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Apocytochrome P-450: reconstitution of functional cytochrome with hemin in vitro
Abstract:
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.
Insights
Cytochrome P-450 synthesis in rat liver involves apoprotein and heme. Phenobarbital induction of apocytochrome P-450 is independent of heme synthesis, suggesting apocytochrome production is the rate-limiting step.
Area of Science:
- Biochemistry
- Cell Biology
- Pharmacology
Background:
- Microsomal cytochrome P-450 synthesis requires both apoprotein and heme.
- Apoprotein is synthesized in the rough endoplasmic reticulum, while heme is produced in mitochondria.
Purpose of the Study:
- To investigate the relationship between apocytochrome P-450 and heme synthesis.
- To determine if cytochrome P-450 induction is dependent on concurrent heme synthesis.
Main Methods:
- Rats were treated with phenobarbital (cytochrome P-450 inducer) and cobalt (heme synthesis inhibitor).
- Holocytochrome P-450 was reconstituted in vitro by adding hemin to cell fractions.
- Spectrophotometric and functional assays were used to detect and measure holocytochrome P-450 activity.
Main Results:
- Concomitant treatment led to an excess of apocytochrome P-450, which could be reconstituted with hemin.
- Heme-mediated reconstitution was most efficient in fractions containing mitochondria-rough endoplasmic reticulum complexes.
- Phenobarbital-induced apocytochrome P-450 synthesis was independent of heme synthesis.
Conclusions:
- The structural association of mitochondria and rough endoplasmic reticulum may be a functional unit for cytochrome P-450 synthesis.
- Apoprotein synthesis appears to be the primary and rate-limiting step in holocytochrome P-450 formation.