The reversible phosphorylation of key enzymes in metabolism is one of the central themes of metabolic regulation. Several classes of protein kinase have now been characterized, that depend on cyclic nucleotides, calcium ions, calmodulin, and derivatives of membrane phospholipids. In addition to these, an additional group of protein kinases are described as """"""""messenger-independent"""""""", because they do not respond to a known second messenger. Messenger- independent protein kinases are capable of phosphorylating numerous substrates in vitro; their substrates include such metabolically important enzymes as glycogen synthase and acetyl CoA carboxylase in the cytoplasm, pyruvate dehydrogenase and branched chain alpha- keto acid dehydrogenase in the mitochondria, and 3-hydroxy-3- methylglutaryl-CoA reductase attached to the microsomes. These enzymes are all targets of insulin action; elucidating the mechanism by which insulin controls the phosphorylation state of these enzymes would have aid greatly in search for the mechanism of insulin action in vivo. This project would examine one hypothesis for polyamine or a molecule whose action is mimicked by polyamines, and that the effector molecule released promotes, depending on the protein substrate, either decreased or increased phosphorylation of the substrate. A combination of approaches will be used. First, measurements of polyamine concentrations in the cytoplasmic and mitochondrial compartments will be made to determine the availability of a pool of potential effectors. Second, enzymes whose phosphorylation states are known to change as a result of insulin action will be systemically screened for susceptibility to polyamine-activated phosphorylation or dephosphorylation. Third, those target enzymes that appear to be susceptible to polyamine regulation and their protein kinases will be purified and characterized kinetically and structurally. Finally, the effect of insulin and diabetes on polyamine distributions in rat liver and adipose tissue will be examined and correlated to effects on enzyme activity, as will the effects of inhibitors of polyamine biosynthesis.
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