Act-2 is a member of a family of small secreted proteins, many members of which have inflammatory or chemotactic activities. This family can be divided into two subfamilies based on whether the first two of four conserved cysteines are adjacent (CC) or separated by one amino acid (CXC). Act-2 is a member of the CC subfamily. Synthesis and secretion of this cytokine are rapidly induced in T cells, B cells, and monocytes following stimulation with antigen or mitogen. Since the last annual report the group published the genomic organization and chromosomal localization of this gene, and the identification of the region of the promoter which regulates expression in response to mitogens, phorbol esters and the transactivator gene of HTLG-I. During the year, earlier preliminary results were confirmed that Act-2 protein has activity as a bone marrow stem cell inhibitor. Having previously expressed Act-2 protein in a baculovirus expression system, more than 15 milligrams of purified Act-2 protein have now been produced. Analysis of this protein by one dimensional NMR indicates that it is of high quality and suggests that it should be possible to produce sufficient protein (including (15)N and (13)C labeled protein) to undertake structural analysis by multidimensional NMR. Such information is vital to eventual drug agonist/antagonist development as well as being scientifically important since no structural information is yet available on any members of the CC subfamily. Pilot studies are being performed to evaluate the feasibility of cDNA cloning of the Act-2 receptor.
