经低浓度镁离子处理而失活的兔网织红细胞核糖体肽基转移酶中心的重新激活。

Re-activation of the peptidyltransferase centre of rabbit reticulocyte ribosomes after inactivation by exposure to low concentrations of magnesium ion.

作者信息

Cox R A, Greenwell P, Hirst W

出版信息

Biochem J. 1976 Dec 15;160(3):521-31. doi: 10.1042/bj1600521.

DOI:10.1042/bj1600521

PMID:1016237

原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC1164268/

Abstract

The larger subrivosomal particles of rabbit reticulocytes retained full activity in the puromycin reaction and in poly(U)-directed polyphenylalanine synthesis after 4h at 0 degrees C when buffered 0.5M-NH4Cl/10-30mM-MgCl2 was the solvent. 2. Activity in the puromycin reaction was diminished to approx 10% after 15-30 min at 0 degrees C when the concentration of MgCl2 was lowered to 2mM. 3. Activity was not restored when the concentration of MgCl2 was raised from 2mM to 10-30 mM at 0 degrees C. However, activity was recovered as measured by both assay systems when the ribosome fraction was heated to 37 degrees C at the higher concentrations of MgCl2. 4. Recovery of activity was noted during the course of the polyphenylalanine synthesis in 50 mM-KCl/5mM-MgCl2/25mM-Tris/HCl, pH 7.6, at 37 degrees C. Re-activation was slow at 20 degrees C and below. 5. No more than about 5% of the protein moiety of the subparticle was lost in 0.5M-NH4Cl on decreasing MgCl2 concentration from 10mM to 2mM. No proteins were detected in the supernatant fractions by gel electrophoresis after ribosomes were separated by differential centrifugation. The supernatant fraction was not essential for the recovery of activity. However, at higher (e.g. 1M) concentrations of NH4Cl, proteins were split from the subparticle. 6. The loss and regain of activity found on lowering and restoring the concentration of MgCl2 at 0.5M-NH4Cl appears to arise from a conformational change that does not seem to be associated with a loss and regain of particular proteins. 7. A 2% decrease in E260 was noticed when the concentration of Mg2+ was restored, and the change in the spectrum indicated a net increase of approx. 100A-U base-pairs per subribosomal particle. 8. When the concentration of Mg2+ was restored, S20,W of the subparticle remained at 52+/- 1S until the sample was incubated at 37 degrees C when S20,W increased to 56 +/- 1S compared with the value of 58 +/- 1S for the subparticle as originally isolated.

摘要

当以0.5M - NH₄Cl/10 - 30mM - MgCl₂作为缓冲溶剂时，兔网织红细胞较大的亚核糖体颗粒在0℃放置4小时后，在嘌呤霉素反应和聚（U）指导的聚苯丙氨酸合成中仍保持全部活性。2. 当MgCl₂浓度降至2mM时，在0℃放置15 - 30分钟后，嘌呤霉素反应中的活性降低至约10%。3. 当在0℃将MgCl₂浓度从2mM提高到10 - 30mM时，活性未恢复。然而，当核糖体组分在较高MgCl₂浓度下加热至37℃时，两种测定系统均测得活性恢复。4. 在37℃，50mM - KCl/5mM - MgCl₂/25mM - Tris/HCl，pH 7.6的条件下，聚苯丙氨酸合成过程中观察到活性恢复。在20℃及以下，重新激活缓慢。5. 当MgCl₂浓度从10mM降至2mM时，亚颗粒蛋白质部分在0.5M - NH₄Cl中损失不超过约5%。通过差速离心分离核糖体后，凝胶电泳未在上清液组分中检测到蛋白质。上清液组分对活性恢复不是必需的。然而，在较高（例如1M）浓度的NH₄Cl中，蛋白质从亚颗粒中分离出来。6. 在0.5M - NH₄Cl中降低和恢复MgCl₂浓度时发现的活性丧失和恢复似乎源于构象变化，这种变化似乎与特定蛋白质的丧失和恢复无关。7. 当恢复Mg²⁺浓度时，观察到260nm处的吸光度下降2%，光谱变化表明每个亚核糖体颗粒净增加约100个A - U碱基对。8. 当恢复Mg²⁺浓度时，亚颗粒的S₂₀,W保持在52±1S，直到样品在37℃孵育，此时S₂₀,W增加到56±1S，而最初分离的亚颗粒的值为58±1S。