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. 1986 Jan;80(1):161–166. doi: 10.1104/pp.80.1.161

Isolation and Characterization of Tonoplast from Chilling-Sensitive Etiolated Seedlings of Vigna radiata L. 1,2

Shizuo Yoshida 1, Takefumi Kawata 1, Matsuo Uemura 1, Teruo Niki 1
PMCID: PMC1075075  PMID: 16664574

Abstract

Tonoplasts were isolated in a high purity from etiolated young seedlings of Vigna radiata L. (mung bean) utilizing a sucrose density gradient system. The excised hypocotyls were homogenized in a sorbitol-buffer system and the 3,600 to 156,000g pellets obtained after the differential centrifugations were suspended in a sorbitol medium and loaded on a linear sucrose density gradient. After centrifugation at 89,000g for 2 hours, tonoplasts were banded at the sample load/sucrose interface. Assessed by electron microscopy and marker enzymes, the purity and the quantity were found to be sufficient for biochemical and biophysical analyses. The tonoplasts were associated with NO3−-sensitive and vana-date-insensitive ATPase. The tonoplast ATPase was stimulated by proton ionophores such as carbonyl cyanide p-trifluoromethoxyphenyl-hydrazone and gramicidin D, suggesting a proton-pumping enzyme. In the presence of ATP and Mg2+, a proton gradient was formed in the isolated tonoplast vesicles as assessed by fluorescence quenching of quinacrine. The tonoplasts contained several kinds of mannosylated or glycosylated glycoproteins and a major protein (65 kilodaltons) which was unique to the membranes.

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Selected References

These references are in PubMed. This may not be the complete list of references from this article.

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