Wesierska-Gadek J, Sauermann G
Eur J Biochem. 1985 Dec 2;153(2):421-8. doi: 10.1111/j.1432-1033.1985.tb09319.x.
Nuclear matrices were isolated by treatment of isolated HeLa cell nuclei with high DNase I, pancreatic RNase and salt concentrations. ADP-ribosylated nuclear matrix proteins were identified by electrophoresis, blotting and autoradiography. In one experimental approach nuclear matrix proteins were labeled by exposure of permeabilized cells to the labeled precursor [32P]NAD. Alternatively, the cellular proteins were prelabeled with [35S]methionine and the ADP-ribosylated nuclear matrix proteins separated by aminophenyl boronate column chromatography. By both methods bands of modified proteins, though with differing intensities, were detected at 41, 43, 46, 51, 60, 64, 69, 73, 116, 140, 220 and 300 kDa. Approximately 2% of the total nuclear ADP-ribosyltransferase activity, but only 0.07% of the nuclear DNA, was tightly associated with the isolated nuclear matrix. The matrix-associated enzyme catalyzes the incorporation of [32P]ADP-ribose into acid-insoluble products of molecular mass 116 kDa and above, in a 3-aminobenzamide-inhibited, time-dependent reaction. The possible function of ADP-ribosylation of nuclear matrix proteins and of the attachment of ADP-ribosyltransferase to the nuclear matrix in the regulation of matrix-associated biochemical processes is discussed.
通过用高浓度的脱氧核糖核酸酶I、胰核糖核酸酶和盐处理分离的海拉细胞核来分离核基质。通过电泳、印迹和放射自显影鉴定ADP-核糖基化的核基质蛋白。在一种实验方法中，通过将通透细胞暴露于标记的前体[32P]NAD来标记核基质蛋白。或者，用[35S]甲硫氨酸对细胞蛋白进行预标记，并用氨基苯基硼酸柱色谱法分离ADP-核糖基化的核基质蛋白。通过这两种方法，在41、43、46、51、60、64、69、73、116、140、220和300 kDa处检测到修饰蛋白条带，尽管强度不同。约2%的总核ADP-核糖基转移酶活性，但仅0.07%的核DNA与分离的核基质紧密相关。与基质相关的酶在3-氨基苯甲酰胺抑制的、时间依赖性反应中催化[32P]ADP-核糖掺入分子量为116 kDa及以上的酸不溶性产物中。讨论了核基质蛋白ADP-核糖基化以及ADP-核糖基转移酶附着于核基质在调节与基质相关的生化过程中的可能功能。
