Pyruvate kinase is one of the key enzymes in controlling the rate of glycolysis and gluconeogenesis. As such, it is subject to close metabolic regulation. We propose to investigate whether the enzymatic activity of pyruvate kinase isozymes could be controlled by their interaction with actin- containing filaments. We will study the interaction of the muscle isozyme with muscle F-actin and reconstituted thin filaments, and the yeast isozyme with yeast F-actin. The effect of relevant metabolites on the interactions will also be studied. The enzymatic properties of the actin-bound enzyme will be compared with those of the free enzyme. The interaction of pyruvate kinase with actin-containing filaments will be performed by separating the free and actin- bound enzyme using ultracentrifugation, and assaying the activity of the free enzyme in the supernatant. The kinetics of the free and actin-bound enzyme will be studied using pH stat. The long term objective of our research is to study the effect of changing the environment and the cytoskeletal components on the regulation of the catalytic activity of cytoplasmic enzymes.
