METABOLISM OF TENTOXIN BY HEPATIC CYTOCHROME-P-450 3A ISOZYMES

The interaction between mt and human liver cytochrome P-450 with tento xin, a natural phytotoxic cyclotetrapeptide having chlorotic propertie s, was studied by difference ultraviolet visible spectroscopy. Tentoxi n interacted with rat liver microsomes and the difference spectrum was characteristic of binding to a protein site close to the heme, The in tensity of this spectrum was clearly dependent on the amounts of P-450 3A in the microsomes and was optimal in dexamethasone-treated rut mic rosomes. Tentoxin exhibited a high affinity for P-450 3A (K approximat ely 10 mu M). Similar results were observed with human P-450 isozymes expressed in yeast, Only P-450 3A4 and 3A5 were able to give spectral interactions with tentoxin. Liver microsomes from rats pretreated with dexamethasone, a specific inducer of P-450 3A, were found to be parti cularly active for the oxidation of tentoxin, which occurs mainly on i ts Ala(Me) function leading to demethylation. Yeast-expressed P-450 3A also exhibited high activity to metabolize tentoxin. The metabolites were identified by their ultraviolet and mass spectra in fast atom bom bardment and collision-activated dissociation modes, In addition to th e major N-demethylated metabolite, other hydroxylated metabolites were formed.Preliminary analysis showed that as tentoxin, some metabolites were still efficient chloroplast ATPase inhibitors, while at least on e of them exhibited even at low concentration stimulatory effects.