2-Macroglobulin (2M) is a proteinase inhibitor that functions by a trapping mechanism which has been exploited such that the receptor-recognized, activated form (2M*) can be employed to target antigens to antigen-presenting cells. kupffer cells of the liver [16-18]. Nucleosides could be potential candidates for 2M* incorporation as amine groups present in the nucleoside could form covalent linkages by nucleophilic substitution at the thiolester as previously observed for other nucleophiles. Synthetic nucleosides derived from guanosine demonstrate immuno – modulatory and immuno-stimulatory properties and provide anti-tumor and -viral activity by the stimulation of endogenous Mouse monoclonal to CD10.COCL reacts with CD10, 100 kDa common acute lymphoblastic leukemia antigen (CALLA), which is expressed on lymphoid precursors, germinal center B cells, and peripheral blood granulocytes. CD10 is a regulator of B cell growth and proliferation. CD10 is used in conjunction with other reagents in the phenotyping of leukemia cytokines [19-26]. The current studies were designed to probe whether guanosine nucleosides can incorporate into 2M* and to develop a general approach demonstrating incorporation of low molecular weight substrates into 2M*. We now report the incorporation into 2M* of fluorescently-labeled guanosine triphosphate (GTP) into 2M*, to date the smallest molecule reported to form complexes with 2M* at buy 475108-18-0 the thiolester. buy 475108-18-0 We demonstrate that the incorporation of the fluorescently-labeled GTP is both SDS and 2-mercaptoethanol resistant and buy 475108-18-0 is not dependent on the fluorescent probe. Furthermore we show that other nucleosides and bases, including guanosine, and imidazole, compete with the fluorescently-labeled GTP for binding with 2M*. This is the first demonstration of incorporation of nucleosides and nucleobases into 2M* and provides a model for studying the incorporation of related compounds of potential therapeutic application. Material and methods Materials Guanosine triphosphate – Texas red (guanosine 5′-triphosphate BODIPY ? TR) and sulforhodamine were from Invitrogen (Carlsbad, CA). Adenosine, adenine, guanosine, guanosine triphosphate guanine, imidazole, iodoacetamide, thymidine, HEPES, NaCl, and porcine pancreatic elastase were from Sigma-Aldrich (St. Louis, MO). Preparation of human 2M and 2M complexes Human 2M was purified using endotoxin-free plasma, columns, and buffers [9,15,27]. Activation to 2M* was achieved using ammonium bicarbonate [27]. 2M was also activated using elastase. Elastase and 2M were incubated at a ratio of 10:1 at room temperature for 45 min. Activated 2M* was carboxamidomethylated with10mM iodoacetamide for 15min at room temperature. GTP-TR (20,50 or 200 M) was incubated with 2M or 2M* at 50C for 5h or 37C for 18h in HEPES buffer. The final concentrations of GTP-TR was 4-, 10-, and 40-fold in excess to 2M. HEPES (25mM) buffers were prepared at pHs of 6.5, 7.0, 8.0 or 9.0 with either 100mM or 200mM NaCl. Following incubation, unincorporated GTP-TR was removed using Micro Bio-Spin? P-30 tris chromatography columns (Bio-Rad, Hercules, CA). The fluorescence of GTP-TR was determined using the detector of a real-time quantitative PCR machine (MX300sp; Stratagene, LaJolla, CA) or a Storm 860 Phosphorimager? (Molecular Devices, Sunnyvale, CA). Fluorescence was converted to GTP concentration using a standard curve. Protein concentration was quantified using the bicinchoninic acid protein assay (Pierce Biotechnology, Rockford, IL) and measuring the absorbance at = 280nm (extinction coefficient 0.893 M?1 cm?1). Competition binding studies GTP-TR was employed as a substrate to investigate whether other low molecular weight molecules form complexes with 2M*. A final concentration of 20M GTP-TR was co-incubated at 50C for 5h with adenosine, adenine, guanosine, guanosine triphosphate, guanine, thymidine, or imidazole at 1.0mM. Following incubation, free GTP-TR was separated from the complexes as described above and the fluorescence was quantified. A decrease in fluorescence indicates competition of the low molecular weight molecule with GTP-TR for complex formation with 2M*. Polyacrylamide gel electrophorersis (PAGE) Proteins were separated by SDS – PAGE, 4 – 20% polyacrylamide gels (pre-cast Ready Gel; Bio-Rad), using 25mM Tris, 192mM glycine, and 0.1% SDS (w/v; Bio-Rad). Non-denaturing 5% polyacrylamide gels (pre-cast Ready Gel; Bio-Rad) buy 475108-18-0 using 130mM tris, 45mM boric acid and 2.5mM ethylenediaminetetraacetic acid were also performed. Data analysis All incorporations were repeated a.