br Results br Discussion Today enzymes play important roles

Results
Discussion Today, enzymes play important roles in various fields. A good assay for enzyme activity is of great value, especially when it has good specificity, sensitivity, reproducibility and economy. ADK is the first identified enzyme in the metabolic pathway of adenosine salvage [15,16]. As one of the most ubiquitous and abundant nucleoside kinases, ADK acts as an important regulator of extracellular adenosine in animal models of epilepsy and ischemia [17,18]. There are two isoforms of ADK in various mammalian organisms including humans, the long isoform (ADK-L) and the short isoform (ADK-S) [19]. ADK-L is localized within the nucleus, whereas ADK-S is found in the Cyclopamine [20]. Previous ADK activity assays are too complicated to directly reflect the activity of ADK and determine the initial velocity of the reaction. These assays require additional expensive enzymes and special equipment, and the radioactive substrates are harmful to human body. Therefore, developing a simple, quick, sensitive and economical assay to detect ADK activity is of great significance. In this study, we established a spectrophotometric method using bromophenol blue as a pH indicator to analyze ADK activity. The initial velocity and product inhibition studies of ADK have been measured at pH 7.4 [21]. Thus, we adjusted the initial absorbance of the reaction system at 614 nm to 2.2, where pH was closed to 7.4. Further, we evaluated the effects of buffer and bromothymol blue on the sensitivity of the reaction, and then analyzed the sensitivity of the assay on ADK contents and adenosine concentration. We calculated the K and Vmax of BmADK using the developed assay. Our results suggested this assay was feasible, convenient, quick, reliable and sensitive for the activity of ADK.
Conflicts of interest
Acknowledgements This project was supported by the National Natural Science Foundation of China (31572465), the Chongqing Research Program of Basic Research and Frontier Technology (cstc2015jcyjA00040, cstc2015jcyjBX0035), Fundamental Research Funds for the Central Universities (XDJK2018B010, XDJK2018 C063), the State Key Program of the National Natural Science of China (31530071), Start-up grant from Southwest University (SWU112111), the Graduate Research and Innovation Project of Chongqing (CYB17069) and the Open Project Program of Chongqing Engineering and Technology Research Center for Novel Silk Materials (silkgczx2016003).
Introduction Adenosine kinase (AdK, ATP: adenosine 5′-phosphotransferase, EC 2.7.1.20) is a key enzyme of the purine salvage pathway, and together with adenosine deaminase (ADA) AdK is responsible for the metabolism of cellular adenosine. AdK is an evolutionarily ancient and highly conserved enzyme. It catalyzes the phosphorylation of adenosine to AMP utilizing ATP as the main phosphate donor in the presence of magnesium. AdK is a low capacity and low km (approx. 1μM) enzyme and is therefore considered to be the primary enzyme involved in adenosine clearance under physiological conditions whereas ADA seems to play a less prominent role. In interplay with the two other enzymes responsible for adenosine formation through dephosphorylation of AMP to adenosine, namely the cytosolic enzyme 5′-nucleotidase (cN-I) and the plasma membrane enzyme ecto-5′-nucleotidase (5′-NT, CD73) AdK regulates intra- and extracellular adenosine concentrations. Adenosine is a neuromodulator and general signaling molecule which activates specific G protein-coupled cell membrane receptors, designated adenosine A1, A2A, A2B and A3 receptors, thereby modulating a variety of physiological responses.2, 3 Adenosine signaling contributes extensively to the tissue and cell protection mechanism of the body under conditions of ischemic stress. Its exogenous use as a therapeutic agent is not possible due its extremely short half-life (<10s). Also the systemic use of direct adenosine receptor agonists is very limited as these molecules cause side effects by activating adenosine receptors also in non-target tissues.