PURIFICATION AND CHARACTERIZATION OF PHOSPHORYLASE A FROM HUMAN-PLATELETS

1. 1. Platelet phosphorylase a was isolated from human platelets by 5'-AMP-Sepharose 4B affinity chromatography to give two peaks of enzyme activity. The specific activities of the two peaks [I and II] were 24 U/mg and 45 U/mg corresponding to 400- and 800-fold purification with respect to the activity in the homogenate supernatant. The molecular weight of the purified enzyme was estimated by Na dodecyl sulphate polyacrylamide gel electrophoresis to be a single component with a molecular weight of 95,000. 2. 2. On the basis of enzyme activity measured in the presence of various ligands Peak I proved to be a partially phosphorylated, hybrid phosphorylase. 3. 3. The kinetic constants for the dephosphorylation of Peak I and Peak II phosphorylase were compared and showed the same Vmax with K = 9 μM and 4μM, respectively. 4. 4. Nucleotides (AMP, IMP, ADP) inhibited the dephosphorylation of platelet phosphorylase a. Glucose. glucose-6-phosphate and caffeine decreased this inhibition. © 1979.