本實驗以 IAEDANS (5-[2-(2-iodoacetamido)ehtyla mino]-1-
naphthalene sulfonic acid利用 化學修飾法來修飾白化綠豆幼苗的液泡
囊膜上,腺核甘三磷酸水解酵素中活化部位的半胱胺酸(cysteine)殘基,進
而研究酵素的結構及功能。IAEDANS能抑制ATP水解酵素的活性及其質子位
移的能力。活性抑制的形式為競爭型且抑制的反應級數為0.84,顯示有一
個半胱氨酸殘基與腺核甘三磷酸水解酵素的去活化有關。在螢光共振能量
轉移法中腺核甘三磷酸水解酵素同時標示上IAEDANS與fluorescein
5''-isothioc yanate (FITC). FITC已知能標示腺核甘三磷酸水解酵素中
活化部位之離胺酸(Tzeng CM,Hsu LH andPan RL. Bioc hem J 1992
285:737-743)且被當為螢光團,而IAEDANS為吸光團。由螢光共振能量轉移
法所測得被FITC標示的離胺酸與被IAEDANS標定的半胱胺酸之間的距離
為27.6o。

5-[2-(2-iodoacetamido)ethylamino]-1-naphthale nesulfonicacid
(IAEDANS)wasused to modify the cyst eine residue in the
activesite of tonoplastH+-ATPa se. IAEDANS inhibited the
activityof both solubili zed ATPase and its associated proton
translocation .The mode of inhibition by IAEDANS was
competitive with respect to ATP. The reaction order of IAEDANS
inhibition was 0.84,indicating that at least one essential
cysteine residue was involved in the in- activation of ATPase.
The stoichiometry of IAEDANS was approximately 1.0 mol IAEDANS/
mol of ATPase. For fluorescence spectroscopic study, ATPase was
s uccessively labeled by IAEDANS and fluorescein 5''-
isothiocyanate (FITC). The FITC, a lysine modifier in the
active site of ATPase (Tzeng CM, Hsu LH and Pan RL(1992)
Biochem J 285:737-743) was used as an fluorescence acceptor,
while IAEDANS as the donor. The distance between IAEDANS and
FITC was deter- mined to be 27.6 o by fluorescence
resonance energy transfer.