Takita H, Kuboki Y
Department of Biochemistry, School of Dentistry, Hokkaido University, Sapporo, Japan.
Calcif Tissue Int. 1995 Jun;56(6):559-65. doi: 10.1007/BF00298590.
To clarify calcium-induced conformational changes in bovine bone osteonectin, the protein was labeled with fluorescein isothiocyanate (FITC) in the presence and absence of calcium. By calcium titration using fluorescence spectrometry, it was demonstrated that FITC-osteonectin labeled in the presence of 2 mM CaCl2 showed a much higher affinity for calcium ions than did that labeled in the absence of calcium ions. The midpoint for completion of the increase in the intrinsic fluorescence (K0.5) of the two were 1 x 10(-7) M and 5 x 10(-7) M, respectively. By tryptic digestion and isolation of the fluorescent peptide of both FITC-osteonectins, the site of FITC-labeling was determined to be Lys174. Furthermore, it was found that the efficacy of labeling in this specific binding site was three times higher in the FITC-osteonectin labeled in the presence of 2 mM CaCl2 than in that labeled in the absence of calcium. The results indicate that in the presence of 2 mM CaCl2 the microenvironment around Lys174 of osteonectin was more open to modification than in the absence of calcium.
为了阐明钙离子诱导的牛骨骨粘连蛋白构象变化，在有钙和无钙的情况下，用异硫氰酸荧光素（FITC）标记该蛋白。通过荧光光谱法进行钙滴定，结果表明，在2 mM CaCl2存在下标记的FITC-骨粘连蛋白对钙离子的亲和力远高于无钙离子时标记的FITC-骨粘连蛋白。两者内在荧光增加的中点（K0.5）分别为1×10（-7）M和5×10（-7）M。通过胰蛋白酶消化和分离两种FITC-骨粘连蛋白的荧光肽，确定FITC标记位点为Lys174。此外，还发现，在2 mM CaCl2存在下标记的FITC-骨粘连蛋白在该特异性结合位点的标记效率比无钙时标记的高3倍。结果表明，在2 mM CaCl2存在下，骨粘连蛋白Lys174周围的微环境比无钙时更容易被修饰。
