Our goal is to elucidate the molecular mechanisms by which insulin regulates metabolism. The acute effects of insulin on glycogen metabolism, activating glycogen synthase and inactivating phosphorylase and phosphorylase kinase, are mediated by the activation of a certain phosphoprotein phosphatase. A chronic effect of insulin is the accumulation of a protein component essential for this acute response. A principle objective is to define the mechanism by which the phosphatase is regulated and determine the molecular modifications in the structure of the phosphatase produced in response to insulin. A second objective is to compare levels of phosphatase components in protein and mRNA of liver and other tissues from diabetic and normal animals. The experimental strategy employs unique anticatalytic and anti-regulatory immunoglobulins as well as biochemical and immunochemical analytical procedures developed in the first term of this project. The proposal presents a new hypothesis for regulation of the phosphatase through intramolecular disulfide interchange. A phosphorylated protein regulator of Mr=60.000, related to inhibitor-2, influences this reaction. Active and inactive forms of the phosphatase will be analyzed by diagonal electrophoresis of cystine peptides. Purified peptides will be sequenced and the appropriate oligonucleotides synthesized to use as hybridization probes for isolating and sequencing the phosphatase gene. Immunoblotting will be used to measure the phosphatase proteins in normal and diabetic rat liver, fat and muscle. The immunoglobulins will also be used to recover the newly-discovered phosphatase catalytic or regulator proteins from in vitro translation of liver mRNA. The molecular defect in alloxan-diabetes may be the diminished production of one of these proteins, so understanding their interactions may prove to be the basis for novel chemotherapeutic approaches to diabetes.
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