Purification and characterization of a soluble and a particulate glutamate dehydrogenase from rat brain.
Glutamate dehydrogenase (GDH) activity was determined in high-speed fractions (100,000 g for 60 min) obtained from whole rat brain homogenates after removal of a low-speed pellet (480 g for 10 min). Approximately 60% of the high-speed GDH activity was particulate (associated with membrane) and the remaining was soluble (probably of mitochondrial matrix origin). Most of the particulate GDH activity resisted extraction by several commonly used detergents, high concentration of salt, and sonication; however, it was largely extractable with the cationic detergent cetyltrimethylammonium bromide (CTAB) in hypotonic buffer solution. The two GDH activities were purified using a combination of hydrophobic interaction, ion exchange, and hydroxyapatite chromatography. Throughout these purification steps the two activities showed similar behavior. Kinetic studies indicated similar Km values for the two GDH fractions for the substrates alpha-ketoglutarate, ammonia, and glutamate; however, there were small but significant differences in Km values for NADH and NADPH. Although the allosteric stimulation by ADP and L-leucine and inhibition by diethylstilbestrol was comparable, the two GDH components differed significantly in their susceptibility to GTP inhibition in the presence of 1 mM ADP, with apparent Ki values of 18.5 and 9.0 microM GTP for the soluble and particulate fractions, respectively. The Hill plot coefficient, binding constant, and cooperativity index for the GTP inhibition were also significantly different, indicating that the two GDH activities differ in their allosteric sites. In addition, enzyme activities of the two purified proteins exhibited a significant difference in thermal stability when inactivated at 45 degrees C and pH 7.4 in 50 mM phosphate buffer.
A D
AD
Colon
A
A
Plaitakis
A
A
Perakis
S
S
Berl
D D
DD
Clarke
D000244:Adenosine Diphosphate / Q000494:pharmacology; D000818:Animals; D001921:Brain / Q000201:enzymology*; D002458:Cell Fractionation; D005969:Glutamate Dehydrogenase / Q000302:isolation & purification*; D006160:Guanosine Triphosphate / Q000494:pharmacology; D007700:Kinetics; D008297:Male; D009243:NAD / Q000378:metabolism; D009249:NADP / Q000378:metabolism; D051381:Rats; D011919:Rats, Inbred Strains; D012995:Solubility
D009243:NAD; D009249:NADP; D000244:Adenosine Diphosphate; D006160:Guanosine Triphosphate; D005969:Glutamate Dehydrogenase