Characterization of rat brain kynurenine aminotransferases I and II
Characterization of rat brain kynurenine aminotransferases I and II
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DOI：
10.1002/(sici)1097-4547(19971101)50:3
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发表时间：
1997-11
影响因子：
4.2
通讯作者：
P. Guidetti;E. Okuno;R. Schwarcz
中科院分区：
文献类型：
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作者：
P. Guidetti;E. Okuno;R. Schwarcz
内源性神经保护剂犬尿氨酸（KYNA）由L-犬尿氨酸（KYN）的不可逆转氨作用产生。在大脑中，两种不同的犬尿氨酸氨基转移酶（KAT I和KAT II）负责KYNA的形成。本实验旨在研究这两种KAT在正常大鼠脑中的各自作用。为此，这两种酶进行了部分纯化，并检查了它们的特性。KAT I（与谷氨酰胺转氨酶K相同）的最适pH为9.5，优选丙酮酸作为共底物，并被谷氨酰胺有效抑制。KAT II（与L-α-氨基己二酸转氨酶相同）具有中性最适pH，对丙酮酸盐无偏好，对谷氨酰胺的抑制作用基本不敏感。使君子酸可选择性抑制KAT II（IC 50：520 μM）。内源性底物3-羟基犬尿氨酸对KAT II的偏好约为10倍。这两种酶的不同性质使得可以通过使用透析的组织匀浆（以去除干扰内源性氨基酸）来平行测量脑KAT I和KAT II。在这些条件下，这两种酶呈现出与部分纯化的酶基本相同的表观Km值。在受损的，神经元耗尽的脑组织和小脑以外的脑区，KYNA主要来自生理pH下的KAT II。总之，本研究描述了一种简单的方法，用于同时测定小鼠脑组织样品中的两种KYNA产生酶，并为实验挑战动物的未来工作提供基线值。神经科学杂志Res. 50：457-465，1997.© 1997 Wiley利斯公司
The endogenous neuroprotectant kynurenic acid (KYNA) is produced by irreversible transamination of L‐kynurenine (KYN). In the brain, two distinct kynurenine aminotransferases (KAT I and KAT II) are responsible for the formation of KYNA. The present experiments were designed to examine the respective roles of the two KATs in the normal rat brain. To this end, the two enzymes were partially purified, and their characteristics were examined. KAT I (identical with glutamine transaminase K) had an optimal pH of 9.5, preferred pyruvate as a cosubstrate and was potently inhibited by glutamine. KAT II (identical with L‐α‐aminoadipate transaminase) had a neutral optimal pH, showed no preference for pyruvate, and was essentially insensitive to inhibition by glutamine. KAT II was selectively inhibited by quisqualic acid (IC50: 520 μM). The endogenous substrate 3‐hydroxykynurenine had an approximately 10‐fold preference for KAT II. The distinct properties of the two enzymes made it possible to measure brain KAT I and KAT II in parallel by using dialyzed tissue homogenate (to remove interfering endogenous amino acids). Under these conditions, both enzymes presented essentially the same apparent Km values as the partially purified enzymes. In lesioned, neuron‐depleted brain tissue and in brain regions other than the cerebellum, KYNA derived primarily from KAT II at physiologic pH. In summary, the present study describes a simple methodology for the simultaneous determination of the two KYNA‐producing enzymes in small rat brain tissue samples and provides baseline values for future work in experimentally challenged animals. J. Neurosci. Res. 50:457–465, 1997. © 1997 Wiley‐Liss, Inc.