Introduction Adenosine kinase ADK ATP adenosine phosphotrans

Introduction Adenosine kinase (ADK; ATP: adenosine 5′-phosphotransferase, EC 2.7.1.20), is isolated from yeast cells by Kornberg for the first time in 1951 [1]. ADK belongs to the ribokinase protein family and is one of the most abundant nucleoside kinases in mammalian tissues [2,3]. As the first identified enzyme in the purine salvage pathway, ADK could catalyze the transfer of γ-phosphate group from GKT137831 to adenosine to produce AMP (Adenosine + ATP→AMP + ADP). ADK plays the role of potent cardioprotective agent and neuromodulator via regulating both intracellular and extracellular adenosine concentration [2,4,5]. In the diabetes model, the decrease of ADK expression impairs the proliferation of T lymphocytes [6]. ADK also plays a significant role in facilitating intracellular methylation. The absence of ADK in the ADK−/--lesioned mice leads to the acute hepatic steatosis in the newborn infants and the death at postnatal day 8 [7]. However, the overexpression of ADK in brain results in frequent electrographic seizures with a frequency of four times per hour [8], and the ADK−/--lesioned mice awakes earlier compared with the wild-type [9]. In addition, ADK also can catalyze the phosphorylation of other nucleosides and their analogs to generate the corresponding monophosphate [10]. Two methods have been developed to measure the enzymatic activity of ADK. One is a coupled multi-enzyme system assay, another one is a radioactive labeling assay [11]. The coupled multi-enzyme system contains three kinds of enzymes: ADK, pyruvate kinase and lactate dehydrogenase. In this assay, ADP formed in the reaction catalyzed by ADK is reconverted to ATP in the presence of excess P-enolpyruvate and pyruvate kinase. At the same time, the produced pyruvate is catalyzed to generate lactate in the presence of excess NADH and lactate dehydrogenase, which will result in the decrease of the absorbance at 340 nm because of the consumption of NADH. It is a time consuming multi-steps process subject to errors at each step, and other two enzymes have different optimal reaction conditions, thus it is difficult to balance the optimum conditions for the three enzymes system. Moreover, it could not accurately determine the initial velocity of ADK reaction. Another assay is dependent on the direct measurements of radioactivity of reaction products after separation of the radioactive nucleoside substrates from nucleotides by chromatograph. It needs specific equipment, and the whole process takes up to 18 h. Also, the radioactive nucleoside substrate is harmful to the human body, and it could not accurately determine the real initial rate of ADK reaction. Therefore, it is necessary to develop a convenient, quick, sensitive and low-cost assay to determine the enzymatic activity of ADK. Acid-base indicators are usually applied to enzymatic assay for their extraordinary sensitivity to pH change. In 2002, Yu et al. have established an assay of arginine kinase activity based on the light absorption of a complex acid-base indicator [12]. Recently, Dhale et al. have developed a rapid and sensitive assay to measure l-asparaginase activity using methyl red as the indicator [13]. Bromothymol blue (BTB) is a good indicator in pH ranges of 6.0–8.0. Its color changes from yellow to blue as pH increases from 6.0 to 8.0, because it can form a highly conjugated structure while deprotonated in alkaline solution [14]. Here, we developed a one-step method to determine ADK activity, using bromothymol blue as the pH indicator, ATP and Adenosine as the substrates. Using this method, we analyzed the activity of adenosine kinase of Bombyx mori (BmADK), and evaluated the effect of buffer and pH indicator on the activity of ADK. The results indicated that this assay was time-saving, convenient, reliable and sensitive. This assay has shown a great potential as an alternative for the conventional ADK activity assay.
Materials and methods