Amino acid sequence of porcine antithrombin III.
Antithrombin III (ATIII) is a member of the serine protease inhibitor (serpin) family. As a step towards a better understanding of the heparin-binding mechanism of mammalian ATIIIs, the amino acid sequence of porcine ATIII was established by sequence analysis of the peptides derived from cyanogen bromide cleavage and enzymatic digestion with lysyl endopeptidase, V8 protease, and trypsin. Porcine ATIII was found to consist of 431 amino acid residues, with a calculated molecular weight of 48,930 without carbohydrate. Its molecular weight with 16.4% carbohydrate was estimated as 56,955, which is in good agreement with the value determined by SDS-PAGE. Porcine ATIII showed high sequence similarity to other mammalian ATIIIs, including the reactive site, heparin-binding basic amino acid residues, and disulfide bonds. The most notable feature of porcine ATIII was that it possesses only three carbohydrate chains, at Asn136, 156, and 193, whereas other mammalian ATIIIs have four, additional chain being at Asn97; this is replaced by Asp in porcine ATIII. In the case of human ATIII, the chains are at Asn96, 135, 155, and 192. The heparin-binding affinities of human and porcine ATIIIs were compared using an immobilized heparin column. Porcine ATIII eluted from the column with a peak at an NaCl concentration of 924 mM while human ATIII eluted at 838 mM NaCl. Neuraminidase treatment of each ATIII enhanced the heparin-affinity to the same extent. These results suggest that in spite of the high degree of amino acid sequence identity between porcine and human ATIIIs (91% identical), porcine ATIII has a higher heparin-binding affinity than human, because it lacks a carbohydrate chain at Asp97.
Tokunaga
F
F
Department of Life Science, Faculty of Science, Himeji Institute of Technology, Hyogo.
Goto
T
T
Wakabayashi
S
S
Koide
T
T
D000595:Amino Acid Sequence; D000818:Animals; D000990:Antithrombin III / Q000737:chemistry* / Q000378:metabolism; D006493:Heparin / Q000378:metabolism; D008969:Molecular Sequence Data; D009439:Neuraminidase; D016415:Sequence Alignment; D013552:Swine
D000990:Antithrombin III; D006493:Heparin; D009439:Neuraminidase