Brain glycogenolysis plays a critical role in meeting the acute energy requirements of the central nervous system during hypoxic-ischemic events, hypoglycemia, and seizures. Recent evidence suggests that brain glycogen phosphorylase has important regulatory differences from the isoenzymes expressed by skeletal muscle and liver. These regulatory differences, in particular inhibition of brain phosphorylase by glucose, has important implications into understanding control of brain glycogenolysis during acute alterations in cellular metabolism of brain tissue. We propose to identify and determine the amino sequence of brain glycogen phosphorylase in rabbit using recombinant DNA techniques. Our laboratory has determined the x-ray crystallographic structure of rabbit muscle phosphorylase a to 2.1 Angstrom resolution. We have co-crystallized regulatory ligands with the enzyme, identified five regulatory sites for this enzyme, and have elucidated many of the molecular mechanisms governing their allosteric control. We will compare the amino acid sequence of rabbit brain phosphorylase with the known sequence and tertiary structure of the rabbit muscle isoenzyme. This should allow us by comparison to account for the maintenance or alteration of regulatory sites for the brain isoenzyme. We will partially purify brain phosphorylase in rabbit from the other phosphorylase isoenzymes and examine the regulatory properties of purine compounds, AMP, ATP, as well as selective gluconeogenic substrates. DNA probes encoding for fragments of brain and skeletal muscle glycogen phosphorylase will be used for in situ hybridization to regions of the neocortex to localize these isoenzymes to neuronal and glial elements within the central nervous system.