Substrate For Hexokinase Research Articles

A radiochemical enzymatic activity assay for glycerol kinase and hexokinase

1. 1. A simple radiochemical sampling assay for hexokinase (ATP: d-hexose 6-phosphotransferase, EC 2.7.1.1) and glycerol kinase (ATP:glycerol phosphotransferase, EC 2.7.1.30) has been developed. The paper describes tests carried out in order to show that this assay is valid for the accurate measurement of enzyme activity. Some of the problems associated with coupled assays of enzyme activity are not encountered with this radiochemical assay. 2. 2. Radioactive substrate was incubated with enzyme for varying periods of time, and the reaction was stopped by addition of alcohol; other methods of stopping the reaction are discussed. Samples of the reaction mixture were pipetted on to discs of DEAE-cellulose paper and the substrate was removed by washing the paper discs with water. The anionic product remained adsorbed on the paper. The radioactivity on the paper, which is proportional to the enzyme activity, was measured in a liquid scintillation counter. 3. 3. The measured rate of product formation by glycerol kinase or hexokinase was the same whether the product was determined radiochemically or enzymatically. 4. 4. When [ 14C]glucose with a specific activity of approx. 120 mC/mmole was used as substrate for hexokinase, the sensitivity of the radiochemical assay was higher than that of an assay depending on the fluorescence of pyridine nucleotides.