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. 1979 Dec;64(6):1114–1120. doi: 10.1104/pp.64.6.1114

Isolation and Partial Characterization of Vacuoles from Tobacco Protoplasts 1

Irvin J Mettler a,2, Robert T Leonard a
PMCID: PMC543202  PMID: 16661103

Abstract

Protoplasts from suspension-cultured cells of Nicotiana glutinosa L. were lysed in 0.3 molar sorbitol in 2 millimolar ethylenediaminetetraacetate-tris(hydroxymethyl) aminomethane (pH 7.5) to release intact vacuoles. The vacuoles were purified by centrifugation in a Ficoll step gradient. About 11% of the vacuoles and 13% of the acid phosphatase activity was recovered in the purified vacuole fraction, suggesting that the vacuole is the major site for acid phosphatase in these cells. NADH-cytochrome c reductase, malate dehydrogenase, and cytochrome c oxidase activities were reduced during vacuole purification. The majority of the adenosine 5′-triphosphate (ATP) hydrolytic activity of purified vacuoles was associated with nonspecific acid phosphatase and not with a transport ATPase. As judged by acid phosphatase distribution and electron microscopy, the effective density of vacuoles in a sucrose gradient was low (less than 1.1 grams per cubic centimeter), although an unequivocal estimate of the vacuole or tonoplast density was not possible from the experiments conducted.

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Selected References

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

  1. Boller T., Kende H. Hydrolytic enzymes in the central vacuole of plant cells. Plant Physiol. 1979 Jun;63(6):1123–1132. doi: 10.1104/pp.63.6.1123. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Butcher H. C., Wagner G. J., Siegelman H. W. Localization of Acid hydrolases in protoplasts: examination of the proposed lysosomal function of the mature vacuole. Plant Physiol. 1977 Jun;59(6):1098–1103. doi: 10.1104/pp.59.6.1098. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Leigh R. A., Branton D. Isolation of Vacuoles from Root Storage Tissue of Beta vulgaris L. Plant Physiol. 1976 Nov;58(5):656–662. doi: 10.1104/pp.58.5.656. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Leonard R. T., Hansen D., Hodges T. K. Membrane-bound Adenosine Triphosphatase Activities of Oat Roots. Plant Physiol. 1973 Apr;51(4):749–754. doi: 10.1104/pp.51.4.749. [DOI] [PMC free article] [PubMed] [Google Scholar]
  5. Leonard R. T., Hodges T. K. Characterization of Plasma Membrane-associated Adenosine Triphosphase Activity of Oat Roots. Plant Physiol. 1973 Jul;52(1):6–12. doi: 10.1104/pp.52.1.6. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Leonard R. T., Hotchkiss C. W. Cation-stimulated Adenosine Triphosphatase Activity and Cation Transport in Corn Roots. Plant Physiol. 1976 Sep;58(3):331–335. doi: 10.1104/pp.58.3.331. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Leonard R. T., Vanderwoude W. J. Isolation of plasma membranes from corn roots by sucrose density gradient centrifugation: an anomalous effect of ficoll. Plant Physiol. 1976 Jan;57(1):105–114. doi: 10.1104/pp.57.1.105. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Lin W., Wagner G. J., Siegelman H. W., Hind G. Membrane-bound ATPase of intact vacuoles and tonoplasts isolated from mature plant tissue. Biochim Biophys Acta. 1977 Feb 14;465(1):110–117. doi: 10.1016/0005-2736(77)90359-5. [DOI] [PubMed] [Google Scholar]
  9. Matile P., Wiemken A. Vacuoles and spherosomes. Methods Enzymol. 1974;31:572–578. [PubMed] [Google Scholar]
  10. Mettler I. J., Leonard R. T. Ion transport in isolated protoplasts from tobacco suspension cells: I. General characteristics. Plant Physiol. 1979 Jan;63(1):183–190. doi: 10.1104/pp.63.1.183. [DOI] [PMC free article] [PubMed] [Google Scholar]
  11. Nagahashi G., Leonard R. T., Thomson W. W. Purification of plasma membranes from roots of barley: specificity of the phosphotungstic Acid-chromic Acid stain. Plant Physiol. 1978 Jun;61(6):993–999. doi: 10.1104/pp.61.6.993. [DOI] [PMC free article] [PubMed] [Google Scholar]
  12. Ray P. M., Shininger T. L., Ray M. M. ISOLATION OF beta-GLUCAN SYNTHETASE PARTICLES FROM PLANT CELLS AND IDENTIFICATION WITH GOLGI MEMBRANES. Proc Natl Acad Sci U S A. 1969 Oct;64(2):605–612. doi: 10.1073/pnas.64.2.605. [DOI] [PMC free article] [PubMed] [Google Scholar]
  13. Walker-Simmons M., Ryan C. A. Immunological Identification of Proteinase Inhibitors I and II in Isolated Tomato Leaf Vacuoles. Plant Physiol. 1977 Jul;60(1):61–63. doi: 10.1104/pp.60.1.61. [DOI] [PMC free article] [PubMed] [Google Scholar]

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