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標題: Purification and subunit determination of H+-pyrophosphatase from endoplasmic reticulum-enriched vesicles of mung bean seedlings
作者: Kuo, S.Y.
Chien, L.F.
Van, R.C.
Yan, K.H.
Liu, P.F.
Chang, W.C.
Wang, J.K.
Pan, R.L.
關鍵字: endoplasmic reticulum;H+-pyrophosphatase;mung bean;functional mass;radiation inactivation;pumping inorganic pyrophosphatases;radiation inactivation;submitochondrial particles;membrane pyrophosphatase;tonoplast;proteins;localization;arabidopsis;bacterial;leaves
Project: Plant Science
期刊/報告no:: Plant Science, Volume 169, Issue 5, Page(s) 847-853.
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 homoditneric. (c) 2005 Elsevier Ireland Ltd. All rights reserved.
ISSN: 0168-9452
DOI: 10.1016/j.plantsci.2005.06.001
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