Abstract
溴化丁二醯亞銨酸(NBS)和溴化二氫基五硝基苯(HNBB)是二種會抑制色銨 酸(Trp)的試劑而抑制Tonoplast ATPase的水解活行，HNBB只修飾Trp而 NBS會修飾Trp以及其它的殘基，在 HNBB對ATPase的抑制與時間及能濃度 皆有關，由抑制時間與活性作圖可以得到反應機級數是0.973，由詞此可 知至少有一個 Trp涉及ATPase的水解活性中，另外由動力分析發現NBS不 改變最大反應速率(Vmax)但酵素對受質的解離度不變(Km)，而HNBB改變Km 但不改變Vmax，受質及其它類似物可保護ATPase抵抗NB S的抑制但不保護 HNBB的抑制可知NBS及HNBB是抑制在不同的位置，更進一步以化學修飾法 顯示ATPase中有七個Trp可被HNBB修飾，但分析後發現只有一個Trp對 ATPase的活行是必須的而個Trp不是位在活化部位 Two modifying reagent, N-bromosuccinimide (NBS) an d 2-hydroxy-5-nitrobenzyl bromide (HNBB), could inhibi t the hydrolysis activity of tonoplast ATPase from eti olated mung bean. Under our condition, HNBB is specifi c for the modification of tryptophan, while NBS could react with several residues besides tryptophan. the in activation of ATPase by HNBB was concentration- and ti me-dependent. From the semilogarithmic plot of time co urse of the inactivation, the reaction order of inacti vation was calculated as 0.973, indicating that at lea st one tryptophan was esstential to the enzymatic acti vity of V-ATPase. Kinetic analysis shows that Km but n ot Vmax of ATPase was changed by NBS, while the Vmax b ut not Km of the enzyme was decreased by HNBB. ATP and its analogs could protect ATPase against NBS but not H NBB, implying that modification sites of NBS and HNBB are not same.