The study of the naturally occurring """"""""cannabinoid"""""""" system in our bodies will help us elucidate any harmful and beneficial effects of marijuana use. The long term goal of this research is to understand how the actions of the endogenous cannabinomimetic (anandamide) are enzymatically terminated (by an amidase) in the nervous system and peripheral tissues. In this proposal, we plan to build upon our progress and capitalize upon recent advances in the area by others (cloning of the enzyme) to answer questions of fundamental importance.
The specific aims are to: 1) Identify the active-site and the transmembrane region (s) of anandamide amidase. The amino acids crucial for the active site of the enzyme (a putative serine) will be identified as well as those regions of the enzyme which may be responsible for insertion or association of the enzyme into the membrane. These studies will employ site-directed mutagenesis, radiolabeling of the active site with an irreversible inhibitor, and deletion mutagenesis. 2) Characterize the enzymatic properties of the amidase. We will answer the fundamental questions: (a) Does the enzyme catalyze the reverse reaction; i.e., the synthesis of anandamide from the condensation of ethanolamine and arachidonic acid; (b) What are the effects of some novel irreversible inhibitors upon the enzyme; and How many molecules of anandamide can the enzyme degrade per unit time (turnover number). These experiments will utilize purified amidase preparations from tissue or from the cloned amidase. 3) Expression of the amidase in tissue. These experiments are designed to determine the levels of the enzyme and its mRNA in organs where the cannabinoid receptors (CB1 and CB2) have been shown to occur. In addition, any variants of the mRNA that are detected will be analyzed by sequencing. In situ hybridization experiments will be undertaken to determine the cell specific expression of the amidase mRNA in some selected organs including uterus, kidney and brain.
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