Purification, Characterization, and Spectral Analyses of histidine-tagged Vacuolar H(superscript +)-pyrophosphatase Expressed in Yeast

液泡無機焦磷酸水解酶(V-PPase)利用水解焦磷酸產生能量來驅動質子傳遞造成細胞膜內外的質子梯度，而V-PPase水解及質子傳遞的活性可被許多的離子所調控，相對高濃度的K(上標 +)可以促進V-PPase的活性而F(上標 -)、Na(上標 +)、Ca(上標 2+)以及過多的PP(下標 i)則會抑制它的活性，在本研究中我們利用酵母菌表達系統表達帶有六個組胺酸尾飾的V-PPase接著利用n-dodecylβ-D-maltoside(DDM)當作介面活性劑來將V-PPase由細胞膜中溶解出來，再利用Ni(上標 2+)-NTA親合性層析管注將V-PPase純化出來得到一個分子量大約是73kDa的純化蛋白質，組胺酸尾飾V-PPase的水解活性大約是86.4±7.4μmol PP(下標 i)/mg.h比在細胞膜上的還要高約6.5倍；比較酵母菌表達的組胺酸尾飾V-PPase的活性只有直接由綠豆中純化出來的59%。進一步的分析組胺酸尾飾V-PPase的特性，發現純化的V-PPase和位在細胞膜上的有相似的特性。再則，利用光譜分析純化的組胺酸尾飾V-PPase，發現影響V-PPase活性的這些離子多會造成V-PPase結構上的改變（特別是二級結構的改變）；這個結果證實影響V-PPase的離子是經由改變其構型（二級結構）來影響其活性。

關鍵字

圓二色譜學；金屬親合性層析；液泡焦磷酸水解酶；液泡

並列摘要

Vacuolar proton-translocating pyrophosphatase (V-PPase) generates a proton electrochemical gradient across the membrane by hydrolyzing pyrophosphate for maintenance of acidic condition of vacuoles and translocation of secondary metabolites, ions, and even toxics. The enzymatic activity of V-PPase could be stimulated by relatively high concentration of K(superscript +), but inhibited by F(superscript -), Na(superscript +), Ca(superscript 2+) and excess PP(subscript i). In this study, we used the yeast expression system to express hexa-histidine tagged mung bean V-PPase and employed detergent n-dodecyl β-D-maltoside (DDM) to solubilize the protein from microsomal membrane, followed by a Ni(superscript 2+)-nitrilotriacetate (Ni(superscript 2+)-NTA) affinity column to yield a highly purified enzyme. The specific activity of purified His-tagged V-PPase was approximately 86.4±7.4 μmol PP(subscript i)/mg.h, at least 6.5 fold purification compared to that on the vesicle membrane. The specific activity of His-tagged purified V-PPase were approximately 59% compare to the mung bean innate one. Further characterization indicates that the His-tagged V-PPase thus obtained resembles primarily those on membrane in most enzymatic features. The spectroscopic analyses including circular dichroism spectroscopy on His-tagged V-PPase revealed variations in conformational change induced by ions, as those inhibitors Na(superscript +), Ca(superscript 2+), and F(superscript -), of this proton translocase. These results confirm the effect of ions are exerted concomitantly with the conformational (secondary structural) changes.

並列關鍵字

Circular dichroism spectroscopy ； Metal affinity chromatography ； Vacuolar H(superscript +)-pyrophosphatase ； tonoplast

國際替代計量

Purification, Characterization, and Spectral Analyses of histidine-tagged Vacuolar H(superscript +)-pyrophosphatase Expressed in Yeast

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