Biomedical subjects
Publications and source records attributed to P Berndt.
Allopurinol generally is considered to be a problematic substance as to its bioavailability. To prove the quality, the in-vivo bioequivalence of an allopurinol (CAS 315-30-0) preparation (Cellidrin 300) in comparison to the standard preparation has been investigated. Both products were bioequivalent with regard to all relevant parameters such as AUC, tmax and Cmax.
The effects of bifunctional cross-linking reagents on the purified hexameric cytochrome P-450LM2 in an aqueous medium and on the proteoliposomal cytochrome P-450LM2 have been compared. In both cases, cross-linking is shown to result in the appearance of a range of additional protein bands in SDS electrophoretograms. The number and the positions of these bands seem to be similar in the solubilized and in the proteoliposomal cytochromes. No additional bands appear when the purified cytochrome P-450 was pretreated with 0.2%. Emulgen 913, which decomposes cytochrome P-450LM2 hexamers. The results indicate that the membrane-bound cytochrome P-450 can exist in the oligomeric (presumably hexameric) form.
Subunit interactions in the purified hexameric cytochrome P-450LM2 have been studied using covalent binding of one of the 6 promoters to an insoluble matrix. High ionic strength, large-scale pH changes, guanidine chloride and sodium cholate taken at membrane-solubilizing concentrations, had no effect on the aggregation state of the immobilized hemoprotein. SDS caused a 6-fold decrease in the amount of the bound cytochrome. Non-ionic detergents (Emulgen 913, octylglucoside, Tritons) induced hexamer dissociation. In the presence of Emulgen 913 (greater than 0.2%), monomers and immobilized dimers were obtained as cytochrome P-450 was studied in an aqueous medium and in the immobilized state, respectively. Immobilized dimers could be reconstituted to hexamers by treatment with an excess of solubilized monomers after removal of the detergent. In the presence of various phospholipids, which increased the immobilized cytochrome P-450LM2 demethylase activity and induced characteristic spectral changes, no hexamer dissociation was shown. The data obtained are thus in agreement with the suggestion that hexameric arrangement is inherent in the cytochrome P-450 when it is bound to the native membranes.
Subunit interactions of highly purified hexameric cytochrome P-450 LM 2 has been studied using covalent binding of one of the six protomers to an insoluble matrix. Immobilized cytochrome was catalytically active in monooxygenase reactions and retained the spectral characteristics of cytochrome P-450 LM 2. High ionic strength, large scale pH changes and addition of guanidine chloride were without effect on the aggregation state of the immobilized hemoprotein. However, several detergents induced hexamer dissociation. The crucial role of hydrophobic forces in hexamer subunit interaction was demonstrated. Incubation of the immobilized cytochrome P-450 LM 2 with sonicated liposomes composed of various phospholipids did not result in oligomer dissociation and protein translocation from the matrix to the lipid phase, although the catalytic activity of the immobilized cytochrome significantly increased in the presence of liposomes. The data suggest that cytochrome P-450 LM 2 may be of hexameric structure in the native membranes.
It was shown that the maximal degree of dissociation of cytochrome P-450 LM2 hexamers in the presence of the nonionic detergent Emulgene 913 (20 degrees C) is observed at the detergent concentration of about 0.2%. Using equilibrium centrifugation in solutions of different density, the molecular mass of the dissociation product minus that of the bound detergent was found to be equal to 50 +/- 8 kDa, thus corresponding to the molecular mass of the monomer. One cytochrome P-450 LM 2 molecule binds 80 +/- 20 molecules of Emulgene 913.
Crosslinking of protein molecules with bifunctional reagents and subsequent electrophoresis of the modified proteins revealed the presence of cytochrome P-450 LM 2 oligomers in proteoliposome membranes obtained in different ways and differing in their phospholipid composition. Data from a comparative analysis of cytochrome P-450 oligomeric structures in solution and in membrane are suggestive of the hexameric organization of cytochrome P-450 LM 2 within proteoliposome membranes.
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The partial specific volume (v) of highly purified membrane protein cytochrome P450 LM2 monooxygenase from rabbit liver endoplasmatic reticulum has been estimated by various independent methods. The values of v obtained through our experiments are practically equal to the value calculated from the amino acid composition of the protein (0.75 cm3/g).
Cytochrome P-450LM2 was isolated from rabbit liver microsomes in a form which was shown to be homogeneous in AcA-22 Ultrogel and ultracentrifugation studies. The molecular mass determined by sedimentation equilibrium roughly corresponded to hexamer composed of 56 kDa monomers. Hexamer structure of the cytochrome was directly demonstrated by electron microscopic study. In the cytochrome P-450LM2 hexamer, monomers seem to be arranged in two layers (three monomers in the layer) in such a way that each monomer occupies a position at the vertices of a triangular antiprism with a 32 point group symmetry.
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