Dunkley P R, Heath J W, Harrison S M, Jarvie P E, Glenfield P J, Rostas J A
Neuroscience Group, Faculty of Medicine, University of Newcastle, New South Wales, Australia.
Brain Res. 1988 Feb 16;441(1-2):59-71. doi: 10.1016/0006-8993(88)91383-2.
A method for preparation of synaptosomes from rat cerebral cortex, on a discontinuous Percoll gradient, was previously developed for use with a P2 pellet (Brain Research, 372 (1986) 115-129). Here the Percoll method has been adapted for use with an S1-supernatant which eliminates a potentially damaging resuspension step and saves over 30 min, representing a third of the total preparation time. The homogeneity of the synaptosomes in each of the 5 subcellular fractions obtained with the S1-Percoll method was determined biochemically by analysis of the distribution of total protein, myelin basic protein, synapsin I and pyruvate dehydrogenase across the gradient. Electron microscopy was also used to determine the homogeneity of the synaptosomes, as well as to determine their morphological characteristics. Fraction 4 was the most enriched in synaptosomes and contained the lowest level of contamination by myelin, extrasynaptosomal mitochondria and plasma membranes. The yield of synaptosomes in fraction 4 with the S1-Percoll method was 1.4-fold greater than with the P2-Percoll method. While all other fractions contained some synaptosomes the major additional content in fractions 1-3 and 5 was, respectively, unidentified small membranes, myelin, synaptic plasma membranes and extrasynaptosomal mitochondria. Fraction 1 was enriched for very small synaptosomes (0.34 micron mean diameter) only 8% of which contained mitochondria, while fractions 2-4 progressively included larger synaptosomes containing more mitochondria. Fraction 5 synaptosomes were approximately the same size as those in fraction 4 (0.63 micron mean diameter), but 83% contained mitochondria, significantly more than in fraction 4. The synaptosomes in fraction 5 were found to be relatively resistant to hypotonic lysis, explaining a previously observed lack of phosphorylation of synapsin I in this fraction. The differences in homogeneity and morphological characteristics of the synaptosomes in fractions 1-5 suggest that the basis for their fractionation on Percoll gradients is different from that achieved with the more traditional procedures for isolating synaptosomes and that unique synaptosomal fractions are obtained with the S1-Percoll procedure.
此前开发了一种在不连续Percoll梯度上从大鼠大脑皮层制备突触体的方法，该方法用于P2沉淀（《脑研究》，372（1986）115 - 129）。在此，Percoll方法已被调整用于S1上清液，这消除了一个潜在的破坏性重悬步骤，并节省了超过30分钟，占总制备时间的三分之一。通过分析总蛋白、髓鞘碱性蛋白、突触素I和丙酮酸脱氢酶在梯度中的分布，对用S1 - Percoll方法获得的5个亚细胞组分中每个组分的突触体同质性进行了生化测定。电子显微镜也被用于确定突触体的同质性以及它们的形态特征。第4组分富含突触体，且髓鞘、突触外线粒体和质膜的污染水平最低。用S1 - Percoll方法在第4组分中获得的突触体产量比用P2 - Percoll方法高1.4倍。虽然所有其他组分都含有一些突触体，但第1 - 3组分和第5组分中的主要额外成分分别是未鉴定的小膜、髓鞘、突触质膜和突触外线粒体。第1组分富含非常小的突触体（平均直径0.34微米），其中只有8%含有线粒体，而第2 - 4组分逐渐包含含有更多线粒体的较大突触体。第5组分的突触体大小与第4组分的大致相同（平均直径0.63微米），但83%含有线粒体，显著多于第4组分。发现第5组分中的突触体对低渗裂解相对抗性较强，这解释了此前观察到的该组分中突触素I缺乏磷酸化的现象。第1 - 5组分中突触体在同质性和形态特征上的差异表明，它们在Percoll梯度上分级分离的基础与更传统的分离突触体程序所实现的基础不同，并且用S1 - Percoll程序可获得独特的突触体组分。
