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

点击翻译按钮获取中文摘要

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.