裂殖性酵母菌 S. pombe 2476 鎘誘導蛋白質之 純化與性質檢定

Abstract

裂殖性酵母菌 Schizosaccharomyces pombe ATCC 2476 生長在 1 mM 鎘濃度環境下，仍能維持細胞生長。經由 HPLC 分析結果，得知 ATCC 2476 在鎘的誘導下，並不會產生 PC 與鎘螯合。將其細胞粗抽液經一連串純化步驟與製備式電泳溶離，再以二維圖譜分離，挖出可與鎘結合的若干色點進行質譜分析 LC-MS/MS，所得到蛋白質身分，與基因庫資料序列比對，得知所誘生之鎘結合蛋白質為：cyclophilin, nuclear transport factor 2 (NTF2), cyclin-dependent kinase regulatory subunit (CKS), 2,3-bisphosphoglycerate -dependent phosphoglycerate mutase, thioredoxin (Trx1) 及WD 蛋白質。推測當裂殖性酵母菌細胞受到外來鎘離子逆境時，thioredoxin 先與鎘離子結合：當過多鎘離子進入細胞質，會在細胞質中產生大量自由基，此時具抗氧化能力之 thioredoxin 及 cyclophilin 可能會形成第二道防線消滅自由基。

Fission yeast (Schizosaccharomyces pombe ATCC 2476) survives the toxic growing condition of 1 mM Cd. However, the HPLC analysis showed that it does not produce the heavy metal binding peptides, phytochelatins. The crude extract of the fission yeast was purified by a series of chromatographic column, and the final step of preparative electrophoresis; several proteins which could bind Cd were further isolated from 2-dimensional electrophoresis, and then subjected to LC/MS/MS. These proteins were identified as cyclophilin, nuclear transport factor 2 (NTF2), cyclin-dependent kinase regulatory subunit (CKS), 2,3-bisphosphoglycerate- dependent phosphoglycerate mutase, thioredoxin (Trx1) and WD protein. It is proposed that the fission yeast might chelate the invading Cd by its thioredoxin first, then the free radical might increase and will be removed by thioredoxin and clyclophilin if the environmental Cd keeps increasing, which serve as the second defense line.