EFFECT OF TEMPERATURE ON IN-VITRO ASSEMBLY OF BACTERIAL FLAGELLA
EFFECT OF TEMPERATURE ON IN-VITRO ASSEMBLY OF BACTERIAL FLAGELLA
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DOI：
10.1016/0022-2836(73)90040-5
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发表时间：
1973-01-01
影响因子：
5.6
通讯作者：
OOSAWA, F
中科院分区：
文献类型：
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作者：
GERBER, BR;ASAKURA, S;OOSAWA, F
使用奥斯特瓦尔德型粘度计测量蛋白质、鞭毛蛋白在中性pH下对鞭毛的重构或聚合(k+)速率的温度依赖性。类似地，测量了逆过程的动力学，即鞭毛丝热诱导解聚为鞭毛蛋白单体（k-）。 k−等于零时的温度用于定义鞭毛丝的热解离温度或熔点。从三种沙门氏菌菌株（分别带有 H 抗原类型 i、1.2 和 e、n、x 的 SJ670、SJ25 和 SJ30）中分离的鞭毛获得的熔点显着相似（36.8 ± 0.2 ℃），这表明这些不同蛋白丝的结构稳定性也相似。在 12 至 28°C 之间升高温度时，k+ 平稳增加，并具有 Q10 1.8.在28.0以上，k+迅速下降，在37℃附近降至零，其精确值因菌株而异。这一结果支持了之前的假设（Gerber & Noguchi，1967），即加热时，蛋白质的不同状态之间会发生可逆的协同转变；在一种状态下，鞭毛蛋白 (M) 可以聚合为鞭毛，而其构象异构体可能很难聚合或根本不能聚合。对于菌株 SJ25 和 SJ30，聚合和解聚速率在 37°C 附近均降至零。因此，所有比例的单体和鞭毛片段（短聚合物或“种子”）的混合物在接近该临界温度的温度下似乎处于平衡，并且鞭毛蛋白聚合为鞭毛或聚合物熔化均不明显。对菌株SJ670的鞭毛进行的测量表明k+和k−分别在45°C和37°C接近于零。在此温度范围内，测定与长丝平衡的单体的浓度。通过零点型实验，将单体和种子的溶液混合以找出粘度随时间既不增加（聚合）也不减少（解聚）的比率。一个意想不到的发现是温度定义了临界单体浓度，该浓度与任何长丝浓度（而不是单体与长丝浓度的比率）处于平衡状态。因此，鞭毛蛋白到鞭毛的聚合对应于类似于结晶或缩合的相变。将克拉佩龙-克劳修斯方程应用于所获得的结果产生70 kcal/mol单体蛋白质的缩合热。与 M ⇌ Mi 相关的焓变估计为 110 kcal/mol 蛋白质。由于这些不同形式的鞭毛蛋白的热含量的顺序为 Mi > F > M，根据差异，我们估计单体转化为聚合物的焓变为 40 kcal/mol 单体。
The temperature dependence for the rate of reconstitution or polymerization (k+) at neutral pH of the protein, flagellin, to flagella was measured using Ostwald-type viscometers. Similarly, the kinetics for the reverse process, the thermally-induced depolymerization of flagella filaments to the flagellin monomer (k−) was measured. The temperature at whichk−equals zero was used to define the thermal dissociation temperature or melting point of flagella filaments. The remarkable similarity of melting points obtained (36.8 ± 0.2 deg. C) for flagella isolated from threeSalmonellastrains (SJ670, SJ25 and SJ30 bearing H-antigen typesi, 1.2 ande,n,x, respectively) suggests that the structural stability of these different protein filaments is also similar.On increasing the temperature between 12 and 28°C,k+increased smoothly and had aQ10of 1.8. Above 28.0,k+decreased rapidly and fell to zero at a temperature near 37°C, its precise value varying with the bacterial strain. This result supports the prior hypothesis (Gerber & Noguchi, 1967) that on heating, a reversible co-operative transconformation occurs between different states of the protein; in one state, flagellin (M) can polymerize to flagella, whereas its conformational isomer(s) may do so with difficulty or not at all.For strains SJ25 and SJ30 the rates of polymerization and depolymerization both fall to zero near 37°C. Therefore, mixtures of monomer and flagella fragments (short polymers or “seeds”),in all ratios, appear to be in equilibrium at temperatures near this critical temperature, and neither polymerization of flagellin to flagella nor melting of polymers is apparent.Measurements made on flagella from strain SJ670 showed thatk+andk−approached zero at 45 and 37°C, respectively. Within this temperature range the conc entration of monomer in equilibrium with filaments was determined. By a null -point type experiment, solutions of monomer and seed were mixed to find the ratio that showed neither increases (polymerization) nor decreases (depolyme rization) in viscosity with time. An unexpected finding was that the temperature defines acritical monomer concentration, which exists in equilibrium with any concentration of filaments (and not the ratio of monomer-to-filament concentrations). Thus, the polymerization of fiagellin to flagella corresponds to a phase change akin to either crystallization or condensation.Application, of the Clapeyron-Clausius equation to the results obtained yields a heat of condensation of 70 kcal/mol of monomeric protein. The enthalpy change associated with M ⇌ Miis estimated as 110 kcal/mol of protein. Since the heat content of these various forms of flagella protein lies in the order Mi> F > M, by difference we estimate the enthalpy change for the conversion of monomers to polymers to be 40 kcal/mol of monomer.