Koide T, Odani S, Takahashi K, Ono T, Sakuragawa N
Proc Natl Acad Sci U S A. 1984 Jan;81(2):289-93. doi: 10.1073/pnas.81.2.289.
Structural analyses of a hereditary abnormal antithrombin III, antithrombin III Toyama, which has normal progressive antithrombin activity but no heparin cofactor activity, have been carried out to elucidate the molecular abnormality causing recurrent thrombophlebitis of a patient and to identify an amino acid residue essential for the binding with heparin. Abnormal antithrombin III was reduced, S-pyridylethylated, and treated with cyanogen bromide. Eleven fragments were isolated by the combination of Sephadex G-50 gel filtration and reversed-phase HPLC and compared with those from normal antithrombin III. One large fragment (CN-III) that appeared to have a different amino acid composition from that of the corresponding fragment from normal antithrombin III was digested with trypsin, and the digests were separated by HPLC. The abnormal peptide was identified by comparing the peptide map with that from normal antithrombin III. Amino acid sequence analysis of the abnormal peptide indicated that the arginine-47 of normal antithrombin III had been replaced by cysteine in antithrombin III Toyama. One base mutation, C leads to T, in the 5' terminal position of the arginine-47 genetic codon (CGT) is probably responsible for this substitution. These results also suggest that arginine-47 is an essential amino acid residue for the binding with heparin.
对遗传性异常抗凝血酶III（富山抗凝血酶III）进行了结构分析，该抗凝血酶III具有正常的进行性抗凝血酶活性，但无肝素辅因子活性，目的是阐明导致一名患者反复发生血栓性静脉炎的分子异常，并确定与肝素结合所必需的氨基酸残基。将异常抗凝血酶III进行还原、S-吡啶基乙基化，并用溴化氰处理。通过Sephadex G-50凝胶过滤和反相高效液相色谱相结合的方法分离出11个片段，并与正常抗凝血酶III的片段进行比较。用胰蛋白酶消化一个似乎与正常抗凝血酶III相应片段氨基酸组成不同的大片段（CN-III），消化产物通过高效液相色谱分离。通过将肽图与正常抗凝血酶III的肽图进行比较来鉴定异常肽。异常肽的氨基酸序列分析表明，富山抗凝血酶III中正常抗凝血酶III的精氨酸-47被半胱氨酸取代。精氨酸-47遗传密码子（CGT）5'末端位置的一个碱基突变，即C突变为T，可能是这种取代的原因。这些结果还表明，精氨酸-47是与肝素结合所必需的氨基酸残基。
