ZERO-LENGTH CROSS-LINKING OF THE BETA-SUBUNIT OF PHOSPHORYLASE-KINASETO THE N-TERMINAL HALF OF ITS REGULATORY ALPHA-SUBUNIT

Phosphorylase kinase, a regulatory enzyme of glycogenolysis in skeleta l muscle, is a hexadecameric oligomer containing four copies each of f our distinct subunits: alpha, beta, gamma, and delta. By intramolecula r zero-length crosslinking with transglutaminase, we have previously d emonstrated that the regulatory alpha and beta subunits abut one anoth er in the holoenzyme [Nadeau, O. W., and Carlson, G. M. (1994) J. Biol . Chem. 269, 29670-29676]. Selective partial proteolysis of the 138 kD a alpha subunit in holoenzyme that had been crosslinked by transglutam inase has revealed a high molecular weight conjugate corresponding to full-length beta subunit crosslinked to a 60 kDa N-terminal fragment o f alpha (determined by SDS-PAGE, Western blotting and N-terminal seque ncing). This conjugate was also observed when the enzyme was first act ivated by partial proteolysis of alpha and then crosslinked by transgl utaminase. Both forms of the kinase, generated by either sequential cr osslinking and proteolysis or the reverse, coeluted with non-crosslink ed hexadecameric control enzyme in size exclusion chromatography, indi cating that the crosslinking was intramolecular, i.e., within hexadeca mers. This is the first demonstration of any intersubunit interaction involving the N-terminal domain of the alpha subunit and the first reg ion of any subunit shown to interact with the beta subunit. The result s are consistent with the predicted path of the polypeptide backbone o f the alpha subunits within the holoenzyme and with the proposed locat ion of the beta subunits. (C) 1998 Academic Press.