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.

中文翻译：

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大鼠脑胞浆酪氨酸转氨酶的纯化，鉴定和鉴定为谷氨酰胺转氨酶-K。

进行当前的研究以研究大鼠脑的胞质部分中酪氨酸转氨酶的光谱，并专门纯化和表征先前鉴定的具有酪氨酸/乙醛酸转氨酶活性的胞质脑酶。根据纯化的脑酪氨酸/乙醛酸转氨酶的广泛生化和免疫化学特征，我们得出结论，纯化的酶是谷氨酰胺转氨酶-K（EC 2.6.1.64）。该结论基于：1.）纯化期间同时富集酪氨酸/乙醛酸和谷氨酰胺/苯丙酮酸转氨酶活性； 2.）证明了氨基酸和α-酮酸的共底物特异性与已发表的结果高度一致谷氨酰胺转氨酶-K的相关信息，3.）详细的动力学分析结果，4。）谷氨酰胺是体外酪氨酸/乙醛酸转氨作用的有效抑制剂，5.）生化特性，包括最适pH值为8.5和分光光度法分析，以及6.）使用针对大鼠肾脏谷氨酰胺转氨酶-k的特异性抗血清进行免疫分析。此外，全脑粗提物的免疫化学特性表明，几种不同的α-酮酸共底物的体外酪氨酸转氨酶活性可能反映了谷氨酰胺转氨酶-K的活性。总之，这项研究证实了大鼠大脑胞质提取物中存在多种酪氨酸转氨酶。此外，我们得出的结论是谷氨酰胺转氨酶-K代表大鼠脑中主要的胞质酶，能够催化对酪氨酸和其他芳香族氨基酸的体外转氨作用，包括神经递质前体L-多巴和5-羟色氨酸。纯化的转氨酶具有广泛的共底物特异性，并且与衍生自中性脂族和芳族氨基酸的α-酮酸具有优先反应性。最后，我们确定了大鼠脑中酪氨酸/乙醛酸转氨酶（谷氨酰胺转氨酶-K）的异质区域分布，其在小脑中的体外活性明显较高。