Expression of two different forms of cDNA for thromboxane synthase in insect cells and site-directed mutagenesis of a critical cysteine residue.
cDNA coding for human placental thromboxane synthase (EC 5.3.99.5) was amplified by PCR from a human placental cDNA library and sequenced. This cDNA and a shorter cDNA isolated from a human lung cDNA library with a deletion of 163 bp near the 3' end were expressed in Spodoptera frugiperda (Sf9) insect cells using a baculovirus expression system. The cDNA from human placenta was expressed as an active enzyme (60 kDa) with a specific activity higher than those reported from other cell types, whereas the shorter cDNA was expressed in an inactive form (52 kDa). The active recombinant enzyme appeared to be unglycosylated as the molecular mass and the enzyme activity were not altered in the presence of tunicamycin. Site-directed mutagenesis was performed to convert a cysteine at position 480 in thromboxane synthase to a serine. This cysteine is found to be highly conserved in related cytochrome P-450 enzymes. The mutant enzyme was found to be inactive, although Western blot, immunoprecipitation and SDS/PAGE analysis indicated that the mutant enzyme was expressed at a level comparable with the wild-type enzyme. These results suggest that Cys-480 is essential for the enzyme catalytic activity and that the short-form cDNA may be a non-functional transcript.
Xia
Z
Z
Division of Medicinal Chemistry and Pharmaceutics, College of Pharmacy, University of Kentucky, Lexington 40536.
Shen
R F
RF
Baek
S J
SJ
Tai
H H
HH
D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae / Q000235:genetics; D001483:Base Sequence; D002478:Cells, Cultured; D003001:Cloning, Molecular; D003545:Cysteine / Q000235:genetics*; D018076:DNA, Complementary; D006801:Humans; D008969:Molecular Sequence Data; D009036:Moths; D016297:Mutagenesis, Site-Directed; D017386:Sequence Homology, Amino Acid; D013930:Thromboxane-A Synthase / Q000235:genetics*
D018076:DNA, Complementary; D013930:Thromboxane-A Synthase; D003545:Cysteine