Structural characteristics of brain glutamate decarboxylase in relation to its interaction and activation

Archives of Biochemistry and Biophysics
C H ChenD L Martin

Abstract

The conformation, stability, cofactor interactions, and activation of a recombinant 65-kDa form of rat brain glutamate decarboxylase (GAD65) were investigated by using UV/visible spectrophotometry, fluorescence spectroscopy, circular dichroism, and differential scanning microcalorimetry. The enzyme was prepared from Sf9 insect cells infected with a recombinant baculovirus containing the entire GAD65 coding region. The UV/visible absorption spectrum of purified holoenzyme (holoGAD) exhibits two peaks in the range of 300-450 nm, which are due to the formation of a Schiff base when pyridoxal phosphate (pyridoxal-P) binds to GAD. Fluorescence emission intensity (excited at 295 or 280 nm) was substantially enhanced when pyridoxal-P was removed from holoGAD and quenched when pyridoxal-P was added to the apoenzyme (apoGAD). These observations implied that a significant enzyme conformational change occurs during the formation of holoGAD. Circular dichroism provided additional evidence for a conformational change, as the ellipticity of both negative (202-242 nm) and positive (188-202 nm) bands decreased when pyridoxal-P was removed from holoGAD. Secondary structure determination estimated that holoGAD contains a higher content of alpha-...Continue Reading

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Citations

Dec 7, 2011·Proceedings of the National Academy of Sciences of the United States of America·Giorgio GiardinaFrancesca Cutruzzolà
Jun 14, 2014·Proceedings of the National Academy of Sciences of the United States of America·Itamar KassAshley M Buckle
Feb 3, 2005·Journal of Neurochemistry·Hong JinJang-Yen Wu
Mar 9, 2006·Comparative Biochemistry and Physiology. Part B, Biochemistry & Molecular Biology·Ravinder K SardanaVance L Trudeau
Sep 29, 2009·Molecular Immunology·Yasir ArafatAshley M Buckle
Sep 21, 2000·The Journal of Biological Chemistry·S BettatiA Mozzarelli
May 25, 2002·Journal of Biomolecular Structure & Dynamics·Natalia P BazhulinaBella S Sukhareva
Apr 13, 2005·The Biochemical Journal·Mariarita BertoldiCarla Borri Voltattorni
Oct 1, 2011·Applied Biochemistry and Biotechnology·Homa TorabizadehMassoud Amanlou
Mar 28, 2017·The Protein Journal·Riccardo MontioliBarbara Cellini
Jan 22, 2003·Biochimica Et Biophysica Acta·Chang Hwei ChenWilfredo Colón

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