Synthesis and Application of the Affinity Labeling Reagent Specific for Nucleotide-Binding Site in Proteins

具有核苷酸结合位点的蛋白质在细胞内的核酸和能量代谢以及细胞间的信号转导中起着重要作用。为了阐明这些蛋白质的功能，我们尝试制备一种新的特异性的蛋白质核苷酸结合位点的亲和标记试剂，选择吡哆醛作为蛋白质中氨基的反应基团，与核苷酸结合（腺苷或鸟苷）。磷酸基团的数目从2到4到c不等;改变反应基团与亲和基团之间的距离，合成了一组核苷多磷酸吡哆醛，并将其应用于兔肌乳酸脱氢酶、兔肌腺苷酸激酶、大肠杆菌F_1-ATP酶、兔骨骼肌肌浆网Ca^<2+>-转运ATP酶、兔肌肉磷酸化酶激酶和Ha-ras癌基因产物p21。发现任何核苷多磷酸吡哆醛都与这些蛋白质中的核苷酸结合位点反应，对修饰蛋白的结构分析结果表明，修饰蛋白中的赖氨酰残基是标记位点，因此我们合成的化合物是核苷酸（ATP和GTP）结合位点的优异亲和标记物。核苷酸结合位点的结构特征，特别是关于富含甘氨酸的区域的参与，可以从这些试剂对蛋白质的反应性的差异来预测。

Protein with nucleotide-binding site play important roles in the nucleic acid and energy metabolisms within cells as well as in the intercellular signal transduction. To elucidate the functions of these proteins,we have tried to prepare the novel affinity labeling reagent specific for the nucleotide-binding site in proteins.Pyridoxal was selected as a reactive group for amino groups in proteins, and was conjugated with nucleotides(adenosine or guanosine).Numbers of the phosphate group was varied from 2 to 4 to c;hange the distance between the reactive and affinity groups.Thus a group of nucleoside polyphosphopyridoxals were synthesized and applied to various proteins including rabbit muscle lactate dehydrogenase, rabbit muscle adenylate kinas,Escherichia coli F_1-ATPase,rabbit skeletal muscle sarcoplasmic reticulum Ca^<2+>-transporting ATPase,rabbit muscle phosphorylase kinase, and Ha-ras oncogene product p21.Any nucleoside polyphosphopyridoxal was found to be reactive for the nucleotide-binding site in these proteins,resulting in the rapid inactivation The results of structural analyses of the modified proteins could identify the specific lysyl residues as labeled sites.The compounds we synthesized are therefore excellent affinity labels for the nucleotide (ATP and GTP) binding sites.The structural feature,especially regarding the involvement of a glycine-rich region,of the nucleotide-binding sites may be predicted from the difference in the reactivities of these reagents to proteins.