Emil Schiltz

Possible function of astrocyte cytochrome P450 in control of xenobiotic phenytoin in the brain: in vitro studies on murine astrocyte primary cultures.

[4-(14)C]Phenytoin underwent a rapid cellular uptake by diffusion within 5 min when applied in a concentration of 10 microM to mouse brain astrocyte cultures. Subsequently, a slow linear increase of intracellular radioactivity indicated metabolic trapping of the drug, with final concentrations reaching 144 pmol phenytoin/mg protein in the astrocytes. Phenytoin levels from 1 to 10 microM decreased cell viability by 15%. The action of cytochrome P450 present in astrocytes in concentrations of 16-17 pmol P450/mg protein could explain these slight cytotoxic effects by generating intermediate metabolites of phenytoin. In contrast, concentrations of 50 microM strongly inhibited cell proliferation. A Cyp2c29 immunorelated P450 isoform was expressed in nearly all astrocytes in culture. Intracellular [4-(14)C]phenytoin was degraded to its major metabolites dihydrodiol, p-HPPH, and m-HPPH through a P450-dependent reaction with a specific activity of 0.66 pmol/min x mg protein, or 0.12 pmol/min x mg protein as measured in cell homogenates. These data underscore the importance of astrocytes as brain cells active in the detoxification of foreign substrates, but also in their toxification due to reactive metabolites generated during these metabolic processes. After diffusionary influx of drugs and other xenobiotics, the astrocyte P450 monooxygenases perform an essential role in the mediation of toxicity most frequently encountered in highly vulnerable neurons.

Association of betaine-homocysteine S-methyltransferase with microtubules.

Abstract In mammals, betaine of the mitochondrial matrix is used in the cytosol by betainehomocysteine Smethyltransferase for methionine synthesis. The resulting dimethylglycine is shuttled back into the mitochondrial matrix for further degradation. Nanospray tandem mass spectrometry and Nterminal amino acid sequencing of microtubuleassociated proteins from rat liver tubulin revealed that betainehomocysteine Smethyltransferase is microtubule associated. This was confirmed by confocal laser scanning microscopy of HepG2 cells labeled with betainehomocysteine Smethyltransferase and αtubulinspecific monoclonal antibodies. The association of betainehomocysteine Smethyltransferase with the cytoskeleton may functionally integrate the mitochondrial and cytoplasmic compartments of choline degradation.

Redox-controlled, in vivo and in vitro phosphorylation of the α subunit of the light-harvesting complex I in Rhodobacter capsulatus

Labelling of Rhodobacter capsulatus cells with (32P)Pi in a phototrophic culture results in phosphorylation of a membrane-bound polypeptide identified as the α subunit of the LHI antenna complex of the photosynthetic apparatus. Phosphorylation of the same polypeptide was also observed by incubation of chromatophores with (32P)ATP or under conditions of photophosphorylation with ADP and (32P)Pi. The identity of the phosphorylated LHI-α subunit was demonstrated by N-terminal protein sequencing of the phosphorylated polypeptide and by failure of labelling in LHI-defective mutants. Pre-aeration of the samples or addition of the oxidant potassium ferrcyanide stimulated the kinase activity whereas the presence of soluble cytoplasmic proteins impaired phosphorylation in an in vitro assay. No effect resulted from addition of reductants to the assay medium. The results indicate the presence of a membrane-bound protein kinase in R. capsulatus that phosphorylates the α subunit of the LHI antenna complex under redox control.

End group analysis of yeast fatty acid synthetase.

The purified yeast fatty acid synthetase complex has been subjected to amino and carboxyl terminal amino acid end group analysis. Amino end groups were studied by Edman degradation and by dansylation of the sodium dodecyl sulfate- or urea-denatured complex. No N-terminal amino acid could be identified by either method. C-terminal amino acids were investigated by tritium labeling and by digestion of the complex with carboxypeptidases A and B. By both methods, the two amino acids valine and lysine were consistently identified as the C-termini of two different polypeptide chains. After separation of the fatty acid synthetase subunits A and B by sodium dodecyl sulfate polyacrylamide gel electrophoresis lysine was identified as the C-terminus of subunit A and valine as that of subunit B. The results are interpreted as evidence that the yeast fatty acid synthetase complex is basically composed of two nonidentical and multifunctional polypeptide chains.