Abstract
Vacuoles were isolated from primary leaves of barley (Hordeum vulgare L.) by mechanical breakage of protoplasts, and their polypeptide composition analyzed by two-dimensional gel electrophoresis. Vacuoplasts which consist of the vacuole, a portion of the plasmalemma and of the cytoplasma were prepared from protoplasts by ultracentrifugation. By comparing the vacuolar polypeptide pattern with polypeptide patterns of isolated chloroplasts and of vacuoplasts, vacuolar polypeptides could clearly be distinguished from polypeptides derived from cross-contaminating cell compartments. At least 14 polypeptides of apparent molecular mass between 12 and 76 kilodaltons and an isoelectric point between 4.5 and 7.6 could be attributed to the tonoplast fraction of the vacuole, and 35 polypeptides to the soluble fraction of the vacuole. Several lectins with different specificity were employed to characterize the degree and nature of glycosylation of vacuolar polypeptides. Concanavalin A bound to a large number of polypeptides. Three out of the 14 tonoplast polypeptides exhibited detectable carbohydrate moieties and almost two-thirds of the surveyed soluble polypeptides were glycosylated.
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Abbreviations
- IEF:
-
isoelectric focussing
- kDa:
-
kilodalton
- SDS-PAGE:
-
sodium dodecyl sulfate-polyacrylamide gel electrophoresis
References
Ansorge, W. (1983) Fast visualization of protein bands by impregnation in potassium permanganate and silver nitrate. In: Proceedings of electrophoresis, pp. 235–242, Stathakos, D., ed. W. de Gruyter, Berlin
Beeley, J.G. (1985) Glycoprotein and proteoglycan techniques. Elsevier Biomedical Press, Amsterdam New York Oxford
Beevers, L. (1982) Post-translational modifications. In: Encyclopedia of Plant Physiology, vol. 14 A: Nucleic acids and proteins in plants I, pp. 136–168, Boulter, D., Parthier, B., eds. Springer, Berlin Heidelberg New York
Boller, T., Wiemken, A. (1986) Dynamics of vacuolar compartmentation. Annu. Rev. Plant Physiol. 37, 137–164
Dietz, K.-J., Bogorad, L. (1987) Plastid development in Pisum sativum leaves during greening. I. A comparison of plastid polypeptide composition and in organello translation characteristics. Plant Physiol. 85, 808–815
Faye, L., Chrispeels, M.J. (1985) Characterization of N-linked oligosaccharides by affinoblotting with concanavalin A-peroxidase and treatment of the blots with glycosidases. Anal. Biochem. 149, 218–224
Holden, D.W., Rohringer, R. (1985) Peroxidases and glycosidases in intercellular fluids from non-inoculated and rustaffected wheat leaves. Isozyme assay on nitrocellulose blots from two-dimensional gels. Plant Physiol. 79, 820–824
Kaiser, G., Martinoia, E., Schmitt, J.M., Hincha, D.K., Heber, U. (1986) Polypeptide pattern and enzymic character of vacuoles isolated from barley mesophyll protoplasts. Planta 169, 345–355
Kaiser, G., Martinoia, E., Wiemken, A. (1982) Rapid appearance of photosynthetic products in the vacuoles isolated from barley mesophyll protoplasts by a new fast method. Z. Pflanzenphysiol. 107, 103–113
Kenyon, W.H., Black, C.C., Jr. (1986) Electrophoretic analysis of protoplast, vacuole, and tonoplast vesicle protein in crassulacean acid metabolism plants. Plant Physiol 82, 916–924
Lörz, H., Harms, C.T., Potrykus, I. (1976) Isolation of vacuoplasts from protoplasts of higher plants. Biochem. Physiol. Pflanz. 169, 617–620
Mandala, S., Taiz, L. (1986) Characterization of the subunit structure of the maize tonoplast ATPase. J. Biol. Chem. 261, 12850–12855
Martinoia, E., Flügge, U.I., Kaiser, G., Heber, U., Heldt, H.W. (1985) Energy-dependent uptake of malate into vacuoles isolated from barley mesophyll protoplasts. Biochim. Biophys. Acta 806, 311–319
Martinoia, E., Heck, U., Wiemken, A. (1981) Vacuoles as storage compartment for nitrate in barley leaves. Nature 289, 292–294
Martinoia, E., Kaiser, G., Schramm, M.J., Heber, U. (1987) Sugar transport across the plasmalemma and the tonoplast of barley mesophyll protoplasts. Evidence for different transport systems. J. Plant Physiol. 131, 467–478
Martinoia, E., Schramm, M.J., Kaiser, G., Kaiser, W.M., Heber, U. (1986) Transport of anions in isolated vacuoles. 1. Permeability to anions and evidence for a Cl--uptake system. Plant Physiol. 80, 895–901
Sze, H. (1985) H+-translocating ATPases: advances using membrane vesicles. Annu. Rev. Plant Physiol. 36, 175–208
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Dietz, KJ., Kaiser, G. & Martinoia, E. Characterization of vacuolar polypeptides of barley mesophyll cells by two-dimensional gel electrophoresis and by their affinity to lectins. Planta 176, 362–367 (1988). https://doi.org/10.1007/BF00395416
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DOI: https://doi.org/10.1007/BF00395416