The porcine thromboxane synthase-encoding cDNA: sequence, mRNA expression and enzyme production in Sf9 insect cells.
A full-length cDNA encoding porcine thromboxane synthase (TS) was isolated and sequenced. The open reading frame encodes a 534-amino acid (aa) protein (M(r) 60,451) which shares more than 75% identity with TS from other species and is 30% homologous to several enzymes of the cytochrome P-450 III family. Sequence comparison among porcine (p), human (h), and murine (m) TS indicated conservation of eight Cys residues and one putative N-glycosylation site. Several highly conserved regions were identified at the near N terminus, middle and C terminus. The most divergent region lies at aa residues 290-325, within which a Lys308 residue was unique to pTS. Between aa residues 70 and 90, considerable divergence was observed in mTS. Northern analysis showed that the pTS gene was expressed as a 2.3-kb transcript primarily in lung, kidney and thymus. A high-titer recombinant (re-) baculovirus containing pTS cDNA was developed to conduct a time course study of enzyme production in Spodoptera frugiperda (Sf9) cells. TS activity was detectable in the microsomes of Sf9 cells 12-h post-infection and reached maximum by 48 h. The produced TS resembles purified pTS in catalysis, as well as inhibition by a substrate analog inhibitor.
Shen
R F
RF
Department of Obstetrics and Gynecology, University of Maryland School of Medicine, Baltimore, MD 21201.
Zhang
L
L
Baek
S J
SJ
Tai
H H
HH
Lee
K D
KD
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D015152:Blotting, Northern; D002460:Cell Line; D003001:Cloning, Molecular; D004247:DNA; D006031:Glycosylation; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D009036:Moths; D016366:Open Reading Frames; D009928:Organ Specificity / Q000235:genetics; D012333:RNA, Messenger / Q000096:biosynthesis / Q000235:genetics; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000737:chemistry; D017386:Sequence Homology, Amino Acid; D013552:Swine; D013930:Thromboxane-A Synthase / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics*
D012333:RNA, Messenger; D011994:Recombinant Proteins; D004247:DNA; D013930:Thromboxane-A Synthase