Acetate kinase, a member of the acetate and sugar kinase-Hsp70-actin (ASKHA) enzyme superfamily [1][2][3][4][5] , is responsible for the reversible phosphorylation of acetate to acetyl phosphate utilizing ATP as a substrate. Acetate kinases are ubiquitous in the Bacteria, found in one genus of Archaea, and are also present in microbes of the Eukarya 6 . The most well characterized acetate kinase is that from the methane-producing archaeon Methanosarcina thermophila [7][8][9][10][11][12][13][14] . An acetate kinase which can only utilize PPi but not ATP in the acetyl phosphate-forming direction has been isolated from Entamoeba histolytica, the causative agent of amoebic dysentery, and has thus far only been found in this genus 15,16 .In the direction of acetyl phosphate formation, acetate kinase activity is typically measured using the hydroxamate assay, first described by Lipmann 17-20 , a coupled assay in which conversion of ATP to ADP is coupled to oxidation of NADH to NAD + by the enzymes pyruvate kinase and lactate dehydrogenase 21,22 , or an assay measuring release of inorganic phosphate after reaction of the acetyl phosphate product with hydroxylamine 23 . Activity in the opposite, acetate-forming direction is measured by coupling ATP formation from ADP to the reduction of NADP + to NADPH by the enzymes hexokinase and glucose 6-phosphate dehydrogenase 24 .Here we describe a method for the detection of acetate kinase activity in the direction of acetate formation that does not require coupling enzymes, but is instead based on direct determination of acetyl phosphate consumption. After the enzymatic reaction, remaining acetyl phosphate is converted to a ferric hydroxamate complex that can be measured spectrophotometrically, as for the hydroxamate assay. Thus, unlike the standard coupled assay for this direction that is dependent on the production of ATP from ADP, this direct assay can be used for acetate kinases that produce ATP or PPi.
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ProtocolThe overall scheme of this protocol is outlined in Figure 1.
Solution Preparation for Standard Curves and AssaysDecember 2011 | 58 | e3474 | Page 1 of 7Journal of Visualized Experiments www.jove.comCopyright © 2011 Journal of Visualized Experiments 1. Prepare 100 mL of a 2 mol/L solution of hydroxylamine-HCl. Weigh out 13.9 g of hydroxylamine hydrochloride (MW 69.49 g/mol) and dissolve in approximately 50 mL distilled-deionized water (ddH2O). Adjust the pH to 7.0 using potassium hydroxide pellets or a concentrated solution. Bring the final volume to 100 mL. The solution can be stored at room temperature for up to 30 days or at 4°C for up to 90 days. 2. Prepare 100 mL of a ferric chloride/ hydrochloric acid solution. Weigh out 13.5 g ferric chloride (MW 270.32 g/mol) and dissolve in approximately 50 mL ddH2O. Add 41.3 mL concentrated hydrochloric acid (12.1 mol/L) and bring to a final volume of 100 mL. The final concentration of ferric chloride will be 0.5 mol/L and t...