The objective of this research is to study the regulation of the enzyme specifically cleaving dynorphin B to give Leu-enkephalin-Arg6 in human spinal cord. As tool to achieve this, we will clone cDNA for this enzyme from human spinal cord astrocyte cells. Enzyme activity in these cells is 100fold higher than in spinal cord. The cloning will be performed by either sequencing of the purified protein and generating oligonucleotide probes, or by expression cloning. Antisera will be raised against purified protein, and this antisera, combined with the cDNA clones obtained, will be used to study the regulation of dynorphin cleavage in human spinal cord cells at the molecular level. The questions to be answered include: (1) what is the mechanism of upregulation of dynorphin converting activity during morphine treatment: is the level of enzyme production raised, or is the enzyme activated by posttranslational modification, e.g. phosphorylation; (2) what are the enzymes carrying out the modification; (3) what are the sites of the possible modification in the enzyme. The means to obtain answers in these studies will include kinetic measurements using hybridization with cDNA probes to follow message levels coding the enzyme, specific kinase inhibitors, immunoprecipitation of the enzyme by antisera.
