Abstract
A method for the isolation in high yield of intact chloroplasts from the unicellular green alga Dunaliella marina (Volvocales) is described. This procedure uses chemically induced lysis of cells with the polycationic macromolecules, DEAE-dextran (M=500,000) or poly-D,l-lysine (M=30,000-70,000). Reaction conditions were optimized with respect to obtaining a high yield of intact chloroplasts, after isopycnic centrifugation in a linear sucrose density gradient, by varying the concentration of polycation and the temperature and pH of incubation. Broken chloroplasts devoid of the stromal marker enzymes fructosebisphosphate phosphatase and ribulosebisphosphate carboxylase, but containing mitochondrial (fumarase) and microbody (catalase) contamination, were banded at a bouyant density of 1.18 g cm-3. Intact chloroplasts, as indicated by their retention of alkaline fructosebisphosphate phosphatase and ribulosebisphosphate carboxylase, were found in 30% yield (chlorophyll in intact cells, 100%) at an equilibrium density of 1.24 g cm-3. Contamination by cytoplasmic material (pyruvate kinase), mitochondria, and microbodies was less than 8% each.
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Abbreviations
- Chl:
-
chlorophyll
- DEAE-dextran:
-
diethylaminoethyl-dextran
- DTT:
-
dithiothreitol
- EDTA:
-
ethylenediamine tetraacetic acid
- FBPase:
-
fructose-1,6-bisphosphate phosphatase, EC 3.1.3.11
- G6P-DH:
-
glucose 6-phosphate dehydrogenase, EC 1.1.1.49
- HEPES:
-
N-2-hydroxyethylpiperazine-N′-ethanesulphonic acid
- MES:
-
2-(N-morpholino)ethanesulphonic acid
- RuBP carboxylase:
-
D-ribulose-1,5-bisphosphate carboxylase or 3-phospho-D-glycerate carboxy-lyase (dimerizing), EC 4.1.1.39
References
Aebi, H. (1974) Katalase. In: Methoden der enzymatischen Analyse, pp. 713–724, Bergmeyer, H.U., ed. Verlag Chemie, Weinheim
Arnon, D.I. (1949) Copper enzymes in isolated chloroplasts. Polyphenol oxidase in Beta vulgaris. Plant Physiol. 24, 1–15
Barber, J. (1976) The intact chloroplast. In: Topics in photosynthesis, vol. I. Elsevier/North Holland Biomedical Press, Amsterdam
Ben-Amotz, A., Avron, M. (1972) Photosynthetic activities of the halophilic alga Dunaliella parva. Plant Physiol. 49, 240–243
Bergmeyer, H.U., Gawehn, K., Graßl, M. (1974) Enzyme als biochemische Reagentien. Fumarase. In: Methoden der enzymatischen Analyse, pp. 479–480, Bergmeyer, H.U., ed. Verlag Chemie, Weinheim
Cockburn, W., Walker, D.A., Baldry, C.W. (1968) The isolation of spinach chloroplasts in pyrophosphate media. Plant Physiol. 43, 1415–1418
Dürr, M., Boller, T., Wiemken, A. (1975) Polybase induced lysis of yeast spheroplasts. A new gentle method for preparation of vacuoles. Arch. Microbiol. 105, 319–327
Hall, D.O. (1972) Nomenclature for isolated chloroplasts. Nature New Biol. 235, 125–126
Hess, B., Wieker, H.-J. (1974) Pyruvat-Kinase. Bestimmung der Aktivität des Hefeenzyms. In: Methoden der enzymatischen Analyse, pp. 804–811, Bergmeyer, H.U., ed. Verlag Chemie, Weinheim
Köller, W., Kindl, H. (1977) Glyoxylate cycle enzymes of the glyoxysomal membrane from cucumber cotyledons. Arch. Biochem. Biophys. 181, 236–248
Kombrink, E., Wöber, G. (1980) Identification and subcellular localization of starch-metabolizing enzymes in the green alga, Dunaliella marina. Planta 149, 130–137
Kombrink, E., Wöber, G., Walker, D.A. (1979) Einfluß von DEAE-Dextran auf den Elektronentransport in Chloroplasten von Dunaliella marina. Ber. Dtsch. Bot. Ges. 92, 379–392
Latzko, E., Gibbs, M. (1968) Distribution and activity of enzymes of the reductive pentose phosphate cycle in spinach leaves and in chloroplasts isolated by different methods. Z. Pflanzenphysiol. 59, 184–194
Latzko, E., Gibbs, M. (1974) “Alkalische” C1-Fructose-1,6-diphosphatase. In: Methoden der enzymatischen Analyse, pp. 914–917, Bergmeyer, H.U., ed. Verlag Chemie, Weinheim
Latzko, E., Zimmermann, G., Feller, U. (1974) Evidence for a hexosediphosphatase from the cytoplasm of spinach leaves. Hoppe-Seyler's Z. Physiol. Chem. 355, 321–326
Löhr, G.W., Waller, H.D. (1974) Glucose-6-Phosphat-Dehydrogenase. In: Methoden der enzymatischen Analyse, pp. 673–681, Bergmeyer, H.U., ed. Verlag Chemie, Weinheim
Lorimer, G.H., Badger, M.R., Andrews, T.J. (1977) D-Ribulose-1,5-bisphosphate carboxylase-oxygenase. Improved methods for the activation and assay of catalytic activities. Anal. Biochem. 78, 66–75
Lowry, O.H., Rosebrough, N.J., Farr, A.L., Randall, R.J. (1951) Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193, 265–275
McLachlan, J. (1960) The culture of Dunaliella tertiolecta Butcher — an euryhaline organism. Can. J. Microbiol. 6, 367–379
Miflin, B.J., Beevers, H. (1974) Isolation of intact plastids from a range of plant tissues. Plant Physiol. 53, 870–874
Nishimura, M., Beevers, H. (1978) Isolation of intact plastids from protoplasts from castor bean endosperm. Plant Physiol. 62, 40–43
Nishimura, M., Graham, D., Akazawa, T. (1976) Isolation of intact chloroplasts and other cell organelles from spinach leaf protoplasts. Plant Physiol. 58, 309–314
Urbach, W. (1977) Eucaryotic alga. In: Encyclopedia of plant physiology, vol. V, pp. 603–624, Pirson, A., Zimmermann, M.H., eds. Springer, Berlin Heidelberg New York
Walker, D.A. (1971) Chloroplasts (and grana): aqueous (including high carbon fixation ability). Methods Enzymol. 23, 211–220
Wright, S.W., Grant, B.R. (1978) Properties of chloroplasts isolated from siphonous algae. Plant Physiol. 61, 768–771
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Kombrink, E., Wöber, G. Preparation of intact chloroplasts by chemically induced lysis of the green alga Dunaliella marina . Planta 149, 123–129 (1980). https://doi.org/10.1007/BF00380872
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DOI: https://doi.org/10.1007/BF00380872