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

Soong Yu Kuo, Lee Feng Chien, Ru Chun Van, Kun Huang Yan, Pei Feng Liu, Wen Chi Chang, Jung Kai Wang, Rong Long Pan

Research output: Contribution to journalArticlepeer-review

1 Citation (Scopus)


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.

Original languageEnglish
Pages (from-to)847-853
Number of pages7
JournalPlant Science
Issue number5
Publication statusPublished - 2005 Nov

All Science Journal Classification (ASJC) codes

  • Genetics
  • Agronomy and Crop Science
  • Plant Science

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