Burtscher H J, Schneider R, Klocker H, Auer B, Hirsch-Kauffmann M, Schweiger M
Institut für Biochemie, Naturwissenschaftliche Fakultät, Universität Innsbruck, Austria.
J Comp Physiol B. 1987;157(5):567-72. doi: 10.1007/BF00700976.
Covalent modification of proteins by ADP-ribosylation is a major mode of protein regulation in eukaryotic cells. ADP-ribosyltransferases have been characterized from mammals but little is known about these enzymes in lower vertebrates. We purified an ADP-ribosyltransferase (E.C. 2.4.2.30) from trout (Salmo trutta faris) by affinity chromatography and characterized it. The 11,700-fold purified activity shows a major protein band at a molecular mass of 75,000 kDa in a SDS-polyacrylamide gel. In situ reactivation of SDS gels showed the 75,000 kDa protein to be enzymatically active, and additional enzymatically active bands at molecular masses of 115,000, 90,000 and 87,000 kDa, respectively. The enzyme is capable of poly-ADP-ribosylation. It crossreacts with affinity purified antibodies raised against human poly(ADP-ribose)synthetase and, except for the temperature optimum, its properties strongly resemble the mammalian enzymes, indicating the conserved character of nuclear ADP-ribosyltransferases. The trout enzyme is DNA- and histone-dependent, has an optimal pH between 8 and 9 and an apparent Km for NAD+ of 24 microM. The temperature optimum is 10 degrees C compared with 25 degrees C for the human enzyme. Known ADP-ribosyltransferase inhibitors also inhibit the enzyme from trout.
蛋白质的 ADP 核糖基化共价修饰是真核细胞中蛋白质调节的主要方式。ADP 核糖基转移酶已在哺乳动物中得到表征，但对于低等脊椎动物中的这些酶知之甚少。我们通过亲和色谱法从鳟鱼（Salmo trutta faris）中纯化了一种 ADP 核糖基转移酶（E.C. 2.4.2.30）并对其进行了表征。经过 11,700 倍纯化后的活性在 SDS - 聚丙烯酰胺凝胶中显示出一条主要的蛋白质条带，分子量为 75,000 kDa。SDS 凝胶的原位复性显示 75,000 kDa 的蛋白质具有酶活性，另外还有分子量分别为 115,000、90,000 和 87,000 kDa 的酶活性条带。该酶能够进行多聚 ADP 核糖基化。它与针对人多聚（ADP - 核糖）合成酶产生的亲和纯化抗体发生交叉反应，并且除了最适温度外，其性质与哺乳动物酶非常相似，表明核 ADP 核糖基转移酶具有保守性。鳟鱼酶依赖于 DNA 和组蛋白，最适 pH 在 8 至 9 之间，对 NAD + 的表观 Km 为 24 μM。其最适温度为 10℃，而人酶的最适温度为 25℃。已知的 ADP 核糖基转移酶抑制剂也能抑制鳟鱼的这种酶。
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