Apocytochrome P-450: reconstitution of functional cytochrome with hemin in vitro.
Synthesis of microsomal cytochrome P-450 in rat liver requires synthesis of apoprotein in rough endoplasmic reticulum and of heme in mitochondria. Dissociation of apoprotein and heme synthesis by concomitant treatment of rats with inducers of cytochrome P-450 (i.e., phenobarbital) and inhibitors of heme synthesis (i.e., cobalt) resulted in a relative excess of apocytochrome P-450. Under these circumstances, it was possible to reconstitute the holocytochrome by addition of hemin in vitro. The holocytochrome was detected spectrophotometrically by its CO-binding properties and functionally by its increased oxidative activity. Heme-mediated reconstitution was most efficient in cell fractions rich in mitochondria-rough endoplasmic reticulum complexes (640 times g fraction), suggesting that the structural association of these two organelles may represent a functional unit essential for the synthesis of holocytochrome P-450. These findings indicate that phenobarbital-mediated induction of apocytochrome P-450 is independent of heme synthesis. It is suggested that synthesis of the apocytochrome may be the primary and rate-limiting event in the formation of cytochrome P-450.
D000818:Animals; D001059:Apoproteins / Q000096:biosynthesis*; D001665:Binding Sites; D003035:Cobalt / Q000494:pharmacology; D003577:Cytochrome P-450 Enzyme System / Q000096:biosynthesis* / Q000378:metabolism; D004721:Endoplasmic Reticulum / Q000201:enzymology / Q000378:metabolism; D004790:Enzyme Induction / Q000187:drug effects; D004791:Enzyme Inhibitors; D004792:Enzyme Precursors / Q000378:metabolism; D006418:Heme / Q000031:analogs & derivatives*; D006427:Hemin / Q000096:biosynthesis* / Q000494:pharmacology; D008297:Male; D008862:Microsomes, Liver / Q000201:enzymology / Q000378:metabolism*; D010634:Phenobarbital / Q000494:pharmacology; D011485:Protein Binding; D051381:Rats
D001059:Apoproteins; D004791:Enzyme Inhibitors; D004792:Enzyme Precursors; D003035:Cobalt; D006418:Heme; D006427:Hemin; D003577:Cytochrome P-450 Enzyme System; D010634:Phenobarbital