An assay for phosphorylase a or b is described in which the rate of release of phosphate from glucose l-phosphate is followed by means of an "instantaneous" method for determination of phosphate.
Glycogen phosphorylase is commonly assayed either in the direction of glycogen breakdown using a complex, coupled spectrophotometric assay system to follow the liberation of glucose l-phosphate (1) or in the direction of glycogen synthesis using a single-point determination of the inorganic phosphate released from glucose l-phosphate at concentrations of 15 (2) and 75 mM (3).
The coupled assay system ( 1) is complex and expensive, involving numerous additions to each reaction cuvette and, in our hands at least, it does not give very reproducible rates of reaction. The single-point assays (2,3) are cheap and relatively easy to perform but both rely on one, endpoint phosphate determination as the measure of phosphorylase activity. This gives little information on the error inherent in each determination. The method of Fiske and Subbarow ( 4) is used to estimate the inorganic phosphate released from glucose l-phosphate. This procedure has several steps which have to be carefully timed and this makes it rather difficult to carry out several assays within fairly short periods of each other.
We have been using phosphorylase as a substrate on which to test the activity of a neutral, trypsin-like protease from rat intestinal muscle (5). This requires measuring the loss of phosphorylase activity with time which in turn demands a simple, reproducible, and convenient assay for phosphorylase. In this paper, we describe a modification of the assay of Hedrick and Fischer (3) using the "instantaneous" inorganic phosphate determination of Parvin and Smith (6). This not only permits the removal of samples at successive time points from the phosphorylase-glucose l-phosphate incubation mixture for phosphate determination, thereby enabling an initial rate of phosphate release to be obtained, but also, since the coloured product is perfectly stable, no critical timing is necessary (for the phosphate determination) so that many samples can be processed easily.
Methods
Phosphorylase b was prepared from rabbit skeletal muscle (7) and was a generous gift from Dr. P. Cohen. Phosphorylase a was obtained from Boehringer Corp., Ltd. Rabbit liver glycogen and glucose l-phosphate