Fernlund P, Stenflo J, Roepstorff P, Thomsen J
J Biol Chem. 1975 Aug 10;250(15):6125-33.
Tryptic peptides obtained from normal prothrombin have been compared with those obtained from prothrombin synthesized by cattle given the vitamin K antagonist dicumarol. Two peptides were found which contain vitamin K-dependent structures. These peptides contain residues 4 through 10 and residues 12 through 44, respectively. One of these (residues 4 through 10) has previously been shown to contain gamma-carboxyglutamic acid residues. Digestion of this peptide with aminopeptidase M and carboxypeptidase B yielded a tetrapeptide (residues 6 through 9). Mass spectra of this peptide showed that it has the structure Leu-Glu(CO2)-Glu(CO2)-Val. The structure of the peptide containing residues 12 through 44 was determined by automated degradation in a peptide sequenator. The modified glutamic acid residues were identified by mass spectrometric comparison with the thiohydantoin derivatives of synthetic gamma-carboxyglutamic acid. This approach unequivocally demonstrated that all of the first 10 glutamic acid residues in prothrombin are carboxylated to form gamma-carboxyglutamic acid residues. Evidence is also presented that indicates that these gamma-carboxyglutamic acid residues constitute the entire vitamin K-dependent modification of prothrombin.
将从正常凝血酶原获得的胰蛋白酶肽段与从给予维生素K拮抗剂双香豆素的牛所合成的凝血酶原获得的肽段进行了比较。发现了两个含有维生素K依赖性结构的肽段。这些肽段分别包含第4至10位残基和第12至44位残基。其中一个（第4至10位残基）先前已被证明含有γ-羧基谷氨酸残基。用氨肽酶M和羧肽酶B消化该肽段产生了一个四肽（第6至9位残基）。该四肽的质谱显示其结构为Leu-Glu(CO2)-Glu(CO2)-Val。通过在肽序列分析仪中进行自动降解确定了含有第12至44位残基的肽段的结构。通过与合成γ-羧基谷氨酸的硫代乙内酰脲衍生物进行质谱比较鉴定了修饰的谷氨酸残基。该方法明确证明凝血酶原中前10个谷氨酸残基全部羧化形成γ-羧基谷氨酸残基。还提供了证据表明这些γ-羧基谷氨酸残基构成了凝血酶原的全部维生素K依赖性修饰。
