ISSN:
1573-4943
Keywords:
Phosphorylase kinase
;
Mg2+
;
quaternary structure
;
activation
Source:
Springer Online Journal Archives 1860-2000
Topics:
Chemistry and Pharmacology
Notes:
Abstract Phosphorylase kinase (PhK) from skeletal muscle is a structurally complex, highly regulated, hexadecameric enzyme of subunit composition (αβγδ)4. Previous studies have revealed that the activity of its catalytic γ subunit is controlled by alterations in quaternary structure initiated at allosteric and covalent modification sites on PhK's three regulatory subunits; however, changes in the conformation of the holoenzyme initiated by the catalytic subunit have been more difficult to document. In this study a monoclonal antibody (mAb γ79) has been generated against isolated γ subunit and used as a conformational probe of that subunit. The epitope recognized by this antibody is within the catalytic core of the γ subunit, between residues 100 and 240, and monovalent fragments of the antibody inhibit the catalytic activity of the holoenzyme, the γ-calmodulin binary complex, and the free γ subunit. Activation of PhK by a variety of mechanisms known or thought to act through its regulatory subunits (phosphorylation, ADP binding, or alkaline pH) increased the binding of the holoenzyme to immobilized mAb γ79, indicating that activation by any of these distinct mechanisms involves repositioning of the portion of the catalytic domain of the γ subunit containing the epitope for mAb γ79. The activating ligand Mg2+ also stimulated the binding of the PhK holoenzyme to immobilized mAb γ79, as well as the binding of mAb γ79 to immobilized γ subunit. Thus, Mg2+ increases the accessibility of the mAb γ79 epitope in both the isolated γ subunit and in the holoenzyme. Our results suggest that previously reported influences of Mg2+ on the quaternary structure of the PhK holoenzyme are directly mediated by the γ subunit.
Type of Medium:
Electronic Resource
URL:
http://dx.doi.org/10.1023/A:1020667720565
Permalink