Sood S M, Slattery C W, Filley S J, Wu M X, Hill K A
Department of Biochemistry, Loma Linda University School of Medicine, California 92350-0001, USA.
Arch Biochem Biophys. 1996 Apr 15;328(2):295-301. doi: 10.1006/abbi.1996.0176.
Selected physical and thermodynamic parameters for Escherichia coli alanyl-tRNA synthetase (AlaRS) have been determined primarily to assess the quaternary structure of this enzyme. The extinction coefficient (epsilon) at 280 nm was determined experimentally to be 0.71 ml mg-1 cm-1, and the partial specific volume (nu) was calculated from the amino acid composition to be 0.73 ml g-1. From viscosity experiments the intrinsic viscosity (eta) of AlaRS was extrapolated to be 3.4 ml g-1 and the degree of hydration (delta 1) estimated to be 0.67 gH2O g(-1)(AlaRS). Laser light-scattering studies indicated some heterogeneity; a radius of 6.3 nm was calculated for the major fraction with a diffusion coefficient (D20,W) of 3.89 x 10(-7) cm2 s-1. In 50 mM Hepes, pH 7.5, 20 mM KCl, 2 mM 2-mercaptoethanol and at a protein concentration of 4.2 mg ml-1 the sedimentation coefficient (S20,W) was 6.36 S; this value increased slightly when the protein concentration was decreased. The combination of S20,W and D20,W under these conditions yielded a molecular weight of approximately 186,000 Da, corresponding to a dimer. The S20,W was virtually independent of temperature in the range of 10-37 degrees C, while an Arrhenius plot of aminoacylation activity was biphasic. The isoelectric point was determined experimentally to be 4.9. Sedimentation equilibrium data were best fit to a decamer association complex in which dimeric AlaRS is the predominant species at 25 degrees C.
已确定大肠杆菌丙氨酰 - tRNA合成酶（AlaRS）的选定物理和热力学参数，主要用于评估该酶的四级结构。通过实验测定，280 nm处的消光系数（ε）为0.71 ml mg-1 cm-1，根据氨基酸组成计算出的偏比容（ν）为0.73 ml g-1。通过粘度实验，AlaRS的特性粘度（η）外推值为3.4 ml g-1，水合度（δ1）估计为0.67 gH2O g(-1)(AlaRS)。激光散射研究表明存在一些异质性；计算出主要组分的半径为6.3 nm，扩散系数（D20,W）为3.89×10(-7) cm2 s-1。在50 mM Hepes，pH 7.5，20 mM KCl，2 mM 2-巯基乙醇且蛋白质浓度为4.2 mg ml-1的条件下，沉降系数（S20,W）为6.36 S；当蛋白质浓度降低时，该值略有增加。在这些条件下，S20,W和D20,W的组合得出分子量约为186,000 Da，对应于二聚体。在10 - 37℃范围内，S20,W实际上与温度无关，而氨酰化活性的阿累尼乌斯图呈双相。通过实验测定等电点为4.9。沉降平衡数据最符合十聚体缔合复合物，其中二聚体AlaRS在25℃时是主要物种。
