ADP-RIBOSYLATION CYCLES

ADP-核糖基化循环

• 批准号: 6162671
• 负责人: J MOSS
• 金额: --
• 依托单位: NATIONAL HEART, LUNG, AND BLOOD INSTITUTE
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6162671
• 关键词: ADP ribosylation; SDS polyacrylamide gel electrophoresis; adenine phosphoribosyltransferase; immunoprecipitation; integrins; laminin; molecular site; myoblasts; northern blottings; posttranslational modifications; protein sequence; protein structure function; receptor binding; tissue /cell culture; western blottings

RT6 is a rat [T cell] alloantigen linked to the surface of T lymphocytes through a glycosylphosphatidylinositol (GPI) anchor. Two alleles of the rat RT6 gene have been reported. RT6.1 consists of a family of non-glycosylated and variably glycosylated proteins with molecular masses of 25 to 35 kDa and RT6.2 is a non glycosylated protein with molecular mass of 24-28 kDa. The two alloantigens possess both NAD glycohydrolase (NADase) and auto-ADP-ribosyltransferase activities. In the BB rat, a defect in RT6 expression has been shown to coincide with susceptibility to autoimmune insulin-dependent diabetes mellitus (IDDM). Treatment of diabetes-resistant (DR) rats with cytotoxic monoclonal antibody directed against RT6.1+ T cells (DS4.23) induces depletion of RT6+ T cells and leads to development of IDDM in two or four weeks. Following injection of antibody, NADase activity is increased in the plasma approximately 3.4-fold. By sodium dodecyl sulfate-polyacrylamide gel electrophoresis under non-reducing conditions followed by detergent extraction and quantification of enzyme activity, the size of the NADase was estimated to be approximately 30-36 kDa, in agreement with the expected size of a glycosylated form of RT6.1. To characterize the plasma distribution of released RT6, plasma was fractionated into very low/low density lipoprotein (VLDL/LDL), high density lipoprotein (HDL) and nonlipoprotein fraction (d>1.21 g/ml). After antibody treatment, a significant increase in NADase activity was found in the nonlipoprotein fraction whereas effects on the activities associated with HDL and VLDL/LDL were much less. The molecular size of this NADase was consistent with a protein of 30-36 kDa. After immunoprecipitation of VLDL/LDL, HDL, and nonlipoprotein fractions with DS4.23 antibody, an immunoreactive protein of 33 kDa was detected in nonlipoprotein fraction by the polyclonal rabbit anti-RT6 antiserum 1126. Effects of antibody on NADase activity in thymectomized and control rats were similar, suggesting that RT6 NADase activity released by antibody was not derived from peripheral lymphocyte population. Removal of the intestine abolished release of NADase activity, consistent with the hypothesis that RT6 NADase may derived from an intestinal source. Since fractionation by centrifugation in KBr might cause proteins to be released from HDL into nonliprotein fraction, separation was also done by FPLC, and which demonstrated that all the antibody-induced increase in NADase activity was associated with HDL.

RT6是一种与T淋巴细胞表面相关的大鼠[T细胞] 通过糖基磷脂酰肌醇（GPI）锚固。两个等位基因 已经报道了大鼠RT6基因。 RT6.1由一个家庭组成 非糖基化和可变的糖基化蛋白质与分子 25至35 kDa和RT6.2的质量是一种非糖基化蛋白， 分子质量为24-28 kDa。这两个同种剂都具有NAD GlyCohOllolase（NADase）和自动ADP-核糖基转移酶活性。在 BB大鼠（RT6表达中的缺陷）已显示与 自身免疫性胰岛素依赖性糖尿病（IDDM）的敏感性。 用细胞毒性单克隆治疗耐糖尿病（DR）大鼠 针对RT6.1+ T细胞（DS4.23）的抗体诱导 RT6+ T细胞并在两到四个星期内导致IDDM的发展。 注射抗体后，NADase活性在 血浆约3.4倍。十二烷基硫酸钠 - 聚丙烯酰胺 在非还原条件下随后洗涤剂的凝胶电泳 酶活性的提取和定量，NADase的大小 据估计约为30-36 kDa，与 RT6.1的糖基化形式的预期大小。表征 释放RT6的血浆分布，等离子体被分级为非常 低密度脂蛋白（VLDL/LDL），高密度脂蛋白（HDL） 和非脂蛋白分数（D> 1.21 g/ml）。抗体处理后 在非脂蛋白中发现了NADase活性的显着增加 分数对与HDL和HDL相关的活动的影响 VLDL/LDL少得多。该NADase的分子大小是 与30-36 kDa的蛋白质一致。免疫沉淀后 vldl/ldl，HDL和非脂蛋白级数ds4.23抗体，一种 在非脂蛋白部分中检测到33 kDa的免疫反应性蛋白 由多克隆兔抗RT6抗血清1126。抗体的作用 对胸腺切除酶和对照大鼠的NADase活性相似， 提示未得出抗体释放的RT6 NADase活性 来自外周淋巴细胞种群。去除肠子 废除NADase活性的释放，与假设一致 该RT6 NADase可能来自肠子源。自从 通过离心在KBR中进行分馏可能会导致蛋白质 也从HDL释放到非脂蛋白分数，也进行了分离 通过FPLC，这证明了所有抗体诱导的增加 在NADase中，活性与HDL有关。