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. 1980 Oct 1;87(1):72–83. doi: 10.1083/jcb.87.1.72

Analytical characterization of beetroot vacuole membrane

PMCID: PMC2110713  PMID: 7419602

Abstract

Vacuoles from beetroot (Beta vulgaris L. var. esculenta Gurke) isolated by a mechanical procedure were osmotically lysed to separate the membrane and sap components for analysis. Approximately 62% of the vacuole proteins, 70% of the nondialyzable carbohydrates and almost all of the phospholipids and sterols were recovered in the membrane fraction. The vacuole membrane had a phospholipid protein ratio of 0.68 and a sterol:phospholipid ratio of 0.21. 17 complex polar lipids including phosphatides and glycolipids have been tentatively identified. Phosphatidylcholine (54%) and phosphatidylethanolamine (24%) were the most prominent phosphoglycerides besides phosphatidylserine, phosphatidylglycerol, phosphatidylinositol, and phosphatidic acid (1, 4, 5, and 12%, respectively). A putative sulfoglycoside and two major ceramide glycoside-like lipids, resembling those of animal lysosomes, were identified by thin-layer chromatography. High-resolution SDS-acrylamide gel electrophoresis of the polypeptides from the vacuole revealed 15 major bands with apparent molecular weights ranging from 91,000 to 12,000. Selective elution experiments delineated those polypeptides that were peripheral membrane proteins or sap proteins adsorbed to the membrane, and those that exhibited hydrophobic interactions with the lipid core. Lectin labeling results indicated that most of the polypeptides from the membrane and from the sap were glycoproteins probably of the high-mannose type characteristic of lysosomal enzymes that have undergone several stages of posttranslational modification.

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Selected References

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  1. BARTLETT G. R. Phosphorus assay in column chromatography. J Biol Chem. 1959 Mar;234(3):466–468. [PubMed] [Google Scholar]
  2. BEISS U. ZUR PAPIERCHROMATOGRAPHISCHEN AUFTRENNUNG VON PFLANZENLIPIDEN. J Chromatogr. 1964 Jan;13:104–110. doi: 10.1016/s0021-9673(01)95079-4. [DOI] [PubMed] [Google Scholar]
  3. Donaldson R. P., Beevers H. Lipid composition of organelles from germinating castor bean endosperm. Plant Physiol. 1977 Feb;59(2):259–263. doi: 10.1104/pp.59.2.259. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. FOLCH J., LEES M., SLOANE STANLEY G. H. A simple method for the isolation and purification of total lipides from animal tissues. J Biol Chem. 1957 May;226(1):497–509. [PubMed] [Google Scholar]
  5. Fisher K. A. Analysis of membrane halves: cholesterol. Proc Natl Acad Sci U S A. 1976 Jan;73(1):173–177. doi: 10.1073/pnas.73.1.173. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Glossmann H., Neville D. M., Jr Glycoproteins of cell surfaces. A comparative study of three different cell surfaces of the rat. J Biol Chem. 1971 Oct 25;246(20):6339–6346. [PubMed] [Google Scholar]
  7. Henning R., Kaulen H. D., Stoffel W. Biochemical analysis of the pinocytotic process. I. Isolation and chemical composition of the lysosomal and the plasma membrane of the rat liver cell. Hoppe Seylers Z Physiol Chem. 1970 Oct;351(10):1191–1199. doi: 10.1515/bchm2.1970.351.2.1191. [DOI] [PubMed] [Google Scholar]
  8. Kornfeld R., Kornfeld S. Comparative aspects of glycoprotein structure. Annu Rev Biochem. 1976;45:217–237. doi: 10.1146/annurev.bi.45.070176.001245. [DOI] [PubMed] [Google Scholar]
  9. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  10. Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
  11. 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]
  12. Lowry R. R. Ferric chloride spray detector for cholesterol and cholesteryl esters on thin-layer chromatograms. J Lipid Res. 1968 May;9(3):397–397. [PubMed] [Google Scholar]
  13. Marty F. Cytochemical studies on GERL, provacuoles, and vacuoles in root meristematic cells of Euphorbia. Proc Natl Acad Sci U S A. 1978 Feb;75(2):852–856. doi: 10.1073/pnas.75.2.852. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Mettler I. J., Beevers H. Isolation and characterization of the protein body membrane of castor beans. Plant Physiol. 1979 Sep;64(3):506–511. doi: 10.1104/pp.64.3.506. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Rodriguez Boulan E., Sabatini D. D., Pereyra B. N., Kreibich G. Spatial orientation of glycoproteins in membranes of rat liver rough microsomes. II. Transmembrane disposition and characterization of glycoproteins. J Cell Biol. 1978 Sep;78(3):894–909. doi: 10.1083/jcb.78.3.894. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. SVENNERHOLM L. The quantitative estimation of cerebrosides in nervous tissue. J Neurochem. 1956 May;1(1):42–53. doi: 10.1111/j.1471-4159.1956.tb12053.x. [DOI] [PubMed] [Google Scholar]
  17. Shaw N. The detection of lipids on thin-layer chromatograms with the periodate-Schiff reagents. Biochim Biophys Acta. 1968 Oct 22;164(2):435–436. doi: 10.1016/0005-2760(68)90171-9. [DOI] [PubMed] [Google Scholar]
  18. Siakotos A. N. Analytical separation of nonlipid water soluble substances and gangliosides from other lipids by dextran gel column chromatography. J Am Oil Chem Soc. 1965 Nov;42(11):913–919. doi: 10.1007/BF02632444. [DOI] [PubMed] [Google Scholar]
  19. Steck T. L. The organization of proteins in the human red blood cell membrane. A review. J Cell Biol. 1974 Jul;62(1):1–19. doi: 10.1083/jcb.62.1.1. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Travis R. L., Booz M. L. Partial Characterization of a Potassium-stimulated Adenosine Triphosphatase from the Plasma Membrane of Meristematic and Mature Soybean Root Tissue. Plant Physiol. 1979 Mar;63(3):573–577. doi: 10.1104/pp.63.3.573. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. WAGNER H., HOERHAMMER L., WOLFF P. [Thin layer chromatography of phosphatides and glycolipids]. Biochem Z. 1961;334:175–184. [PubMed] [Google Scholar]

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