Date of Award

4-1-1995

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Biochemistry and Molecular Biology

Abstract

Dynamic protein-protein interactions may be a basis for regulation of enzymes that share cross-road metabolites. This type of control could direct metabolites through different pathways depending on which groups of enzymes are associated. Enzymes at the point of convergence of gluconeogenesis, glycolysis and the aspartate-malate shuttle were investigated for interactions. Phosphoenolpyruvate is a product of enolase and a substrate for phosphoenolpyruvate carboxykinase, and pyruvate kinase. Oxalacetate is shared with malate dehydrogenase, aspartate transaminase and phosphoenolpyruvate carboxykinase. Guanine nucleotides are shared between nucleoside diphosphate kinase and phosphoenolpyruvate carboxykinase.The rabbit liver cytosolic enzymes mentioned above were either purified or commercially purchased to be sure that the appropriate isozymes were used. These enzymes were incubated in 14% polyethylene glycol (used as a crowding agent to mimic intracellular conditions). Enzyme activities of both the supernate and the pellet were assayed after centrifugation.Interactions between two enzymes were tested by incubating each enzyme alone and or with another enzyme. A non-interacting protein was also incubated with each enzyme to determine the effect of protein concentration and the specificity of co-pelleting. Similar experiments were also conducted with the structural protein tubulin, which polymerizes to form microtubules. Eleven of the 15 possible pairs of liver enzymes resulted in significantly increased in pelleting of both enzymes. Both nucleoside diphosphate kinase and pyruvate kinase showed evidence of interaction with microtubules.Incubation of all of the enzymes together resulted in a large increase in pelleting over the controls. Removal of any one of the enzymes from the mixture did not dramatically change the amount of pelleting. When a combination of all six enzymes was incubated with microtubules, pelleting of each enzyme increased. These results show evidence for interactions between previously unknown interacting enzymes. This work also suggests that the enzymes tested are possibly interacting as a complex with multiple contact points between enzymes in the cluster.

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