SYNAPTIC PLASMA MEMBRANES FROM RAT BRAIN SYNAPTOSOMES - ISOLATION AND PARTIAL CHARACTERIZATION
SYNAPTIC PLASMA MEMBRANES FROM RAT BRAIN SYNAPTOSOMES - ISOLATION AND PARTIAL CHARACTERIZATION
复制标题
DOI：
10.1016/0005-2736(71)90117-9
复制
发表时间：
1971-01-01
期刊：
影响因子：
--
通讯作者：
MATTHEWS, DA
中科院分区：
文献类型：
--
作者：
COTMAN, CW;MATTHEWS, DA
我们直接从通过聚蔗糖-蔗糖梯度离心制备的突触体中分离出高度纯化的质膜组分。该制备方法的一个关键特征是使用碱性条件来实现有效的渗透压休克，从而使膜有效地从线粒体中分离出来。如果在碱性 pH 下进行渗透压休克，突触体的 (Na+-K+) 激活哇巴因敏感 ATP 酶 (EC 3.6. 1.4) 的 70% 可以被回收，而突触体膜组分中的总细胞色素氧化酶 (EC 1.9. 3.1) 仅占 7%。相反，如果在中性 pH 条件下进行渗透休克，则 50% 的线粒体覆盖 85% 的膜。富含突触质膜的级分（SPM 级分）可以通过简单的不连续梯度以相对较高的产率制备。 SPM 分数带介于 25-32.5% 蔗糖之间；每克组织湿重的产量约为 1.5 毫克蛋白质。 SPM 级分中发现碱性磷酸酶 (EC 3.1. 3.1)。碱性磷酸酶的分布模式与Na+-K+)-ATP酶的分布模式相似。 5'-核苷酸酶 (EC 3.1. 3.5) 和乙酰胆碱酯酶 (EC 3.1. 1.7) 的沉积密度比 (Na+-K+)-ATP 酶或碱性磷酸酶更轻，尽管存在相当大的重叠。 SPM 级分含有 β-N-乙酰氨基葡萄糖苷酶。这种酶不会通过盐处理完全从 SPM 级分中洗掉，而是通过低浓度的 Triton X-100 释放。
We have isolated a highly purified plasma membrane fraction directly from synaptosomes prepared by centrifugation on a Ficoll-sucrose gradient. A critical feature of the preparation method is use of alkaline conditions to achieve effective osmotic shock so that membranes effectively resolve from mitochondria. If osmotic shock is carried out at alkaline pH, 70% of the (Na+-K+)-activated-ouabain-sensitive ATPase (EC 3.6. 1.4) of the synaptosomes can be recovered with only 7% of total cytochrome oxidase (EC 1.9. 3.1) in synaptosomal membrane fractions. In contrast, if osmotic shock is carried out at neutral pH, 50% of the mitochondria overlays 85% of the membranes. A fraction enriched in synaptic plasma membranes (SPM fraction) can be prepared on a simple discontinuous gradient in relatively good yield. The SPM fraction bands between 25–32.5% sucrose; the yield is about 1.5 mg of protein per g tissue wet wt. An alkaline phosphatase (EC 3.1. 3.1) is found in the SPM fraction. The distribution pattern of alkaline phosphatase parallels that of Na+-K+)-ATPase. 5′-Nucleotidase (EC 3.1. 3.5) and acetylcholinesterase (EC 3.1. 1.7) sediment at lighter densities than (Na+-K+)-ATPase or alkaline phosphatase although there is considerable overlap. The SPM fraction contains β-N-acetylglucosaminidase. This enzyme is not completely washed out of the SPM fraction by salt treatment but is released by low concentrations of Triton X-100.