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Synthesis and insertion of cytochrome P-450 into endoplasmic reticulum membranes
Abstract:
Treatment of rats with phenobarbital leads to a substantial increase in levels of translatable liver cytochrome P-450 mRNA. This mRNA is primarily associated with ribosomes bound to endoplasmic reticulum membranes which in an in vitro system synthesized approximately 10 times more cytochrome P-450 than did free polysomes from the same animals. Cytochrome P-450 synthesized by rough microsomes in vitro appears to be directly inserted into the membranes because it was not released by a treatment with low detergent concentrations that released albumin and other microsomal content proteins. The amino-terminal amino acid sequence of cytochrome P-450 synthesized in an mRNA-dependent system resembles in hydrophobicity the signal segment of presecretory proteins and therefore may serve to insert the polypeptide into the membrane during synthesis. In contrast to the situation with secretory proteins and several other membrane proteins, however, the putative insertion signal of cytochrome P-450 is not removed by a membrane-associated peptidase and remains in the mature polypeptide.
Insights
Phenobarbital treatment boosts liver cytochrome P-450 mRNA, enhancing protein synthesis on rough endoplasmic reticulum. This cytochrome P-450 is directly inserted into membranes and its signal peptide remains in the mature protein.
Area of Science:
- Biochemistry
- Molecular Biology
- Cell Biology
Background:
- Liver cytochrome P-450 enzymes are crucial for drug metabolism.
- Phenobarbital is a known inducer of cytochrome P-450 expression.
- Understanding the synthesis and membrane insertion of cytochrome P-450 is vital for pharmacology.
Purpose of the Study:
- To investigate the synthesis of cytochrome P-450 in response to phenobarbital treatment.
- To determine the site of cytochrome P-450 synthesis and its insertion into cellular membranes.
- To characterize the role of the amino-terminal sequence in cytochrome P-450 membrane association.
Main Methods:
- Treatment of rats with phenobarbital.
- Isolation of liver microsomes and polysomes.
- In vitro synthesis assays using isolated mRNA and ribosomes.
- Analysis of protein insertion into membranes via detergent treatment.
- Amino acid sequencing of synthesized cytochrome P-450.
Main Results:
- Phenobarbital significantly increased translatable liver cytochrome P-450 mRNA levels.
- Ribosomes bound to endoplasmic reticulum synthesized ~10 times more cytochrome P-450 than free polysomes.
- Synthesized cytochrome P-450 was directly inserted into membranes and not released by mild detergent treatment.
- The amino-terminal sequence showed hydrophobicity, suggesting a role in membrane insertion.
- Unlike secretory proteins, the cytochrome P-450 signal sequence was not cleaved by peptidases.
Conclusions:
- Phenobarbital induces cytochrome P-450 mRNA, leading to increased synthesis on rough endoplasmic reticulum.
- Cytochrome P-450 is directly inserted into the endoplasmic reticulum membrane via its N-terminal sequence.
- The signal sequence for cytochrome P-450 insertion remains in the mature protein, distinguishing it from secretory proteins.