title: Comparison of microsomal and solubilized monooxygenases from rat and rabbit by proton magnetic relaxation.
abstract: The paper presents results of a comparative study of the haem environment, by proton magnetic relaxation, in P-450 and P-448 monooxygenases from rat and rabbit, induced by phenobarbital and 3-methylcholanthrene, in both species. It was established that the method yields information on the accessibility of the haem iron for solvent molecules (protons), both in microsomes and in solubilized samples of various degrees of purification, i.e. association. The state of micelles in the solutions does not alter the haem iron accessibility. A slight difference was found for the microsomes suspended in a phosphate vs. pyrophosphate buffer, but this is without any consequence with regard to the species and form differences. The correlation time for the highly purified LM2 fraction of rabbit P-450 could not be determined more precisely than before for a sample of lower purity, because the relaxation rates are frequency independent. The correlation time for the rat P-448 monooxygenase was determined by dispersion measurements to be (4.1 +/- 0.4) x 10(-11) s. It was found that the PMRx behaviours of rabbit and rat monooxygenases are more alike in microsomes than in the partially purified solubilized form. The solubilization produces a pronounced alteration of the PMRx temperature dependence only for the rat 3-MC induced monooxygenase P-448. For the P-450 form the haem iron becomes less accessible on solubilization, both for the rabbit and the rat liver monooxygenases, whereas in case of rat liver P-448 the accessibility is considerably enhanced on solubilization. There is a substantial structural specificity of the haem environments from the two animal species, the one from rat being tighter. The reduced, NO-bound rabbit liver monooxygenase was studied also, but the results are not yet conclusive, except the fact that the unpaired spin from NO is thoroughly shielded from the solvent compared with the haem iron from the original sample. The following series of increased haem-iron accessibility emerges from the PMRx studies known so far: rat (P-448) less than rabbit (P-448) less than rat (P-450) less than rabbit (P-450) in microsomes, and rabbit (P-448, with 3-MC bound?) less than Pseudomonas putida (P-450) rat less than (P-448), less than rat (P-450) less than rabbit (P-450) from solubilized samples. For the latter, it appears that increased enzymic specificity goes along with a closing of the haem cleft.
mesh_terms: D000818:Animals; D003577:Cytochrome P-450 Enzyme System / Q000302:isolation & purification; D004578:Electron Spin Resonance Spectroscopy; D006418:Heme; D007501:Iron; D007700:Kinetics; D008024:Ligands; D008297:Male; D008748:Methylcholanthrene / Q000494:pharmacology; D008862:Microsomes, Liver / Q000187:drug effects / Q000378:metabolism*; D009569:Nitric Oxide; D010105:Oxygenases / Q000302:isolation & purification; D010634:Phenobarbital / Q000494:pharmacology; D011817:Rabbits; D051381:Rats; D012995:Solubility; D013045:Species Specificity; D013696:Temperature
chemical_list: D008024:Ligands; D009569:Nitric Oxide; D006418:Heme; D008748:Methylcholanthrene; D003577:Cytochrome P-450 Enzyme System; D007501:Iron; D010105:Oxygenases; D010634:Phenobarbital