Phosphofructokinase (PFK) is a rate-limiting enzyme that regulates carbohydrate metabolism in the heart and skeletal muscle. It is also involved in the effect of epinephrine and of insulin on cellular metabolism. The enzyme appears to be regulated during development to meet cellular needs for energy. In spite of the vast knowledge obtained in many laboratories including our own on the kinetic and biophysical properties of PFK, details of its in vivo regulation in intact animals and tissues remain elusive. We plan to use a muscle cell line that is more amenable to experimental manipulation than intact animals or isolated tissues to study cellular regulation of PFK and how it is influenced by insulin and epinephrine. We will consider the regulatory role of subcellular distribution of PFK, its property to undergo association-dissociation conversion, and the effect of macromolecular crowding on its activity. PFK will be studied in two stages of muscle cell development, the mononucleated proliferating myoblasts and the differentiated multinucleated myotubes. We plan (1) to investigate the nature of isozymes of PFK in the two muscle cell types and the mechanism of the marked increase of PFK in the myotubes compared to the myoblasts; (2) to investigate the effect of phosphorylation of pure muscle and liver PFK isozymes by different protein kinases and by a combination of these enzymes on PFK activity, and to identify the phosphorylated peptides in each isozyme as a result of phosphorylation; (3) to characterize the phosphopeptides of PFK from myoblasts and myotubes and compare their phosphopeptide patterns with those of pure liver and muscle isozymes, with the ultimate goal of identifying the protein kinase(s) involved in phosphorylation of PFK in these cells; (4) to investigate the effect of epinephrine and of insulin on glycolysis on PFK activity and on phosphopeptide patterns of PFK in myoblasts and myotubes; and (5) to determine the role of fructose-2,6-P2 in the regulation of PFK during development from myoblasts to myotubes and to assess changes in the activity of 6-phosphofructo-2-kinase and fructose-2,6-biphosphatase during development; and (6) to study the effect of epinephrine and of insulin on fructose-26-P2 levels in both muscle cell types, and the possible effects of both hormones on 6-phosphofructo-2-kinase and fructose-2,6- biphosphatase. Because of the critical role that PFK plays in regulation of metabolism in heart and skeletal muscle, information on PFK modulation during development and by hormones should contribute to a better understanding of heart and muscle diseases.