Isolation and characterization of delta-subspecies of protein kinase C from rat brain.

The delta-subspecies of protein kinase C (delta PKC) was purified to near homogeneity from the Triton X-100 extract of the rat brain particulate fraction by successive chromatographies on S-Sepharose fast flow, phenyl 5PW, heparin 5PW, hydroxyapatite, and Mono Q columns. The purified enzyme was a doublet with molecular masses of 78 and 76 kDa on SDS/PAGE. The doublet proteins were separated partially by Mono Q column chromatography; both were recognized by the antibodies raised against synthetic oligopeptides, parts of the deduced amino acid sequence of the rat delta PKC. Protein phosphatase 2A treatment suggested that the 78-kDa protein was a phosphorylated form of the 76-kDa protein. To confirm the structural and genetic identity of the doublet proteins, delta PKC was expressed in COS 7 cells by transfecting its cDNA-constructed plasmid and was purified for comparison. This recombinant enzyme was also a doublet. The enzymes isolated from the brain and COS 7 cells showed identical reactivities with delta PKC-specific antibodies, chromatographic behaviors, and V8 protease peptide mappings. In addition, these two enzyme preparations were indistinguishable from each other in their responses to phosphatidylserine, diacylglycerol, phorbol esters, free fatty acids, Ca2+, and enzyme inhibitors. Comparison was also made between the enzymologic properties of delta PKC and alpha PKC, which were distinctly different from each other. IMAGES:

本研究从大鼠脑微粒体组分的曲拉通X-100（Triton X-100）提取液中，通过依次经过S-琼脂糖快速流柱（S-Sepharose fast flow）、苯基5PW柱（phenyl 5PW）、肝素5PW柱（heparin 5PW）、羟基磷灰石柱（hydroxyapatite）以及Mono Q柱的连续层析步骤，将蛋白激酶Cδ亚型（delta PKC）纯化至接近均一的纯度。经十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS/PAGE）检测，纯化得到的酶呈现分子量分别为78 kDa和76 kDa的双条带。该双条带蛋白可通过Mono Q柱层析实现部分分离；两者均可被针对大鼠delta PKC推导氨基酸序列中部分合成寡肽所制备的抗体识别。经蛋白磷酸酶2A处理后的实验结果表明，78 kDa的蛋白为76 kDa蛋白的磷酸化形式。为验证该双条带蛋白的结构与遗传同一性，通过转染其经互补DNA（cDNA）构建的质粒，在COS-7细胞中表达delta PKC并纯化以用于对照比较。该重组酶同样呈现双条带特征。从大鼠脑组织与COS-7细胞中分离得到的酶，在针对delta PKC的特异性抗体反应性、层析行为以及V8蛋白酶肽谱分析方面均表现出一致性。此外，这两份酶制剂在对磷脂酰丝氨酸、二酰甘油、佛波酯、游离脂肪酸、Ca²+以及酶抑制剂的响应特性上也无显著差异。本研究还对delta PKC与蛋白激酶Cα亚型（alpha PKC）的酶学特性进行了比较，二者的酶学特性存在显著差异。图片：