Molecular cloning of guinea pig CYP1A1: complete primary structure and fast mobility of expressed protein on electrophoresis.
Guinea pig CYP1A1 cDNA was isolated from a liver cDNA library of guinea pig treated with 3-methylcholanthrene. The cDNA, named GPc1, was 2674 bp long and contained an entire coding region for 516 amino acids. The amino acid sequence of guinea pig CYP1A1 shared 74-78% identity with those of the other mammalian CYP1A1s. RNA blot and immunoblot analyses revealed that CYP1A1 was constitutively expressed and was induced by 3-methylcholanthrene in guinea pig liver. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, guinea pig CYP1A1 expressed in yeast had a significantly smaller apparent molecular mass than expressed mouse CYP1A1. An alignment of the amino acid sequences of mammalian CYP1A1s demonstrated that guinea pig CYP1A1 was several residues shorter than the counterparts in the N-terminal region. Thus, to clarify the contribution of the N-terminal sequence of guinea pig CYP1A1 to the fast mobility on the electrophoresis, mouse-guinea pig chimeric CYP1A1 was prepared through cDNA-directed expression in yeast. The chimeric CYP1A1 protein had an intermediate molecular mass between mouse and guinea pig CYP1A1s indicating that the anomalous mobility of guinea pig CYP1A1 is in part due to the shortened N-terminal amino acid sequence of the protein.
Ohgiya
S
S
Government Industrial Development Laboratory-Hokkaido, Agency of Industrial Science and Technology, Sapporo, Japan.
Ishizaki
K
K
Shinriki
N
N
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001483:Base Sequence; D002678:Chimera; D003001:Cloning, Molecular; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry / Q000235:genetics*; D018076:DNA, Complementary / Q000032:analysis / Q000302:isolation & purification; D004591:Electrophoresis, Polyacrylamide Gel; D004790:Enzyme Induction / Q000187:drug effects; D015870:Gene Expression; D006168:Guinea Pigs; D008099:Liver / Q000187:drug effects / Q000201:enzymology; D008748:Methylcholanthrene / Q000494:pharmacology; D051379:Mice; D008969:Molecular Sequence Data; D012441:Saccharomyces cerevisiae / Q000235:genetics
D000596:Amino Acids; D018076:DNA, Complementary; D008748:Methylcholanthrene; D003577:Cytochrome P-450 Enzyme System