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Cloning of a cDNA coding for P-450 LM3c from rabbit liver microsomes and regulation of its expression
Abstract:
Liver cytochromes P-450 LM3c in the rabbit and P-450p in the rat are two related forms, inducible by macrolide antibiotics such as triacetyloleandomycin (TAO) and glucocorticoids such as dexamethasone. We prepared a cDNA library from TAO induced rabbit liver mRNA and characterized a cDNA (pLM3c-4.1) that hybridized to pDex 3.22, a cDNA complementary to cytochrome P-450p mRNA. Northern blots of liver poly(A)RNA from untreated or TAO, erythromycin and rifampicin treated animals, revealed two mRNA species of approximately 1700 and 1850 nucleotides in length, that hybridized to LM3c cDNA and to pDEX 3.22. The level of both mRNAs was increased five fold over control by macrolide antibiotics but unaffected by both phenobarbital and B-naphthoflavone. After 5 days of TAO treatment LM3c mRNA had increased 5 fold while LM3c protein had increased 25 fold. However, the rate of P-450 LM3c gene transcription measured in isolated liver nuclei remained unchanged throughout five days of TAO treatment. We conclude that TAO may induce cytochrome P-450 LM3c by post-transcriptional effects.
Insights
Macrolide antibiotics like triacetyloleandomycin (TAO) increase rabbit liver cytochrome P-450 LM3c protein levels significantly. This induction appears to occur via post-transcriptional effects, not increased gene transcription.
Area of Science:
- Biochemistry
- Molecular Biology
- Pharmacology
Background:
- Cytochromes P-450 (CYPs) are crucial enzymes in drug metabolism.
- Liver CYPs LM3c (rabbit) and P-450p (rat) are related, inducible forms.
- Induction is triggered by macrolide antibiotics and glucocorticoids.
Purpose of the Study:
- To investigate the mechanism of cytochrome P-450 LM3c induction by TAO.
- To characterize the cDNA for rabbit liver cytochrome P-450 LM3c.
- To determine if induction involves transcriptional or post-transcriptional regulation.
Main Methods:
- Preparation of a cDNA library from TAO-induced rabbit liver mRNA.
- Characterization of LM3c cDNA (pLM3c-4.1) and hybridization studies.
- Northern blot analysis of liver poly(A)RNA from treated and untreated rabbits.
- Measurement of P-450 LM3c gene transcription rates in isolated liver nuclei.
Main Results:
- A cDNA (pLM3c-4.1) for LM3c was identified and hybridized with a P-450p cDNA probe.
- Northern blots revealed two mRNA species (~1700 and ~1850 nucleotides) that hybridized to LM3c cDNA.
- Macrolide antibiotics increased both mRNA species fivefold, while phenobarbital and B-naphthoflavone had no effect.
- LM3c protein increased 25-fold after 5 days of TAO treatment, while mRNA increased only fivefold.
- Gene transcription rates remained unchanged during TAO treatment.
Conclusions:
- Triacetyloleandomycin (TAO) induces cytochrome P-450 LM3c protein in rabbit liver.
- The induction mechanism involves post-transcriptional regulation rather than increased gene transcription.
- This study elucidates a key regulatory pathway for drug-metabolizing enzymes.