大頭鰱腦脊髓液鈣離子結合蛋白p64的純化與特性分析

Abstract

鯉科魚類腦脊髓液中蛋白質的濃度（23mg／ml）約為哺乳類腦脊髓液蛋白質濃度（0.2mg／ml-0.5mg／ml）的五十到一百倍，因此鯉科魚類腦脊髓液是研究腦脊髓液蛋白質一個很好的系統．本文利用硫酸胺沉澱，Sephacryl - S200分子篩選管柱色層分析，Phenyl-Sepharose CL-4B親水性作用色層分析從大頭鰱腦脊髓液中純化出一蛋白（p64），p64蛋白質為醣蛋白，醣類分析結果以葡萄糖含量最高。用西方點墨法發現p64蛋白質也存在於血液及大腦萃取液中。跑完SDS聚丙烯醯胺膠後將蛋白質轉移到NC paper上，以Ca2＋當探針（probe），結果發現p64為一鈣離子結合蛋白，以biontinylated p64當探針（probe），在腦脊髓液及血液中有一分子質量約為90kDa的蛋白質，在大腦萃取液中一分子質量約為70kDa的蛋白質為p64的標的分子。以synaptosome作為蛋白質磷酸化實驗的材料，不論在有Ca2＋或是沒有Ca2＋的情形下，加入p64都會加強蛋白質磷酸化的程度。目前仍不清楚p64蛋白質是經由那些機制來活化蛋白質磷酸化反應。此蛋白質在腦脊髓液的作用可能是將鈣離子運送進入腦脊髓液，或是負責鈣離子在腦脊髓液中的運輸。封p64蛋白質活化蛋白質磷酸化機制的闡明可使我們對細胞內訊息的傳遞更加瞭解。

The protein concentration in the carp CSF, 23 mg/ml, is much higher than that in the mammalian CSF, 0.2-0.5 mg/ml. Therefore, carp CSF serves as a good system for the studies of CSF proteins. A Ca2+-binding protein was purified from carp CSF with a molecular mass of 64 kDa (p64) on SDS polyacrylamide gel. Using ammonium sulfate precipitation, Sepharcyl S200 gel filtration chromatography, and Phenyl-Sepharose CL-4B hydrophobic interaction chromatography, we have purified this p64. we have found that p64 also exists in serum and brain cytosol by Western blotting analysis. Ca2+-binding assay and protein-protein interaction assay with biotinylated p64 as a probe were performed after electrophoretic transfer of proteins from SDS ployacrylamide gel to nitricellulose paper. We have found purified p64 has the Ca2+-binding ability. In addition, there are two target proteins of p64, one with a molecularmass of 90 kDa present in serum and CSF, another with a molecularmass of 70 kDa present in brain cytosol. In the protein phosphorylation assay, p64 can enhance the level of protein phosphorylation in the synaptosome, in the presence or absence of Ca2+. Therefore, p64 may play an important role in signal transduction within the brain cells.