Mishima K, Terashima M, Obara S, Yamada K, Imai K, Shimoyama M
Department of Oral and Maxillofacial Surgery, Shimane Medical University.
J Biochem. 1991 Sep;110(3):388-94. doi: 10.1093/oxfordjournals.jbchem.a123591.
We have reported the purification and characterization of arginine-specific ADP-ribosyltransferase from hen liver nuclei [Tanigawa, Y. et al. (1984) J. Biol. Chem. 259, 2022-2029] and the DNA-dependent mono(ADP-ribosyl)ation of p33, an acceptor protein in the nuclei [Mishima, K. et al. (1989) Eur. J. Biochem. 179, 267-273]. In the present study, we obtained evidence that among various tissues and cells from chicken, polymorphonuclear cells, so-called heterophils, possess both the ADP-ribosyltransferase and p33 at high levels. Percoll density gradient centrifugation of the postnuclear fraction of the heterophils revealed the co-localization of ADP-ribosyltransferase with p33 in the granule fraction. The enzyme and p33 were purified approximately 219- and 3.77-fold, respectively, from postnuclear pellet fraction to apparent homogeneity. The properties of heterophil ADP-ribosyltransferase and p33 were compared with those of the liver enzyme and p33. The molecular mass of the heterophil enzyme was estimated by SDS-polyacrylamide gel electrophoresis to be 27.5 kDa. The enzyme activity was stimulated by a sulfhydryl agent and inhibited by lysolecithin, NaCl, and inorganic phosphate. The mono(ADP-ribosyl)ation of p33 was markedly enhanced by polyanion, such as DNA, RNA, or poly(L-glutamate). SDS-polyacrylamide gel electrophoretic analysis after limited trypsin proteolysis of p33s, purified from chicken heterophils and liver, showed much the same pattern. Thus, it appears that ADP-ribosyltransferase and p33 present in heterophils are identical to those in the liver, respectively. p33 is considered to be an in situ substrate for ADP-ribosyltransferase, since it was specifically mono(ADP-ribosyl)ated in permeabilized heterophils.(ABSTRACT TRUNCATED AT 250 WORDS)
我们已经报道了从鸡肝细胞核中纯化并鉴定精氨酸特异性 ADP - 核糖基转移酶[谷川洋等人（1984 年）《生物化学杂志》259 卷，2022 - 2029 页]以及细胞核中受体蛋白 p33 的 DNA 依赖性单（ADP - 核糖基）化[三岛克等人（1989 年）《欧洲生物化学杂志》179 卷，267 - 273 页]。在本研究中，我们获得的证据表明，在鸡的各种组织和细胞中，多形核细胞，即所谓的异嗜细胞，同时高水平地拥有 ADP - 核糖基转移酶和 p33。对异嗜细胞的核后部分进行 Percoll 密度梯度离心，结果显示 ADP - 核糖基转移酶与 p33 共定位于颗粒部分。该酶和 p33 分别从核后沉淀部分纯化至表观均一性，纯化倍数约为 219 倍和 3.77 倍。将异嗜细胞的 ADP - 核糖基转移酶和 p33 的性质与肝酶和 p33 的性质进行了比较。通过 SDS - 聚丙烯酰胺凝胶电泳估计异嗜细胞酶的分子量为 27.5 kDa。该酶活性受到巯基试剂的刺激，并受到溶血卵磷脂、NaCl 和无机磷酸盐的抑制。p33 的单（ADP - 核糖基）化被多聚阴离子如 DNA、RNA 或聚（L - 谷氨酸）显著增强。对从鸡异嗜细胞和肝脏中纯化的 p33 进行有限胰蛋白酶水解后的 SDS - 聚丙烯酰胺凝胶电泳分析显示出大致相同的模式。因此，看来异嗜细胞中存在的 ADP - 核糖基转移酶和 p33 分别与肝脏中的相同。p33 被认为是 ADP - 核糖基转移酶的原位底物，因为它在通透的异嗜细胞中被特异性地单（ADP - 核糖基）化。（摘要截短于 250 字）
