Görisch H, Goss D J, Parkhurst L J
Biochemistry. 1976 Dec 28;15(26):5743-53. doi: 10.1021/bi00671a010.
The association-dissociation kinetics of ribosomes from Escherichia coli have been studied under various conditions in a light-scattering stopped-flow apparatus. The dissociation reaction at 2 mg/ml at 25 degrees C, induced by lowering the MgCl2 concentration from 18 to 3 mM, can best be described by three independent first-order processes with rate constants of 15 s-1, 0.9 s-u, and 3 X 10(-2) s-1, the slowest process comprising about 60% of the overall reaction. The fraction of ribosomes dissociating with the fastest rate (15 s-1) is concentration dependent and becomes negligible at 0.1 mg/ml9 Ribosomes treated with puromycin also show three dissociation rates with essentially the same rate constants as the nontreated samples. The dissociation induced by a high KCl concentration (0.85 MKCl, 18 mM MgCl) also shows three first-order phases with the same rate constants as for the dissociation induced by lowering the MgCl2 concentration. The formation of 70S ribosomes from 30S and 50S subunits, induced by increasing the MgCl2 concentration from 2 to 21 mM, follows second-order biphasic kinetics. A detailed analysis of the kinetic results shows that the two principal ribosomal forms must have one type of subunit in common. When the association data are analyzed assuming that the kinetic heterogeneity arises from two forms of only one subunit, the rate constants are found to be 6.4 X 10(6) and 1.05 X 10(6) M-1 s-1. Sequential flow experiments show that the rapid and slow association species are to be identified, respectively, with phases II (0.9 s-1) and III 0.03 s-1) of dissociation. Relaxation measurements show that these correspond to type B ("loose") and A ("tight") ribosomes, respectively. Tight and loose ribosomes were isolated by sucrose density centrifugation, and dissociation and association kinetic studies confirmed the above assignments. Furthermore, the rate constants for these ribosoems agreed within experimental error with rate constants derived from analysis of the multiphasic kinetic data. The association rate constants are for ribosomes dissociated by dilution with the appropriate buffer immediately before recording the kinetics of association. Ribosomes dissociated by dialysis overnight against 2 mM MgCl2 show an association rate constant for the slower association reaction (type-A ribosome) that is about four times smaller, whereas the rate constant for the faster process is roughly the same. The activation energies of the dissociation reactions, whether induced by lowering the MgCl2 concentration or increasing the KCl concentration, and the association raaction induced by increasing the MgCl2 concentration are less than 3.5 kcal/mol. The rate constants of the dissociation at 3 mM MgCl2 and of the association reaction at 21 mM MgCl2 do not vary between pH 7.2 and 8.4. When 30S and 50S subunits are flowed against buffer containing 20 mM spermidine, the association process is monophasic, with an association constant k - 6 X 10(6) M-1 s-1...
在光散射停流装置中，研究了不同条件下大肠杆菌核糖体的缔合-解离动力学。在25℃下，通过将MgCl₂浓度从18 mM降至3 mM，诱导2 mg/ml核糖体的解离反应，该反应最好用三个独立的一级过程来描述，速率常数分别为15 s⁻¹、0.9 s⁻¹和3×10⁻² s⁻¹，最慢的过程约占总反应的60%。以最快速率（15 s⁻¹）解离的核糖体比例与浓度有关，在0.1 mg/ml时可忽略不计。用嘌呤霉素处理的核糖体也显示出三个解离速率，其速率常数与未处理样品基本相同。由高KCl浓度（0.85 M KCl，18 mM MgCl₂）诱导的解离也显示出三个一级相，其速率常数与降低MgCl₂浓度诱导的解离相同。通过将MgCl₂浓度从2 mM增加到21 mM，由30S和50S亚基形成70S核糖体遵循二级双相动力学。对动力学结果的详细分析表明，两种主要的核糖体形式必须有一个共同的亚基类型。当假设动力学异质性源于仅一种亚基的两种形式来分析缔合数据时，速率常数分别为6.4×10⁶和1.05×10⁶ M⁻¹ s⁻¹。顺序流动实验表明，快速和慢速缔合物种分别对应于解离的II期（0.9 s⁻¹）和III期（0.03 s⁻¹）。弛豫测量表明，这些分别对应于B型（“松散”）和A型（“紧密”）核糖体。通过蔗糖密度离心分离紧密和松散核糖体，解离和缔合动力学研究证实了上述归属。此外，这些核糖体的速率常数在实验误差范围内与从多相动力学数据分析得出的速率常数一致。缔合速率常数是针对在记录缔合动力学之前立即用适当缓冲液稀释解离的核糖体。通过对2 mM MgCl₂透析过夜解离的核糖体，其较慢缔合反应（A型核糖体）的缔合速率常数约小四倍，而较快过程的速率常数大致相同。无论是通过降低MgCl₂浓度还是增加KCl浓度诱导的解离反应以及通过增加MgCl₂浓度诱导的缔合反应，其活化能均小于3.5 kcal/mol。在3 mM MgCl₂下的解离速率常数和在21 mM MgCl₂下的缔合反应速率常数在pH 7.2至8.4之间不变。当30S和50S亚基与含有20 mM亚精胺的缓冲液流动时，缔合过程是单相的，缔合常数k = 6×10⁶ M⁻¹ s⁻¹...
