Molecular cloning and cell-free expression of mouse antithrombin III.
The homology between antithrombin III (AT-III) of mouse, of man, and that of other species was investigated. Preliminary experiments showed that mouse AT-III inhibited human alpha-thrombin efficiently (second order rate constant [K2nd] 5.8 x 10(3) M-1 s-1) as compared to human AT-III (K2nd 6.7 x 10(3) M-1), but was not recognized on immunoblots by antibodies that recognized both human and rabbit AT-III. In order to compare AT-III from different species at the molecular level, a cDNA clone for murine AT-III was isolated from a lambda ZAP mouse liver cDNA library on the basis of hybridization to a rabbit AT-III cDNA probe. The 1509 bp murine AT-III cDNA consists of a 1398 bp open reading frame, preceded by a 15 bp 5' untranslated region, followed by a 75 bp 3' untranslated region. The deduced primary protein structure consists of a 32 amino acid signal sequence, with a mature portion of 433 residues. Mature murine AT-III is 89% identical to its human counterpart, 86% identical to bovine AT-III, and 82% identical to that of the rabbit. Constructs lacking the nucleotides encoding the signal sequence were engineered and expressed in a cell-free system. The resulting 47 kDa non-glycosylated translation product was capable of being cleaved by human alpha-thrombin, of forming SDS-stable complexes with the protease, and of binding to immobilized heparin. Isolation of the murine AT-III cDNA will make feasible molecularly defined experiments with murine AT-III in the mouse system.
Wu
J K
JK
Canadian Red Cross Society Blood Transfusion Service, McMaster University, Hamilton, Ontario.
Sheffield
W P
WP
Blajchman
M A
MA
D000595:Amino Acid Sequence; D000818:Animals; D000990:Antithrombin III / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D001483:Base Sequence; D002474:Cell-Free System / Q000378:metabolism; D003001:Cloning, Molecular; D004247:DNA / Q000032:analysis / Q000096:biosynthesis*; D015723:Gene Library; D051379:Mice; D008808:Mice, Inbred CBA; D008822:Mice, Transgenic; D008969:Molecular Sequence Data; D010957:Plasmids / Q000235:genetics; D014176:Protein Biosynthesis / Q000235:genetics; D017386:Sequence Homology, Amino Acid; D013045:Species Specificity; D014158:Transcription, Genetic / Q000235:genetics
D000990:Antithrombin III; D004247:DNA