Volker Ullrich

Multiple forms of cytochrome P450

The microsomal monooxygenase system is characterized by its broad substrate specificity which includes endogenous substrates as well as lipophilic drugs and chemicals. From in vitro investigations it was known that the relative reactivities and the pattern of products varied greatly with species, sex, age, diet or pretreatment with drugs of the animal. The suggestion that this was possibly due to a variety of cytochrome P450 enzymes rather than a single monooxygenase was recently confirmed by the isolation of several cytochrome P450 species with different although overlapping substrate specificities. In view of the consequences of a genetic and environment-dependent pattern of monooxygenases for drug metabolism and drug-mediated toxicity the methods of a quantitative assessment of the various forms are discussed.

In vitro induction by phenobarbital of drug monooxygenase activity in mouse isolated small intestine

Abstract Induction of drug monooxygenase activity by phenobarbital was studied in the mouse isolated small intestine. Incubation for 2 hours in the presence of 5 × 10 −4 M phenobarbital resulted in a 2–3 fold increase of the O -dealkylation activity for 7-ethoxycoumarin based on the protein content of the mucosal cell homogenate. The highest activity was located in the jejunum between 4 and 12 cm distal to the pylorus. The induction started after a lag phase of about 30 min and could be prevented by the simultaneous addition of 1·5 × 10 −5 M puromycin or 5 × 10 −7 M actinomycin D. Puromycin decreased the basal rate of O -dealkylation, but actinomycin D at this concentration did not. Actinomycin D, 2 × 10 −5 M, caused a “superinduction”, i.e. stimulation of the induced O -dealkylation activity 30 per cent greater than that with phenobarbital alone. Incorporation of 14 C-leucine into mucosal proteins was doubled in the presence of phenobarbital but the effect was abolished by inhibitors of protein synthesis. Spectroscopic measurements did not indicate major changes in the content of cytochrome b 5 and cytochrome P-450. However, the activity of NADPH-cytochrome c reductase increased in parallel with the O -dealkylation activity. Hence, the induction can probably be attributed to a more rapid turnover of the monooxygenase cycle.

Conjugation of 1-naphthol and transport of 1-naphthol-conjugates in the vascularly perfused small intestine of the mouse

A method is described which allows the simultaneous vascular and luminal perfusion of the murine small intestine. This preparation was used for the investigation of 1-naphthol conjugation in the gut and the sidedness of conjugate release. The viability of this preparation can be maintained for more than 1 hr as indicated by morphological controls, measurement of tissue metabolism and the transport of 3-O-methyl-glucose against a concentration gradient. When 100 microM 1-naphthol was administered on the luminal side, it was conjugated at a constant rate, yielding 1-naphthyl-glucuronide and 1-naphthyl-sulfate in a molar ratio of 1:2. Both metabolites were excreted into the blood at the contraluminal side of the epithelium. The results are discussed with respect to the sidedness of intestinal transport systems for anionic conjugates of xenobiotics and drugs.

A direct test for mono-oxygenase activity of intact small intestine using surface reflectance fluorimetry

Abstract The O -dealkylation activity for 7-ethoxycoumarin of mouse small intestine was determined by recording the surface fluorescence of the umbelliferone formed in everted intestinal segments. A simple holding device suitable for commercially available cuvettes has been designed. After hypotonic shocking of the mucosal ccells the substrate was added in 0·1 M Tris buffer pH 7·9 causing a linear increase in fluorescence for more than 15 min. Pretreatment of mice with phenobarbital increased the activity several fold, the induction following a biphasic course. The maximal rate of O -dealkylation by the entire small intestine was calculated as 19 nmoles min −1 . In agreement with previous results obtained with homogenates of small intestine the highest activity was located at a distance of 8–12 cm from the pylorus. Inhibition experiments indicate the involvement of cytochrome P-450.

Binding of bromosulphthalein to serum albumins

Abstract The binding of bromosulphthalein to human and bovine serum albumin was studied by infrared spectroscopy, laser-Raman spectroscopy, visible spectroscopy and pH measurements in order to obtain information on the binding forces involved. No conformational change of the proteins was observed during the tight binding of the first three bromosulphthalein molecules as indicated by the kinetics of the H- 2 H exchange and infrared spectroscopy in 2 H 2 O. Subsequent occupation of the low affinity binding sites causes a partial unfolding of the proteins. Binding of the dye at the high affinity sites is accompanied by a change in intensity and a shift of the lactone carbonyl band in infrared and laser-Raman spectra as well as a decrease of the visible absorption at 580 nm suggesting a hydrophobic environment. Binding at these sites is caused by Van der Waal or hydrophobic forces since the charge of the proteins remains unchanged during this process. It may be concluded that the main binding forces at the 14 low affinity binding sites consist of electrostatic interactions as indicated by pH shift studies and model studies for the bathochromic shift of the quinoic dye.