Ravel P, Garel M C, Toullec D
U 91, Inserm, hôpital Henri-Mondor, Créteil, France.
C R Acad Sci III. 1997 Jan;320(1):27-33. doi: 10.1016/s0764-4469(99)80083-3.
In red blood cells, a modulation of the level of the allosteric effector of hemoglobin, 2,3-diphosphoglycerate (2,3-DPG) would have implications in the treatment of ischemia and sickle cell anemia. Its concentration is determined by the relative activities of the synthase and phosphatase reactions of the multifunctional bisphosphoglycerate mutase (BPGM). In this report we develop first a more direct synthase assay which uses glyceraldehyde phosphate to suppress the aldolase and triose phosphate isomerase reactions. Secondly we propose a radioactive phosphatase assay coupled to chromatographic separation and identification of the reaction products by paper electrophoresis. Such identification of these products allow us to show that the multifunctional BPGM expresses its mutase instead of its phosphatase activity in conditions of competition between the 3-phosphoglycerate and the 2-phosphoglycolate activator in the phosphatase reaction. These two more precise procedures could be used to study the effects of substrate and cofactor analogues regarding potential therapeutic approaches and could be used for clinical analyses to detect deficiency of BPGM.
在红细胞中，血红蛋白变构效应物2,3-二磷酸甘油酸（2,3-DPG）水平的调节对缺血和镰状细胞贫血的治疗具有重要意义。其浓度由多功能双磷酸甘油酸变位酶（BPGM）的合酶和磷酸酶反应的相对活性决定。在本报告中，我们首先开发了一种更直接的合酶测定方法，该方法使用磷酸甘油醛来抑制醛缩酶和磷酸丙糖异构酶反应。其次，我们提出了一种放射性磷酸酶测定方法，该方法与色谱分离相结合，并通过纸电泳对反应产物进行鉴定。对这些产物的鉴定使我们能够表明，在磷酸酶反应中，当3-磷酸甘油酸和2-磷酸乙醇酸激活剂之间存在竞争时，多功能BPGM表现出其变位酶活性而非磷酸酶活性。这两种更精确的方法可用于研究底物和辅因子类似物对潜在治疗方法的影响，也可用于临床分析以检测BPGM缺乏症。
