Sensitive, nonradioactive assay of phosphorylase kinase through measurement of enhanced phosphorylase activity towards fluorogenic dextrin

In this study, we developed a highly sensitive and nonradioactive assay for PhK activity by measuring the enhanced GP activity towards a pyridylaminated maltohexaose. The enhanced GP activity (A) was calculated by the following formula: A = A+ – A0, where A+ and A0 represent the GP activities of the PhK-treated and PhK-nontreated samples, respectively. Using a high-performance liquid chromatograph equipped with a fluorescence spectrophotometer, the product of GP activity could be isolated and quantified at 10 fmol. This method does not require the use of any radioactive compounds and only 1 µg of GPb per sample was needed to obtain A+ and A0 values. The remarkable reduction in GPb concentration enabled us to discuss an interesting new role for glycogen in PhK activity.

Source: Journal of Biochemistry - January 25, 2016 Category: Biochemistry Authors: Miyagawa, D., Makino, Y., Sato, M. Tags: Regular Papers Source Type: research