The subunit structure of rabbit-skeletal-muscle phosphorylase kinase, and the molecular basis of its activation reactions.

Abstract

Phosphorylase kinase was isolated from rabbit skeletal muscle in a state approaching homogeneity as judged by the criteria of ultracentrifugal analysis, ion‐exchange chromatography, and antigen‐antibody precipitation in agar. The purified enzyme showed four protein‐staining components upon acrylamide gel electrophoresis in the presence of sodium dodecylsulphate, termed α, α′, β and γ. A variety of precautions designed to eliminate the possible action of proteases and ensure complete reduction and denaturation of the protein had no influence on the gel pattern. The molecular weights of the four components determined by gel electrophoresis, and supported by sedimentation equilibrium in the presence of 6 M guanidinium chloride, following partial separation of the chains by gel filtration on Sephadex G‐200 in the presence of sodium dodecylsulphate, were:–α= 145000, α′= 140000, β= 128000 and γ= 45000. The α' component was present only in trace amounts and the evidence suggests it may be derived from the α component. The three subunits α, β, and γ, were found to exist in equimolar quantities by densitometric analysis of acrylamide gels, by gel filtration on Sephadex G‐200 in the presence of sodium dodecylsulphate, and by carboxymethylation with iodo[14C]acetate, suggesting a minimal binding molecular weight, αβγ, of 318000. The molecular weight of the native enzyme was determined as 1.28 × 106, demonstrating that phosphorylase kinase is composed of 4.0 αβγ units.Activation of the enzyme by incubation with adenosine‐3′:5′‐phosphate‐dependent protein kinase, adenosine 3′:5′‐phosphate and Mg‐ATP, was accompanied by the phosphorylation of one site on the β subunit, although a second site on the α subunit was phosphorylated at a slower rate. Activation of the enzyme by proteolysis resulted from a limited cleavage of the α subunit. The products of proteolytic attack suggest that the α and β subunits may be structurally related. The γ subunit was not phosphorylated, was resistant to proteolysis and distinct from the (α+β) subunits in its amino acid composition. The possible functions of the chains, and the implications of the activation reactions to the nervous and hormonal control of glycogenolysis in vivo are discussed.