Affinity‐Purified Anti‐B‐50 Protein Antibody: Interference with the Function of the Phosphoprotein B‐50 in Synaptic Plasma Membranes
Affinity‐Purified Anti‐B‐50 Protein Antibody: Interference with the Function of the Phosphoprotein B‐50 in Synaptic Plasma Membranes
复制标题
亲和纯化的抗 B-50 蛋白抗体：干扰突触质膜中磷蛋白 B-50 的功能
DOI：
10.1111/j.1471-4159.1983.tb04747.x
复制
发表时间：
1983
影响因子：
4.7
通讯作者：
W. Gispen
中科院分区：
文献类型：
--
作者：
A. Oestreicher;C. J. Dongen;H. Zwiers;W. Gispen
摘要：利用亲和纯化的抗B‐50蛋白抗体，研究了先前提出的B‐50蛋白磷酸化状态与突触质膜多磷肌醇代谢的关系。抗体是针对从大鼠脑中获得的富含B - 50蛋白及其促肾上腺皮质激素敏感蛋白激酶的膜提取物产生的。Anti - B - 50蛋白免疫球蛋白通过亲和层析纯化，该固体免疫吸附剂由改进的方法分离的B - 50蛋白制备而成。纯化抗体仅与B - 50和B - 60蛋白（B - 50的蛋白水解衍生物）反应，通过十二烷基硫酸钠凝胶免疫过氧化物酶法进行评估。这些抗体特异性抑制突触质膜中B‐50蛋白的内源性磷酸化，而不显著影响其他膜蛋白的磷酸化。这种抑制作用伴随着突触质膜中磷脂酰肌醇4,5 -二磷酸和磷脂酸形成的变化，而磷脂酰肌醇4 -磷酸的形成没有改变。ACTH 1-24抑制膜中B - 50蛋白内源性磷酸化仅与磷脂酰肌醇4 -磷酸磷酸化增强为磷脂酰肌醇4,5 -二磷酸相关。这些数据支持了我们关于大鼠脑膜中B - 50蛋白和磷脂酰肌醇4 -磷酸激酶功能相互作用的假设。证据表明，纯化的抗B‐50蛋白抗体可用于特异性探测B‐50蛋白在膜中的功能。
Abstract: Affinity‐purified anti‐B‐50 protein antibodies were used to study the previously proposed relationship of the phosphorylation state of B‐50 protein and polyphosphoinositide metabolism in synaptic plasma membranes. Antibodies were raised against a membrane extract enriched in the B‐50 protein and its adrenocorticotropin‐sensitive protein kinase, obtained from rat brain. Anti‐B‐50 protein immunoglobulins were purified by affinity chromatography on a solid immunosorbent prepared from B‐50 protein isolated by an improved procedure. The purified antibodies reacted only with the B‐50 and B‐60 protein, a proteolysis derivative (of B‐50), as assessed by the sodium dodecyl sulfate‐gel immunoperoxidase method. These antibodies inhibited specifically the endogenous phosphorylation of B‐50 protein in synaptic plasma membranes, without affecting notably the phosphorylation of other membrane proteins. This inhibition was accompanied by changes of the formation of phosphatidylinositol 4,5‐diphosphate and phosphatidic acid in synaptic plasma membranes, whereas formation of phosphatidylinositol 4‐phosphate was not altered. Inhibition by ACTH 1–24 of the endogenous phosphorylation of B‐50 protein in membranes was associated only with an enhancement of the phosphorylation of phosphatidylinositol 4‐phosphate to phosphatidylinositol 4,5‐diphosphate. These data support our hypothesis on the functional interaction of B‐50 protein and phosphatidylinositol 4‐phosphate kinase in rat brain membranes. The evidence shows that purified anti‐B‐50 protein antibodies can be used to probe specifically the function of B‐50 protein in membranes.