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

蛋白质核苷酸结合位点特异性亲和标记试剂的合成及应用

基本信息

批准号：
61470158
负责人：
FUKUI Toshio
金额：
$ 3.84万
依托单位：
Institute of Scientific and Industrial Research
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (B)
财政年份：
1986
资助国家：
日本
起止时间：
1986 至 1987
项目状态：
已结题

来源：
https://kaken.nii.ac.jp/en/grant/KAKENHI-PROJECT-61470158/
关键词：
Affinity label Lysine Nucleotide-binding site pyridoxal phosphate ATP ATPアーゼリシン

项目摘要

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.

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信号转导中起着重要作用。为了阐明这些蛋白的功能，我们试图制备一种针对蛋白质中核苷酸结合位点的新型亲和标记试剂。吡哆醛被选为蛋白质中氨基的反应基团，并与核苷酸（腺苷或鸟苷）偶联。磷酸基团的数目从2到4到c不等；改变反应基团和亲和基团之间的距离。因此，我们合成了一组核苷类多磷酸吡咯酮，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、兔肌磷酸化酶激酶和Ha-ras癌基因产物p21等多种蛋白。在这些蛋白中，任何核苷多磷吡哆醛都能对核苷酸结合位点产生反应，从而导致快速失活。对修饰蛋白的结构分析结果可以识别出特定的赖氨酸残基作为标记位点。因此，我们合成的化合物是核苷酸（ATP和GTP）结合位点的优秀亲和力标签。核苷酸结合位点的结构特征，特别是关于甘氨酸丰富区域的参与，可以从这些试剂对蛋白质的反应性的差异来预测。