Isolation and Properties of the Catalytically Active

Nonactivated phosphorylase kinase was dissociated to catalytically active lower molecular weight species by incubation with either ATP or LiBr at 0°C. Sucrose density gradient analysis showed that incubation with 100 m~ ATP produced 14 S and 7.5 S active forms and incubation with 1 M LiBr produced a 5 S active species. Large increases in the pH 6.8/8.2 activity ratio and loss of inhibition by EGTA were characteristics of the dissociated forms. After native phosphorylase kinase was dissociated optimally by incubation with 1.8 M LiBr, a catalytically active subunit was purified to near homogeneity by gel filtration and blue dextran-Sepharose affinity chromatography. This catalytically active subunit of phosphorylase kinase was identified as the y subunit (M, = 45,800) by using sodium dodecyl sulfate disc gel electrophoresis. In addition, by using radioactively labeled activated phosphorylase kinase, the distribution of the other subunits was determined while isolating the catalytic subunit. Some properties of the purified catalytically active y subunit were examined. The apparent molecular weight as determined by gel filtration is 86,000, indicating that the active species is a dimer (yz). The pH 6.8/8.2 activity is 0.67 to 1.0. The enzyme is also active when assayed in the presence of EGTA, indicating a loss of the absolute requirement of Ca2+ for activity. Like the holoenzyme, the purified y subunit did not phosphorylate casein and histone. The kinetic parameters for the purified y subunit were determined at pH 6.8 and 8.2 for MgATP and phosphorylase b.