Abstract
Glucose-6-phosphate dehydrogenase was purified from rabbit brain cortex using a single immunoaffinity chromatographic step and was contaminated only by a 50 kDa protein. The proteins, separated by SDS-PAGE, were sequenced: the glucose-6-phosphate dehydrogenase was blocked at the N-terminal, the co-eluted protein was similar to α-tubulin. Our technique can be applied to purification and sequencing of the enzyme from brain areas or to measure its turnover rate in cultured cells.
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Baquer NZ, Hothersall JS, McLean P (1988) Current topics cellular regulation. In: Horeker B & Stadtman ER, eds. Function and Regulation of the Pentose Phosphate Pathway in Brain. New York: Academic Press, pp. 265-289.
Bergman T, Gheorghe MT, Hjelmqvist L, Jornvall H (1996) Alcoholytic deblocking of N-terminal acetylated peptides and proteins for sequence analysis. FEBS Lett. 390: 199-202.
Bradford M (1976) A rapid sensitive method for the quantitation of microgram quantities of protein utilizing the principles of protein-dye binding. Anal. Biochem. 72: 248-254.
Camardella L, Damonte G, Carratore V, Benatti U, Tonetti M, Moneti G (1995) Glucose-6-phosphate dehydrogenase from human erythrocytes: identification of N-acetylalanine at the N-terminus of the mature protein. Biochem. Biophys. Res. Commun. 207: 331-338.
Coligan JE, Dunn BM, Ploegh HL, Speicher DW, Wingfield PT (1996) Current Protocols in Protein Science. New York: Wiley & Sons, Inc.
De Flora A, Morelli A, Benatti U, Giuliano F (1975) An improved procedure for the rapid isolation of glucose-6-phosphate dehydrogenase from human erythrocytes. Arch. Biochem. Biophys. 169: 364-369.
Jeffrey J, Barros-Soderling J, Murray L, Wood I, Hansen R, Szepesi B, Jornvall H (1989) Glucose-6-phosphate dehydrogenase: caracteristics revealed by the rat liver enzyme structure. Eur. J. Biochem. 186: 551-556.
Kletzien RF, Harris PKW, Foellmi LA (1994) Glucose-6-phosphate dehydrogenase: a ‘housekeeping’ enzyme subject to tissuespecific regulation by hormones, nutrients and oxidant stress. FASEB J. 8: 174-181.
Kugler P (1994) Glucose-6-phosphate dehydrogenase is enriched in oligodendrocytes of the rat spinal cord. Enzyme histochemical and immunocytochemical studies. Histochemistry 101: 43-153.
Laemmli UK (1970) Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227: 680-685.
Morelli A, Benatti U, Gaetani GF, De Flora A (1978) Biochemical mechanisms of glucose-6-phosphate dehydrogenase deficiency. Proc. Natl. Acad. Sci. USA 75: 1979-1983.
Ninfali P, Palma F (1990) Comparative study on glucose-6-phosphate dehydrogenase from rabbit tissues. J. Exp. Zool. 254: 6-12.
Ninfali P, Aluigi G, Pompella A (1997) Methods for the study of glucose-6-phosphate dehydrogenase activity in brain areas. Brain Res. Prot. 1: 357-363.
Ninfali P, Aluigi G, Pompella A (1998) Postnatal expression of glucose-6-phosphate dehydrogenase in different brain areas. Neurochem. Res. 23: 1199-1206.
Ninfali P, Baronciani L, Rapa S, Marzioni D, Mannello F (1994) Goat immunoglobulin purification on phosphocellulose and DEAE Affy-gel Blue. Prep. Biochem. 24: 1-13.
Pandolfi PP, Sonati S, Rivi R, Mason P, Grosveld F, Luzzatto L (1995) Targeted disruption of the housekeeping gene encoding glucose-6-phosphate dehydrogenase (G6PD): G6PD is dispensable for pentose synthesis but essential for defense against oxidative stress. EMBO J. 14: 5209-5215.
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Capellacci, S., Aluigi, G., Tabellini, L. et al. One step purification of glucose-6-phosphate dehydrogenase from brain areas by immunoaffinity chromatography. Biotechnology Letters 23, 353–357 (2001). https://doi.org/10.1023/A:1005645321854
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DOI: https://doi.org/10.1023/A:1005645321854