KINETIC STUDY OF GLUTAMATE TRANSPORT IN RAT-BRAIN MITOCHONDRIA
KINETIC STUDY OF GLUTAMATE TRANSPORT IN RAT-BRAIN MITOCHONDRIA
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DOI：
10.1111/j.1471-4159.1977.tb10731.x
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发表时间：
1977-01-01
影响因子：
4.7
通讯作者：
GAYET, J
中科院分区：
文献类型：
--
作者：
MINN, A;GAYET, J
谷氨酸穿透离体大鼠脑非突触体线粒体的内膜，无论是在谷氨酸-羟基逆向转运蛋白或谷氨酸-天冬氨酸逆向转运蛋白。在燕麦酰胺或N-乙基马来酰亚胺存在下，以谷氨酸为底物的线粒体呼吸活性得到抑制。在等渗NH 4 +-L-谷氨酸的线粒体肿胀抑制avenaciline和N-乙基马来酰亚胺的存在下，但mersalyl，红藻氨酸，glisoxepide和amino-oxyacetic acid上的谷氨酸-羟基交换没有影响。谷氨酸诱导线粒体内NAD（P）的减少，如双光束分光光度法所估计的，这种减少被N-乙基马来酰亚胺，avenacidin或褐质，和氨氧乙酸抑制。通过使用离心停止程序直接估计L-[14 C]谷氨酸进入脑线粒体的渗透。这种渗透遵循饱和动力学，在pH 7.4和20 ℃时平均表观Km为1.56mM。C时，Vmax为4.34nmol/min/mg蛋白。N-乙基马来酰亚胺减慢了谷氨酸渗透的初始速率，并且这种抑制似乎是非竞争性的，在pH 7.4和20 ℃时Ki为0.7mM。C.谷氨酸的进入是pH依赖性的，并且在7.4-6.4的pH范围内增加2倍。谷氨酸转运的温度依赖性也显示在2 - 25 ℃之间。C; Arrhenius图是直线，计算的EA为12.8 kcal/mol谷氨酸盐，Q10为2.16。γ的活性-谷氨酰转肽酶在这些大鼠脑线粒体中几乎不存在。在大鼠脑非突触体线粒体中，通过体外重组的苹果酸-天冬氨酸穿梭来氧化线粒体外的NADH。在线粒体外的苹果酸或谷氨酸的情况下，穿梭没有功能，在线粒体外的天冬氨酸的情况下，NADH氧化的速率低。谷氨酰胺或γ-氨基丁酸不能有效地替代谷氨酸。在存在avenacilline或mersalyl的情况下，苹果酸-天冬氨酸穿梭的抑制作用较高，在存在N-乙基马来酰亚胺、格列索派或丙二酸正丁酯的情况下，苹果酸-天冬氨酸穿梭的抑制作用中等。在线粒体外谷氨酸存在的情况下，完整脑线粒体中的谷氨酰胺酶活性受到抑制。
Glutamate penetrated the inner membrane of isolated rat brain non-synaptomosomal mitochondria, either on a glutamate-hydroxyl antiporter or on a glutamate-aspartate antiporter. An inhibition of respiratory activity of mitochondria with glutamate as substrate was obtained in the presence of avenaciolide or N-ethylmaleimide. Swelling of the mitochondria in iso-osmotic NH4+-L-glutamate was inhibited in the presence of avenaciolide and N-ethylmaleimide, but mersalyl, kainic acid, glisoxepide and amino-oxyacetic acid had no effect on the glutamate-hydroxyl exchange. Glutamate induced the reduction of intramitochondrial NAD(P), as estimated by double-beam spectrophotometry, and this reduction was inhibited by N-ethylmaleimide, avenaciolide or fuscin, and by aminooxyacetic acid. A direct estimation of the penetration of L-[14C]glutamate into brain mitochondria was performed by using the centrifugation-stop procedure. This penetration followed saturation kinetics, with a mean apparent Km of 1.56 mM at pH 7.4 and at 20.degree. C, the value of Vmax was 4.34 nmol/min per mg protein in the same conditions. N-Ethylmaleimide slowed down the initial rate of glutamate penetration, and this inhibition appeared to be non-competitive with a Ki of 0.7 mM at pH 7.4 and at 20.degree. C. The entry of glutamate was pH-dependent and it increased 2-fold in the pH range of 7.4-6.4. A temperature-dependence of glutamate transport was also shown between 2 and 25.degree. C; the Arrhenius plot was a straight line, with a calculated EA of 12.8 kcal/mol of glutamate and a Q10 of 2.16. The activity of .gamma.-glutamyl transpeptidase was practically absent in these rat brain mitochondria. Oxidation of extramitochondrial NADH by the malate-aspartate shuttle reconstituted in vitro was followed in rat brain non-synaptosomal mitochondria. In the absence of extramitochondrial malate or glutamate the shuttle did not function, and in the absence of extramitochondrial aspartate the rate of NADH oxidation was low. Glutamine or .gamma.-aminobutyrate did not replace glutamate efficiently. A high inhibition of the malate-aspartate shuttle occurred in the presence of avenaciolide or mersalyl, and a moderate one in the presence of N-ethylmaleimide, glisoxepide or n-butylmalonate. Glutaminase activity in intact brain mitochondria was inhibited in the presence of extramitochondrial glutamate.