Phosphorylase kinase is an enzyme involved in carbohydrate metabolism in muscle whose activity is regulated by physiological concentrations of Ca2+ ions. We have found that Mg2+ acts in concert with Ca2+ to cause a time-dependent activation of the enzyme. One of the goals of this project is to gain an understanding of the mechanism underlying this synergistic activation. We will determine whether time-dependent conformational changes occur in response to these metals and, if so, whether the rate of activation corresponds to an identifiable conformational state. In order to determine whether there is a salient conformational change associated with activation in general, we will examine the conformational changes produced by other factors that cause activation (phosphorylation and slightly alkaline pH). We will also perform a topological analysis in an attempt to determine the overall conformation of the molecule, the spatial relationships of its sixteen subunits (Alpha4Beta4Gamma4Delta4), and whether those spatial relationships are altered by effectors of the enzyme, such as Ca2+ plus Mg2+. Another Ca2+ dependent process to be studied is autoactivation resulting from autophosphorylation. We will characterize the properties of the autophosphorylated enzyme and will attempt to determine whether autophosphorylation occurs in vivo. The sum of all these data should provide information concerning regulation of glycogen breakdown in muscle and how that breakdown, with subsequent energy production, is coupled to muscle contraction.
| Cheng, A; Carlson, G M (1988) Competition between nucleoside diphosphates and triphosphates at the catalytic and allosteric sites of phosphorylase kinase. J Biol Chem 263:5543-9 |
| Cheng, A; Fitzgerald, T J; Bhatnagar, D et al. (1988) Allosteric nucleotide specificity of phosphorylase kinase: correlation of binding, conformational transitions, and activation. Utilization of lin-benzo-ADP to measure the binding of other nucleoside diphosphates, including the phosphorothioates of ADP. J Biol Chem 263:5534-42 |
| Paudel, H K; Carlson, G M (1987) Inhibition of the catalytic subunit of phosphorylase kinase by its alpha/beta subunits. J Biol Chem 262:11912-5 |
| Fitzgerald, T J; Trempe, M R; Carlson, G M (1987) Autophosphorylation of the alpha subunit of phosphorylase kinase from rabbit skeletal muscle. J Biol Chem 262:11239-46 |
