Glutamate decarboxylase: Loss of N-terminal segment does not affect homodimerization and determination of the oxidation state of cysteine residues
Glutamate decarboxylase: Loss of N-terminal segment does not affect homodimerization and determination of the oxidation state of cysteine residues
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DOI：
10.1007/s11064-005-6772-0
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发表时间：
2005-08-01
影响因子：
4.4
通讯作者：
Martin, DL
中科院分区：
文献类型：
--
作者：
Battaglioli, G;Liu, HC;Martin, DL
谷氨酸脱羧酶（GAD）产生GABA，这是成年哺乳动物大脑中主要的抑制性神经递质。采用质谱法和部分蛋白消化法研究GAD的物理特性。两个主要亚型GAD（65）和GAD（67）的n端通过去除初始蛋氨酸残基和乙酰化倒数第二的丙氨酸进行处理。天然重组GAD（65）和GAD（67）作为同型二聚体存在，可以用非还原方法解离，表明同型二聚体不涉及分子间二硫键。用胰蛋白酶酶切截断n端片段不影响同型二聚化，但通过降低GAD的K-m和增加两种同型异构体的V-max来增加活性（67）。在GAD的15个半胱氨酸（65）中，在n端段发现的6个半胱氨酸可以形成二硫键，在GAD的13个半胱氨酸（67）中，半胱氨酸32和38可以形成二硫键。在体外条件下，在n端形成二硫键，并以相对少量的胰蛋白酶去除末端，表明GAD（65）和GAD（67）的n端片段是暴露的和柔性的。GAD的半胱氨酸30和半胱氨酸45之间形成二硫桥（65）表明，正常氧化还原条件的改变可能影响GAD的靶向性。
Glutamate decarboxylase (GAD) produces GABA, the main inhibitory neurotransmitter in adult mammalian brain. The physical characteristics of GAD were studied using mass spectrometry and partial protein digests. The N-termini of the two main isoforms, GAD(65) and GAD(67), were processed by removal of the initial methionine residues and acetylation of the penultimate alanines. Native recombinant GAD(65) and GAD(67) exist as homodimers that can be dissociated with non-reducing methods, indicating that homodimerization does not involve intermolecular disulfide bonds. Truncation of the N-terminal segment with trypsin digestion did not affect homodimerization but increased activity by decreasing the K-m of GAD(67) and increasing the V-max of both isoforms. Of the 15 cysteines in GAD(65), the six found in the N-terminal segment can form disulfide bonds and of the 13 cysteines in GAD(67), cysteines 32 and 38 can form a disulfide bond. The in vitro formation of disulfide bonds in the N-termini, and the removal of the termini with relatively low amounts of trypsin, indicate that the N-terminal segments of GAD(65) and GAD(67) are exposed and flexible. The formation of a disulfide bridge between cysteines 30 and 45 of GAD(65) suggests that alteration of normal redox conditions could affect GAD targeting.