The aims of this research are to determine if glycolytic enzymes are truly compartmented in the brain and, if so, to identify the location of the """"""""compartments"""""""" and to determine the mechanisms that permit compartmentation. The purpose of such compartmentation is to permit cells a localized source of energy production. The synaptic plasma membrane is one site where the enzymes are proposed to be enriched. It is hypothesized herein that a diversity of interactions involving the cytoskeleton and certain membrane proteins are available for compartmenting or anchoring the glycolytic enzymes both at the plasma membrane and elsewhere such as in the microtrabeculae, the microtrabeculae being a mesh work of cytoplasmic proteins. This proposal will focus on interactions of glycolytic enzymes with the cytoskeletal components actin and clathrin, and the anion porter. Each of these proteins shows enrichment at the plasma membrane consistent with the hypothesis that the glycolytic enzymes show enrichment at the membrane. Another aspect of the proposal is to investigate enzyme-enzyme-cytoskeleton interactions, i.e. one enzyme must be associated with a cytoskeletal component before a second enzyme will bind. This concept, derived from our earlier studies with phosphoglycerate mutase (PGM) and lactate dehydrogenase (LDH) and F-actin, is now being referred to in the literature as piggyback binding. Such interactions may be representative of microtrabecular interactions. Experiments have been designed to investigate two specific interactions of this type. Glyceraldehyde phosphate dehydrogenase (G3PDH), is known to bind to membranes and to cytoskeletal components while purified phosphoglycerate kinase (PGK) does not bind. However, in impure mixtures, PGK does bind. Because PGK has been recently reported to bind to G3PDH, we hypothesize that PGK binds to G3PDH which in turn binds to the cytoskeleton in agreement with the piggyback model. Along with studying the proposed G3PDH-cytoskeleton interactions we will continue to study the PGM-LDH interaction with F-actin. Perhaps in this way prototype interactions pertinent to the microtrabecular system will be characterized.
