Size and shape of isolated proteins of the small ribosomal subunit of rat liver.
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Animals↗
Biomedical subjects
J Behlke
Publications and source records attributed to J Behlke.
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↗
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↗
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↗
The N-terminal sequence protein of L7/L 12 is responsible for its dimerization.
Ultracentrifuge studies of intact protein L7/L12, of its fragments 27--120, 1--74 and 74--120 and of protein L7/L12 with oxidized methionine residues, indicate that the N-terminal sequence of the protein L7/L12 is responsible for its dimerization. The symmetry model of the dimer is discussed.
Amino Acid Sequence↗