Agmatine is synthesized by a mitochondrial arginine decarboxylase in rat brain.
Agmatine is synthesized by a mitochondrial arginine decarboxylase in rat brain.
复制标题
DOI：
10.1111/j.1749-6632.1995.tb32418.x
复制
发表时间：
1995-07-12
影响因子：
5.2
通讯作者：
Reis, D J
中科院分区：
文献类型：
--
作者：
Li, G;Regunathan, S;Reis, D J
材料与方法大鼠脑在HEPES-蔗糖缓冲液（5 mM HEPES, pH 7.4; 2 mM DTT; 0.5 mM PMSF; 0.2 mM EDTA）中用特氟龙玻璃均质机均质，1000 x g离心10分钟。保存含有核部分的颗粒（Pl）用于酶分析。将上清液在12,000 x g离心得到粗粒线粒体突触体颗粒（P2）。将得到的上清液在30,000 x g下离心20分钟，生成含有质粒微粒体膜部分的颗粒（P3）。这是用于酶分析。用Percoll密度梯度法从P2颗粒中分离出线粒体或突触体。每个部分的膜用于酶分析。除了研究酶在每个亚细胞间室的分布外，还在含有线粒体-突触体膜的P2颗粒中测量了ADC活性。采用Wu和Moms测定14c -精氨酸转化为14co2的方法测定精氨酸脱羧酶。简而言之，将组织颗粒悬浮在不含蔗糖的预冷亚细胞分离缓冲液中，用Polytron （Brinkman，设置6,2 × 15 s）均质，并在100,000 x g下离心
MATERIALS AND METHODSRat brains were homogenized in HEPES-sucrose buffer (5 mM HEPES, pH 7.4; 2 mM DTT; 0.5 mM PMSF; 0.2 mM EDTA) with a Teflon-glass homogenizer and centrifuged at 1,000 x g for 10 minutes. The pellet (Pl) containing the nuclear fraction was saved for enzyme assays. The supernatant was centrifuged at 12,000 x g to produce a crude mitochondriallsynaptosomal pellet (P2). The resulting supernatant was centrifuged at 30,000 x g for 20 minutes generating a pellet (P3) containing the plasmdmicrosomal membrane fraction. This was used for enzyme assays. Separate mitochondria1 or synaptosomal fractions were isolated from the P2 pellet by Percoll density gradients.'Membranes from each fraction were used for enzyme assays. Except for studies on enzyme distribution in each subcellular compartment, ADC activity was measured in the P2 pellet which contained mitochondriallsynaptosomal membranes. Arginine decarboxylase was assayed by the method of Wu and Moms' measuring conversion of 14C-arginine to 14C02. In brief, the tissue pellet was suspended in pre-chilled subcellular fractionation buffer without sucrose, homogenized by a Polytron (Brinkman, setting 6 for 2 x 15 s), and centrifuged at 100,000 x g for