Zero-Length Crosslinking of the β Subunit of Phosphorylase Kinase to the N-Terminal Half of its Regulatory α Subunit

Abstract

Phosphorylase kinase, a regulatory enzyme of glycogenolysis in skeletal muscle, is a hexadecameric oligomer containing four copies each of four distinct subunits: α, β, γ, and δ. By intramolecular zero-length crosslinking with transglutaminase, we have previously demonstrated that the regulatory α and β subunits abut one another in the holoenzyme [Nadeau, O. W., and Carlson, G. M. (1994)J. Biol. Chem.269, 29670–29676]. Selective partial proteolysis of the 138 kDa α subunit in holoenzyme that had been crosslinked by transglutaminase has revealed a high molecular weight conjugate corresponding to full-length β subunit crosslinked to a 60 kDa N-terminal fragment of α (determined by SDS-PAGE, Western blotting and N-terminal sequencing). This conjugate was also observed when the enzyme was first activated by partial proteolysis of α and then crosslinked by transglutaminase. Both forms of the kinase, generated by either sequential crosslinking and proteolysis or the reverse, coeluted with non-crosslinked hexadecameric control enzyme in size exclusion chromatography, indicating that the crosslinking was intramolecular, i.e., within hexadecamers. This is the first demonstration of any intersubunit interaction involving the N-terminal domain of the α subunit and the first region of any subunit shown to interact with the β subunit. The results are consistent with the predicted path of the polypeptide backbone of the α subunits within the holoenzyme and with the proposed location of the β subunits.