RESUMO
The current study was undertaken to investigate the spectrum of tyrosine transaminases enzymes in a cytosolic fraction of rat brain and to specifically purify and characterize a previously identified cytosolic brain enzyme possessing tyrosine/glyoxylate transaminase activity. Based upon extensive biochemical and immunochemical characterization of purified brain tyrosine/glyoxylate transaminase, we concluded the purified enzyme is glutamine transaminase-K (EC 2.6.1.64). This conclusion was based on: 1.) a concurrent enrichment in the tyrosine/glyoxylate and glutamine/phenylpyruvate transaminase activities during purification, 2.) demonstration of a co-substrate specificity for amino acids and α-keto acids that was highly consistent with published information for glutamine transaminase-K, 3.) results from detailed kinetic analysis, 4.) glutamine was a potent inhibitor of in vitro tyrosine/glyoxylate transamination, 5.) biochemical characterization, including pH optimum of 8.5 and spectrophotometric analysis and 6.) immunoanalytical analysis using a specific antiserum to rat renal glutamine transaminase-k. In addition, immunochemical characterization of a crude soluble extract of whole brain suggests that the in vitro tyrosine transaminase activity for several different α-keto acid co-substrates likely reflect the activity of glutamine transaminase-K. In conclusion, this investigation confirmed the presence of multiple tyrosine transaminase enzymes in a cytosolic extract of rat brain. Moreover, we concluded glutamine transaminase-K represents a predominant cytosolic enzyme in rat brain that's capable of catalyzing in vitro transamination of p-tyrosine and other aromatic amino acids, including the neurotransmitter precursors L-dopa and 5-hydroxytryptophan. The purified transaminase possesses a broad co-substrate specificity with preferential reactivity with α-keto acids derived from neutral aliphatic and aromatic amino acids. Lastly, we identified a heterogeneous regional distribution of tyrosine/glyoxylate transaminase (glutamine transaminase-K) in rat brain with a significantly higher level of in vitro activity in cerebellum.
