title: Characterization of monomeric 4-aminobutyrate aminotransferase at low pH.
abstract: 4-Aminobutyrate aminotransferase undergoes a reversible process of association/dissociation at low pH. At pH 5.0, monomeric species exist predominantly in solution as revealed by FPLC and time-dependent emission anisotropy measurements. The observed rotational correlation time at pH 5.0, phi obs = 25 ns, corresponds to a compact spherical unit of 52 kDa. An increase in the net charge of the macromolecule at pH 5.0 is responsible for destabilization of the dimeric structure, (WEL approximately 41.84 kJ/mol), but the dissociation of the protein does not perturb the secondary structure as revealed by CD measurements. The fluorescent probe 1-anilinonaphthalene-8-sulfonate (ANS), bound to hydrophobic sites of the enzyme, was used to monitor the kinetics of protein dissociation by stopped-flow spectroscopy. The dissociation of the dimeric structure at pH 5.0 was characterized by a relaxation time of 18 ms. The rate of association of monomeric subunits at pH 7.0 was too fast to be detected in the stopped-flow instrument. These observations have some bearing on the mechanism of reconstitution of dimeric structures of 4-aminobutyrate aminotransferase in the cell.
authors.affiliation: Department of Biochemistry, University of Tennessee, Knoxville, USA.
authors.forename: T
authors.initials: T
authors.lastname: Pineda
authors.forename: Y D
authors.initials: YD
authors.lastname: Osei
authors.forename: J E
authors.initials: JE
authors.lastname: Churchich
mesh_terms: D000612:4-Aminobutyrate Transaminase / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D000817:Anilino Naphthalenesulfonates; D000818:Animals; D001665:Binding Sites; D002850:Chromatography, Gel; D002942:Circular Dichroism; D004795:Enzyme Stability; D005456:Fluorescent Dyes; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008099:Liver / Q000201:enzymology; D017433:Protein Structure, Secondary; D013552:Swine
chemical_list: D000817:Anilino Naphthalenesulfonates; D005456:Fluorescent Dyes; C027132:1-anilino-8-naphthalenesulfonate; D000612:4-Aminobutyrate Transaminase