Isolation and physicochemical properties of active complexes of rabbit muscle phosphorylase kinase.

A catalytically active species composed of a, y, and 8 subunits was isolated from phosphorylase kinase. Upon incubation of the holoenzyme (cusyS)4 with 1.8 M LiBr, 10 m~ LiBr, 10 m~ M&+ at 0 “C for 6-7 h and subsequent gel filtration and ion exchange chromatography, an active ayS complex was purified to apparent homogeneity by the criteria of gel filtration, disc gel electrophoresis under denaturing and nondenaturing conditions, and sucrose density gradient sedimentation. The ayS complex was shown to have a molecular weight of 243,000 by gel filtration and stoichiometric amounts of a, y, and S subunits by densitometric tracings, suggesting that it is a monomer. A species containing the catalytically active y subunit isolated previously (Skuster, J. R., Chan, K.-F. J., and Graves, D. J. (1980) J. Biol. Chem 255,2203-2210) was found to contain the 6 subunit in near stoichiometric amounts. A molecular weight of 66,000 was determined by using gel filtration, indicating that this species also exists as a monomer. A Stokes radius of 53.2 A and a szo,u, of 11.8 S were estimated for a#, whereas those for yS were 35.2 A and 4.2 S, respectively. The W spectrum of ayS was quite distinct from the holoenzyme, having an estimated Azso /Azeo ratio of 1.06 and an A:&of 7.3 Circular dichroic spectropolarimetry revealed that phosphorylase kinase contains approximately 38% a-helical structure, whereas only 30% was found for the ayS complex. The far ultraviolet circular dichroism of the holoenzyme was almost unaffected by EGTA, but substantial changes in the helical content occurred in the ayS complex. Individual a, @, and y subunits were also purified in the presence of sodium dodecyl sulfate and their amino acid composition analyzed. The ayS and yS complexes are active and show considerable differences in pH-activity profiles from nonactivated phosphorylase kinase.