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D Pfeil

Publications and source records attributed to D Pfeil.

6 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

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

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

Hydrodynamic studies on interactions between the components of the liver microsomal cytochrome P-450 system.

To understand the different behaviour of cytochrome P-450 systems in kinetics as well as in the demethylase activity, sedimentation and molecular weight experiments have been carried out with the following results: 1) Sedimentation coefficients of solubilized P-450 and P-450 LM2 fractions amount to 24 +/- 4 [S] and 12.8 +/- 1.2 [S], respectively. Molecular weights were determined to be 1.0 +/- 0.2 . 10(6) and 3.0 +/- 0.5 . 10(5) Dalton. 2) Triton N-101 provokes splitting of the associated structure both of solubilized P-450 and P-450 LM2; this effect is reversible. 3) The dissociation depends not only on the absolute concentration of Triton but rather on the Triton P-450 ratio. The dissociation curves of solubilized P-450 and P-450 LM2 are similar in shape and in the Triton/P-450 ratio dependence. 4) In the presence of small concentrations of Triton a more complicated dissociation behaviour was observed with broad integral distribution of the sedimentation coefficients. 5) The ionic detergent cholate splits the associated structure of P-450 LM2 at considerably higher concentrations in comparison with Triton-N 101. 6) Addition of reductase causes a decrease of sedimentation coefficients and molecular weights of solubilized P-450. The same effect in P-450 LM2 could be observed only in the presence of phospholipids.

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↗