Purification of the catalytically active phosphorylated form of insulin receptor kinase by affinity chromatography with O-phosphotyrosyl-binding antibodies.
Purification of the catalytically active phosphorylated form of insulin receptor kinase by affinity chromatography with O-phosphotyrosyl-binding antibodies.
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使用 O-磷酸酪氨酰结合抗体通过亲和层析纯化催化活性磷酸化形式的胰岛素受体激酶。
DOI：
10.1016/0003-9861(85)90491-6
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发表时间：
1985
影响因子：
3.9
通讯作者：
Shafer,JA
中科院分区：
文献类型：
--
作者：
Pang,DT;Sharma,BR;Shafer,JA
从人胎盘中分离出具有催化活性的酪氨酸磷酸化形式的胰岛素受体激酶，其方法是利用完整的α2β2形式的胰岛素受体在胰岛素促进的酪氨酸残基自磷酸化和伴随的酪氨酸激酶活化的倾向。固定化蛋白a - sepharose （Ab-protein a）上的o -磷酸酪氨酸结合抗体柱吸附和半抗原洗脱纯化酪氨酸磷酸化胰岛素受体。以胎盘膜溶解后的蛋白为原料，用固定化小麦胚芽凝集素层析纯化。在Ab-protein A层析去除预先存在的含有go -磷酸酪氨酸的蛋白后，未吸附到Ab-protein A柱上的部分与胰岛素孵育，并短暂用ATP处理，以最大限度地选择性胰岛素受体的自磷酸化。该材料随后在ab蛋白a上进行层析，尽管起始材料中存在的完整α2β2形式的胰岛素受体仅占蛋白质的一小部分（~0.2%），仅占胰岛素受体结合形式的~20%，但从柱中以≥80%的纯度洗脱（用10 mmp-nitrophenyl phosphate）。与利用固定胰岛素或抗胰岛素受体抗体吸附胰岛素受体的亲和层析方法相比，b-蛋白A的层析方法似乎具有优势，因为这些方法不能将完整的α2β2形式的胰岛素受体与不经历胰岛素促进的自磷酸化的胰岛素结合形式的受体分离。
The catalytically active, tyrosyl-phosphorylated form of insulin receptor kinase was isolated from human placenta by a procedure which exploits the propensity for the intactα2β2form of insulin receptor to undergo insulin-promoted autophosphorylation at tyrosyl residues and concomitant activation as a tyrosyl kinase. Purification of tyrosylphosphorylated insulin receptor was effected by adsorption on and elution (with a hapten) from a column ofO-phosphotyrosyl-binding antibody immobilized on protein A-Sepharose (Ab-protein A). The starting material for the purification process was protein which had been solubilized from placental membranes and purified by chromatography on immobilized wheat germ agglutinin. After chromatography on Ab-protein A to remove preexistingO-phosphotyrosyl-containing proteins, the fraction which did not adsorb to the Ab-protein A column was incubated with insulin and briefly treated with ATP so as to maximize selective autophosphorylation of insulin receptor. This material was then subjected to chromatography on Ab-protein A. Although the amount of the intactα2β2form of insulin receptor present in the starting material was only a small fraction of the protein (~0.2%) and only ~20% of the insulin-binding forms of the receptor present, it was eluted (with 10 mmp-nitrophenyl phosphate) from the column in ≥80% purity. Chromatography on Ab-protein A appears to have an advantage over the alternative affinity chromatographic procedures which utilize immobilized insulin or antiinsulin receptor antibody to adsorb insulin receptor, since these procedures do not resolve the intactα2β2form of insulin receptor from the nicked insulin-binding forms of the receptor which do not undergo insulin promoted autophosphorylation.
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影响因子：
4
作者：
RUFF, HA;LAWSON, KR
通讯作者：
LAWSON, KR
6 个月大的孩子视觉和触觉探索之间的关系。
DOI：
--
发表时间：
1985
期刊：
影响因子：
--
作者：
E. Bushnell;L. Shaw;David Strauss
通讯作者：
David Strauss
正常婴儿
DOI：
--
发表时间：
1967
期刊：
影响因子：
--
作者：
Jerome Hellmuth
通讯作者：
Jerome Hellmuth
活动水平作为幼儿气质的一个维度：其与有机体特异性假设的相关性。
影响因子：
4.6
作者：
M. Gandour
通讯作者：
M. Gandour
视觉、触觉和操作体验对婴儿物体视觉识别记忆的影响。
影响因子：
4
作者：
A. Gottfried;S. Rose;W. Bridger
通讯作者：
W. Bridger