Synthesis of affinity chromatography resins for the purification of brain glutamate decarboxylase
In the present paper we describe the synthesis of Sepharose-boundN-(5′-phosphopyridoxyl)-amino-oxyacetic acid,N-(5′-phosphopyridoxyl)-canaline, andN-(5′-phosphopyridoxyl)-α,γ-diaminobutyric acid (Seph-DAB-PLP), designed for binding specifically brain glutamate decarboxylase (GAD). The three Sepharose-N-(phosphopyridoxyl)-amino acids were capable of binding GAD from a mouse brain soluble preparation. The enzyme can be purified up to 25-fold in one step by affinity chromatography on Seph-DAB-PLP.
KeywordsGlutamate Mouse Brain Affinity Chromatography Canaline Glutamate Decarboxylase
Unable to display preview. Download preview PDF.
- 1.Tapia, R. 1975. Biochemical pharmacology of GABA in CNS.In Iversen, L. L., Iversen, S. D., and Snyder, S. H. (eds.), Handbook of Psychopharmacology, Volume 4, Plenum Press, New York, pp. 1–58.Google Scholar
- 2.Tapia, R., Sandoval, M. E., andContreras, P. 1975. Evidence for a role of glutamate decarboxylase activity as a regulatory mechanism of cerebral excitability.J. Neurochem. 24:1283–1285.Google Scholar
- 3.Tower, D. B. 1976. GABA and seizures: Clinical correlates in man.In Roberts, E., Chase, T. N., and Tower, D. B. (eds.), GABA in Nervous System Function, Raven Press, New York, pp. 461–478.Google Scholar
- 4.Wu, J.-Y., Matsuda, T., andRoberts, E. 1973. Purification and characterization of glutamate decarboxylase from mouse brain.J. Biol. Chem. 248:3029–3034.Google Scholar
- 5.Wu, J.-Y. 1976. Purification, characterization, and kinetic studies of GAD and GABA-T from mouse brain.In Roberts, E., Chase, T. N., and Tower, D. B. (eds.), GABA in Nervous System Function, Raven Press, New York, pp. 7–55.Google Scholar
- 6.Tapia, R., andAwapara, J. 1969. Effects of various substituted hydrazones and hydrazines of pyridoxal 5′-phosphate on brain glutamate decarboxylase.Biochem. Pharmacol. 18:145–152.Google Scholar
- 7.Tapia, R., andSandoval, M. E. 1971. Study on the inhibition of brain glutamate decarboxylase by pyridoxal phosphate oxime-O-acetic acid.J. Neurochem. 18:2051–2059.Google Scholar
- 8.Wada, H., andSnell, E. E. 1961. The enzymatic oxidation of pyridoxine and pyridoxamine phosphates.J. Biol. Chem. 236:2089–2095.Google Scholar
- 9.Axén, R., Porath, J., andErnback, S. 1967. Chemical coupling of peptides and proteins to polysaccharides by means of cyanogen halides.Nature 214:1302–1304.Google Scholar
- 10.Erickson, B. W., andMerrifield, R. B. 1973. Use of chlorinated benzyloxycarbonyl protecting groups to eliminate N∈-branching at lysine during solid-phase peptide synthesis.J. Am. Chem. Soc. 95:3757–3763.Google Scholar
- 11.Possani, L. D., andTapia, R. 1975. An affinity chromatography column for the purification of brain glutamate decarboxylase.Trans. Am. Soc. Neurochem. 6:168.Google Scholar
- 12.Lowry, O. H., Rosebrough, N. J., Farr, A. L., andRandall, R. J. 1951. Protein measurement with the Folin phenol reagent.J. Biol. Chem. 193:265–275.Google Scholar