Summary
This communication confirms the validity of two marker enzymes, NADH-cytochrome c reductase and succinate dehydrogenase (as succinate-INT reductase). for quantitation of the content of endoplasmic reticulum and mitochondria of plant cell fractions. Membrane fractions enriched to varying degrees in rough endoplasmic reticulum and mitochondria as well as reference fractions containing smooth membranes and dictyosomes were prepared from homogenates of etiolated soybean (Glycine max L.) hypocotyls. The NADPH-cytochrome c reductase activities of the fractions correlated with the content of rough endoplasmic reticulum as determined by morphometry; succinate-INT reductase activity correlated with the content of mitochondria. The absolute specific activity for pure endoplasmic reticulum was calculated from the regression analysis to be 17.8 Μmoles cytochrome c reduced hr−1 mg−1 protein and for mitochondria was determined to be 37.1 Μmoles INT reduced hr−1 mg−1 protein. These values permit estimation of endoplasmic reticulum and mitochondrial contamination of soybean fractions in the absence of pure reference fractions.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Depierre, J. W., Dallner, G., 1975: Structural aspects of the membrane of the endoplasmic reticulum. Biochim. biophys. Acta415, 411–472.
Douce, R., Christenson, E. L., Bonner, W. D., 1972: Preparation of intact plant mitochondria. Biochim. biophys. Acta275, 148–160.
Hodges, T. K., Leonard, R. T., 1974: Purification of a plasma membrane-bound adenosine triphosphatase from plant roots. Methods Enzymol.32, 392–406.
Hurkman, W. J., Morré, D. J., Bracker, C. E., Mollenhauer, H. H., 1979: Identification of etioplast fragments in fractions from soybean hypocotyls. Plant Physiol.64, 398–403.
Lembi, C. A., Morré, D. J., St.-Thomson, K., Hertel, R., 1971: N-1-naphthyl-phthalamic acid-binding activity of a coleoptiles. Planta99, 37–45.
Lord, J. M., Kagawa, T., Moore, T. S., Beevers, W., 1973: Endoplasmic reticulum as the site of lecithin formation in castor bean endosperm. J. Cell Biol.57, 659–667.
Loud, A. V., 1962: A method for the quantitative estimation of cytoplasmic structures. J. Cell Biol.15, 481–487.
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.
Luft, J. M., 1961: Improvements in epoxy resin embedding methods. J. biophys. biochem. Cytol.9, 409–414.
Mills, W. R., Joy, K. W., 1980: A rapid method for isolation of purified physiologically active chloroplasts used to study the intracellular distribution of amino acids in pea leaves. Planta148, 75–83.
Morré, D. J., 1973: Isolation and purification of organelles and endomembrane components from rat liver. In: Molecular techniques and approaches in developmental biology (Chrispeels, M. J., ed.), pp. 1–27. New York: Wiley.
—, 1971: Isolation of Golgi apparatus. Methods Enzymol.22, 130–148.
—,Buckhout, T. J., 1979: Isolation of Golgi apparatus. In: Plant organelles (Reid, E., ed.). Meth. Surveys Biochem.9, 117–134. Chichester: Ellis Horwood.
Pennington, R. J., 1961: Biochemistry of distrophic muscle: mitochondrial succinate—tetrazolium reductase and adenine triphosphatase. Biochem. J.80, 649–654.
Pomeroy, M. K., McMurchie, E. J., 1982: Comparison of the properties of ATPase associated with wheat and cauliflower plasma membranes. Plant Physiol.69, 1241–1246.
Quail, P. H., 1979: Plant cell fractionation. Ann. Rev. Plant Physiol.30, 425–484.
Reynolds, E. S., 1963: The use of lead citrate at high pH as an electron opaque stain in electron microscopy. J. Cell Biol.17, 208–212.
Roland, J.-C., Lembi, C. A., Morré, D. J., 1972: Phosphotungstic acid-chromic acid as a selective stain for plasma membranes of plant cells. Stain Technol.47, 195–200.
Sedmak, J. J., Grossberg, S. E., 1977: A rapid, sensitive and versatile assay for protein using Coomassie Brilliant Blue G 250. Anal. Biochem.79, 544–552.
Widell, S., Lundborg, T., Larson, C., 1982: Plasma membranes from oats prepared by partition in an aqueous polymer two phase system. Plant Physiol.70, 1429–1435.
Williamson, F. A., Morré, D. J., Jaffe, M. J., 1975: Associations of phytochrome with rough-surfaced endoplasmic reticulum fractions from soybean hypocotyls. Plant Physiol.56, 738–743.
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Lem, N.W., Morré, D.J. NADPH-cytochrome c reductase and succinate-INT reductase of soybean membranes: An evaluation of two enzyme markers. Protoplasma 128, 14–21 (1985). https://doi.org/10.1007/BF01273230
Received:
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF01273230