An epitope proximal to the carboxyl terminus of the α-subunit is located near the lobe tips of the phosphorylase kinase hexadecamer

Deborah A. Wilkinson, Tony Marion, David M. Tillman, Mona T. Norcum, James F. Hainfeld, Jerome M. Seyer, Gerald M. Carlson

Research output: Contribution to journalArticle

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Abstract

An epitope of the α-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 α-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 α-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-α 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 α-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 α-snbvmit 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 α-subunit, and thus to the lobe tips of the hexadecamer, may have a role other than catalytic or regulatory.

Original languageEnglish (US)
Pages (from-to)974-982
Number of pages9
JournalJournal of Molecular Biology
Volume235
Issue number3
DOIs
StatePublished - Jan 20 1994

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Phosphorylase Kinase
Epitopes
Peptide Hydrolases
Enzymes
Holoenzymes
Immunoglobulin Fab Fragments
Scanning Transmission Electron Microscopy
Immunoelectron Microscopy
Calmodulin
Transmission Electron Microscopy
Protein Isoforms
Skeletal Muscle
Phosphotransferases
Immunoglobulin G
Western Blotting
Monoclonal Antibodies
Phosphorylation

All Science Journal Classification (ASJC) codes

  • Structural Biology
  • Molecular Biology

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An epitope proximal to the carboxyl terminus of the α-subunit is located near the lobe tips of the phosphorylase kinase hexadecamer. / Wilkinson, Deborah A.; Marion, Tony; Tillman, David M.; Norcum, Mona T.; Hainfeld, James F.; Seyer, Jerome M.; Carlson, Gerald M.

In: Journal of Molecular Biology, Vol. 235, No. 3, 20.01.1994, p. 974-982.

Research output: Contribution to journalArticle

Wilkinson, Deborah A. ; Marion, Tony ; Tillman, David M. ; Norcum, Mona T. ; Hainfeld, James F. ; Seyer, Jerome M. ; Carlson, Gerald M. / An epitope proximal to the carboxyl terminus of the α-subunit is located near the lobe tips of the phosphorylase kinase hexadecamer. In: Journal of Molecular Biology. 1994 ; Vol. 235, No. 3. pp. 974-982.
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abstract = "An epitope of the α-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 α-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 α-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-α 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 α-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 α-snbvmit 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 α-subunit, and thus to the lobe tips of the hexadecamer, may have a role other than catalytic or regulatory.",
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AU - Marion, Tony

AU - Tillman, David M.

AU - Norcum, Mona T.

AU - Hainfeld, James F.

AU - Seyer, Jerome M.

AU - Carlson, Gerald M.

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N2 - An epitope of the α-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 α-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 α-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-α 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 α-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 α-snbvmit 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 α-subunit, and thus to the lobe tips of the hexadecamer, may have a role other than catalytic or regulatory.

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