Skip to main content
NCBI home page
Biochemical Journal logoLink to Biochemical Journal
. 1997 Jun 1;324(Pt 2):555–563. doi: 10.1042/bj3240555

Purification of a dichlorophenol-indophenol oxidoreductase from rat and bovine synaptic membranes: tight complex association of a glyceraldehyde-3-phosphate dehydrogenase isoform, TOAD64, enolase-gamma and aldolase C.

C Bulliard 1, R Zurbriggen 1, J Tornare 1, M Faty 1, Z Dastoor 1, J L Dreyer 1
PMCID: PMC1218466  PMID: 9182718

Abstract

NADH-dichlorophenol-indophenol oxidoreductases (PMOs) were purified from synaptic plasma membranes or synaptic vesicles (small recycling vesicles) from both bovine and rat brains and from a neuroblastoma cell line, NB41A3. Several isoforms could be identified in purified plasma membranes and vesicles. Purification of the enzyme activity involved protein extraction with detergents, (NH4)2SO4 precipitation, chromatography under stringent conditions and native PAGE. PMO activity could be attributed to a very tight complex of several proteins that could not be separated except by SDS/PAGE. SDS/PAGE resolved the purified complex into at least five proteins, which could be micro-sequenced and identified unambiguously as hsc70, TOAD64 and glyceraldehyde-3-phosphate dehydrogenase tightly associated with the brain-specific proteins aldolase C and enolase-gamma. Enzyme activity could be purified from both synaptic plasma membranes and recycling vesicles, yields being much greater from the latter source. Highly purified plasma membranes (prepared from a neuroblastoma cell line NB41A3 by iminobiotinylation of intact cells and affinity purification with avidin and anti-avidin antibodies under very stringent conditions) also displayed PMO activity tightly associated with TOAD64. The association of PMO in a tight complex was confirmed by its immunoprecipitation from cellular and membrane extracts of NB41A3 using antibodies directed against any component protein of the complex followed by immunodetection with antibodies directed against the other members. Antibodies also inhibited the enzyme activity synergistically. In addition, induction of the different components of the complex during dichlorophenol-indophenol stress was demonstrated by the S1 RNase-protection assay in synchronized NB41A3 cells. The role of the complex in membrane fusion and cellular response to extracellular oxidative stress during growth and development is discussed.

Full Text

The Full Text of this article is available as a PDF (437.4 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Ahn A. H., Dziennis S., Hawkes R., Herrup K. The cloning of zebrin II reveals its identity with aldolase C. Development. 1994 Aug;120(8):2081–2090. doi: 10.1242/dev.120.8.2081. [DOI] [PubMed] [Google Scholar]
  2. Alouani S., Ketchum S., Rambosson C., Eistetter H. R. Transcriptional activity of the neuron-specific enolase (NSE) promoter in murine embryonic stem (ES) cells and preimplantation embryos. Eur J Cell Biol. 1993 Dec;62(2):324–332. [PubMed] [Google Scholar]
  3. Arcari P., Martinelli R., Salvatore F. Human glyceraldehyde-3-phosphate dehydrogenase pseudogenes: molecular evolution and a possible mechanism for amplification. Biochem Genet. 1989 Aug;27(7-8):439–450. doi: 10.1007/BF02399673. [DOI] [PubMed] [Google Scholar]
  4. Balaban N., Goldman R. The association of glycosomal enzymes and microtubules: a physiological phenomenon or an experimental artifact? Exp Cell Res. 1990 Dec;191(2):219–226. doi: 10.1016/0014-4827(90)90008-x. [DOI] [PubMed] [Google Scholar]
  5. Bennett M. K., Calakos N., Kreiner T., Scheller R. H. Synaptic vesicle membrane proteins interact to form a multimeric complex. J Cell Biol. 1992 Feb;116(3):761–775. doi: 10.1083/jcb.116.3.761. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Boucherié H., Bataille N., Fitch I. T., Perrot M., Tuite M. F. Differential synthesis of glyceraldehyde-3-phosphate dehydrogenase polypeptides in stressed yeast cells. FEMS Microbiol Lett. 1995 Jan 15;125(2-3):127–133. doi: 10.1111/j.1574-6968.1995.tb07348.x. [DOI] [PubMed] [Google Scholar]
  7. Byk T., Dobransky T., Cifuentes-Diaz C., Sobel A. Identification and molecular characterization of Unc-33-like phosphoprotein (Ulip), a putative mammalian homolog of the axonal guidance-associated unc-33 gene product. J Neurosci. 1996 Jan 15;16(2):688–701. doi: 10.1523/JNEUROSCI.16-02-00688.1996. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Bähler M., Klein R. L., Wang J. K., Benfenati F., Greengard P. A novel synaptic vesicle-associated phosphoprotein: SVAPP-120. J Neurochem. 1991 Aug;57(2):423–430. doi: 10.1111/j.1471-4159.1991.tb03769.x. [DOI] [PubMed] [Google Scholar]
  9. Cotman C. W., Matthews D. A. Synaptic plasma membranes from rat brain synaptosomes: isolation and partial characterization. Biochim Biophys Acta. 1971 Dec 3;249(2):380–394. doi: 10.1016/0005-2736(71)90117-9. [DOI] [PubMed] [Google Scholar]
  10. Coyle J. T., Puttfarcken P. Oxidative stress, glutamate, and neurodegenerative disorders. Science. 1993 Oct 29;262(5134):689–695. doi: 10.1126/science.7901908. [DOI] [PubMed] [Google Scholar]
  11. DeLorenzo R. J., Freedman S. D. Calcium dependent neurotransmitter release and protein phosphorylation in synaptic vesicles. Biochem Biophys Res Commun. 1978 Jan 13;80(1):183–192. doi: 10.1016/0006-291x(78)91121-x. [DOI] [PubMed] [Google Scholar]
  12. Deloulme J. C., Lucas M., Gaber C., Bouillon P., Keller A., Eclancher F., Sensenbrenner M. Expression of the neuron-specific enolase gene by rat oligodendroglial cells during their differentiation. J Neurochem. 1996 Mar;66(3):936–945. doi: 10.1046/j.1471-4159.1996.66030936.x. [DOI] [PubMed] [Google Scholar]
  13. Fuhrmann G. F., Fehlau R., Schneider H., Knauf P. A. The effect of ferricyanide with iodoacetate in calcium-free solution on passive cation permeability in human red blood cells: comparison with the Gardos-effect and with the influence of PCMBS on passive cation permeability. Biochim Biophys Acta. 1989 Aug 7;983(2):179–185. doi: 10.1016/0005-2736(89)90231-9. [DOI] [PubMed] [Google Scholar]
  14. Gautron S., Maire P., Hakim V., Kahn A. Regulation of the multiple promoters of the human aldolase A gene: response of its two ubiquitous promoters to agents promoting cell proliferation. Nucleic Acids Res. 1991 Feb 25;19(4):767–774. doi: 10.1093/nar/19.4.767. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Glaser P. E., Gross R. W. Rapid plasmenylethanolamine-selective fusion of membrane bilayers catalyzed by an isoform of glyceraldehyde-3-phosphate dehydrogenase: discrimination between glycolytic and fusogenic roles of individual isoforms. Biochemistry. 1995 Sep 26;34(38):12193–12203. doi: 10.1021/bi00038a013. [DOI] [PubMed] [Google Scholar]
  16. Goshima Y., Nakamura F., Strittmatter P., Strittmatter S. M. Collapsin-induced growth cone collapse mediated by an intracellular protein related to UNC-33. Nature. 1995 Aug 10;376(6540):509–514. doi: 10.1038/376509a0. [DOI] [PubMed] [Google Scholar]
  17. Graven K. K., Troxler R. F., Kornfeld H., Panchenko M. V., Farber H. W. Regulation of endothelial cell glyceraldehyde-3-phosphate dehydrogenase expression by hypoxia. J Biol Chem. 1994 Sep 30;269(39):24446–24453. [PubMed] [Google Scholar]
  18. Gupta S., Hollenstein R., Kochhar S., Christen P. Paracatalytic self-inactivation of fructose-1,6-bisphosphate aldolase. Structure of the crosslink formed at the active site. Eur J Biochem. 1993 Jun 1;214(2):515–519. doi: 10.1111/j.1432-1033.1993.tb17949.x. [DOI] [PubMed] [Google Scholar]
  19. Hanauer A., Mandel J. L. The glyceraldehyde 3 phosphate dehydrogenase gene family: structure of a human cDNA and of an X chromosome linked pseudogene; amazing complexity of the gene family in mouse. EMBO J. 1984 Nov;3(11):2627–2633. doi: 10.1002/j.1460-2075.1984.tb02185.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. Hawkes R., Mascher C. The development of molecular compartmentation in the cerebellar cortex. Acta Anat (Basel) 1994;151(3):139–149. doi: 10.1159/000147656. [DOI] [PubMed] [Google Scholar]
  21. Huitorel P., Pantaloni D. Bundling of microtubules by glyceraldehyde-3-phosphate dehydrogenase and its modulation by ATP. Eur J Biochem. 1985 Jul 15;150(2):265–269. doi: 10.1111/j.1432-1033.1985.tb09016.x. [DOI] [PubMed] [Google Scholar]
  22. Ishitani R., Sunaga K., Hirano A., Saunders P., Katsube N., Chuang D. M. Evidence that glyceraldehyde-3-phosphate dehydrogenase is involved in age-induced apoptosis in mature cerebellar neurons in culture. J Neurochem. 1996 Mar;66(3):928–935. doi: 10.1046/j.1471-4159.1996.66030928.x. [DOI] [PubMed] [Google Scholar]
  23. Jimenez-Asensio J., Gonzalez P., Zigler J. S., Jr, Garland D. L. Glyceraldehyde 3-phosphate dehydrogenase is an enzyme-crystallin in diurnal geckos of the genus Phelsuma. Biochem Biophys Res Commun. 1995 Apr 26;209(3):796–802. doi: 10.1006/bbrc.1995.1570. [DOI] [PubMed] [Google Scholar]
  24. Leighton I. A., Curmi P., Campbell D. G., Cohen P., Sobel A. The phosphorylation of stathmin by MAP kinase. Mol Cell Biochem. 1993 Nov;127-128:151–156. doi: 10.1007/BF01076766. [DOI] [PubMed] [Google Scholar]
  25. Li Y., Jaiswal A. K. Human antioxidant-response-element-mediated regulation of type 1 NAD(P)H:quinone oxidoreductase gene expression. Effect of sulfhydryl modifying agents. Eur J Biochem. 1994 Nov 15;226(1):31–39. doi: 10.1111/j.1432-1033.1994.tb20023.x. [DOI] [PubMed] [Google Scholar]
  26. Luo Y., Raible D., Raper J. A. Collapsin: a protein in brain that induces the collapse and paralysis of neuronal growth cones. Cell. 1993 Oct 22;75(2):217–227. doi: 10.1016/0092-8674(93)80064-l. [DOI] [PubMed] [Google Scholar]
  27. Maine A. B., Ciejek-Baez E. Distinct developmental regulatory mechanisms for two members of the aldolase gene family. Dev Genet. 1991;12(6):431–436. doi: 10.1002/dvg.1020120608. [DOI] [PubMed] [Google Scholar]
  28. Marangos P. J., Schmechel D. E., Parma A. M., Goodwin F. K. Developmental profile of neuron-specific (NSE) and non-neuronal (NNE) enolase. Brain Res. 1980 May 19;190(1):185–193. doi: 10.1016/0006-8993(80)91168-3. [DOI] [PubMed] [Google Scholar]
  29. Maucuer A., Camonis J. H., Sobel A. Stathmin interaction with a putative kinase and coiled-coil-forming protein domains. Proc Natl Acad Sci U S A. 1995 Apr 11;92(8):3100–3104. doi: 10.1073/pnas.92.8.3100. [DOI] [PMC free article] [PubMed] [Google Scholar]
  30. McDonald L. J., Moss J. Stimulation by nitric oxide of an NAD linkage to glyceraldehyde-3-phosphate dehydrogenase. Proc Natl Acad Sci U S A. 1993 Jul 1;90(13):6238–6241. doi: 10.1073/pnas.90.13.6238. [DOI] [PMC free article] [PubMed] [Google Scholar]
  31. Meyer-Siegler K., Mauro D. J., Seal G., Wurzer J., deRiel J. K., Sirover M. A. A human nuclear uracil DNA glycosylase is the 37-kDa subunit of glyceraldehyde-3-phosphate dehydrogenase. Proc Natl Acad Sci U S A. 1991 Oct 1;88(19):8460–8464. doi: 10.1073/pnas.88.19.8460. [DOI] [PMC free article] [PubMed] [Google Scholar]
  32. Minturn J. E., Fryer H. J., Geschwind D. H., Hockfield S. TOAD-64, a gene expressed early in neuronal differentiation in the rat, is related to unc-33, a C. elegans gene involved in axon outgrowth. J Neurosci. 1995 Oct;15(10):6757–6766. doi: 10.1523/JNEUROSCI.15-10-06757.1995. [DOI] [PMC free article] [PubMed] [Google Scholar]
  33. Moullet O., Dreyer J. L. Selective inhibition of adenylate cyclase in bovine cortex by quinones: a novel cellular substrate for quinone cytotoxicity. Biochem J. 1994 May 15;300(Pt 1):99–106. doi: 10.1042/bj3000099. [DOI] [PMC free article] [PubMed] [Google Scholar]
  34. Nakai A., Satoh M., Hirayoshi K., Nagata K. Identification of the ATP-binding heat-inducible protein of MR = 37,000 as glyceraldehyde-3-phosphate dehydrogenase. Biochem Biophys Res Commun. 1991 Apr 15;176(1):59–64. doi: 10.1016/0006-291x(91)90889-f. [DOI] [PubMed] [Google Scholar]
  35. Napolitano E. W., Pachter J. S., Liem R. K. Intracellular distribution of mammalian stress proteins. Effects of cytoskeletal-specific agents. J Biol Chem. 1987 Feb 5;262(4):1493–1504. [PubMed] [Google Scholar]
  36. Njus D., Knoth J., Cook C., Kelly P. M. Electron transfer across the chromaffin granule membrane. J Biol Chem. 1983 Jan 10;258(1):27–30. [PubMed] [Google Scholar]
  37. O'Reilly G., Clarke F. Identification of an actin binding region in aldolase. FEBS Lett. 1993 Apr 19;321(1):69–72. doi: 10.1016/0014-5793(93)80623-3. [DOI] [PubMed] [Google Scholar]
  38. Pelham H. R. Hsp70 accelerates the recovery of nucleolar morphology after heat shock. EMBO J. 1984 Dec 20;3(13):3095–3100. doi: 10.1002/j.1460-2075.1984.tb02264.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
  39. Püschel A. W., Adams R. H., Betz H. Murine semaphorin D/collapsin is a member of a diverse gene family and creates domains inhibitory for axonal extension. Neuron. 1995 May;14(5):941–948. doi: 10.1016/0896-6273(95)90332-1. [DOI] [PubMed] [Google Scholar]
  40. Rogalski A. A., Steck T. L., Waseem A. Association of glyceraldehyde-3-phosphate dehydrogenase with the plasma membrane of the intact human red blood cell. J Biol Chem. 1989 Apr 15;264(11):6438–6446. [PubMed] [Google Scholar]
  41. Schläfer M., Volknandt W., Zimmermann H. Putative synaptic vesicle nucleotide transporter identified as glyceraldehyde-3-phosphate dehydrogenase. J Neurochem. 1994 Nov;63(5):1924–1931. doi: 10.1046/j.1471-4159.1994.63051924.x. [DOI] [PubMed] [Google Scholar]
  42. Seipp S., Buselmaier W. Isolation of glyceraldehyde 3-phosphate dehydrogenase (Gapdh) cDNA from the distal half of mouse chromosome 16: further indication of a link between Alzheimer's disease and glycolysis. Neurosci Lett. 1994 Nov 21;182(1):91–94. doi: 10.1016/0304-3940(94)90214-3. [DOI] [PubMed] [Google Scholar]
  43. Sobel A., Boutterin M. C., Beretta L., Chneiweiss H., Doye V., Peyro-Saint-Paul H. Intracellular substrates for extracellular signaling. Characterization of a ubiquitous, neuron-enriched phosphoprotein (stathmin). J Biol Chem. 1989 Mar 5;264(7):3765–3772. [PubMed] [Google Scholar]
  44. Sytnik A. I., Chumachenko Y. V., Demchenko A. P. Spectroscopic evidence for NADH-induced conformational changes in rabbit muscle aldolase. Biochim Biophys Acta. 1991 Aug 30;1079(2):123–127. doi: 10.1016/0167-4838(91)90116-h. [DOI] [PubMed] [Google Scholar]
  45. Toole-Simms W., Sun I. L., Faulk W. P., Löw H., Lindgren A., Crane F. L., Morré D. J. Inhibition of transplasma membrane electron transport by monoclonal antibodies to the transferrin receptor. Biochem Biophys Res Commun. 1991 May 15;176(3):1437–1442. doi: 10.1016/0006-291x(91)90447-f. [DOI] [PubMed] [Google Scholar]
  46. Walsh J. L., Keith T. J., Knull H. R. Glycolytic enzyme interactions with tubulin and microtubules. Biochim Biophys Acta. 1989 Nov 9;999(1):64–70. doi: 10.1016/0167-4838(89)90031-9. [DOI] [PubMed] [Google Scholar]
  47. Wang J., Morris A. J., Tolan D. R., Pagliaro L. The molecular nature of the F-actin binding activity of aldolase revealed with site-directed mutants. J Biol Chem. 1996 Mar 22;271(12):6861–6865. [PubMed] [Google Scholar]
  48. Weatherbee J. A., Luftig R. B., Weihing R. R. Purification and reconstitution of HeLa cell microtubules. Biochemistry. 1980 Aug 19;19(17):4116–4123. doi: 10.1021/bi00558a033. [DOI] [PubMed] [Google Scholar]
  49. Weber B., Weber W., Buck F., Hilz H. Isolation of the myc transcription factor nucleoside diphosphate kinase and the multifunctional enzyme glyceraldehyde-3-phosphate dehydrogenase by cAMP affinity chromatography. Int J Biochem Cell Biol. 1995 Feb;27(2):215–224. doi: 10.1016/1357-2725(94)00078-p. [DOI] [PubMed] [Google Scholar]
  50. Whatley S. A., Leung T., Hall C., Lim L. The brain 68-kilodalton microtubule-associated protein is a cognate form of the 70-kilodalton mammalian heat-shock protein and is present as a specific isoform in synaptosomal membranes. J Neurochem. 1986 Nov;47(5):1576–1583. doi: 10.1111/j.1471-4159.1986.tb00797.x. [DOI] [PubMed] [Google Scholar]
  51. Whittaker V. P., Michaelson I. A., Kirkland R. J. The separation of synaptic vesicles from nerve-ending particles ('synaptosomes'). Biochem J. 1964 Feb;90(2):293–303. doi: 10.1042/bj0900293. [DOI] [PMC free article] [PubMed] [Google Scholar]
  52. Wistow G. J., Lietman T., Williams L. A., Stapel S. O., de Jong W. W., Horwitz J., Piatigorsky J. Tau-crystallin/alpha-enolase: one gene encodes both an enzyme and a lens structural protein. J Cell Biol. 1988 Dec;107(6 Pt 2):2729–2736. doi: 10.1083/jcb.107.6.2729. [DOI] [PMC free article] [PubMed] [Google Scholar]
  53. Yong Y., Dreyer J. L. Developmental changes in the localization of the transplasma membrane NADH-dehydrogenases in the rat brain. Brain Res Dev Brain Res. 1995 Nov 21;89(2):253–263. doi: 10.1016/0165-3806(95)00125-w. [DOI] [PubMed] [Google Scholar]
  54. Yong Y., Dreyer J. L. Distribution of six transplasma membrane NADH-dehydrogenases in rat brain tissue. Brain Res Dev Brain Res. 1995 Nov 21;89(2):235–252. doi: 10.1016/0165-3806(95)00124-v. [DOI] [PubMed] [Google Scholar]
  55. Zurbriggen R., Dreyer J. L. An NADH-diaphorase is located at the cell plasma membrane in a mouse neuroblastoma cell line NB41A3. Biochim Biophys Acta. 1994 Jan 4;1183(3):513–520. doi: 10.1016/0005-2728(94)90079-5. [DOI] [PubMed] [Google Scholar]
  56. Zurbriggen R., Dreyer J. L. The plasma membrane NADH-diaphorase is active during selective phases of the cell cycle in mouse neuroblastoma cell line NB41A3. Its relation to cell growth and differentiation. Biochim Biophys Acta. 1996 Jul 24;1312(3):215–222. doi: 10.1016/0167-4889(96)00037-7. [DOI] [PubMed] [Google Scholar]

Articles from Biochemical Journal are provided here courtesy of The Biochemical Society

RESOURCES