Formation of cytochrome P-450 containing haem or cobalt-protoporphyrin in liver homogenates of rats treated with phenobarbital and allylisopropylacetamide.
The potent porphyrogen allylisopropylacetamide and related compounds decrease hepatic concentrations of cytochrome P-450. This decrease occurs particularly in phenobarbital-induced cytochrome P-450 and is caused by suicidal breakdown of the haem of cytochrome P-450. Quantitative rocket immunoelectrophoresis showed that the protein moiety of the major phenobarbital-inducible form of hepatic cytochrome P-450 was not diminished up to 1 h, but was markedly decreased (to 43% of that of the phenobarbital-treated control) at 20 h after allylisopropylacetamide treatment. In contrast, the concentration of total cytochrome P-450, measured spectrophotometrically, decreased to 30-40% of the control at both 1 and 20 h after allylisopropylacetamide. Cytochrome P-450-dependent demethylations of ethylmorphine and benzphetamine decreased to a similar extent. When liver homogenates from rats treated with allylisopropylacetamide 1 h before being killed were incubated with haem, functional holocytochrome P-450 could be reconstituted from the apoprotein. Incubation with haem increased spectrophotometrically measurable cytochrome P-450 to 69%, ethylmorphine demethylase to 64% and benzphetamine demethylase to 93% of the activities in rats treated with phenobarbital alone. At 20 h after allylisopropylacetamide treatment, however, little or no reconstitution of cytochrome P-450 occurred after incubation with haem. When liver homogenates were incubated with cobalt and protoporphyrin, and microsomal proteins were then subjected to polyacrylamide-gel electrophoresis, cobalt-protoporphyrin was found specifically associated with proteins of Mr 50 000-53 000. When homogenates from rats given allylisopropylacetamide for 1 h or 20 h were compared, it was found that the extent of this association was higher in livers from the rats containing more apocytochrome P-450, suggesting that cobalt-protoporphyrin had associated with the apocytochrome. The data provide insight into the association of haem with the protein moiety of cytochrome P-450 and factors affecting breakdown of this protein.
D000081:Acetamides / Q000494:pharmacology*; D000502:Allylisopropylacetamide / Q000494:pharmacology*; D000818:Animals; D001051:Apoenzymes / Q000378:metabolism; D003577:Cytochrome P-450 Enzyme System / Q000096:biosynthesis*; D006418:Heme / Q000378:metabolism* / Q000494:pharmacology; D007122:Immunoelectrophoresis; D066298:In Vitro Techniques; D008099:Liver / Q000187:drug effects / Q000201:enzymology*; D008297:Male; D010634:Phenobarbital / Q000494:pharmacology*; D011166:Porphyrins / Q000378:metabolism*; D011524:Protoporphyrins / Q000378:metabolism*; D051381:Rats; D011919:Rats, Inbred Strains; D013053:Spectrophotometry
D000081:Acetamides; D001051:Apoenzymes; D011166:Porphyrins; D011524:Protoporphyrins; C017803:zinc protoporphyrin; D000502:Allylisopropylacetamide; D006418:Heme; C007095:cobaltiprotoporphyrin; D003577:Cytochrome P-450 Enzyme System; D010634:Phenobarbital