Abstract
綠豆液泡囊上的腺核甘三磷酸水解酵素(ATPase)是從綠豆中的白化下胚軸 經過兩個步驟的純化而得到的。它包含有使質子移位和水解腺核甘三磷 酸 (ATP) 的活性。 本實驗是以 Phenylglyoxal (PGO) 和 2,3-Butanedione (BD) 為指標， 利用化學修飾法來修飾白化綠豆幼苗的 液泡囊膜上的腺核三磷酸水解酵素中活化部位的精胺酸 (Arginine) 殘基 ，進而研究酵素的結構和功能。 結果顯示 PGO 和 BD 能抑制腺核甘三磷 酸水解酵素的活性及質子位移的能力。而其活性抑制的形式為競爭型形式 且抑制的反應級數為 0.94， 0,89， 這顯示至少有一個精胺酸與腺核甘 三磷酸水解酵素的去活化有關。 然而由螢光光譜圖中發現，被 PGO 和 BD 標示上去的腺核甘三磷酸水解酵素所發出來的螢光有往短波長方向移 動的現像。另外的由圓形雙色光譜儀中也發現不管有沒有抑制劑的存在， 其二級結構似乎沒什麼太大的改變。在先前的研究指出， Fluorescein 5'-isothiocyanat ( FITC) 是和離胺酸 (Lysine) 作用的，其專一性很 強，且也已知這個離胺酸是位於腺核甘三磷酸的活化位置，跟酵素的活性 有密切的關連。從本篇論文中可以得知被 FITC 所標示的那一段生太 (peptide)已被定序出來了。其順序為 Ile-Ser-Gly-Trp-Asn-Tyr-Pro- Val-Val-x-Lys-x-Leu-， 而其中的離胺酸 (Lysine) 就是 FITC 標示的 位置。 The tonoplast ATPase was purified by two-step detergent solu- bilization from etiolated mung bean seedlings (Vigna radiata L.) . The tonoplast contains electrogenic proton- translocating and ATP hydrolytic activity of ATPase. Phenylglyoxal (PGO) and 2,3-butanedione (BD) were used to modify the arginine residue of enzyme. The result showed that PGO and BD inhibited the activities of both solubilized and membrane bound ATPase and its associated proton translocation. The mode of inhibition by PGO and BD were competitive with respect to ATP. The reaction order of PGO and BD inhibition were 0.94 and 0.89, repectively. This indicates that at least one essential arginine residue was involved in the inactivation of ATPase. The blue shift of guanidino-modified ATPase suggested that the labeled arginine residue was in average located in a relatively hydrophobic environment. The circular dichroism (CD) spectra indicated that the secondary structure of ATPase were similar between labeled and control ATPase. In earlier reports, it was suggested that fluorescein 5'-isothiocyanat (FITC) reacted with lysine and residue(s) which was essential for enzymatic activity at substrate binding site. The amino acid sequence of the peptide is Ile-Ser-Gly- Trp-Asn-Tyr-Pro-Val-Val-x-Lys-x-Leu-. It is suggested that FITC probably reacts with this lysine.