Purification and subunit determination of H⁺-pyrophosphatase from endoplasmic reticulum-enriched vesicles of mung bean seedlings

Endoplasmic reticulum (ER)-enriched vesicles from etiolated hypocotyls of mung bean seedlings (Vigna radiata L.) were isolated by Ficoll gradient and two-polymer phase partition. These ER-enriched vesicles contain a new type of H⁺-pyrophosphatase (H⁺-PPase) distinct from that of tonoplasts in higher plants. H⁺-PPase was then solubilized differentially by deoxycholic acid and lyso-phosphatidylcholine. The solubilized fraction was then subjected to Sephacryl S-200 gel filtration and Mono-Q anion exchange chromatography. The final purified protein complex of ER H ⁺-PPase (ER-PPase) was successfully obtained to high homogeneity. An approximate molecular mass of 170 kDa was determined for the purified ER-PPase by size-exclusion gel filtration chromatography. However, only a single polypeptide of 74 kDa was observed on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Moreover, radiation inactivation analysis of ER-enriched vesicles and purified ER-PPase yielded functional masses of 178.6 ± 9.2 and 143.4 ± 4.7 kDa for inorganic pyrophosphate hydrolysis activity, respectively, indicating that ER-PPase was functionally homodimeric.