Preparation of tissue extracts for glycogen phosphorylase assay

The conditions for preparing heart and skeletal muscle extracts for phosphorylase assay following homogenization and centrifugation have been investigated. It is concluded that extracts should be kept at room temperature to avoid cold inactivation of the phosphorylase b component of total phosphorylase activity. Prolonged delay in assaying total phosphorylase activity especially in dilute solution also results in the loss of phosphorylase b activity but not phosphorylase a activity. The accurate determination of phosphorylase a activity is dependent on an efficient means to reduce the AMP concentration. Although dilution of extracts several-fold has been effective in the past for this purpose, Dowex 1 treatment is recommended. Norit A, another agent used to remove AMP, is less satisfactory because it results in a loss of enzyme activity. Several buffers have been investigated and MES or maleate appear to give the best results.