Naito S, Ueda T
J Biol Chem. 1981 Oct 25;256(20):10657-63.
Protein I is a synaptic protein which serves as an endogenous substrate for cyclic AMP- and calcium-dependent protein kinases. Antibodies raised against purified Protein I have been isolated from rabbit antiserum by affinity chromatography on Protein I-conjugated agarose column. The purified antibodies were identified as immunoglobulin G by sodium dodecyl sulfate polyacrylamide gel electrophoresis and Ouchterlony double immunodiffusion precipitation test. The purified antibodies not only inhibited the phosphorylation of purified Protein I by exogenous cyclic AMP-dependent protein kinase, but also inhibited specifically the phosphorylation of Protein I by endogenous cyclic AMP-dependent protein kinase in a synaptic vesicle fraction, synaptic junctional complex fraction, synaptic membrane fraction, and crude homogenate of cerebrum. The purified anti-Protein I antibodies also inhibited with a similar potency calcium-dependent phosphorylation of Protein I without affecting the phosphorylation of other proteins. The Fab(t) fragment of anti-Protein I immunoglobulin G, which was produced by tryptic digestion, retained the ability to inhibit specifically the phosphorylation of Protein I. This substrate-directed, specific inhibitor of the phosphorylation of Protein I may provide a unique probe for investigating the function of Protein I phosphorylation.
蛋白I是一种突触蛋白，可作为环磷酸腺苷（cAMP）和钙依赖性蛋白激酶的内源性底物。通过在蛋白I偶联琼脂糖柱上进行亲和层析，已从兔抗血清中分离出针对纯化蛋白I产生的抗体。通过十二烷基硫酸钠聚丙烯酰胺凝胶电泳和双向免疫扩散沉淀试验，将纯化的抗体鉴定为免疫球蛋白G。纯化的抗体不仅抑制外源性cAMP依赖性蛋白激酶对纯化蛋白I的磷酸化作用，还特异性抑制突触小泡组分、突触连接复合体组分、突触膜组分和大脑粗匀浆中内源性cAMP依赖性蛋白激酶对蛋白I的磷酸化作用。纯化的抗蛋白I抗体还能以相似的效力抑制蛋白I的钙依赖性磷酸化，而不影响其他蛋白的磷酸化。通过胰蛋白酶消化产生的抗蛋白I免疫球蛋白G的Fab(t)片段，保留了特异性抑制蛋白I磷酸化的能力。这种针对底物的蛋白I磷酸化特异性抑制剂可能为研究蛋白I磷酸化的功能提供一种独特的探针。
