RESUMO
Ran is a small GTPase that cycles between a guanosine diphosphate (GDP)-bound form (RanGDP) and a guanosine triphosphate (GTP)-bound form (RanGTP) and plays important roles in nuclear transport and mitosis. For studies of Ran function and its interactions with partner proteins, pure RanGDP and RanGTP complexes are critical. Ran complexed with the nonhydrolyzable GTP analog, GMPPNP (RanGMPPNP), is used instead of RanGTP when inhibition of hydrolysis is required. In this study, we demonstrate that the binding of Ran to a UNO Q ion exchange column is remarkably sensitive to small shifts in MgCl(2) concentration, and we use this property to purify recombinant RanGTP, RanGMPPNP, and RanGDP complexes. At 10 mM MgCl(2), Ran was found predominantly in the flow-through and, thus, was separated from the vast majority of bacterial proteins. After reducing the concentration of MgCl(2) to 5 mM, further purification of RanGTP, RanGMPPNP, and RanGDP was achieved by loading onto ion exchange columns and elution with an NaCl gradient. Purity of the resulting preparations was confirmed by releasing the bound nucleotide and checking it against a known nucleotide by high-performance liquid chromatography (HPLC). To further confirm the purity and function of the Ran preparations, appropriate protein-binding, enzymatic, and nuclear import assays were carried out. These methods should facilitate studies of cellular processes involving Ran by providing pure functional Ran-nucleotide complexes.
Assuntos
Cromatografia por Troca Iônica/métodos , Nucleotídeos de Guanina/química , Proteína ran de Ligação ao GTP/química , Proteína ran de Ligação ao GTP/isolamento & purificação , Cromatografia Líquida de Alta Pressão , Nucleotídeos de Guanina/metabolismo , Humanos , Cloreto de Magnésio/química , Ligação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Proteína ran de Ligação ao GTP/genética , Proteína ran de Ligação ao GTP/metabolismoRESUMO
CD45, a receptor-like protein tyrosine phosphatase (PTP), plays an essential role in lymphocyte development and immune responses. Recent evidence suggests that dimerization of CD45 down-regulates its function. However, the mechanisms by which CD45 dimerization is regulated remain unclear, and there is no direct evidence that the PTP activity of CD45 dimers is less than that of monomers. CD45 in lymphocytes associates with CD45-AP (CD45-associated protein). Here we show that T cells from CD45-AP-null mice have a much higher level of CD45 dimers than those of wild-type mice, suggesting that CD45-AP inhibits CD45 dimer formation. This was confirmed with the use of a novel CD45-AP-null T-cell line, ALST-1, that we established from a spontaneous thymic tumor found in a CD45-AP-null mouse. Transfected CD45-AP inhibited CD45 dimer formation in ALST-1 cells in proportion to the amount of CD45-AP expressed. Finally, with the use of microsomal fractions from both mouse thymocytes and ALST-1 transfectants, the PTP activity of CD45 was found to be significantly lower in CD45-AP-negative cells than in CD45-AP-positive cells. Therefore, our results support a model in which binding of CD45-AP to inactive CD45 dimers converts them to active monomers.
