Single amino acid substitutions in the reactive site of antithrombin leading to thrombosis. Congenital substitution of arginine 393 to cysteine in antithrombin Northwick Park and to histidine in antithrombin Glasgow.
Antithrombin Northwick Park and antithrombin Glasgow are functionally variant antithrombins with impaired abilities to interact with thrombin. Thrombosis is associated with their inheritance. Both of the purified, reduced, and S-carboxymethylated variant antithrombins were treated with cyanogen bromide and the major pools of each containing the amino acid sequence Gly339-Met423 were isolated. Following treatment of these pools with trypsin, fast atom bombardment mass spectrometry identified tryptic peptides (found also in normal antithrombin treated in the same way) that corresponded to amino acid sequences Gly339-Lys370 and Val400-Met423. The tryptic peptides, corresponding to amino acid sequences Ala371-Arg393 and Ser394-Arg399 were present in both variant preparations in greatly reduced amounts compared to a normal antithrombin preparation. However, two novel tryptic peptides of molecular mass (M + H)+ 2976 and 2952 were identified in the digests of antithrombin Northwick Park and Glasgow, respectively. Further analyses of these novel tryptic peptides were carried out by V8 protease treatment and sequential Edman degradation coupled with mass spectrometric analysis of the shortened peptides. This established that these peptides comprised the amino acid sequence Ala371-Arg399, but with single amino acid substitutions at the reactive site, Arg393 replaced by Cys (in antithrombin Northwick Park) and by His (in antithrombin Glasgow).
Erdjument
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Department of Haematology, Charing Cross and Westminster Medical School, Hammersmith, London, United Kingdom.
Lane
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Panico
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Di Marzo
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Morris
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HR
D000595:Amino Acid Sequence; D000990:Antithrombin III / Q000235:genetics; D001120:Arginine; D003488:Cyanogen Bromide; D003545:Cysteine; D014644:Genetic Variation; D006639:Histidine; D006801:Humans; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D010446:Peptide Fragments; D012697:Serine Endopeptidases; D013927:Thrombosis / Q000235:genetics; D014357:Trypsin
D010446:Peptide Fragments; C054089:antithrombin III Glasgow; C046707:antithrombin III Northwick Park; D006639:Histidine; D000990:Antithrombin III; D001120:Arginine; D012697:Serine Endopeptidases; C028344:glutamyl endopeptidase; D014357:Trypsin; D003545:Cysteine; D003488:Cyanogen Bromide