Walker F J
Biochemistry. 1986 Oct 7;25(20):6305-11. doi: 10.1021/bi00368a071.
Protein S, the protein cofactor for activated protein C in the proteolytic inactivation of factor Va, was chemically modified with a mixture of morpholine and formaldehyde. This treatment resulted in the conversion of the gamma-carboxyglutamic acid (Gla) residues of this vitamin K dependent protein to gamma-methyleneglutamic acid. With a 10,000-fold molar excess of morpholine and formaldehyde over protein S it was found that between 10 and 11 Gla residues could be modified. The degree of modification was proportional to the concentration of the modifying reagents used. The modification of as few as two residues resulted in the 70% loss of activity. Calcium inhibited the modification of several residues. In the presence of 3.2 mM calcium ion, a derivative with 2.5 residues modified was prepared that appeared to have full activity. Modification of protein S resulted in the alteration of a number of its properties. The quenching of intrinsic fluorescence by calcium decreased. The quenching effect of terbium ions was also decreased. However, the modified protein and the native protein were equivalent when protein-dependent terbium fluorescence was measured. When modified, protein S would no longer bind to phospholipid vesicles. Finally, the ability of protein S to self-associate was decreased by modification. These findings suggest that the gamma-carboxyglutamic acid residues of protein S may play several roles in the maintenance of structure.
蛋白S是活化蛋白C在蛋白水解失活因子Va过程中的蛋白辅因子，用吗啉和甲醛的混合物对其进行化学修饰。这种处理导致这种维生素K依赖性蛋白的γ-羧基谷氨酸（Gla）残基转化为γ-亚甲基谷氨酸。当吗啉和甲醛的摩尔量比蛋白S过量10000倍时，发现10至11个Gla残基可被修饰。修饰程度与所用修饰试剂的浓度成正比。仅两个残基的修饰就导致70%的活性丧失。钙抑制了几个残基的修饰。在3.2 mM钙离子存在下，制备了一种修饰了2.5个残基的衍生物，其似乎具有完全活性。蛋白S的修饰导致其许多性质发生改变。钙对内在荧光的猝灭作用降低。铽离子的猝灭作用也降低。然而，在测量蛋白依赖性铽荧光时，修饰后的蛋白和天然蛋白相当。修饰后，蛋白S不再与磷脂囊泡结合。最后，修饰降低了蛋白S自我缔合的能力。这些发现表明，蛋白S的γ-羧基谷氨酸残基可能在维持结构中发挥多种作用。
