Cytosolic activation of hematin-dependent microsomal monooxygenase activity in the lung.
Additions of micromolar concentrations of hematin to washed rat pulmonary microsomal preparations resulted in marked (5-7-fold) increases in the NADPH-dependent generation of phenolic metabolites of benzo[a]pyrene (BaP). 9-Hydroxy-BaP was identified as the major reaction product. Additions of pulmonary cytosolic fractions to microsomes produced no measurable effect but cytosol and hematin added together elicited 25-30-fold increases in total phenolic products. Cytosolic fractions from other tissues, including rat kidneys and perfused rat livers, were also highly effective in enhancing the hematin-mediated increases in monooxygenase activity. However, cytosol from human placental tissues was only minimally effective when either pulmonary or placental microsomes were utilized as enzyme source. Superoxide dismutase and catalase (alone or in combination) had no measurable effect on hematin-mediated increases. Horseradish peroxidase effectively inhibited the hematin-dependent reactions but hematin-independent reactions were inhibited with equal effectiveness. Carbon monoxide profoundly inhibited all hematin-mediated increases in metabolite formation. The activating cytosolic component was non-dialyzable, inactivated by trypsin and heat, and eluted in the void volume from Sephadex G-150 columns. This suggested that the cytosolic factor(s) responsible for the increased hematin-dependent oxidation was a protein(s) with a high molecular weight or perhaps an aggregate or oligomer of proteinaceous material. HPLC profiles indicated a major effect on the generation of phenolics; quinones were also increased but only minimal increases in diols were observed. Results were consistent with the hypothesis that hematin-mediated increases in pulmonary monooxygenase activity result from an increased association of a small pool of pulmonary P-450-apoprotein(s) with the hematin prosthetic group to result in increased levels of an unidentified holocytochrome(s) with a relatively high substrate turnover number. The current data suggest a quaternary interaction among P-450 apoprotein(s), heme prosthetic group, reaction products (particularly 3-hydroxy-BaP) and a cytosolic protein(s). We postulate that the mechanism of action of the cytosolic factor is to facilitate the interaction of hematin with the apocytochrome.
Dean
P A
PA
Rettie
A E
AE
Turnblom
S M
SM
Namkung
M J
MJ
Juchau
M R
MR
D000818:Animals; D001564:Benzo(a)pyrene / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid; D003600:Cytosol / Q000378:metabolism; D004789:Enzyme Activation; D005260:Female; D006418:Heme / Q000494:pharmacology*; D007700:Kinetics; D008168:Lung / Q000201:enzymology*; D008297:Male; D008861:Microsomes / Q000201:enzymology*; D006899:Mixed Function Oxygenases / Q000378:metabolism*; D011247:Pregnancy; D051381:Rats; D011919:Rats, Inbred Strains
D001564:Benzo(a)pyrene; D006418:Heme; D006899:Mixed Function Oxygenases