Phosphorylase kinase is a pivotal enzyme in the regulation of glycogen metabolism in response to hormonal and neural stimulation and the metabolic demands of the cell. It is a protein of complex structure, alpha 4, beta 4, gamma 4, delta 4, that is regulated by covalent modification, by Ca2+ (involving both the intrinsic delta subunit and extrinsic calmodulin), and by allosteric regulators, including ADP and glycogen. Within the cell, it interacts with at least five proteins that include the cAMP- dependent protein kinase, selective alpha and beta subunit phosphatases calmodulin (and/or TnC) and phosphorylase. In the continuing understanding of the physiological control of phosphorylase kinase, a detailing of the structure- function relationships of each subunit is planned. This will be approached from two perspectives. Methods are outline to obtain the cDNAs for the alpha, beta and gamma subunits, to establish suitable expression vectors for each, and then by deletion mapping and cassette and site-directed mutagenesis to explore and characterize the domains and regions in each that are associated with catalytic competency and regulation. In complementary studies it is planned to isolate and characterize partial complexes and individual subunits that may be obtained by dissociation of the holoenzyme using physicochemical methodologies; several possible strategies to achieve this are proposed. It is also planned to determine methods for reconstitution of the holoenzyme (and intermediary complexes). Typical questions that could then be probed include a characterization of the regulatory properties, catalytic activity and efficacy as a substrate of holoenzyme forms having precisely defined subunit phosphorylation stoichiometries and/or where selected regions have been deleted or mutated. In corollary studies, and in particular using avenues that would be opened up by the proposed cloning experiments, it is planned to determine the complementary coordination of subunit synthesis and the in vivo assembly of phosphorylase kinase holoenzyme. This will be examined in myocytes in culture. We also plan to explore the expression of isozymes and the reasons for the absence of expressed protein in the phoskin- I strain mouse.
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