温度对天冬氨酸转氨酶稳态动力学及测量的影响。

Effects of temperature on the steady-state kinetics and measurement of aspartate aminotransferases.

作者信息

Rej R, Vanderlinde R E

出版信息

Clin Chem. 1981 Feb;27(2):213-9.

Abstract

We examined the effects of temperature on the activity and steady-state kinetics of aspartate aminotransferase (EC 2.6.1.1), using purified human soluble (s-AspAT) and mitochondrial (m-AspAT) isoenzymes, human serum, and porcine s-AspAT. All enzymes obeyed similar linear Arrhenius relationships over the range 20-40 degrees C. Apparent energies of activation (52.3 kJ.mol-1) and ratios of activity between 30 and 37 degrees C (0.626) were identical for the human s- and m-AspAT. This ratio was 0.623 (SEM 0.004) for human sera; deviation from the predicted ratio by individual sera was within analytical error. Similar activity/temperature relationships were observed for porcine s-AspAT. The use of factors to convert AspAT activities at 30 and 37 degrees C influenced neither precision of measurement of frequency distributions of results. The apparent Michaelis constants for the human isoenzymes increased with temperature. The least-influenced Km was for 2-oxoglutarate and s-AspAT: K2-oxoglutarate was 0.24 mmol.L-1 at 25 degrees C and 0.29 mmol.L-1 at 37 degrees C; apparent enthalpy change for substrate binding (delta HS) was 12.1 kJ.mol-1. The largest variation was for 2-oxoglutarate and m-AspAT: K2-oxoglutarate was 0.46 mmol.L-1 at 25 degrees C and 1.02 mmol.L-1 at 37 degrees C; delta HS was 50.8 kJ.mol-1. Incubation of the human isoenzymes with substrate mixture (without 2-oxoglutarate) at 23 and 37 degrees C did not affect activity during 60 min if tris(hydroxymethyl)aminomethane buffer was used. When the isoenzymes were diluted to 10 nmol-L-1 (about 200 U.L-1) in buffer alone and incubated at 50 degrees C, m-AspAT activity was decreased by 20% after 120 min; the cytoplasmic enzyme was unaffected.

摘要

我们使用纯化的人可溶性（s - AspAT）和线粒体（m - AspAT）同工酶、人血清以及猪s - AspAT，研究了温度对天冬氨酸转氨酶（EC 2.6.1.1）活性和稳态动力学的影响。在20 - 40摄氏度范围内，所有酶均遵循相似的线性阿伦尼乌斯关系。人s - AspAT和m - AspAT的表观活化能（52.3 kJ·mol⁻¹）以及30和37摄氏度之间的活性比（0.626）相同。人血清的该比值为0.623（标准误0.004）；各血清与预测比值的偏差在分析误差范围内。猪s - AspAT也观察到类似的活性/温度关系。使用在30和37摄氏度下转换AspAT活性的因子，既不影响测量精度，也不影响结果频率分布。人同工酶的表观米氏常数随温度升高而增加。受影响最小的Km是2 - 氧代戊二酸与s - AspAT的：25摄氏度时K₂ - 氧代戊二酸为0.24 mmol·L⁻¹，37摄氏度时为0.29 mmol·L⁻¹；底物结合的表观焓变（ΔHS）为12.1 kJ·mol⁻¹。变化最大的是2 - 氧代戊二酸与m - AspAT的：25摄氏度时K₂ - 氧代戊二酸为0.46 mmol·L⁻¹，37摄氏度时为1.02 mmol·L⁻¹；ΔHS为50.8 kJ·mol⁻¹。如果使用三（羟甲基）氨基甲烷缓冲液，在23和37摄氏度下将人同工酶与底物混合物（不含2 - 氧代戊二酸）孵育60分钟不会影响活性。当同工酶在单独的缓冲液中稀释至10 nmol·L⁻¹（约200 U·L⁻¹）并在50摄氏度下孵育时，120分钟后m - AspAT活性降低20%；细胞质酶不受影响。