Kim H J, Okazaki I J, Takada T, Moss J
Pulmonary-Critical Care Medicine Branch, NHLBI, National Institutes of Health, Bethesda, Maryland 20892, USA.
J Biol Chem. 1997 Apr 4;272(14):8918-23. doi: 10.1074/jbc.272.14.8918.
Transfection of NMU (rat mammary adenocarcinoma) cells with NAD:arginine ADP-ribosyltransferase cDNAs from Yac-1 murine lymphoma cells or rabbit muscle increased NAD glycohydrolase and ADP-ribosyltransferase activities. The ADP-ribosyltransferase activity was released from transformed NMU cells by phosphatidylinositol-specific phospholipase C (PI-PLC) and hence glycosylphosphatidylinositol (GPI)-anchored, whereas the NAD glycohydrolase (NADase) activity remained cell-associated. By gel permeation chromatography, the size of the PI-PLC-released transferase was approximately 40 kDa and that of the detergent-solubilized NADase was approximately 100 kDa. Using polyclonal antibodies against rabbit muscle transferase on Western blots, approximately 18- and approximately 30-kDa band were visualized among proteins from the NADase fractions and 38-40-kDa bands with protein from the transferase fractions. Incubation of blots with [32P]NAD led to the incorporation of radioactivity into the immunoreactive transferase bands of 38 kDa and the immunoreactive NADase band of approximately 18 kDa. These data suggest that proteolysis of ADP-ribosyltransferase synthesized in transformed NMU cells might result in the formation of aggregates of an 18-kDa NAD glycohydrolase. A fusion protein with glutathione S-transferase linked to the amino terminus of Yac-1 transferase, from which the amino-terminal 121 amino acids had been deleted (GST-Yac-1-delta121), exhibited NADase, but not transferase, activity. The size of the recombinant fusion protein was similar to that of the proteolytic fragment seen in NMU cells transformed with transferase cDNA. These results are compatible with the conclusion that the NAD glycohydrolase activity was generated in NMU cells by proteolysis of ADP-ribosyltransferase, with release of a carboxyl-terminal fragment that possesses glycohydrolase but not transferase activity, i.e. the carboxyl-terminal portion of the transferase can exist as a catalytically active NADase.
用来自Yac-1鼠淋巴瘤细胞或兔肌肉的NAD：精氨酸ADP-核糖基转移酶cDNA转染NMU（大鼠乳腺腺癌）细胞，可增加NAD糖水解酶和ADP-核糖基转移酶活性。ADP-核糖基转移酶活性可被磷脂酰肌醇特异性磷脂酶C（PI-PLC）从转化的NMU细胞中释放出来，因此是糖基磷脂酰肌醇（GPI）锚定的，而NAD糖水解酶（NADase）活性仍与细胞相关。通过凝胶渗透色谱法，PI-PLC释放的转移酶大小约为40 kDa，去污剂溶解的NADase大小约为100 kDa。在蛋白质印迹中使用抗兔肌肉转移酶的多克隆抗体，在NADase组分的蛋白质中可见约18 kDa和约30 kDa的条带，在转移酶组分的蛋白质中可见38 - 40 kDa的条带。用[32P]NAD孵育印迹，导致放射性掺入38 kDa的免疫反应性转移酶条带和约18 kDa的免疫反应性NADase条带中。这些数据表明，在转化的NMU细胞中合成的ADP-核糖基转移酶的蛋白水解可能导致形成18 kDa NAD糖水解酶的聚集体。一种与谷胱甘肽S-转移酶融合的蛋白，连接到Yac-1转移酶的氨基末端，其中氨基末端的121个氨基酸已被删除（GST-Yac-1-delta121），表现出NADase活性，但没有转移酶活性。重组融合蛋白的大小与用转移酶cDNA转化的NMU细胞中看到的蛋白水解片段相似。这些结果与以下结论一致，即NAD糖水解酶活性是通过ADP-核糖基转移酶的蛋白水解在NMU细胞中产生的，释放出具有糖水解酶但没有转移酶活性的羧基末端片段，即转移酶的羧基末端部分可以作为具有催化活性的NADase存在。
