Glycogen phosphorylase from flight muscle of the hawk moth,Manduca sexta: purification and properties of three interconvertible forms and the effect of flight on their interconversion

Glycogen phosphorylase (EC 2.4.1.1) of Manduca sexta flight muscle was separated into three distinct peaks of activity on diethylaminoethyl-Sephacel. The three fractions of phosphorylase activity were further purified by affinity chromatography on AMP-Sepharose and shown to have the same relative molecular mass (=178000) on polyacrylamide gradient gel electrophoresis under non-denaturating conditions and to produce subunits of molecular mass =92000 on SDS gelelectrophoresis. On the basis of their kinetic properties with respect to the activator AMP and the inhibitor caffeine, the three fractions of phosphorylase activity were assigned as follows: peak 1=phosphorylase b (unphosphorylated form), peak 3=phosphorylase a (phosphorylated form); peak 2 represented a phospho-dephospho hybrid in which only one subunit of the dimeric enzyme was phosphorylated. This hypothesis was corroborated as the various forms could be interconverted in vitro by either dephosphorylation by an endogenous protein phosphatase producing the b form, or by phosphorylation catalyzed by purified phosphorylase kinase from rabbit muscle producing phosphorylase ab and a. From muscle of resting moths more phosphorylase was isolated in the b form (41%) than in the forms ab (28%) and a (31%), respectively. This proportion was changed in favour of the fully phosphorylated a form after a brief interval of flight when 68% of the phosphorylase activity was represented by the a form and only 13% by the b form. Unlike the phosphorylated forms a and ab of phosphorylase, the b form had low affinities for the substrates and for the activator AMP, and was virtually inactive if near-physiological concentrations of substrates and effectors were employed in the assays. The results demonstrate that in Manduca flight muscle three forms of phosphorylase coexist and that their interconversion is a mechanism for regulating phosphorylase activity in vivo.Abbreviations DEAE diethylaminoethyl - EDTA ethylenediamine tetraacetate - EGTA ethyleneglycol-bis(-aminoethylether)N,N-tetra-acetic acid - M_r relative molecular mass - NMR nuclear magnetic resonance - PAGGE polyacrylamide gradient gel electrophoresis - P_i morganic phosphate - SDS sodium dodecylsulphate - TRIS tris(hydroxymethyl)-aminomethane - V_max maximum activity