Abstract
從文獻得知,無機焦磷酸水解酵素存在於高等植物粒線體膜上,並且具有質 子傳遞的能力.在本論文中,我們首先將無機焦磷酸水解酵素從白化綠荳下 胚軸的葉綠體膜上分離出,所用的試劑為丙酮,再經由QA-52, S-100及 Mono-Q等管柱柱層分析,作部份純化,其專一活性由1.33單位提昇到30.4單 位,大約有23倍的純度,而蛋白質的產率為0.89%.我們將此部份純化的無機 焦磷酸水解酵素與在膜上的此酵素作特性分析,比較結果發現幾乎沒有差 異:其最適水解條件在酸鹼值8.0;鎂離子為水解所必須的輔因子,且鎂離子 與無機焦磷酸鹽的比例必須是3,才有最高活性 .大部份的陰陽離子對純化 出的無機焦磷酸水解酵素之水解活性沒有多大影響,但氟離子卻可完全抑 制其活性.此酵素對於修飾劑NEM,DEP和PGO敏感,據此推論半胱銨酸,組織 銨酸和精銨酸可能參與無機焦磷酸水解酵素的水解活性,至於作用的機制 則有待更進一步的探討. It has been known that H+ -PPase, which is able to couple to Pi hydrolysis with proton translocation, was present on thetochondrial membrane of pea stem. In this study, we attempted to isolate this PPase from mung bean mitochondria. First of all, the PPase was solubilized from the mitochondrial membrane by ice- cold acetone and further purified by QA-52, S-100 and Mono-Q chromatographies. Specific activity of the solubilized enzyme increased 23 fold after purification and the yield was 0.9%. We further compared the properties of the solubilized PPaseith membrane-bound PPase. It has found that the solubilizedzyme functioned exactly in the same way as the membrane-bound protein. The optimal pH for the enzyme reaction was 8.0, andagnesium ion was required for PPase activity at a Mg2+/PPi ratio of 3. However, fluoride ion could completely inhibit PPasectivity. The enzyme was sensitive to NEM, DEP and PGO suggesting that cysteine, histidine and arginine residues were involved inhe PPi hydrolysis activity.