Purification and properties of adenosine kinase from rat liver: separation from deoxyadenosine kinase activity.

A K Drabikowska, L Halec, D Shugar
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引用次数: 10

Abstract

Ion exchange and affinity chromatography techniques, similar to those previously reported for purification of adenosine kinase from human placenta, were applied to purification of rat liver adenosine kinase. The enzyme, purified 400-fold in 41% yield, was homogeneous on SDS-polyacrylamide gel electrophoresis, with a molecular weight of 52000. It specific activity, 18 mumol/min/mg protein, is the highest hitherto reported for this enzyme from mammalian sources. Chromatography on DEAE-cellulose removed about 98% of the phosphorylating activity towards 2'-deoxyadenosine present in the initial pH-treated liver extract. The final preparation exhibited only minimal activity (approximately 1.5%) under optimal conditions (pH 7.5) vs 2'-deoxy-adenosine, the lowest yet reported for such a preparation, with a Km of 670 microM, as compared to 0.3 microM for adenosine. The residual activity towards deoxyadenosine is considered an intrinsic property of the purified adenosine kinase and, in fact, phosphorylation of adenosine was inhibited competitively by deoxyadenosine, with a Ki of 70 microM. Competitive inhibition was also exhibited by cordycepin (3'-deoxyadenosine) with a Ki of 150 microM. A more potent competitive inhibitor was tubercidin, the Ki for which was 1.9 microM.

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大鼠肝中腺苷激酶的纯化及性质:脱氧腺苷激酶活性的分离。
离子交换和亲和层析技术,类似于先前报道的纯化人胎盘中腺苷激酶的技术,被用于纯化大鼠肝腺苷激酶。该酶纯化400倍,产率41%,sds -聚丙烯酰胺凝胶电泳均相,分子量为52000。它的比活性为18 μ mol/min/mg蛋白,是迄今为止报道的该酶在哺乳动物中最高的。deae -纤维素层析去除初始ph处理的肝脏提取物中约98%的对2'-脱氧腺苷的磷酸化活性。与2'-脱氧腺苷相比,最终的制剂在最佳条件下(pH 7.5)仅表现出最小的活性(约1.5%),这是迄今为止报道的最低的此类制剂,Km为670微米,而腺苷为0.3微米。对脱氧腺苷的残留活性被认为是纯化的腺苷激酶的固有特性,事实上,腺苷的磷酸化被脱氧腺苷竞争性地抑制,Ki为70微米。虫草素(3′-脱氧腺苷)在Ki为150 μ m时也表现出竞争性抑制作用。一种更有效的竞争性抑制剂是结核菌素,其Ki值为1.9微米。
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