| Abstract: | A catalytically active gamma subunit of phosphorylase kinase was prepared from pure, but inactive, gamma subunit obtained by reverse-phase high pressure liquid chromatography (HPLC). The HPLC procedure (Crabb, J. W., and Heilmeyer, L. M. J., Jr. (1984) J. Biol. Chem. 259, 6346-6350) leaves the isolated gamma subunit in 50% acetonitrile and 0.09% trifluoroacetic acid (pH 2.5) and assay of this species at pH 8.2 indicates that it is inactive. Reactivation occurred, however, when the HPLC-isolated gamma subunit was diluted into an ice-cold, pH 8.2 buffer containing both calcium and calmodulin. Optimum reactivation depended on time, temperature, concentration of the HPLC solvent components, gamma subunit concentration, pH, the presence of both calcium and calmodulin, and an additional protein such as bovine serum albumin or phosphorylase b. Studies of the reactivated gamma subunit in the presence of the reactivation mixture indicate that it may be equivalent to a gamma delta subunit complex previously isolated (Chan, K.-F. J., and Graves, D. J. (1982) J. Biol. Chem. 257, 5939-5947). Like the gamma delta subunit complex, the catalytic activity of the reactivated gamma subunit species is not significantly affected by pH within the range of pH 6.8-8.2 and is inhibited 70% by removal of Ca2+. A reactivated gamma subunit free of calmodulin was also obtained. This was done by first substituting agarose-bound calmodulin for free calmodulin in the reactivation procedure described above and, then, elution of the gamma subunit from the calmodulin-agarose with a solution containing 1.0 M Tris-Cl (pH 7.0), 1% Triton X-100, 1 mM EGTA, and 5 mM dithiothreitol. The activity of the isolated, active gamma subunit is insensitive to Ca2+ and is stimulated 1.4-fold in a calcium-dependent manner by the addition of calmodulin. |
