经区带离心分离的脑细胞核中脱氧核糖核酸聚合酶的活性及脱氧核糖核酸合成

The activity of deoxyribonucleic acid polymerase and deoxyribonucleic acid synthesis in nuclei from brain fractionated by zonal centrifugation.

作者信息

Stambolova M A, Cox D, Mathias A P

出版信息

Biochem J. 1973 Nov;136(3):685-95. doi: 10.1042/bj1360685.

DOI:10.1042/bj1360685

PMID:4780694

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC1166004/

Abstract

The DNA polymerase (EC 2.7.7.7) activity in purified intact brain nuclei from infant rats was investigated. The effects of pH, Mg(2+), glycerol, sonication and storage of the nuclei under different conditions were examined and a suitable assay system was established. 2. The nuclei from infant brain cells were fractionated by zonal centrifugation in a discontinuous sucrose gradient into five zones: zone (I) contained neuronal nuclei (59%) and astrocytic nuclei (41%); zone (II) contained astrocytic nuclei (81%) and neuronal nuclei (19%); zone (III) contained astrocytic nuclei (82%) and oligodendrocytic nuclei (18%); zone (IV) contained oligodendrocytic nuclei (92%) and zone (V) contained oligodendrocytic nuclei (100%). 3. The content of DNA, RNA and protein for each fraction was measured. 4. The distribution of DNA polymerase activity in the fractionated infant and adult rat brain nuclei was determined. The highest activity was found in the neuronal nuclei from zone (I) and the following zones exhibited a progressive decline. In contrast with the nuclei from infant rats those from adults had a much higher activity and expressed a preference for native DNA as template. 5. The deoxyribonuclease activity in all classes of nuclei was measured with [(3)H]DNA as substrate. A general correspondence in the pattern of the relative activities in the nuclear fractions with the distribution of DNA polymerase was found. 6. The incorporation of [(3)H]thymidine into nuclear DNA in infant and adult rat brain was investigated. The specific radioactivity of the DNA in the 10-day-old rats was highest in zone (V) whereas in the nuclei of adult rats, which exhibited a comparatively low incorporation, the highest specific radioactivity was associated with zones (I) and (V).

摘要

对幼鼠纯化的完整脑细胞核中的DNA聚合酶（EC 2.7.7.7）活性进行了研究。考察了pH、镁离子、甘油、超声处理以及在不同条件下细胞核的储存对其的影响，并建立了合适的检测系统。2. 通过在不连续蔗糖梯度中进行区带离心，将幼鼠脑细胞的细胞核分离为五个区带：区带（I）含有神经元细胞核（59%）和星形胶质细胞核（41%）；区带（II）含有星形胶质细胞核（81%）和神经元细胞核（19%）；区带（III）含有星形胶质细胞核（82%）和少突胶质细胞核（18%）；区带（IV）含有少突胶质细胞核（92%），区带（V）含有少突胶质细胞核（100%）。3. 测定了每个组分的DNA、RNA和蛋白质含量。4. 确定了分离后的幼鼠和成年大鼠脑细胞核中DNA聚合酶活性的分布。在区带（I）的神经元细胞核中发现活性最高，随后的区带活性逐渐下降。与幼鼠的细胞核相比，成年大鼠的细胞核活性更高，并且更倾向于以天然DNA作为模板。5. 以[³H]DNA为底物，测定了所有类型细胞核中的脱氧核糖核酸酶活性。发现细胞核组分中相对活性模式与DNA聚合酶的分布总体一致。6. 研究了[³H]胸腺嘧啶核苷掺入幼鼠和成年大鼠脑细胞核DNA的情况。10日龄大鼠DNA的比放射性在区带（V）中最高，而在掺入量相对较低的成年大鼠细胞核中，最高比放射性与区带（I）和（V）相关。