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A different cytochrome P450 form is induced in primary cultures of rat hepatocytes
1Department of Molecular Biology, Graduate School of Medical Science, Kyushu University, Fukuoka, Japan.
Abstract:
A 49-kDa protein (P49) was discovered in the primary cultures of rat hepatocytes. P49 cross-reacted with the antibodies against purified P450IIC11 [formerly P-450(M-1)]. P49 was located in microsomes and highly induced after plating of isolated hepatocytes on collagen-coated culture dishes. To characterize P49, cDNA clones were screened from a rat liver lambda gt11 expression library. From sequence analysis of the cloned cDNAs, the amino acid sequence of P49 was deduced, and the protein was identified as a previously uncharacterized form of cytochrome P450. P49 consists of 489 amino acids and shows approximately 60% similarity with the members of class IIC subfamily of rat cytochrome P450, such as P450IIC11 and P450IIC12 [formerly P-450(F-1)]. RNA blot analysis indicates that the mRNA translating P49 was induced approximately 20- to 30-fold at 70 hr in the primary cultures compared with the liver of adult rats. Induction of P49 was not affected by density of the plated cells and the presence or absence of several hormones, serum, or antibiotics in the culture medium. On the other hand, lower induction of P49 was seen when the hepatocytes were cultured on Matrigel-coated plates. Expression of P49 mRNA was low in the liver of adult rats and was not detectable in the livers of 1- and 2-week-old male and female rats. P49 is an additional form of cytochrome P450, which is induced in the primary culture of rat hepatocytes.
Insights
A novel cytochrome P450 protein, P49, was identified in rat liver cells. This protein is significantly induced in primary hepatocyte cultures, suggesting its role in cellular responses.
Area of Science:
- Biochemistry
- Molecular Biology
- Cell Biology
Background:
- Primary cultures of rat hepatocytes were used to study protein expression.
- A 49-kDa protein (P49) was detected and showed cross-reactivity with antibodies against P450IIC11.
- P49 was localized to microsomes and its expression was significantly altered upon plating.
Purpose of the Study:
- To characterize the newly discovered 49-kDa protein (P49) found in rat hepatocytes.
- To identify P49 and understand its expression patterns in primary cell cultures.
- To determine the relationship of P49 to known cytochrome P450 enzymes.
Main Methods:
- Screening of a rat liver lambda gt11 expression library using cDNA clones.
- Sequence analysis of cloned cDNAs to deduce the amino acid sequence of P49.
- RNA blot analysis to assess mRNA levels and induction patterns.
Main Results:
- P49 was identified as a previously uncharacterized form of cytochrome P450, comprising 489 amino acids.
- P49 shares approximately 60% similarity with rat cytochrome P450 class IIC subfamily members (e.g., P450IIC11, P450IIC12).
- P49 mRNA levels were induced 20-30 fold in primary hepatocyte cultures after 70 hours, with minimal influence from culture conditions (cell density, hormones, serum, antibiotics) but lower induction on Matrigel.
Conclusions:
- P49 represents a novel cytochrome P450 isoform.
- The expression of P49 is significantly induced in primary rat hepatocyte cultures, particularly on collagen-coated surfaces.
- P49 mRNA is minimally expressed in adult rat liver and undetectable in young rats, indicating its specific induction in cultured hepatocytes.