Summary
A method is described for the isolation of lysosomal fractions from dark-grown potato shoots using a single stage separation on a Ficoll gradient. Peaks of acid hydrolase activity consisting of acid phosphatase, phosphodiesterase, ribonuclease, carboxylic esterase and β-glycerophosphatase were well separated from peaks of mitochondrial and glyoxysomal enzymes. A heavy lysosomal fraction with particle diameters from 0.1 to 1.6 μ and density of 1.10 g cm-3 containing relatively low hydrolase activity was distinguishable from a light fraction with diameters 0.025 to 0.6 μ and density of 1.07 g cm-3 with a higher level of hydrolase activity. Both fractions appeared heterogeneous by electron microscopy, but the fine structure of the membranes of both heavy and light lysosomes was similar. The heavy lysosomal fraction was rich in autophagic vacuoles (secondary lysosomes) containing organelles and amorphous cytoplasmic material. Both fractions were rich in ribonucleic acid.
Freezing and thawing, high speed blending and ultrasonication either singly or in combination solubilised a maximum of ca. 30% of the acid phosphatase from crude lysosomal fractions derived from dark-grown potato shoots. Treatment with Triton X-100 and deoxycholate released appreciably more enzyme activity but acetone and carbon tetrachloride failed to solubilise any acid phosphatase. Only detergent treatments gave marked overrecovery of enzyme and indicated structure-linked latency. Liberation of enzyme from lysosomes varied with pH and was almost complete at both extremes of pH. Crude snake venom was rapid and effective in solubilising acid phosphatase from lysosomal preparations, purified phospholipase A was less effective and phospholipases C and D had negligible effects. Phospholipase and venom mediated release of acid phosphatase was accompanied by the coincident release of an acid end-product. Gel filtration of acid phosphatase liberated from heavy and light lysosomal fractions by snake venom digestion revealed that each of these fractions was characterised by the presence of distinct molecular forms of the enzyme. The nature of the association of acid phosphatase with potato shoot lysosomes is discussed.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Allmann, D. W., Bachmann, E., Green, D. E.: The membrane system of the mitochondrion. II. The K factor of the outer membrane of beef heart mitochondria. Arch. Biochem. 115, 165–171 (1966).
Andrews, P.: The gel-filtration behaviour of proteins related to their molecular weights over a wide range. Biochem. J. 96, 595–606 (1965).
Appelmans, F., de Duve, C.: Tissue fractionation studies. 3. Further observations on the binding of acid phosphatase by rat liver particles. Biochem. J. 59, 426–433 (1955).
Bachmann, E., Allmann, D. W., Green, D. E.: The membrane systems of the mitochondrion. 1. The S fraction of the outer membrane of beef heart mitochondria. Arch. Biochem. 115, 153–164 (1966).
Balz, H. P.: Intrazelluläre Lokalisation und Funktion von hydrolytischen Enzymen bei Tabak. Planta (Berl.) 70, 207–236 (1966).
Beaufay, H.: Methods for the isolation of lysosomes. In: Frontiers of biology, 14 B. Lysosomes in biology and pathology, vol. 2, ch. 18, p. 515–546, eds. J. T. Dingle and H. B. Fell. Amsterdam and London: North Holland Publishing Co. 1969.
Beaufay, H., Duve, C. de: Tissue fractionation studies. 9. Enzymic release of bound hydrolases. Biochem. J. 73, 604–609 (1959).
Berthet, J., Duve, C. de: Tissue fractionation studies. 1. The existence of a mitochondrial-linked enzymically inactive form of acid phosphatase in rat-liver tissue. Biochem. J. 50, 174–181 (1951).
Bonnichsen, R. K., Chance, B., Theorell, H.: Catalase activity. Acta chem. scand. 1, 685–709 (1947).
Bowen, I. D.: A high resolution technique for the fine-structural localisation of acid hydrolases. J. Microscopie 94, 25–38 (1971).
Chakravorty, A. K.: Ribosomal RNA synthesis in the germinating black eye pea (Vigna unguiculata). 1. The effect of cycloheximide on RNA synthesis in the early stages of germination. Biochim. biophys. Acta (Amst.) 179, 67–82 (1969).
Cohen, E., Shain, Y., Ben-Shaul, Y., Meyer, A. M.: Structural and enzymatic characterisation of plant zymogen bodies. Canad. J. Bot. 49, 2053–2057 (1971).
Cooper, T. G., Beevers, H.: Mitochondria and glyoxysomes from castor bean endosperm. J. biol. Chem. 244, 3507–3513 (1969).
Corbett, J. R., Price, C. A.: Intracellular distribution of p-nitrophenylphosphatase in plants. Plant Physiol. 42, 827–830 (1967).
Dixon, G. H., Kornberg, H. L.: Assay methods for key enzymes of the glyoxylate cycle. Biochem. J. 72, 3 P (1959).
Fiske, C. H., Subba Row, Y.: The colorimetric determination of phosphorus. J. biol. Chem. 66, 375–400 (1925).
Gregory, R. P. F.: A rapid assay for peroxidase activity. Biochem. J. 101, 582–583 (1966).
Grossman, I. E., Heitkamp, D. H.: Electron microscopic localisation of mitochondrial adenosine triphosphatase activity. J. Histochem. Catochem. 16, 645–653 (1968).
Hack, M. H.: Estimation of the phospholipides in human blood. J. biol. Chem. 169, 137–143 (1947).
Heftmann, E.: Lysosomes in tomatoes. Cytobios 3, 129–136 (1971).
Kalckar, H. M.: Differential spectrophotometry of purine compounds by means of specific enzymes. 1. Determination of hydroxy-purine compounds. J. biol. Chem. 167, 429–443 (1947).
Koenig, H.: Histological distribution of brain gangliosides in lysosomes as glycolipoprotein granules. Nature (Lond.) 195, 782–784 (1962).
Koenig, H.: Lysosomes in the nervous system. In: Frontiers of biology, 14 B. Lysosomes in biology and pathology, vol. 2, ch. 6, p. 111–162, eds. J. T. Dingle and H. B. Fell. Amsterdam and London: North Holland Publishing Co. 1969.
Koenig, H., Gray, R.: Action of phospholipase C on lysosomes. J. Cell Biol. 23, 50 A (1964).
Kramer, R., Lambrechter, R., Kaiser, E.: Release of acid hydrolases from lysosomes by animal venoms. Toxicon 9, 125–129 (1971).
Lowry, O. H., Rosebrough, N. J., Farr, A. L., Randall, R. J.: Protein measurement with the Folin phenol reagent. J. biol. Chem. 193, 265–275 (1951).
Luft, J. H.: Improvements in epoxy resin embedding methods. J. biophys. biochem. Cytol. 9, 409–414 (1961).
Matile, P.: Lysosomes of root tip cells in corn seedlings. Planta (Berl.) 79, 181–196 (1968).
Matile, P.: Plant Lysosomes. In: Frontiers of biology, 14 A. Lysosomes in biology and pathology, vol. 1, ch. 15, p. 406–430, eds. J. T. Dingle and H. B. Fell. Amsterdam and London: North Holland Publishing Co. 1969.
Matile, P., Balz, J. P., Semadeni, E., Jost, M.: Isolation of sphersomes with lysosome characteristics from seedlings. Z. Naturw. 20b, 693–698 (1965).
Matile, P., Moor, H.: Vacuolation: Origin and development of the lysosomal apparatus in root-tip cells. Planta (Berl.) 80, 159–175 (1968).
Matile, P., Winkenbach, F.: Function of lysosomes and lysosomal enzymes in senescing corolla of the morning glory (Ipomoea purpurea). J. exp. Bot. 22, 759–771 (1971).
McClurkin, I. T., McClurkin, D. C.: Cytochemical demonstration of a sodium-activated and a potassium-activated adenosine triphosphatase in loblolly pine seedling root tips. Plant Physiol. 42, 1103–1110 (1967).
Mejbaum, W.: Über die Bestimmung kleiner Pentosemengen insbesondere in Derivaten der Adenylsäure. Hoppe-Seylers Z. physiol. Chem. 258, 117–120 (1939).
Nachlas, M. M., Seligman, A. M.: Evidence for the specificity of esterase and lipase by the use of three chromogenic substrates. J. biol. Chem. 181, 343–345 (1949).
Nesvadba, H.: Aminosäure-β-naphthylamide zur Aktivitätsbestimmung proteolytischer Fermente. Mh. Chem. 93, 386–396 (1962).
Novikoff, A. B., Essner, E., Quintana, N.: Golgi apparatus and lysosomes. Fedn. Proc. Fedn. Amer. Socs. exp. Biol. 23, 1010–1022 (1964).
Pitt, D., Coombes, C.: The disruption of lysosome-like particles of Solanum tuberosum cells during infection by Phytophthora erythroseplica Pethybr. J. gen. Microbiol. 53, 197–204 (1968).
Pitt, D., Coombes, C.: Release of hydrolytic enzymes from cytoplasmic particles of Solanum tuber tissues during infection by tuber-rotting fungi. J. gen. Microbiol. 56, 321–329 (1969).
Pitt, D., Galpin, M.: Increase in ribonuclease activity following mechanical damage to leaf and tuber tissues of Solanum tuberosum L. Planta (Berl.) 101, 317–332 (1971).
Pitt, D., Galpin, M.: Role of lysosomal enzymes in pathogenicity. In: Fungal pathogenicity and the plant's response. 3rd Long Ashton Symposium, eds. R. J. W. Byrde and C. C. V. Cutting. London and New York: Academic Press Inc. 1972.
Shibko, S., Pangborn, J., Tappel, A. L.: Studies on the release of lysosomal enzymes from kidney lysosomes. J. Cell Biol. 25, 479–482 (1965).
Snyder, F., Stephens, N.: A simplified spectrophotometric determination of ester groups in lipids. Biochim. biophys. Acta (Amst.) 34, 244–245 (1959).
Shaw, J. G.: Acid phosphatase from tobacco leaves. Arch. Biochem. 117, 1–9 (1966).
Tolbert, N. E., Yamazaki, R. K.: Leaf peroxisomes and their relation to photorespiration and photosynthesis. Ann. N.Y. Acad. Sci. 168, 325–341 (1969).
Waters, L. C., Dure, L. S.: Ribonucleic acid synthesis in germinating cotton seeds. J. molec. Biol. 19, 1–27 (1966).
Watson, B. F., Dworkin, M.: Comparative intermediary metabolism of vegetative cells and microcysts of Myxococcus xanthus. J. Bact. 96, 1465–1473 (1968).
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Pitt, D., Galpin, M. Isolation and properties of lysosomes from dark-grown potato shoots. Planta 109, 233–258 (1972). https://doi.org/10.1007/BF00387087
Received:
Issue Date:
DOI: https://doi.org/10.1007/BF00387087