Purification, characterization, and spectral analyses of histidine-tagged vacuolar H⁺-pyrophosphatase expressed in yeast

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⁺, but inhibited by F^-, Na⁺, Ca²⁺ and excess PP_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 Ni²⁺-nitrilotriacetate (Ni²⁺-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_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⁺, Ca²⁺, and F^-, of this proton translocase. These results confirm the effect of ions are exerted concomitantly with the conformational (secondary structural) changes.