Juskevich J C, Kuhn D M, Lovenberg W
J Biol Chem. 1983 Feb 10;258(3):1950-3.
Calmodulin was removed from brain cytosol by DEAE-52 chromatography or by affinity chromatography employing fluphenazine-Sepharose. The substrates phosphorylated by endogenous protein kinase after chromatography differed depending on the method used, and both chromatographic methods altered the phosphorylation pattern as compared to untreated cytosol. Cytosol, chromatographed on fluphenazine-Sepharose, retained most of the characteristics of untreated cytosol. Both calmodulin and phospholipids increased the phosphorylation of specific but separate brain cytosol proteins in a Ca2+-dependent manner. The effects of phospholipids could be mimicked by the detergent, sodium dodecyl sulfate, and the hydrophobic probe, 8-anilino-1-naphthalenesulfonate. Furthermore, the calmodulin-induced increase in phosphorylation, but not that produced by phospholipids, was blocked by 8-anilino-1-naphthalenesulfonate. These results suggest that the effects of phospholipids may not be due to the presence of a specific phospholipid-sensitive protein kinase in cytosol, but rather to a general interaction of hydrophobic probes with either specific substrate proteins or with the Ca2+-calmodulin-dependent protein kinase itself.
通过DEAE - 52柱色谱法或使用氟奋乃静 - 琼脂糖亲和色谱法从脑细胞质溶胶中去除钙调蛋白。色谱分离后，内源性蛋白激酶磷酸化的底物因所用方法而异，与未处理的细胞质溶胶相比，两种色谱方法都改变了磷酸化模式。在氟奋乃静 - 琼脂糖上进行色谱分离的细胞质溶胶保留了未处理细胞质溶胶的大部分特性。钙调蛋白和磷脂均以Ca2 + 依赖的方式增加特定但不同的脑细胞质溶胶蛋白的磷酸化。磷脂的作用可以被去污剂十二烷基硫酸钠和疏水探针8 - 苯胺基 - 1 - 萘磺酸盐模拟。此外，8 - 苯胺基 - 1 - 萘磺酸盐可阻断钙调蛋白诱导的磷酸化增加，但不能阻断磷脂产生的磷酸化增加。这些结果表明，磷脂的作用可能不是由于细胞质溶胶中存在特定的磷脂敏感蛋白激酶，而是由于疏水探针与特定底物蛋白或与Ca2 + - 钙调蛋白依赖性蛋白激酶本身的一般相互作用。
