微囊藻毒與蛋白質磷酸酵素間之共價鍵形成的動力學分析

Abstract

在稍早的研究中，已知微囊藻毒與蛋白質磷酸酵素PP-1/PP-2A 間會形成共價鍵，導致 兩者永久性的結合，然在其共價鍵結形成動力學方面的研究結果，卻因分析方法及使用毒 素種類的差異而有顯著的不同。在本研究中，嘗試開發基質輔助脫附-飛行時間質譜儀的方 法，用以偵測蛋白質磷酸酵素在與微囊藻毒形成共價鍵結時，所出現分子量加成的訊號， 以此分析PP-1 與MCYST-LR 共價鍵形成之動力學。研究結果顯示，共價鍵結是以十分緩 慢的方式形成，每小時形成共價產物的PP-1 百分比低於5 %。此與Craig et al (1996) 的觀 察結果近似，同時支持其用以解釋微囊藻毒與PP-1 作用之“two-step mechanism” 假說，亦 即毒素先以非共價鍵力量，快速與PP-1 結合、抑制後，再逐漸形成共價鍵結的說法；共價 鍵結與酵素失活並無直接的關連存在。

Formation of microcystin-phosphatase adducts formation is a clear indication of microcystin attach on phosphatases. However, the kinetics of microcystin-phosphatase adduct formation has been controversial due to the uses of different microcystin isoforms and measuring methodologies. Here, we used matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) to measure the changes in relative intensities of molecular weight signals upon mixing of phosphatase and microcystin, which allowed us to monitor the kinetics of covalent adduct formation. Our data clearly indicated that the shifts of signals upon adduct formation and a slow and steady covalent interaction between MCYST-LR and PP-1c were observed. These observations are in agreement with the results of Craig et al (1996) and support their “two-step mechanism” proposed to elucidate the interaction between PP-1c and microcystins.