Preparation of tissue extracts for glycogen phosphorylase assay

✍ Scribed by D.P. Gilboe; F.Q. Nuttall

• Publisher: Elsevier Science
• Year: 1972
• Tongue: English
• Weight: 651 KB
• Volume: 47
• Category: Article
• ISSN: 0003-2697
• DOI: 10.1016/0003-2697(72)90275-8

✦ Synopsis

In the preceding paper (1) we report the development of a new rapid method for the assay of glycogen phosphorylase.

During the development of this method a number of conditions were observed to influence the measurement of enzyme activity in the crude extracts. These have been studied systematically and the results are presented in this report. Recommendations are given for the preparation of tissue extracts for phosphorylase assay which are based on the results obtained.

MBTERIALS AND METHODS

Materials. Rats were purchased from the Holtzman Rat Company (Madison, Wisconsin). Rabbit liver glycogen was purchased from Sigma Chemical Company and purified as described previously (2). Glucose l-phosphate-'% was purchased from New England Nuclear Corporation. Glucose l-phosphate and adenosine 5'-monophosphate (AMP) were obtained from Sigma. (Y-(N-Morpholino) ethanesulfonic acid (MES) was obtained from Calbiochem. 2-Mercaptoethanol was purchased from Eastman Chemical Company. Dowex 1 (AGl-X4, lo@200 mesh) was purchased from Bio-Rad Laboratories. Norit A, obtained from Matheson Coleman & Bell, was acid washed. MES buffer refers to a solution at pH 6.1 of 50 mM MES, 50 mM KF, and 60 mM 2mercaptoethanoI.

Methods. Rat tissue preparation methods have been described (1,3,4). Phosphorylase activity was assayed by the radioactive method previously described (1) and by a modification of the calorimetric method of Danforth et cl. (3). Units used are pmoles glucose incorporated into glycogen per minute. Total phosphorylase activity refers to phosphorylase activity assayed in the presence of saturating amounts of AMP. Phosphorylase a activity refers to activity assayed in the absence of added AMP.