The purpose of the proposed research is to learn more about the structure of the membrane anchoring domain of 5'-nucleotidase (5'-NT), a protein attached to the plasma membrane via covalent bonds to phosphatidylinositol (PI) and to determine whether the expression of this protein on the cell surface is regulated, at least in part, by an endogenous phospholipase C (PLC). A specific goal is to determine the inositol, glucosamine, ethanolamine, neutral sugar, and fatty acid composition of the membrane anchoring domain of human placental 5'-NT. We wish to determine whether all PI-linked proteins have structural features in common which allow them to share a common machinery for biosynthesis and insertion into the membrane. We also plan to determine the structural relationship between the three major molecular forms of placental 5'-NT: a membrane- bound form releasable by a PI-specific PLC (PI-PLC), a membrane-bound form resistant to PI-PLC, and a soluble form. All three forms are recognized by antibodies made to the PI-PLC releasable form, suggesting at least some common structural features. Our experiments will determine whether there is heterogeneity in the mode of membrane attachment between the PI-PLC sensitive and resistant forms and whether the soluble form is derived from the membrane-bound form via the action of an endogenous PI-PLC. To learn more about mechanisms controlling 5'-NT expression, we will treat human lymphocytes with agents that might cause release of 5'-NT from the cell surface or change its intracellular distribution via activation of an endogenous PLC. Specifically, human T and B cells will be treated with anti-T3 and anti-IgM, respectively, to cause cellular activation via PLC-catalyzed phosphoinositide hydrolysis and to see if 5'-NT is released from the cell surface. Human B lymphoblasts will be treated with reagents which cause the intracellular accumulation of substrates for 5'-NT (rotenone or 2-deoxyglucose to cause AMP accumulation and deoxyadenosine + 2'-deoxycoformycin to cause dAMP accumulation) to determine whether 5'-NT might be released inside the cell rather than transported to the outer surface of the plasma membrane. These experiments will reveal whether the expression or intracellular location of 5'-NT is altered to meet the metabolic needs of the cell. Our long-term goal is to understand the function of 5' NT and the mechanisms responsible for the changes in 5'-NT activity during human lymphocyte maturation and differentiation.
