Phosphofructokinase (PFK) is a rate-limiting enzyme that regulates the glycolytic flux in the heart and in skeletal muscle. Because of the critical role it plays during anoxia, information about its modulation contributes to a better understanding of many cardiovascular and muscular abnormalities such as myocardial ischemia and muscle hypoxia. The impetus for the present proposal is our discovery that epinephrine and isoproteronol increase PFK activity in perfused heart, isolated rat diaphragm, and skeletal of intact rabbits. Both agents are known to stimulate glycolysis in these organs. This laboratory has been studying the different regulatory mechanisms of PFK for the past 25 years. In spite of the expensive knowledge gained, the molecular details of the in vivo action of adrenergic agents on PFK to study the molecular and pharmacological events involved in activation of PFK by adrenergic agents. We intend to use three biological systems that offer different experimental advantages: (1) Myotubes derived from a myogenic cell line are more amenable to experimental manipulation (e.g., labeling with 32P) that intact animals and isolated organs; (2) Perfused guinea pig hearts are used to demonstrate activation of cardiac PFK by adrenergic agents; (3) Skeletal muscles from rabbits are used for studying activation of muscle PFK. The effects of adrenergic agents on the following regulatory parameters of PFK in these systems will be studied. First, we will study the effects of adrenergic agents on phosphorylation sites in PFK. These sites will be compared with phosphorylation sites in pure PFK following its phosphorylation by known protein kinases. The possibility that PFK is phosphorylated by as yet unknown protein kinases will be examined. Secondly, we will study the effects of adrenergic agents on PFK activity using both conventional assay methods that favor enzyme deaggregation and newly reported assay methods that favor enzyme aggregation. Third, we will determine whether adrenergic agents affect the levels of fru-2,6-P2, the most potent activator of PFK, and the activity of the kinase that catalyzes its synthesis, fru-6-P,2-P kinase. Finally, we will study whether adrenergic agents influence the translocation of PFK from cytosol to the particulate fraction (myofibril-rich) of heart and of muscle. Through the use of specific alpha and beta adrenergic blockers we intend to ascertain the pharmacological nature of activation of this enzyme by epinephrine and its congeners.
| Mahrenholz, A M; Hefta, S A; Mansour, T E (1991) Phosphofructokinase from Fasciola hepatica: sequence of the cAMP-dependent protein kinase phosphorylation site. Arch Biochem Biophys 288:463-7 |
| Mahrenholz, A M; Lan, L; Mansour, T E (1991) Phosphorylation of heart phosphofructokinase by Ca2+/calmodulin protein kinase. Biochem Biophys Res Commun 174:1255-9 |
| Wills, K N; Mansour, T E (1990) Changes in phosphofructokinase isozymes during development of myoblasts to myotubes. Arch Biochem Biophys 278:81-7 |
