THE PORCINE THROMBOXANE SYNTHASE-ENCODING CDNA - SEQUENCE MESSENGER-RNA EXPRESSION AND ENZYME-PRODUCTION IN SF9 INSECT CELLS

A full-length cDNA encoding porcine thromboxane synthase (TS) was isol ated and sequenced. The open reading frame encodes a 534-amino acid (a a) protein (M(r) 60451) which shares more than 75% identity with TS fr om other species and is 30% homologous to several enzymes of the cytoc hrome 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 w ere identified at the near N terminus, middle and C terminus. The most divergent region lies at aa residues 290-325, within which a Lys308 r esidue 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 an d thymus. A high-titer recombinant (re-) baculovirus containing pTS cD NA was developed to conduct a time course study of enzyme production i n 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 in hibition by a substrate analog inhibitor.