The normal intracellular degradation of ornithine decarboxylase (ODC) in Physarum polycephalum will be studied using direct microinjection of labeled enzyme into plasmodia of this organism. A battery of monoclonal antibodies directed against specific regions of the ODC polypeptide chain will be developed. This battery will enable the establishment of specific structural relationships amongst the metabolic intermediates formed during ODC degradation and provide a basis for assay of the individual metabolic steps. Structure-function relationships in Physarum ODC will be examined using specific amino acid modification reagents and suicide inhibitors. Effects on coenzyme and substrate binding and catalytic competence will be compared to effects on intracellular degradation rate and the formation of degradative intermediates. The relationship of observed ODC degradation to a possible ATP-ubiquitin proteolytic system will be examined. Purification of ubiquitin from Physarum will be undertaken and antibodies to the purified polypeptide raised in rabbits. Potential in vivo ATP-dependent conjugation of ODC and ubiquitin will be studied. Experiments similar to the above will be begun following purification of other proteins, including tyrosine aminotransferase, glutamate dehydrogenase, actin, and pyridoxamine phosphate oxidase from Physarum. The detailed biochemical information developed with ODC and these proteins in Physarum will provide a basis for understanding the molecular events involved in intracellular protein degradation in other systems.
