Summary
From mature green leaves of Spinacia oleracea and Beta vulgaris a fraction has been obtained which is enriched in mitochondria. Washed leaves were crushed in a meat-mill in an isotonic or slightly hypertonic medium containing sucrose, EDTA, MgSO4, bovine serum albumin, mercaptoethanol, KH2PO4, and HEPES as a buffer substance. After squeezing through nylon the suspension was centrifuged first at 1500xg, and following removal of the sediment a second time at 25000xg. After resuspension in a washing medium, the pellet was centrifuged in a swing-out rotor for 30 min at 35000xg on a density gradient generated from a silica sol (“Ludox HS 40”). A temperature of 2°C was maintained during the whole procedure.
Whereas the usual isolation techniques are unsatisfactory for the preparation of intact leaf cell mitochondria, the above procedure enabled us to obtain a fraction from the gradient which had the following properties. The respiration rate was 29.5 μatoms O/mg protein-N/h with α-ketoglutarate as a substrate. Phosphorylation rates as calculated from the phosphate measurements and P/O ratios were low. This has been interpreted as a consequence of ATPase activity in the fraction. In polarographic studies ADP/O ratios of 1.32–2.15 with succinate as a substrate were measured. Respiratory control was also observed. This indicates the presence of tightly coupled mitochondria in the fraction.
The fine structure of the mitochondria was shown to be intact as far as electron microscopical evidence can be used as a criterion. The procedure appears to be suitable for isolating active mitochondria and intact chloroplasts from green leaves. It may permit the study of interrelationships between these organelles.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
Literatur
Arnon, D. I.: Copper enzymes in isolated chloroplasts. Polyphenoloxydase in Beta vulgaris. Plant Physiol. 24, 1–15 (1949).
Bonner, W. D., Jr.: Mitochondria and electron transport. In: Plant biochemistry, ed. J. Bonner and J. E. Varner. New York: Academic Press 1965.
Ducet, G., Rosenberg, A. J., Vandewalle, G.: Phophorylation oxydative par les mitochondries de feuilles et de racines. I. Mitochondries de feuilles. Ann. Phys. Végét. 1, 209–222 (1959).
Fiske, C. H., SubbaRow, Y.: The colorimetric determination of phosphorus. J. biol. Chem. 66, 375 (1925).
Hobson, G. E., Lance, C., Young, R. E., Biale, J. B.: The isolation of active subcellular particles from avocado fruit at various stages of ripeness. Nature (Lond.) 209, 1242–1243 (1966).
Hohorst, H. J.: D-glucose-6-phosphat und D-fructose-6-phosphat. In: Methoden der enzymatischen Analyse, ed. H. U. Bergmeyer. Weinheim: Verlag Chemie 1962.
Ikuma, H.: Necessary conditions for isolation of tightly coupled higher plant mitochondria. Plant Physiol. 45, 773–781 (1970).
Jensen, R. G., Bassham, J. A.: Photosynthesis by isolated chloroplasts. Proc. nat. Acad. Sci. (Wash.) 56, 1095–1101 (1966).
Ku, H. S., Pratt, H. K., Spurr, A. R., Harris, W. M.: Isolation of active mitochondria from tomato fruit. Plant Physiol. 43, 883–887 (1968).
Lowry, O. H., Rosebrough, N. J., Farr, A. L., Randall, R. J.: Protein measurement with the Folin reagent. J. biol. Chem. 193, 265–276 (1951).
Mateyko, G. M., Kopac, M. J.: Isopycnotic cushioning for density gradient centrifugation. Exp. Cell Res. 17, 524–526 (1959).
Packer, L., Murakami, S., Mehard, C. W.: Ion transport in chloroplasts and plant mitochondria. Ann. Rev. Plant Physiol. 21, 271–304 (1970).
Palmer, J. M.: Rapid isolation of active mitochondria from plant tissue. Nature (Lond.) 216, 1208 (1967).
Pertoft, H.: Gradient centrifugation in colloidal silicapolysaccharide media. Biochim. biophys. Acta (Amst.) 126, 594–596 (1966).
—: The separation of rat liver cells in colloidal silica-polyethylene glycol gradients. Exp. Cell Res. 57, 338–350 (1969).
— Philipson, L., Oxelfeld, P., Höglund, S.: Gradient centrifugation of viruses in colloidal silica. Virology 33, 185–196 (1967).
Pierpoint, W. S.: Mitochondrial preparations from the leaves of Nicotiana tabacum. IV. Separation of some components by density-gradient centrifuging. Biochem. J. 82, 143–148 (1962).
Smillie, R. M.: Enzymatic activity of particles from various tissues of the pea plant. Aust. J. biol. Sci. 8, 186–194 (1955).
Stinson, R. A., Spencer, M.: An evaluation of the effects of five buffers on respiratory parameters of isolated mitochondria. Canad. J. Biochem. 46, 43–49 (1968).
Turck, K., Willenbrink, J.: Isolation of intact mitochondria from sugar beet leaves. Naturwissenschaften 56, 222–223 (1969).
Wiskich, J. T., Young, R. E., Biale, J. B.: Metabolic processes in cytoplasmic particles of the avocado fruit. VI. Controlled oxidations and coupled phosphorylations. Plant Physiol. 39, 312–322 (1964).
Zelitch, I., Barber, G. A.: Oxidative phosphorylation by particles from spinach leaves. Plant Physiol. 35, 205–209 (1960).
Author information
Authors and Affiliations
Additional information
Herrn Prof. Dr. W. Schumacher zum 70. Geburtstag gewidmet.
Auszug aus einer Dissertationsschrift (Math.-Nat. Fakultät der Universität Bonn) von K. Gronebaum-Turck.
Rights and permissions
About this article
Cite this article
Gronebaum-Turck, K., Willenbrink, J. Isolierung und Eigenschaften von Mitochondrien aus Blättern von Spinacia oleracea und Beta vulgaris . Planta 100, 337–346 (1971). https://doi.org/10.1007/BF00385198
Received:
Issue Date:
DOI: https://doi.org/10.1007/BF00385198