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

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1 Citation (Scopus)

Abstract

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
Volume169
Issue number5
DOIs
Publication statusPublished - 2005 Nov

All Science Journal Classification (ASJC) codes

  • Genetics
  • Agronomy and Crop Science
  • Plant Science

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