Fan M Z, Chan W Y, Rennert O M
Physiol Chem Phys Med NMR. 1983;15(1):69-79.
Incubation of McCoy cultures with medium containing 14C-putrescine resulted in the incorporation of 14C-polyamine into intracellular proteins. A greater than 100,000 dalton 14C-polyamine conjugated protein was present in the McCoy cell lysate supernatant (CLSP). CLSP was heterogeneous containing proteins with pIs ranging from 4.55 to 5.50. The major proteins had pIs of 4.55 and 5.20. Electrophoresis of solubilized McCoy cell lysate pellet revealed a 14C-polyamine conjugated protein peak with Mr approximately or equal to 70,000 (CLPP). Both CLSP and CLPP contained bound polyamine. The major CLSP polyamine was spermidine while spermine exceeded the other two polyamines (putrescine and spermidine) in CLPP. About 25% of the polyamines associated with CLSP and CLPP were covalently bound with the exception of CLSP putrescine where 62.1% was covalently bound. Results suggested the presence of a polyamine protein isopeptide bond in CLSP. Sephadex gel filtration of cultured medium resulted in the identification of two macromolecular polyamine-containing fractions (MP1, Mr greater than 100,000 and MP2, M = 60,000-70,000). Antibody raised in rabbits against a membrane-organelle preparation cross-reacted with Sephadex gel filtration derived MP1 but not with peak MP2 suggesting that MP1 but not MP2 might be a membrane constituent. Antibody raised against medium polyamine conjugated protein peak 2 (MP2) cross-reacted with the cell lysate supernatant indicating that MP2 was present in the cytosol. It did not cross-react with the cell lysate pellet preparation. Antibody against MP2 also formed a precipitation band with MP1 indicating that there might be a common antigenic site on MP1 and MP2.
用含有14C-腐胺的培养基培养 McCoy 细胞，结果14C-多胺被整合到细胞内蛋白质中。在 McCoy 细胞裂解物上清液（CLSP）中存在一种分子量大于100,000道尔顿的14C-多胺结合蛋白。CLSP 具有异质性，含有等电点范围为4.55至5.50的蛋白质。主要蛋白质的等电点为4.55和5.20。对溶解的 McCoy 细胞裂解物沉淀进行电泳，显示出一个分子量约为70,000（CLPP）的14C-多胺结合蛋白峰。CLSP 和 CLPP 都含有结合的多胺。CLSP 中的主要多胺是亚精胺，而在 CLPP 中，精胺的含量超过了其他两种多胺（腐胺和亚精胺）。与 CLSP 和 CLPP 相关的多胺中约25%是共价结合的，CLSP 中的腐胺除外，其62.1%是共价结合的。结果表明 CLSP 中存在多胺蛋白异肽键。对培养基进行 Sephadex 凝胶过滤，鉴定出两个含大分子多胺的组分（MP1，分子量大于100,000；MP2，分子量为60,000 - 70,000）。用针对膜细胞器制剂免疫兔子产生的抗体与 Sephadex 凝胶过滤得到的 MP1 发生交叉反应，但与 MP2 峰不发生交叉反应，这表明 MP1 可能是膜成分，而 MP2 不是。针对培养基多胺结合蛋白峰2（MP2）产生的抗体与细胞裂解物上清液发生交叉反应，表明 MP2 存在于细胞质中。它与细胞裂解物沉淀制剂不发生交叉反应。针对 MP2 的抗体也与 MP1 形成沉淀带，表明 MP1 和 MP2 上可能存在共同的抗原位点。
