COMPARISON OF CYSTEINE SULFINIC ACID DECARBOXYLASE ISOENZYMES AND GLUTAMIC-ACID DECARBOXYLASE IN RAT-LIVER AND BRAIN
COMPARISON OF CYSTEINE SULFINIC ACID DECARBOXYLASE ISOENZYMES AND GLUTAMIC-ACID DECARBOXYLASE IN RAT-LIVER AND BRAIN
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DOI：
10.1016/0306-4522(81)90114-7
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发表时间：
1981-01-01
期刊：
影响因子：
3.3
通讯作者：
KOPIN, IJ
中科院分区：
文献类型：
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作者：
OERTEL, WH;SCHMECHEL, DE;KOPIN, IJ
我们测试了以下假设：脑中存在半胱氨酸亚磺酸脱羧酶 (CSD) 的两种同工酶 CSD I 和 CSD II，并且脑 CSD I 与肝脏 CSD 相同，脑 CSD II 具有谷氨酸脱羧酶。 大鼠脑半胱氨酸亚磺酸脱羧酶活性，该酶将半胱氨酸转化为牛磺酸，将半胱氨酸转化为亚牛磺酸通过氨基乙基琼脂糖凝胶色谱分离成两个峰，称为 CSD I 和 CSD II。发现CSD活性的第二个峰（CSD II）具有谷氨酸脱羧酶活性，这是γ-氨基丁酸的生物合成酶。当在平行实验中对大鼠脑 CSD 和肝 CSD 活性进行色谱分析时，肝 CSD 以与脑 CSD I 相同的分数洗脱。谷氨酸脱羧酶的抗血清用于不同酶活性的免疫学研究。在与中间凝胶的交叉免疫电泳中，该抗血清改变了脑匀浆上清液中一种抗原-抗体沉淀素线的迁移率。在串联交叉免疫电泳和线-线免疫电泳中，在肝匀浆上清液中未检测到该抗原，因此似乎是脑特异性的。抗血清沉淀了脑匀浆上清液中 85% 的谷氨酸脱羧酶活性和 75% 的 CSD 活性。然而，脑CSD I活性和肝脏CSD活性均不受抗血清影响。在脑匀浆上清液中，谷氨酸脱羧酶不可逆抑制剂γ-乙炔γ-氨基丁酸酯比CSD更有效地抑制谷氨酸脱羧酶，但不影响肝匀浆上清液中的CSD。以半胱氨酸亚磺酸为底物，部分纯化的肝脏 CSD 的表观 Km 为 0.045-0.050 mm，脑 CSD I 为 0.050-0.100 mm，脑 CSD II 为 6mm；而以谷氨酸为底物的脑谷氨酸脱羧酶为1.5毫米。将脑匀浆上清液与 γ-乙炔 γ-氨基丁酸盐一起温育后保留的脑 CSD 活性的 Km 为 0.080 mm 半胱氨酸亚磺酸，因此在肝 CSD 和脑 CSD I 的 Km 值范围内。肝 CSD 和脑 CSD I 的最适 pH 值实际上相同，具有 7.4 和 7.8 之间相当宽的最大值。谷氨酸十酰化酶和 CSD II 表现出更清晰的 pH 最适值，pH 值分别为 6.9-7.1 和 7.4-7.5。谷氨酸（8 mm）抑制脑 CSD II 活性高达 75%，但不影响脑 CSD I 或肝脏 CSD。从这些数据中，我们得出结论，在大鼠脑中存在一种同时具有谷氨酸脱羧酶和脑 CSD II 酶活性的蛋白质，或者谷氨酸脱羧酶和脑 CSD II 活性由两个分子代表，其特征非常相似，以至于目前使用的生化或免疫学技术无法将它们分开。此外，大鼠大脑中存在第二种 CSD 酶（脑 CSD I），其特征与肝 CSD 相同或相似。
We have tested the hypothesis that two isoenzymes of cysteine sulfinic acid decar☐ylase (CSD), CSD I and CSD II, are present in brain, and that brain CSD I is identical with liver CSD and brain CSD II with glutamic acid decar☐ylase.Rat brain cysteine sulphinic acid decar☐ylase activity, the enzyme which converts cysteic acid to taurine and cysteine sulphinic acid to hypotaurine, was separated by chromatography on aminoethylSepharose into two peaks, termed CSD I and CSD II. The second peak of CSD activity (CSD II) was found to possess glutamic acid decar☐ylase activity which is the biosynthetic enzyme of γ-aminobutyric acid. When rat brain CSD and liver CSD activity were chromatographed in parallel experiments, liver CSD was eluted at the same fraction as brain CSD I. An antiserum to glutamate decar☐ylase was used for immunological studies of the different enzyme activities. This antiserum altered the mobility of one antigen-antibody precipitin line in brain homogenate supernatant in crossed-immunoelectrophoresis with intermediate gel. The antigen was not detectable in liver homogenate supernatant in tandem crossed- and line-line-immunoelectrophoresis and thus appeared to be brain specific. The antiserum precipitated 85% of the glutamate decar☐ylase activity and 75% of the CSD activity in brain homogenate supernatant. However, neither brain CSD I activity nor liver CSD activity was affected by the antiserum.In brain homogenate supernatant, γ-acetylenic γ-aminobutyrate, an irreversible inhibitor of glutamate decar☐ylase, inhibited glutamate decar☐ylase more effectively than CSD, but did not affect CSD in liver homogenate supernatant. Using cysteine sulfinic acid as substrate, the apparentKmfor the partially purified liver CSD was 0.045–0.050 mm, for brain CSD I 0.050–0.100 mm, and for brain CSD II 6mm; while that for brain glutamate decar☐ylase with glutamate as substrate was 1.5 mm. TheKmof brain CSD activity, which remained after incubation of brain homogenate supernatant with γ-acetylenic γ-aminobutyrate, was 0.080 mmcysteine sulfinic acid and was thus in the range of theKmvalues of liver CSD and brain CSD I. The pH optima of liver CSD and brain CSD I were virtually identical with a fairly broad maximum between 7.4 and 7.8. Glutamate decar☐ylase and CSD II exhibited sharper pH optima with pH values of 6.9–7.1 and 7.4–7.5 respectively. Glutamate (8 mm) inhibited brain CSD II activity up to 75% but did not affect brain CSD I nor liver CSD.From these data, we conclude that in rat brain there is a protein with both glutamate decar☐ylase and brain CSD II enzyme activity or glutamate decar☐ylase and brain CSD II activities are represented by two molecules with characteristics so similar that they cannot be separated by the biochemical or immunological techniques so far employed. Furthermore, a second CSD enzyme (brain CSD I) exists in rat brain which has characteristics identical, or similar to, liver CSD.