Purification and characterization of human lymphoid poly(adenosine diphosphate ribose) polymerase.
Purification and characterization of human lymphoid poly(adenosine diphosphate ribose) polymerase.
复制标题
人淋巴聚（腺苷二磷酸核糖）聚合酶的纯化和表征。
DOI：
10.1021/bi00265a015
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发表时间：
1982
期刊：
影响因子：
2.9
通讯作者：
Berger,NA
中科院分区：
文献类型：
--
作者：
Carter,SG;Berger,NA
Susan Gear Carter* 和 Nathan A. Berger 摘要：聚 (ADP-核糖) 聚合酶已从人扁桃体中纯化了 12000 倍，相对于初始匀浆，酶活性恢复了 83%。纯化酶的比活性为862单位/毫克蛋白质。通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳测定，分离的蛋白质的分子量约为116 000。在 pH 8.0 和 37°C 下，NAD+ 的表观 Km 估计为 185/uM。纯化的酶绝对需要外源 DNA 来发挥催化活性，并且通过添加纯化的组蛋白 HI 可以增强反应。该酶不需要镁或其他二价阳离子来发挥活性。 I^)ly（二磷酸腺苷核糖）聚合酶是一种染色体酶，其催化NAD+的ADP-核糖1*部分掺入均聚物聚（ADP-核糖）（Hilz & Stone, 1976; Purnell et al., 1980; Hayaishi & Ueda, 1977）。该酶能够启动聚（ADP-核糖）合成，聚合物与其自身共价连接（Kawaichi et al., 1981；Jump & Smulson, 1980；Yoshihara et al., 1977；Ogata et al., 1981）或与染色体蛋白组蛋白 HI（Kawaichi et al., 1980）共价连接。据报道，聚（ADP-核糖）聚合酶及其产物在 DNA 合成（Burzio 和 Koide，1970）、细胞分化（Caplan 和 Rosenberg，1975）、DNA 修复（Smulson 等，1977；Berger 等，1979）和 DNA 转录（Gartemann 等，1981）中发挥作用。聚（ADP-核糖）聚合酶已从多种组织来源和哺乳动物细胞系中不同程度地纯化（Ogata 等人，1981；Yoshihara 等人，1978；Petzold 等人，1981；Okayama 等人，1977；Mandel 等人，1977；Ito 等人，1979；Holtlund 等人，1980）。在本文中，我们报道了从正常人扁桃体和腺样体中纯化聚（ADP-核糖）聚合酶。选择人扁桃体作为组织来源是因为大量组织的可用性以及淋巴组织，特别是动物胸腺（Yoshihara 等人，1978；Petzold 等人，1981；Mandel 等人，1977；Ito 等人，1979；Tsopanakis 等人，1978）是酶的丰富来源。据我们所知，这是从正常人淋巴组织中纯化该酶的第一份报告。纯化方案的初始方面与其他方案类似（Yoshihara 等人，1978 年；Petzold 等人，1981 年），并采用硫酸铵从组织匀浆中沉淀蛋白质，然后在 DNA 纤维素和羟基磷灰石上进行连续色谱法。在以前的纯化过程中，来自羟基磷灰石柱的活性级分根据其分子大小在 Sephadex G-200 柱（Yoshihara 等，1978）或
Susan Gear Carter* and Nathan A. Berger abstract: Poly (ADP-ribose) polymerase has been purified 12000-fold from human tonsils with an 83% recovery of en-zymatic activity relative to that of the initial homogenate. The specific activity of the purified enzyme is 862 units/mg of protein. The isolated protein has a molecular weight of ap-proximately 116 000 as determined by sodium dodecyl sul-fate-polyacrylamide gel electrophoresis. The apparent Km for NAD+ is estimated to be 185/uM at pH 8.0 and 37 C. The purified enzyme has an absolute requirement for exogenous DNA for catalytic activity, and the reaction is enhanced by the addition of purified histone HI. The enzyme does not require magnesium or other divalent cations for activity.I^) ly (adenosine diphosphate ribose) polymerase is a chromosomal enzyme which catalyzes the incorporation of the ADP-ribose1* moiety of NAD+ into a homopolymer, poly-(ADP-ribose)(Hilz & Stone, 1976; Purnell et al., 1980; Hayaishi & Ueda, 1977). This enzyme is able to initiate poly (ADP-ribose) synthesis with the polymer covalently linked to itself (Kawaichi et al., 1981; Jump & Smulson, 1980; Yoshihara et al., 1977; Ogata et al., 1981) or to the chro-mosomal protein, histone HI (Kawaichi et al., 1980). Poly-(ADP-ribose) polymerase and its productshave been reported to function in DNA synthesis (Burzio & Koide, 1970), cellular differentiation (Caplan & Rosenberg, 1975), DNA repair (Smulson et al., 1977; Berger et al., 1979), and DNA tran-scription (Gartemann et al., 1981). Poly (ADP-ribose) polymerase has been purified to variable degrees from several tissue sources and mammalian cell lines (Ogata et al., 1981; Yoshihara et al., 1978; Petzold et al., 1981; Okayama et al., 1977; Mandel et al., 1977; Ito et al., 1979; Holtlund et al., 1980). In this paper we report the purification of poly (ADP-ribose) polymerase from normal human tonsils and adenoids. Human tonsils were selected as a tissue source because of the availability of bulk tissue and because lymphoid tissue, particularly animalthymus (Yoshihara et al., 1978; Petzold et al., 1981; Mandel et al., 1977; Ito et al., 1979; Tsopanakis et al., 1978), is a rich source of the enzyme. To our knowledge, this is the first report of purification of the enzyme from normal human lymphoid tissue. The initial aspects of the purification scheme were similar to others (Yoshihara et al., 1978; Petzold et al., 1981) and employed ammonium sulfate precipitation of protein from a tissue homogenate followed by successive chromatography on DNA-cellulose and hydroxylapatite. In previous purification procedures, the active fraction from the hydroxylapatite column was further purified according to its molecular size on a Sephadex G-200 column (Yoshihara et al., 1978) or a