A LYMPHOBLAST MODEL FOR DISEASES OF PURINE METABOLISM

我们已经确定了含有nad的s -腺苷型同型半胱氨酸酶的作用

We have identified a role for the NAD-containing enzyme S-adenosylhomocysteine hydrolase (AdoHcyase), which is also an adenosine (Ado) and cAMP binding protein, in mediating inhibitory effects of Ado and 2'-dAdo on S-adenosylmethionine (AdoMet)-dependent transmethylation reactions. The objectives of this proposal are to extend our studies of purified human AdoHcyase; to assess inhibition of RNA methylation in Ado deaminase (ADA)-inhibited cells treated with 2'-dAdo (which irreversibly inactivates AdoHcyase); and to assess the contribution of AdoHcyase to the immune defect in genetic or drug induced ADA deficiency. Specific studies are planned in 3 areas: a) To use limited proteolytic mapping techniques in order to further define the organization and function of the nucleoside, cAMP, and NAD binding domains of AdoHcyase. Proteolytic mapping and domain-specific monoclonal antibodies will be used to determine whether these regions are related to analogous domains of the cAMP binding subunit of protein kinase, or the NAD binding domains of other NAD containing enzymes. b) To study the effect of inactivation of intracellular AdoHcyase by 2'-dAdo on methylation of ribosomal RNA precursor in mitogen-stimulated human peripheral blood lymphocytes, and in cells obtained from patients undergoing treatment with the ADA inhibitor deoxycoformycin, or with the antiviral agent adenine arabinoside. We will assess the rates of AdoHcyase synthesis in different cell types as a possible basis for selective inactivation of AdoHcyase in lymphoid cells. Alternative pathways of AdoHcy catabolism in various cell types will also be studied as a basis for selective effects of AdoHcyase inactivation. c) Mutant lymphoid cell lines will be used or selected to define the basis for toxicity of adenine, 5'-methylthioadenosine, and other purine analogs; and for determining the identity of enzymes in T cells responsible for phosphorylating 2'-dAdo. We will attempt to isolate a mutant with altered activity or production of AdoHcyase.