An epitope proximal to the carboxyl terminus of the alpha-subunit is located near the lobe tips of the phosphorylase kinase hexadecamer.
An epitope of the alpha-subunit of phosphorylase kinase from fast-twitch skeletal muscle was localized to the tips of the bilobal kinase molecule by two types of immunoelectron microscopy. This is the first direct evidence identifying the location of any of the enzyme's 16 subunits within the phosphorylase kinase molecule. Negatively stained complexes of phosphorylase kinase with an immunoglobulin G monoclonal antibody specific for the alpha-subunit (mAb 157) were observed by conventional transmission electron microscopy, and complexes of the unstained enzyme with undecagold-labeled Fab' fragments derived from mAb 157 were visualized by scanning transmission electron microscopy. Images from both techniques indicate a symmetrical arrangement of the epitope, consistent with a "head-to-head" packing arrangement of the four alpha-subunits. In Western blots, mAb 157 crossreacted with comigrating fragments obtained by digesting non-denatured phosphorylase kinase with a variety of proteases, suggesting that the epitope for the anti-alpha mAb is contained within a protease-resistant domain. Partial sequencing of a 24.1 kDa immunoreactive chymotryptic fragment narrowed the epitope to somewhere within the carboxyl-terminal one-sixth of the alpha-subunit. Studies of the crossreactivity of mAb 157 with the holoenzyme in the presence of calmodulin, after phosphorylation or with different isoforms (all with known alpha-subunit sequence targets or differences), suggest that the epitope is even more proximal to the carboxyl terminus. This epitope was not implicated in any known function or activity of the enzyme, suggesting that the region proximal to the carboxyl terminus of the alpha-subunit, and thus to the lobe tips of the hexadecamer, may have a role other than catalytic or regulatory.
Wilkinson
D A
DA
Department of Biochemistry, University of Tennessee, Memphis 38163.
Marion
T N
TN
Tillman
D M
DM
Norcum
M T
MT
Hainfeld
J F
JF
Seyer
J M
JM
Carlson
G M
GM
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D001666:Binding Sites, Antibody; D000939:Epitopes; D005260:Female; D008854:Microscopy, Electron; D017348:Microscopy, Electron, Scanning Transmission; D008969:Molecular Sequence Data; D009132:Muscles / Q000201:enzymology*; D010764:Phosphorylase Kinase / Q000737:chemistry / Q000276:immunology*; D011817:Rabbits
D000911:Antibodies, Monoclonal; D000939:Epitopes; D010764:Phosphorylase Kinase