cDNA cloning and expression of rat leukotriene C(4) synthase: elevated expression in rat basophilic leukemia-1 cells after treatment with retinoic acid.
Leukotriene C(4) synthase (LTC(4) S) is considered a pivotal enzyme for generation of potent proinflammatory mediators, cysteinyl-leukotrienes (cysLTs). LTC(4) S cDNA was cloned in rat basophilic leukemia-1 (RBL-1) cells, and exhibited 84.8% and 94.5% identity with the reported human and mouse LTC(4) S cDNA sequences, respectively. Homology between the rat LTC(4) S amino acid sequence and the corresponding sequences from the other species was 86.5% and 95.3% with human and mouse sequences, respectively. Rat LTC(4) S thus showed extensive homology with both mouse and human cDNA sequences. The active enzyme as assessed by LTC(4) S activity was expressed in COS-7 cells. While RBL-1 cells after the culture for 48 h in the presence of 0.1 microg/ml all trans -retinoic acid (RA) exhibited 27 times higher LTC(4) S activity than control cells, Northern-blot analysis of RA-treated cells showed upregulation of LTC(4) S mRNA. Polyclonal antibody was raised against the synthesized peptide deduced from the nucleotide sequence. Thus, Western-blot analysis of RBL-1 cells treated with RA and COS-7 cells transfected with pcDNA-LTC(4) S commonly showed a band at approximately 18 kDa in each solubilized enzyme solution, but either control cells did not. This cDNA probe and antibody may be useful for investigating the roles of cysLTs in various experimental models of rats.
Abe
M
M
Department of Pharmacology, School of Medicine, Fukuoka University, Japan. abemasa@fukuoka-u.ac.jp
Shibata
K
K
Saruwatar
S
S
Soeda
S
S
Shimeno
H
H
Katsuragi
T
T
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015153:Blotting, Western; D045744:Cell Line, Tumor; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D015972:Gene Expression Regulation, Neoplastic / Q000187:drug effects*; D005982:Glutathione Transferase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D006801:Humans; D007938:Leukemia / Q000201:enzymology* / Q000235:genetics*; D051379:Mice; D008969:Molecular Sequence Data; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D051381:Rats; D017386:Sequence Homology, Amino Acid; D012995:Solubility; D014212:Tretinoin / Q000494:pharmacology*
D018076:DNA, Complementary; D012333:RNA, Messenger; D014212:Tretinoin; D005982:Glutathione Transferase; C041116:leukotriene-C4 synthase