9/26/2023 0 Comments NucleoplasminSecond, we immunoabsorbed Np from Xenopus oocyte nuclei obtained by manual isolation under oil, a method which minimally disturbs in vivo nuclear composition and functions ( Paine et al., 1992). First, we generated a highly specific polyclonal antibody to purified Np and coupled this antibody to Sepharose to construct immunoaffinity beads. We isolated Np from nuclei using a two-pronged experimental approach designed to not disturb intact Np’s in vivo associations. Additionally, we found that phosphorylation of nucleoplasmin by its associated casein kinase II is strongly inhibited by histones and that, in addition to nucleoplasmin, another protein (p100) in the nucleoplasmin-complex is phosphorylated by casein kinase II. Moreover, a polyclonal antibody to the α (38 kDa) and α′ (36 kDa) catalytic subunits of casein kinase II specifically recognizes 38 and 36 kDa polypeptides in the nucleoplasmin-complex, and a specific inhibitor of casein kinase II inhibits nucleoplasmin’s nuclear transport. The co-purifying kinase is casein kinase II-like because: (i) it phosphorylates casein (ii) its phospho-transferase activity can be competed out by GTP (iii) it is stimulated by polylysine and (iv) it is inhibited by heparin. We discovered that nucleoplasmin co-isolates by two independent methods (immunoabsorption and chromatography) in a complex including a kinase which phosphorylates nucleoplasmin. Therefore, we sought to identify any protein kinase which specifically associates with nucleoplasmin. We report herein that the kinetics of its cytoplasm r nucleus transport are affected by its degree of phosphorylation. ![]() Nucleoplasmin is a phosphorylated nuclear-accumulating protein.
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