Expressed Phosphorylase b Kinase and Its αγδ Subcomplex as Regulatory Models for the Rabbit Skeletal Muscle Holoenzyme

Understanding the regulatory interactions among the 16 subunits of the (alpha beta gamma delta)(4) phosphorylase b kinase (PhK) complex can only be achieved through reconstructing the holoenzyme or its subcomplexes from the individual subunits. In this study, recombinant baculovirus carrying a vector containing a multigene cassette was created to coexpress in insect cells alpha,beta,gamma, and delta subunits corresponding to rabbit skeletal muscle PhK. The hexadecameric recombinant PhK (rPhK) and its corresponding (alpha gamma delta trimeric subcomplex were purified to homogeneity with proper subunit stoichiometries. The catalytic activity of rPhK at pH 8.2 and its ratio of activities at pH 6.8 versus pH 8.2 were comparable to those of PhK purified from rabbit muscle (RM PhK), as was the hysteresis (autoactivation) in the rate of product formation at pH 6.8. Both the rPhK and (alpha gamma delta exhibited only a very low Ca2+-independent activity and a Ca2+-dependent activity similar to that of the native holoenzyme with [Ca2+](0.5) of 0.4 mu M for the RM PhK, 0.7 mu M for the rPhK, and 1.5 mu M for the alpha gamma delta trimer. The RM PhK, rPhK, and alpha gamma delta subcomplex were also all activated through self-phosphorylation. Using cross-linking and limited proteolysis, the alpha-gamma intersubunit contacts previously observed within the intact RM PhK complex were also observed within the recombinant alpha gamma delta subcomplex. Our results indicate that both the rPhK and alpha gamma delta subcomplex are promising models for future structure-function studies on the regulation of PhK activity through intersubunit contacts, because both retained the regulatory properties of the enzyme purified from skeletal muscle.