Ppase-1 activity is now recognized as having critical roles in a number of cellular functions. Ppase-1 exists in the cell in holoenzyme complexes with at least three regulatory proteins, inhibitor-2, the glycogen binding subunit, and the nuclear inhibitory protein, NIPP. It is inhibited by inhibitor-1, and by several aquatic tumor promoting toxins, including okadaic acid and the microcystins. Nothing is known of the structural elements of ppase-1 responsible for the binding of these ligands.
Aim 1 is directed toward identifying the structural determinants of the binding sites of ppase-1 by classical biochemical methods. Trace-labeling methods will be used to identify the protein-protein interaction sites of ppase-1 and inhibitor-2, while photoaffinity labeling methods will be used to identify binding sites of inhibitor-1, phosphorylase a, and microcystin.
Aim 2 is directed toward the study of the interaction sites of ppase-1 site directed mutagenesis. Mutagenesis methods will also be used to investigate the roles of specific histidines in the catalytic action of ppase-1, and of a putative okadaic acid binding region. The question of whether interaction with its ligands induces conformational changes in latent ppase-1 will be studied by CD spectroscopy; the studies will focus on the effects of Mn++, and on the inhibition of ppase-1 by the Thr-35-Glu mutant of inhibitor-1. Evidence for binding of Mn++ to ppase-1 will be sought by EPR methods.
Aim 3 deals with issues raised by the existence of two stable conformers of ppase-1. These questions are addressed by studies of """"""""trafficking"""""""" of ppase-1 between its holoenzyme forms, and the effects of such exchange on the activation state of ppase-1. The refolding of ppase-1 will be studied in vitro, to determine the specificity and requirement of regulatory proteins. The synthesis and folding of ppase-1 will be studied in a cell free reticulocyte translation system to determine if this process requires the presence of a chaperone, and to evaluate the effects of the regulatory proteins on ppase-1 synthesis and folding.
In aim 5, the long term goals of isolating other regulatory proteins of ppase-1 by affinity chromatography methods are addressed. It is hoped that these studies will materially advance our understanding of the complex regulation of this important enzyme system.
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