Regulation of phosphofructokinase by reversible inactivation Public Deposited

http://ir.library.oregonstate.edu/concern/graduate_thesis_or_dissertations/9z903230t

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  • The interaction of skeletal muscle phosphofructokinase with a variety of acidic proteins including calmodulin and troponin C and with nucleic acids (RNA and DNA) is manifest in a reversible, time-dependent loss of catalytic activity. The inactivation is affected by a number of factors. Substrates (fructose-6-phosphate and ATP), positive effectors (fructose-2,6-bisphosphate, ADP, AMP, and inorganic phosphate), and a negative effector (citrate) of phosphofructokinase all diminish inactivation while a decrease in pH or an increase in temperature favor the inactivation process. Proteins which bind calmodulin or troponin C -- such as troponin I, melittin, and protamine -- reduce the inactivation, possibly by competing with the enzyme. The inactivation can be reversed by the addition of fructose-2,6-bisphosphate, ATP, or ADP. Studies with specific proteolytic fragments of troponin C indicate that the residues near calcium-binding site III are involved in the inactivation of phosphofructokinase. A general kinetic model for the inactivation has been developed mathematically and applied to the data. A novel electrophoresis procedure demonstrates directly the specific interaction of phosphofructokinase with calmodulin or troponin C. The specific calcium-dependent association of phosphofructokinase with calmodulin or troponin C is manifest in large increases in the rate of and extent of in vitro phosphorylation of the enzyme catalyzed by cAMP-dependent protein kinase. Two distinct phosphorylation sites in phosphofructokinase have been identified. The first, corresponding to the known C-terminal site -- which has the sequence His-Ile-Ser-Arg-Lys-Arg-Ser(P)-Gly-Glu -- is demonstrated both in the control and in enzyme samples which have been phosphorylated in the presence of calmodulin or troponin C. The second is a novel site found in the middle of the phosphofructokinase molecule with the sequence Lys- Leu-Arg-Gly-Arg-Ser(P)-Phe-Met. Phosphorylation at this site is induced by calmodulin or troponin C in the presence of calcium. Preliminary studies indicate that phosphorylation has little effect on the activity of phosphofructokinase as determined by standard assays on samples taken from the phosphorylation mixtures.
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