Uchida T, Filburn C R
J Biol Chem. 1984 Oct 25;259(20):12311-4.
An affinity column, prepared by immobilizing phosphatidylserine and cholesterol in polyacrylamide, was utilized in the purification of protein kinase C. Protein kinase activity and phorbol ester binding were monitored by assaying Ca2+ plus phosphatidylserine-dependent phosphorylation of histone H1 and [3H]phorbol dibutyrate binding, respectively. Both activities were present in a cytosolic extract of rabbit renal cortex, eluted together from a DEAE-cellulose column, bound to the affinity column in the presence of Ca2+, and eluted symmetrically upon application of EGTA. Recovery from the affinity column was high (30-50%) and resulted in as much as a 6000-7700-fold purification, depending on the region of the DEAE-cellulose peak that was applied. Following affinity column purification, protein kinase and phorbol ester binding activity eluted symmetrically upon gel filtration, with a molecular weight of approximately 80 kDa. A protein of the same size was present in silver-stained gels following sodium dodecyl sulfate-polyacrylamide gel electrophoresis of affinity column purified samples from the DEAE-cellulose peak. From 2-4 other, smaller proteins were also present, their number and relative amounts depending on the region of the DEAE-cellulose peak used. These data indicate that Ca2+-dependent/binding to a polyacrylamide-immobilized phospholipid provides a useful technique for purification of protein kinase C as well as other, unidentified proteins exhibiting a Ca2+ plus phospholipid-dependent interaction.
通过将磷脂酰丝氨酸和胆固醇固定在聚丙烯酰胺中制备的亲和柱，用于蛋白激酶C的纯化。分别通过检测Ca2+加磷脂酰丝氨酸依赖性组蛋白H1磷酸化和[3H]佛波醇二丁酸酯结合来监测蛋白激酶活性和佛波醇酯结合。两种活性均存在于兔肾皮质的胞质提取物中，从DEAE-纤维素柱上一起洗脱，在Ca2+存在下与亲和柱结合，并在加入EGTA后对称洗脱。从亲和柱上的回收率很高（30-50%），根据所应用的DEAE-纤维素峰的区域，可实现高达6000-7700倍的纯化。亲和柱纯化后，蛋白激酶和佛波醇酯结合活性在凝胶过滤时对称洗脱，分子量约为80 kDa。对来自DEAE-纤维素峰的亲和柱纯化样品进行十二烷基硫酸钠-聚丙烯酰胺凝胶电泳后，银染凝胶中存在相同大小的蛋白质。此外还存在2-4种其他较小的蛋白质，其数量和相对含量取决于所使用的DEAE-纤维素峰的区域。这些数据表明，Ca2+依赖性/与固定在聚丙烯酰胺上的磷脂结合为纯化蛋白激酶C以及其他表现出Ca2+加磷脂依赖性相互作用的未鉴定蛋白质提供了一种有用的技术。
