Biomedical subjects

K Rohde

Publications and source records attributed to K Rohde.

3 recordsLinked to original sources

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↗

PubMed1978

Effect of protein-protein interactions on binding of ligands by proteins.

In general, saturation curves for ligand binding by proteins are described by the Adair-equation. The approach to ligand binding with methods of statistical mechanics leads not only to expressions for the Adair-constants, but gives also the possibility of describing the effect of protein-protein interactions on the binding of ligands by proteins. Under the assumption of the superposition-approximation for the potential of mean force between proteins in a solution, which contains also ligand molecules, the variations of the second and third virial coefficient with ligand activity are calculated for several simple model-pontentials. Finally the pair potential of hemoglobin molecules known from X-ray measurements will be approximated by such a simple square-well potential. With that the effect of hemoglobin density on oxygenation will be estimated. One finds for our model system that at relatively high protein density a further increase in density should be accompanied by a decrease in affinity (hindrance of saturation) and an increasing steepness of the slope of the saturation curve.

Calorimetry↗