Abstract
Phosphate-activated glutaminase (PAG) and glutamic acid decarboxylase (GAD) were assayed in homogenates and synaptosomes obtained from starved (48 hr or 120 hr) and diabetic (streptozotocin) rat brain cortex. Glutamine synthetase (GS) was assayed in homogenates, microsomal and soluble fractions, from brain cortex of similarly treated rats.l-Glutamate uptake and exit rates were determined in cortex slices and synaptosomes under the same conditions. The specific activity (s.a.) of PAG, a glutamate producing enzyme, decreased (50%) in the homogenate after 120-hr starvation. In synaptosomes it decreased (25%) only after 48-hr starvation. The s.a of GAD and GS, which are glutamate-consuming enzymes, were progressively increased with time of starvation, reaching 39% and 55% respectively after 120 hr. GS in the microsomes or the soluble fraction and GAD in the synaptosomes showed no change in s.a. under these conditions. Diabetes increased (40%) microsomal GS s.a. and decreased GAD s.a. (18%) in the homogenate. Thel-glutamate uptake rate was decreased (48%) by diabetes in slices but not in synaptosomes. It is suggested that a) enzymes of the glutamate system respond differently in different subcellular fractions towards diabetes or deprivation of food and b) diabetes may affect the uptake system in glial cells but not in neurons.
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Abbreviations
- AET:
-
2-aminoethylisourethonium bromide
- GAD:
-
glutamic acid decarboxylase
- GS:
-
glutamine synthetase
- GSH:
-
glutathione
- PAG:
-
phosphate-activated glutaminase
- PLP:
-
pyridoxal phosphate
- r.c.f.:
-
relative centrifugal force
- s.a.:
-
specific activity
References
Palaiologos, G., Philippids, H., Chomatas, H., Iakovou, D., and Linardou, A. 1987. Effects of branched chain amino acids, pyruvate or ketone bodies on the free amino acid pool and release from brain cortex slices of normal and streptozotocin-diabetic rats. Neurochem. Res. 12:1–7.
Ward, H. K., Thanki, C. M., and Bradford, H. F. 1983. Glutamine and glucose as precursors of transmitter amino acids: ex vivo studies. J. Neurochem. 40:855–860.
Berl, S. and Clarke, D. D. 1983. The Metabolic compartmentation concept. Pages 205–217in Hertz, L., Kvamme, E., McGeer, E. G., and Schousboe, A. (eds.), Glutamine, Glutamate, and GABA in the Central Nervous System Alan R. Liss, Inc., New York.
Quastel, J. H. 1979. The role of amino acids in the brain. Pages 1–48in Marks, V., and Hales, C. N. (eds.), “Essays in Medical Biochemistry”, Vol. 4, The Biochemical Society and the Association of Clinical Biochemists.
Palaiologos, G., Galanopoulos, E., Lellos, V., Papadakis, M., and Philippidis, H. 1985. Glutamate metabolizing enzymes and its transport in brain cortex from normal and diabetic rats. 13th International Congress of Biochemistry. Abstract. Mo-287:98.
Buse, M. G., Herlong, H. F. and Weigand, D. A. 1976. The effect of diabetes, insulin and the redox potential on leucine metabolism in isolated rat hemidiaphragm. Endocrinology 98:1166–1175.
Palaiologos, G., Koivisto, A. V., and Felig, P. 1979. Interaction of leucine, glucose and ketone metabolism in rat brain in vitro. J. Neurochem. 32:67–72.
Krueger, B. K., Forn, J., and Greengard, P. 1977. Depolarization-induced phosphorylation of specific proteins, mediated by calcium ion influx, in rat brain synaptosomes. J. Biol. Chem. 252:2764–2773.
Elliott, W. H. 1955. Glutamine Synthesis. Pages 337–342in Colowick, S. P., and Kaplan, N. O. (eds.), Methods in Enzymology. Vol. II. Academic Press, New York.
Dierks-Ventling, C., Cone, A. L., and Bressman, S. P. 1975. The attachment of glutamine synthetase to brain membranes. Biochem. Med. 13:213–223.
Lund, P. 1970. A radiochemical assay for glutamine synthetase, and activity of the enzyme in rat tissues. Biochem. J. 118:35–39.
Ward, H. K., and Bradford, H. F. 1979. Relative activities of glutamine synthetase and glutaminase in mammalian synaptosomes. J. Neurochem. 33:339–342.
Bradford, H. F., Ward, H. K., and Thomas, A. J. 1978. Glutamine—A major substrate for nerve endings. J. Neurochem. 30:1453–1459.
Sandberg, M., Ward, H. K., and Bradford, H. F. 1985. Effect of cortico-striate pathway lesion on the activities of enzymes involved in the synthesis and metabolism of amino acid neurotransmitters in the striatum. J. Neurochem. 44:42–47.
Chude, O., and Wu, J. Y. 1976. A rapid method for assaying enzymes whose substrates and products differ by charge. Application to brainl-glutamate decarboxylase. J. Neurochem. 27:83–86.
Balcar, V. J., and Johnston, G. A. R. 1972. The structural specificity of the high affinity uptake ofl-glutamate andl-aspartate by rat brain slices. J. Neurochem. 19:2657–2666.
Weiler, C. T., Nyström, B., and Hamberger, A. 1979. Characteristics of glutamine vs. glutamate transport in isolated glia and synaptosomes. J. Neurochem. 32:559–565.
Blasberg, R., Levi, G., and Lajtha, A. 1970. A comparison of inhibition of steady state, net transport and exchange fluxes of amino acids in brain slices. Biochim. Biophys. Acta 203:464–483.
Davies, L. P., Johnston, G. A. R., and Stephanson, A. L. 1975. Postnatal changes in the potassium-stimulated calcium-dependent release of radioactive GABA and glycine from slices of rat central nervous tissue. J. Neurochem. 25:387–392.
Strong, R., and Wood Gibson, W. 1984. Membrane properties and aging. In vivo and in vitro effects of ethanol on synaptosomal γ-aminobutyric acid (GABA) release. J. Pharmacol. Expt. Ther. 229:726–730.
Troeger, M. B., Wilson, D. F., and Erecinska, M. 1984. The effect of thiol reagents on GABA transport in rat brain synaptosomes. FEBS Lett. 171:303–308.
Lowry, O. W., Rosenbrough, N. J., Farr, A. L., and Randal, R. J. 1951. Protein measurement with Folin phenol reagent. J. Biol. Chem. 193:265–275.
Bergmeyer, H. U., Bernt, E., Gawehn, K. and Michel, G. 1974. Pages 172–174in Bergmeyer, H. V. (ed.), Methods of Enzymatic Analysis, 2nd edition. Academic Press, New York.
Prusiner, S., and Milner, L. 1970. A rapid radioactive assay for glutamine synthetase, glutaminase, asparagine synthetase and asparaginase. Anal. Biochem. 37:429–438.
Martinez-Hernandez, A., Bell, K. P., Norenberg, M. D. 1977. Glutamine synthetase: Glial localization in brain. Science 195:1356–1385.
Norenberg, M. D., and Martinez-Hernandez, A. 1979. Fine structural localization of glutamine synthetase in astrocytes of rat brain. Brain Res. 161:303–310.
Norenberg, M. D. 1983. Immunohistochemistry of glutamine synthetase. Pages 95–111in Hertz, L., Kvamme, E., McGeer, E. G., and Schousboe, A. (eds.), Glutamine, Glutamate, and GABA in the Central Nervous System. Alan R. Liss, Inc., New York.
Weiler, C. T., Nyström, B., and Hamberger A. 1979. Glutaminase and glutamine synthetase activity in synaptosomes bulk-isolated glia and neurons. Brain Res. 160:539–543.
Dennis, S. C., Lai, J. C. K., and Clark, J. B. 1980. The distribution of glutamine synthetase in subcellular fractions of rat brain. Brain Res. 197:469–475.
Hovhannisyan, V. S., Ambartsumyan, V. G., Bedjamian, K. D. 1984. Regulatory properties of membrane form of phosphate-dependent glutaminase of synaptosomes and mitochondria in rat brain. Neurochimia 3:372–381.
Bedjamian, K. D., Badalyan, L. L., Oganesyan, V. C. 1985. Phosphate-dependent soluble glutaminase from rat brain. Role of different agents in the modulation of its activity. Neurochimia 4:379–387.
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Galanopoulos, E., Lellos, V., Papadakis, M. et al. Effects of fasting and diabetes on some enzymes and transport of glutamate in cortex slices or synaptosomes from rat brain. Neurochem Res 13, 243–248 (1988). https://doi.org/10.1007/BF00971540
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DOI: https://doi.org/10.1007/BF00971540