ldentification of CD 45 ligand

淋巴细胞的活化、增殖和细胞凋亡均受蛋白酪氨酸磷酸化的调控。受体型蛋白酪氨酸磷酸酶（PTP） cd45在这些过程中起着关键作用。了解cd45在调节细胞间相互作用和PTP活性方面的功能，鉴定cd45的生理配体至关重要。在这个项目中，我们试图鉴定cd45结合蛋白，作为实现这一目标的第一步。1)从WEHI-231 B细胞系中纯化天然CD45，通过分子筛、小麦胚芽凝集素和谱蛋白亲和层析纯化了天然CD45。纯化后的CD45经SDS-PAGE金胶体染色，仅可见Mr为bb0 200 kDa的一条条带，表明CD45已被纯化至均匀性。2)脾脏裂解液中cd45结合蛋白的鉴定小鼠脾脏均质化并分离成可溶性和膜结合部分。将每个部分应用于cd45结合柱，然后从结合部分中洗脱，分别含有70kDa蛋白和54 kDa、60 kDa和66 kDa三种蛋白。3) CD45结合蛋白对CD45 PTP活性的影响下一步，分析DC45与CD45结合蛋白相互作用对CD45酶活性的影响。纯化后的CD45与每个部分的CD45结合蛋白在冰上孵育1小时，以含磷酸酪氨酸的寡肽作为底物测量CD45 PTP活性。4)能与CD45结合的细胞。最后，确定与CD45结合的脾细胞群。用FITC-CD45和PE-alpha-Thy-1进行的FACS分析表明，CD45可以结合T细胞和B细胞的一部分。

Lymphocyte activation, proliferation, and cell death by apoptosis are regulated by protein tyrosine phosphorylation.One of the key playrs in these processes is a receptor-type protein tyrosine phosphatase (PTP), CD 45. To understand the function of CD 45 in terms of the regulation of cell-cell interaction and of the PTP activity identification of the physiological ligand (s) for CD45is crucial.In this proiect we tried to identify CD45-binding proteins as a first step toward this goal.1) Purification of native CD45 from the WEHI-231 B cell lineNative CD45 was purified from the WEHI-231 B cell line, with molecular-sieve, wheat germ agglutinin and spectrin affinity chromatographies.The gold-colloid staining of the purified CD45 separated by SDS-PAGE revealed that only one band with Mr of >200 kDa is visualized, suggesting that CD45 has been purified to homogeneity.2) ldentification of CD45-binding proteins in spleen lysatesMouse spleen was homogenized and separated into soluble and membrane-bound fractions.Each fraction was applied to a CD45-bound column, and then eluted from the bound fractions, respectively, contain 70kDa protein and three proteins of 54 kDa, 60 kdA and 66 kDa.3) Effect of CD45-binding proteins on CD45 PTP activityNext, how the interaction of DC45 and CD45-binding protein affects enzymatic activity of CD45 was analyzed.Purified CD45 was incubated 1 hr on ice with CD45-binding proteins from each fraction and the CD45 PTP activity was measured sing a phosphotyrosine-containing oligopeptide as a substrate.4) Cells that can bind to CD45Finally, it was determined which spleen cell population is bound to CD45.FACS analysis with FITC-CD45 and PE-alpha-Thy-1 demonstrated that CD45 can bind a part of both T and B cells.