Szebeni A, Herrera J E, Olson M O
Department of Biochemistry, University of Mississippi Medical Center, Jackson 39216-4505, USA.
Biochemistry. 1995 Jun 27;34(25):8037-42. doi: 10.1021/bi00025a009.
Nucleolar protein B23 is a putative ribosome assembly factor with a high affinity for peptides containing nuclear localization signals (NLSs). The interactions of various NLS-containing peptides with two B23 isoforms (B23.1 and B23.2) were examined using equilibrium dialysis and Scatchard analyses. The KD for protein B23 binding to a peptide containing the SV40 T-antigen NLS sequence was approximately 1 microM with a stoichiometry of 1:1 (peptide:protein). No significant differences were seen between the two B23 isoforms in their affinities for any of the peptides tested. Binding by a reverse sequence SV40 T-NLS peptide showed a nonlinear Scatchard plot: this peptide was unable displace the correct sequence peptide, suggesting that the reverse sequence peptide binds to a different site on the protein. A peptide containing the sequence required for nucleolar localization of the HIV-1 Rev protein had an affinity for B23 approximately 10-fold greater than that of the SV40 T-NLS. However, with a sequence sufficient only for Rev location in the nucleoplasm, the affinity for B23 was diminished to a level between that of the longer Rev sequence and the SV40 T-NLS. In competition binding assays, the Rev NLS peptide was able to displace the SV40 T NLS, indicating that both peptides bind to the same site on protein B23. There was no detectable binding to protein B23 by a peptide containing the bipartite NLS of nucleoplasmin. Phosphorylation of protein B23 by casein kinase II enhanced its affinity for the SV40 T- and Rev-derived peptides approximately 2-fold.(ABSTRACT TRUNCATED AT 250 WORDS)
核仁蛋白B23是一种假定的核糖体组装因子，对含有核定位信号（NLSs）的肽具有高亲和力。使用平衡透析和Scatchard分析研究了各种含NLS的肽与两种B23亚型（B23.1和B23.2）的相互作用。蛋白B23与含有SV40 T抗原NLS序列的肽结合的解离常数（KD）约为1微摩尔，化学计量比为1:1（肽:蛋白）。在所测试的任何肽的亲和力方面，两种B23亚型之间未观察到显著差异。反向序列SV40 T-NLS肽的结合显示出非线性Scatchard图：该肽无法取代正确序列的肽，表明反向序列肽与蛋白质上的不同位点结合。含有HIV-1 Rev蛋白核仁定位所需序列的肽对B23的亲和力比SV40 T-NLS大约高10倍。然而，对于仅足以使Rev定位于核质的序列，对B23的亲和力降低到较长Rev序列和SV40 T-NLS之间的水平。在竞争结合试验中，Rev NLS肽能够取代SV40 T NLS，表明两种肽都与蛋白B23上的同一位点结合。含有核纤层蛋白双分NLS的肽与蛋白B23没有可检测到的结合。酪蛋白激酶II对蛋白B23的磷酸化使其对SV40 T和Rev衍生肽的亲和力提高了约2倍。（摘要截短于250字）
