectors inducing the formation of a low spin ligand at room temperature. At low temperature, however, the distal histidine is directly bound to the heme iron. As the formation of the low spin complexes depends on allosteric effectors it is suggested that via the distal histidine the affinity of heme iron ligands is modified.

Allosteric Site↗
PubMed1975

[Studies on the magneto-optical rotation of porphyrins, hemins and methemoglobin compounds].

Using the method of magneto-optical rotation (MOR) various porphyrin derivatives, hemin and heme compounds, and a number of methemoglobin complexes were investigated. The spectra were recorded from 450-600 nm; with methemoglobin also in the Soret region. 1. The metalfree porphyrin derivatives (deutero-, meso-, hemato- and protoporphyrins) were measured in strongly acidic aqueous solution. The derivatives thus present as di-cations yield highly resolved MORspectra, where the Q-bands (Oo leads to; Oo leads to 1) originated from the pi-pi transitions of the porphyrin display the curve shape characteristic of an A-term, this proving the presence of the D4h symmetry. An exception is the protoporphyrin, in which the pi-electron system of the porphyrin is perturbed by the influence of pi-electrons of the vinyl group, causing poor resolution, line broadening, and shift of the Q-bands into the lower-energy spectral region. 2. With iron porphyrins (hemin, heme and their complexes) the charge of the iron and the nature of axial ligands determine the position and intensity of the O-bands in the MOR spectrum. Low-spin complexes have a higher symmetry than the high-spin complexes. Whereas with hemin (S = 5/2), the iron located outside the heme plane strongly disturbs the porphyrin pi-system, the high symmetry of porphyrin is greatly retained in the case of heme. This can be explained by the enhanced binding distance between the bivalent iron and the porphyrin to great for a strong coupling between the microsymmetry of the iron and the macrosymmetry of the porphyrin pi-system.

Heme↗
PubMed1975

[Comparison of the activities of free and carrier fixed horseradish peroxidase].

Commerical horseradish-peroxidase was covalently bound to BrCN-activated sepharose. The activity parameters Vmax and Km were determined by the leukomalachite green reaction. Compared with the soluble enzyme, the immobilized POD has a relative residual activity of 22%. Dimethylsulfoxide and formamide were found to diminish the enzymatic activity in a concentration-dependent manner. The activity of the free enzyme in aqueous formamide solution (10%) is reduced by 43%, that of the insolubilized enzyme by 68%. Dimethylsulfoxide (15%) does not alter the LMG conversion rate of free POD, whilst a rate loss by 60% was observed for the immobilized enzyme.

Binding Sites↗
PubMed1975

[Interaction of the components of the cytochrome P-450 monooxygenase system from liver microsomes. I. Immobilization of the solubilized and partially purified protein components].

The method of matrix fixation has been used to study the interaction between the components of the cytochrome P-450 monooxygenases from rat liver microsomes. The solubilized, isolated protein components were covalently bound to BrCN-activated. Sepharose in different ways and subsequently the N-demethylase activity was determined. It has been proved that in each case of fixation a certain amount of activity could be determined. However the degree of activity varied in dependence on the sequence and number of bound components. The activity compared with the reconstituted soluble system decreased in the following sequence: single fixation of NADPH-cytochrome P-450 reductase (40%), of cytochrome P-450 (23%); sequential fixation: first component cytochrome P-450 (33%), first component NADPH-cytochrome P-450 reductase (8%). Simultaneous fixation of both components yielded a lower activity. From the results it was concluded that the activity is influenced by some kind of self-assembly.

Animals↗
PubMed1975

[Interaction of substrate with cytochrome P-450 in microsomal and solubilized form].

In order to characterize the substrate binding sites, difference spectroscopic titrations in microsomal and solubilized cytochrome P-450 from induced and non-induced rat liver microsomes were performed. The binding constants determined show differences depending on the physicochemical nature of the substrate and the degree of integration of the enzyme system. In hydrophilic substrates the differences of the binding to the microsomal or solubilized form are less pronounced than in lipophilic ones. From the comparison of the parameters obtained at various levels of integration it is concluded that the micromilieu of the binding site is of great importance for the binding of the substrate of cytochrome P-450.

Animals↗