Purification and partial characterization of 4-aminobutyrate:2-oxoglutarate aminotransferase from sheep brain and locust ganglia.
We report here the first purification to homogeneity of 4-aminobutyrate: 2-oxoglutarate aminotransferase (EC 2.6.1.19) (GABA-T) from an invertebrate source (locust) and its initial comparison with that of GABA-T from mammalian brain (sheep). The enzyme from both organisms was found to be a dimer of similar-sized subunits, with a native Mr of approx. 97,000. The pI of GABA-T from the locust was 6.7 and that of the sheep enzyme was 5.5. Michaelis constants for 4-aminobutyric acid (GABA) and 2-oxoglutarate were respectively 0.79 +/- 0.16 mM and 0.27 +/- 0.08 mM for the locust enzyme and 2.2 +/- 0.24 mM and 0.22 +/- 0.11 mM for the sheep enzyme. 5-(Aminomethyl)-3-isoxazolol (muscimol) was a competitive inhibitor of both enzymes, whereas 5-amino-1,3-cyclohexadienylcarboxylic acid (gabaculine) acted as a potent suicide substrate. However, 3-aminopropane-1-sulphonic acid, diaminobutyric acid, 1,2,3,4-tetrahydro-1-methyl-3-pyridinecarboxylic acid (isoguvacine), beta-(aminomethyl)-4-chlorobenzenepropanoic acid (baclofen), bicuculline and picrotoxin did not inhibit either enzyme at concentrations below 100 mM. Polyclonal antisera raised against GABA-T from the sheep failed to cross-react with the enzyme from locust in either an Ouchterlony immunodiffusion plate or a competitive enzyme-linked immunosorbent assay. The purification procedures differed considerably. Ion-exchange chromatography, which was found suitable for the purification of GABA-T from the sheep, was ineffective with locust enzyme, which was finally purified by hydrophobic-interaction chromatography and chromatofocusing.
Jeffery
D
D
Department of Biochemistry, University of Bath, Claverton Down, U.K.
Rutherford
D M
DM
Weitzman
P D
PD
Lunt
G G
GG
D000612:4-Aminobutyrate Transaminase / Q000037:antagonists & inhibitors / Q000302:isolation & purification*; D000818:Animals; D001921:Brain / Q000201:enzymology*; D002499:Centrifugation, Density Gradient; D002846:Chromatography, Affinity; D003509:Cyclohexanecarboxylic Acids / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D005724:Ganglia / Q000201:enzymology*; D006110:Grasshoppers / Q000201:enzymology*; D007525:Isoelectric Focusing; D007700:Kinetics; D012756:Sheep
D003509:Cyclohexanecarboxylic Acids; C012987:gabaculine; D000612:4-Aminobutyrate Transaminase