Bryant D T, Critch S
Biochem J. 1986 Aug 1;237(3):781-7. doi: 10.1042/bj2370781.
Vitamin D-dependent Ca2+-binding protein from pig duodenum was hydrolysed with trypsin in the presence of Ca2+ and two products were obtained: T1, which differed from the native protein by loss of Ac-Ser-Ala-Gln-Lys from the N-terminus and Ile-Ser-Gln-OH from the C-terminus, and T2, which differed from T1 by loss of a C-terminal lysine. The hydrolysis inactivated one of the two high-affinity Ca2+-binding sites on the native protein, and the remaining site was stable in T1 but labile in T2 when the proteins were Ca2+-free. Binding studies showed that T1 had Kd values of 2.8 +/- 0.1 nM, 57 +/- 13 microM and 0.8 +/- 0.3 microM for Ca2+, Mg2+ and Mn2+ respectively, and T2 had Kd 2.2 +/- 0.3 nM for Ca2+. The affinity for Mn2+, together with the other Kd values, identified the site on T1 as the site on the native protein previously found to have Kd 0.6 microM for Mn2+, rather than one with Kd 50 microM for Mn2+. In contrast with both the native protein and another form of the protein with a single Ca2+-binding site, the intrinsic fluorescence of T1 and T2 was little affected by the addition of Ca2+. It was concluded that the active binding site in T1 and T2, and also the site in the native protein with the higher affinity for Mn2+, was probably in the C-terminal half of the molecule.
猪十二指肠中维生素D依赖性钙结合蛋白在钙离子存在的情况下用胰蛋白酶水解，得到两种产物：T1，其与天然蛋白的不同之处在于N端失去了Ac-Ser-Ala-Gln-Lys，C端失去了Ile-Ser-Gln-OH；以及T2，其与T1的不同之处在于C端失去了一个赖氨酸。这种水解使天然蛋白上两个高亲和力钙结合位点中的一个失活，当蛋白处于无钙状态时，剩余的位点在T1中稳定，但在T2中不稳定。结合研究表明，T1对钙离子、镁离子和锰离子的解离常数（Kd）分别为2.8±0.1 nM、57±13 μM和0.8±0.3 μM，T2对钙离子的Kd为2.2±0.3 nM。对锰离子的亲和力以及其他Kd值表明，T1上的位点是天然蛋白上先前发现对锰离子Kd为0.6 μM的位点，而不是对锰离子Kd为50 μM的位点。与天然蛋白和另一种具有单个钙结合位点的蛋白形式不同，添加钙离子对T
