Cercignani G, Allegrini S
Department of Physiology and Biochemistry, University of Pisa, Italy.
Anal Biochem. 1991 Feb 1;192(2):312-5. doi: 10.1016/0003-2697(91)90541-z.
Kinetic investigations on adenosine deaminase from calf intestinal mucosa by spectrophotometric monitoring of the reaction at 264, 270, or 228 nm show that this method does not produce artifactual inhibition by substrate excess up to 0.7 mM concentration, when either adenosine or 2'-deoxyadenosine are employed with calf adenosine deaminase. The evaluation of kinetic parameters for this system was carried out both by initial rate measurements and by numerical differentiation of time progress curves according to a recently published method (S. C. Koerber and A. L. Fink, 1987, Anal. Biochem. 165, 75-87). The following results were obtained by the latter method at pH 7.0 and 30 degrees C: for the conversion of adenosine to inosine, kcat = 251 +/- 15 s-1, KMs = 29.7 +/- 2.8 microM, KMp = 613 +/- 62 microM; for the conversion of 2'-deoxyadenosine to 2'-deoxyinosine, kcat = 283 +/- 17 s-1, KMs = 22.4 +/- 2.2 microM, KMp = 331 +/- 35 microM. At 285 nm, a slight negative deviation from Beer's law was observed for adenosine at concentrations higher than 0.9 mM. No deviation was found for inosine up to 2.0 mM at the same wavelength.
通过在264、270或228nm处对反应进行分光光度监测，对来自小牛肠粘膜的腺苷脱氨酶进行动力学研究表明，当使用腺苷或2'-脱氧腺苷与小牛腺苷脱氨酶反应时，在浓度高达0.7mM时，该方法不会因底物过量而产生人为抑制。该系统动力学参数的评估通过初始速率测量以及根据最近发表的方法（S.C. Koerber和A.L. Fink，1987，《分析生物化学》165，75 - 87）对时间进程曲线进行数值微分来进行。通过后一种方法在pH 7.0和30℃下获得了以下结果：对于腺苷转化为次黄苷，kcat = 251±15 s-1，KMs = 29.7±2.8μM，KMp = 613±62μM；对于2'-脱氧腺苷转化为2'-脱氧次黄苷，kcat = 283±17 s-1，KMs = 22.4±2.2μM，KMp = 331±35μM。在285nm处，当腺苷浓度高于0.9mM时，观察到与比尔定律有轻微负偏差。在相同波长下，次黄苷浓度高达2.0mM时未发现偏差。
