Skip to main content
SpringerLink
Log in

A critique on the preparation and enzymatic characterization of synaptic and nonsynaptic mitochondria from hippocampus

  • Published:
Cellular and Molecular Neurobiology Aims and scope Submit manuscript

Summary

  1. 1.

    In literature two interesting methods are described to obtain from whole pooled brains or areas three types of mitochondria, namely, those of perikaryal origin and those contained in synaptosomes.

  2. 2.

    However, for many types of studies, such “preparative” preparations are not useful; for example, in pharmacological studies only data from a singlen number of animals may be of statistical usefulness and may be correctly analyzed by statistical tests.

  3. 3.

    Thus a method is described by which it was possible to characterize by enzyme activities three populations from single rat brain hippocampus.

  4. 4.

    During preparative “analytical” procedure, it was noted that the 10% Ficoll gradients previously used in the literature were unable to separate purified mitochondria-free mitochondria. This gradient should be 12% Ficoll for single areas.

  5. 5.

    In addition, when results are compared using the more appropriateω 2 t for calculations of gravity forces to be applied instead of the maximum or averageg for different rotors, enzymatic characterization differed considerably among the various mitochondrial populations.

  6. 6.

    The above considerations are also true when different pestle clearances and/or pestle rotations speeds are used during omogenizations; also lysis conditions are essential.

  7. 7.

    Results showed that selected experimental conditions are to be used when subcellular fractions are to be analyzed biochemically.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

References

  • Benzi, G. (1981). Drug-induced changes in some cerebral enzymatic activities related to energy transduction. InHandbook of Neurochemistry (A. Lajtha, Ed.), Plenum Press, New York, pp. 531–542.

    Google Scholar 

  • Bergmeyer, H. U., and Bernt, E. (1974). Lactate dehydrogenase: UV-assay with pyruvate and NADH. InMethods of Enzymatic Analysis (H. U. Bergmeyer, Ed.), Academic Press, New York and London, pp. 573–574.

    Google Scholar 

  • Booth, R. F. G., and Clark, J. B. (1978). A rapid method for the preparation of relatively pure metabolically competent synaptosomes from rat brain.Biochem. J. 176365–370.

    Google Scholar 

  • Clark, J. B., and Nicklas, W. J. (1970). The metabolism of rat brain mitochondria.J. Biol. Chem. 2454724–4731.

    Google Scholar 

  • Cotman, C. W., and Matthews, D. A. (1971). Synaptic plasma membranes from rat brain synaptosomes: Isolation and partial characterization.Biochim. Biophys. Acta 249380–394.

    Google Scholar 

  • Cotman, C. W., and Nadler, J. V. (1981). Glutamate and aspartate as hippocampal transmitters: Biochemical and pharmacological evidence. InGlutamate: Transmitter in the Central Nervous System (P. J. Roberts, J. Storm-Mathisen, and G. A. R. Johnston, Eds.), Wiley & Sons, New York, pp. 117–154.

    Google Scholar 

  • Cotman, C. W., Brown, D. H., Harrell, B. W., and Anderson, N. G. (1970). Analytical differential centrifugation: An analysis of the sedimentation properties of synaptosomes, mitochondria and lysosomes from rat brain homogenates.Arch. Biochem. Biophys. 136436–447.

    Google Scholar 

  • Dennis, S. G. C., and vlark, J. B. (1978). The synthesis of glutamate by rat brain mitochondria.J. Neurochem. 31673–680.

    Google Scholar 

  • De Robertis, E., Pellegrino De Iraldi, A., Rodriguez De Lores Arnaiz, G., and Salganicoff, L. (1962). Cholinergic and non-cholinergic nerve endings in rat brain. I.J. Neurochem. 923–25.

    Google Scholar 

  • Deshmukh, D. R., Owen, O. E., and Patel, M. S. (1980). Effect of aging on the metabolism of pyruvate and 3-hydroxybutyrate in nonsynaptic and synaptic mitochondria from rat brain.J. Neurochem. 341219–1224.

    Google Scholar 

  • Dienel, G., Ryder, E., and Greengard O. (1977). Distribution of mitochondrial enzymes between the perikayal and synaptic fractions of immature and adult rat brain.Biochim. Biophys. Acta 496484–494.

    Google Scholar 

  • Ellman, G. L., Courtney, K. D., Andres, V., and Featherstone, R. M. (1961). A new and rapid colorimetric determination of acetylcholinesterase activity.Biochem. Pharmacol. 788–95.

    Google Scholar 

  • Gray, E. C., and Whittaker, V. P. (1962). This isolation of nerve endings from brain: An electron-microscopic study of cell fragments derived by homogenization and centrifugation.J. Anat. 9679–88.

    Google Scholar 

  • Gregg, M. R., Spanner, S., and Ansell, G. B. (1982). The subcellular fractionation of the bovine caudate nucleus.Neurochem. Res. 71045–1058.

    Google Scholar 

  • Gurd, J. W., Jones, L. R., Mahler, H. R., and Moore, W. J. (1974). Isolation and partial characterization of rat brain synaptic plasma membranes.J. Neurochem. 22281–290.

    Google Scholar 

  • Hajos, F., and Kerpel-Fronius, S. (1969). Electron histochemical observation of succinic dehydrogenase activity in various parts of neurons.Exp. Brain Res. 866–78.

    Google Scholar 

  • Johnson, H. K., and Whittaker, V. P. (1963). Lactate dehydrogenase as a cytoplasmic marker in brain.Biochem. J. 88404–409.

    Google Scholar 

  • Klingenberg, M. (1967). Enzyme profiles in mitochondria. InMethods in Enzymology (R. W. Estabrook and M. E. Pullman, Eds.), Academic Press, New York and London, pp. 3–6.

    Google Scholar 

  • Kurokawa, M., Sakamoto, T., and Kato, M. (1965). Distribution of sodium plus potassium stimulated adenosine triphosphatase activity in isolated nerve ending particles.Biochem. J. 97833–844.

    Google Scholar 

  • Lai, J. C. K., and Clark, J. B. (1976). Preparations and properties of mitochondria derived from synaptosomes.Biochem. J. 154423–432.

    Google Scholar 

  • Lai, J. C. K., and Clark, J. B. (1978). Preparation of synaptic and nonsynaptic mitochondria from mammalian brain. InMethods in Enzymology (S. Fleisher and L. Packer, Eds.), Academic Press, New York and London, pp. 51–60.

    Google Scholar 

  • Lai, J. C. K., Walsh, J. M., Dennis, S. C., and Clark, J. B. (1977). Synaptic and non-synaptic mitochondria from rat brain: Isolation and characterization.J. Neurochem. 28625–631.

    Google Scholar 

  • Leong, S. F., Lai, J. C. K., Lim, L., and Clark, J. B. (1984). The activities of some energy-metabolising enzymes in nonsynaptic free, and synaptic mitochondria derived from selected brain regions.J. Neurochem. 421306–1312.

    Google Scholar 

  • Lowry, O. H., Rosebrough, N. J., Farr, A. L., and Randall, R. J. (1951). Protein measurement with the Folin phenol reagent.J. Biol. Chem. 193265–275.

    Google Scholar 

  • Morgan, I. G., Wolfe, L. S., Mandel, P., and Gombos, G. (1971). Isolation of plasma membranes from rat brain.Biochim. Biophys. Acta 241737–751.

    Google Scholar 

  • Nason, A., and Vasington, F. D. (1973). Lipid dependent DPNH-cytochrome c reductase from mammalian skeletal and heart muscle. InMethods in Enzymology (S. P. Colowick and N. O. Kaplan, Eds), Academic Press, New York and London, pp. 409–415.

    Google Scholar 

  • Ochoa, S. (1955). Malic dehydrogenase from pig heart. InMethods in Enzymology (S. P. Colowick and N. O. Kaplan, Eds.), Academic Press, New York and London, pp. 735–739.

    Google Scholar 

  • Pevzner, L. (1981). Multiple forms of enzymes. InHandbook of Neurochemistry (A. Lajtha, Ed.), Plenum Press, New York, pp. 461–491.

    Google Scholar 

  • Raynolds, E. S. (1962). The use of lead citrate at high pH as an electron-opaque stain in electron miscroscopy.J. Cell. Biol. 17208–212.

    Google Scholar 

  • Reijnierse, G. L. A., Veldstra, H., and Van Den Berg, C. J. (1975). Subcellular localization of gamma-aminobutyrate transaminase and glutamate dehydrogenase in adult rat brain.Biochem. J. 152469–475.

    Google Scholar 

  • Salganicoff, L., and De Robertis, E. (1965). Subcellular distribution of the enzymes of the glutamic acid glutamine and gamma-aminobutyric acid cycles in rat brain.J. Neurochem. 12287–309.

    Google Scholar 

  • Salganicoff, L., and Koeppe, R. E. (1968). Subcellular distribution of pyruvate carboxylase, diphosphopyridine nucleotide and triphosphopyridine nucleotide isocitrate dehydrogenases and malate enzyme in rat brain.J. Biol. Chem. 2432416–2420.

    Google Scholar 

  • Smith, L. (1955). Spectrophotometric assay of cytochrome c oxidase. InMethods of Biochemical Analysis (D. Glick, Ed.), Wiley Interscience, New York, pp. 427–434.

    Google Scholar 

  • Sottocasa, G. L., Kuylenstierna, B., Ernster, L., and Bergstrand, A. (1967). An electron-transport system associated with the outer membrane of liver mitochondria.J. Cell Biol. 32415–438.

    Google Scholar 

  • Sugden, P. H., and Newsholme, E. A. (1975). Activities of citrate synthase, NAD+-linked and NADP+-linked isocitrate dehydrogenase, glutamate dehydrogenase, aspartate aminotransferase and alanine aminotransferase in nervous tissues from vertebrates and invertebrates.Biochem. J. 150105–111.

    Google Scholar 

  • Szutowicz, A., Harris, N. F., Srere, P. A., and Crawford, I. L. (1983). ATP-citrate lyase and other enzymes of acetyl-CoA metabolism in fractions of small and large synaptosomes from rat brain hippocampus and cerebellum.J. Neurochem. 411502–1505.

    Google Scholar 

  • Villa, R. F., Strada, P., Dagani, F., and Benzi, G. (1978). Magnification of some enzymatic activities of brain cortex subfractions.Biochem. Pharmacol. 272278–2280.

    Google Scholar 

  • Villa, R. F., Benzi, G., and Curti, D. (1981). The effect of ischemic and pharmacological treatment evaluated on synaptosomes and purified mitochondria from rat cerebral cortex.Biochem. Pharmacol. 302399–2408.

    Google Scholar 

  • Villa, R. F., Curti, D., Polgatti, M., and Benzi, G. (1982). Synaptosomes and mitochondria from rat brain cerebral cortex: In vivo interference on some enzymatic activities by SAMe and CDP-choline.J. Neurosci. Res. 7341–348.

    Google Scholar 

  • Wharton, D. C., and Tzagoloff, A. (1977). Cytochrome oxidase from beef heart mitochondria. InMethods in Enzymology (R. W. Estabrook and M. E. Pullman, Eds.), Academic Press, New York and London, pp. 245–250.

    Google Scholar 

  • Whittaker, V. P. (1968). The morphology of fractions of rat forebrain synaptosomes separated on continuous sucrose density gradients.Biochem. J. 106412–417.

    Google Scholar 

  • Whittaker, V. P., Michaelson, I. A., and Kirkland, R. J. (1964). The separation of synaptic vesicles from nerve ending particles (synaptosomes).Biochem. J. 90293–303.

    Google Scholar 

  • Wilson, J. E. (1972). The location of latent brain hexokinase on synaptosomal mitochondria.Arch. Biochem. Biophys. 15096–104.

    Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Faculty of Science, Institute of Pharmacology, University of Pavia, Pavia, Italy

    R. F. Villa & A. Gorini

  2. Institute of Anatomy and C.N.R. Centre of Histochemistry, Faculty of Medicine, University of Pavia, Pavia, Italy

    A. Lo Faro & C. Dell'Orbo

Authors
  1. R. F. Villa
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. A. Gorini
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. A. Lo Faro
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. C. Dell'Orbo
    View author publications

    You can also search for this author in PubMed Google Scholar

Rights and permissions

Reprints and permissions

About this article

Cite this article

Villa, R.F., Gorini, A., Lo Faro, A. et al. A critique on the preparation and enzymatic characterization of synaptic and nonsynaptic mitochondria from hippocampus. Cell Mol Neurobiol 9, 247–262 (1989). https://doi.org/10.1007/BF00713032

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF00713032

Key words

Search

Navigation