Biomedical subjects

J Blanck

Publications and source records attributed to J Blanck.

8 recordsLinked to original sources

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↗

PubMed1978

The cytochrome P-450 reaction mechanism--kinetic aspects.

The reviewed kinetic investigations at the experimentally accessible partial reactions of the reaction sequence clearly evidence that the P-450 system represents a complex reaction system, the main control points of which are shown in Scheme 3: As can be seen, control elements are the substrate, the cytochrome species (P-450/448), the reducing agent, the reductase, phospholipid and parts of the b5 system as well. Formula (see text): All of these steps can be rate limiting (rate interfering), depending on the distinct substrate and oxygen donor. The ternary complex evidently has a key position however in that is structural specificities determine rate and product distribution of the respective substrate conversion. But unfortunately the ternary complex decay (including the e2 transfer) is kinetically unresolved till now.

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↗

PubMed1976

A quench-flow kinetic investigation of calcium ion accumulation by isolated cardiac sarcoplasmic reticulum. Dependence of initial velocity on free calcium ion concentration and influence of preincubation with a protein kinase, MgATP, and cyclic AMP.

Ca2+ accumulation at pH 6.8 by isolated rabbit heart microsomes derived chiefly from sarcoplasmic reticulum was investigated by a quench-flow technique. The reaction was terminated at preset times by addition to the reaction mixture of an equal volume of 10 to 50 mM ethyleneglycol-bis-(beta-aminoethyl ether)-N,N'-tetraacetic acid buffered at pH 6.0. The initial velocity of Ca2+ accumulation by microsomal preparations exhibiting a steady state Ca2+ accumulation of 25.6 nmol Ca2+/mg increased from 3.67 to 33.4 nmol Ca2+/mg - s as the free Ca2+ concentration was raised from 0.2 to 18.9 muM. Preincubation of the cardiac microsomes with a partly purified soluble cardiac cyclic AMP-dependent protein kinase, MgATP, and cyclic AMP lead to a significant increase in the initial Ca2+ accumulation rate. The amounts of Ca2+ that were found to accumulate in the first 200 ms of the reaction are comparable to the quantities of the ion that according to literature data need to be removed from the myofilaments and the myoplasm for induction of relaxation of the myocardial fibers.

Adenosine Triphosphate↗

PubMed1975

Studies on electron transfer between mercury electrode and hemoprotein.

The electrochemical behaviour of ferricytochrome c, metmyoglobin and methemoglobin was studied using d.c., a.c. and differential pulse polarography, and controlled potential electrolysis. 1. The three hemoproteins yield d.c. polarographic steps, and peaks in differential pulse polarograms, the height of which is proportional to concentration. The charge transfer is influenced by strong adsorption. 2. The concentration dependence of the a.c. polarograms indicates structural changes in the adsorbed molecules. 3. The reduction products of controlled potential electrolysis of metmyoglobin and methemoglobin have absorption spectra identical with the native control samples. The affinity for oxygen and the cooperativity in hemoglobin are not affected by the reaction at the electrode. 4. The charge transfer proceeds via adsorbed, already reduced, molecules to freely diffusible proteins.

Binding Sites↗

PubMedDate not supplied

Quench-flow measurements of initial rates of Ca2+ accumulation by isolated cardiac sarcoplasmic reticulum.

Using a quench-flow technique, the initial velocity of Ca2+ accumulation by isolated cardiac sarcoplasmic reticulum was estimated at free Ca2+ ion concentrations in the range encountered in the myoplasm during the cardiac contraction cycle. With cardiac microsomes exhibiting a Ca2+ accumulative capacity of 25.6 nmol Ca2+/mg protein, initial rates were found to increase from 3.7 to 33.4 nmol Ca2+/mg protein/sec, when the free Ca2+ ion concentration was raised from 0.2 to 18.0 muM. Preincubation of the cardiac microsomes with a party purified soluble cardiac protein kinase, MgATP, and cAMP led to a significant increase in the initial Ca2+ accumulation rate.

Animals↗