Zero-length crosslinking of the beta subunit of phosphorylase kinase to the N-terminal half of its regulatory alpha subunit.
Phosphorylase kinase, a regulatory enzyme of glycogenolysis in skeletal muscle, is a hexadecameric oligomer containing four copies each of four distinct subunits: alpha, beta, gamma, and delta. By intramolecular zero-length crosslinking with transglutaminase, we have previously demonstrated that the regulatory alpha and beta 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 alpha subunit in holoenzyme that had been crosslinked by transglutaminase has revealed a high molecular weight conjugate corresponding to full-length beta subunit crosslinked to a 60 kDa N-terminal fragment of alpha (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 alpha 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 alpha subunit and the first region of any subunit shown to interact with the beta subunit. The results are consistent with the predicted path of the polypeptide backbone of the alpha subunits within the holoenzyme and with the proposed location of the beta subunits.
Nadeau
O W
OW
Department of Biochemistry, College of Medicine, Memphis, Tennesee, 38163, USA.
Traxler
K W
KW
Carlson
G M
GM
D000818:Animals; D002918:Chymotrypsin; D003432:Cross-Linking Reagents; D004591:Electrophoresis, Polyacrylamide Gel; D020035:Holoenzymes / Q000737:chemistry / Q000378:metabolism; D046911:Macromolecular Substances; D008970:Molecular Weight; D018656:Muscle Fibers, Fast-Twitch / Q000201:enzymology; D018482:Muscle, Skeletal / Q000201:enzymology; D010446:Peptide Fragments / Q000302:isolation & purification; D010764:Phosphorylase Kinase / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D011817:Rabbits; D011503:Transglutaminases / Q000378:metabolism*
D003432:Cross-Linking Reagents; D020035:Holoenzymes; D046911:Macromolecular Substances; D010446:Peptide Fragments; D011503:Transglutaminases; D010764:Phosphorylase Kinase; D002918:Chymotrypsin