CLONING AND EXPRESSION OF HUMAN DEOXYCYTIDINE KINASE CDNA
CLONING AND EXPRESSION OF HUMAN DEOXYCYTIDINE KINASE CDNA
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DOI：
10.1073/pnas.88.4.1531
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发表时间：
1991-02-01
影响因子：
11.1
通讯作者：
MITCHELL, BS
中科院分区：
文献类型：
--
作者：
CHOTTINER, EG;SHEWACH, DS;MITCHELL, BS
多种脱氧核糖核苷和某些广泛用作抗病毒和化疗药物的核苷类似物的磷酸化需要脱氧胞苷 (dCyd) 激酶。 然而，由于其丰度低且不稳定，对该酶的详细分析受到限制。 使用基于纯化的 dCyd 激酶的一级氨基酸序列的寡核苷酸，我们筛选了 T 淋巴细胞 cDNA 文库，并鉴定了编码与纯化蛋白的亚基分子质量相对应的 30.5-kDa 蛋白的 cDNA 序列。 cDNA 在大肠杆菌中的表达导致 dCyd 激酶活性比对照水平增加 40 倍。 在缺乏 dCyd 激酶的小鼠 L 细胞中，用哺乳动物表达载体中的 dCyd 激酶 cDNA 转染，dCyd 磷酸化活性比对照增加 400 倍。 表达的酶对 dCyd 的表观 K(m) 为 1.0-mu-M，并且还能够磷酸化 dAdo 和 dGuo。 Northern 印迹分析显示，T 淋巴细胞中单个 2.8 kb mRNA 的表达水平比 B 淋巴细胞高 5 至 10 倍，并且对阿拉伯呋喃糖基胞嘧啶和双脱氧胞苷耐药的 T 淋巴细胞细胞系中 dCyd 激酶 mRNA 水平降低。 这些发现证明该 cDNA 编码负责 dAdo 和 dGuo 以及 dCyd 和阿拉伯呋喃糖基胞嘧啶磷酸化的 T 淋巴细胞 dCyd 激酶。
Deoxycytidine (dCyd) kinase is required for the phosphorylation of several deoxyribonucleosides and certain nucleoside analogs widely employed as antiviral and chemotherapeutic agents. Detailed analysis of this enzyme has been limited, however, by its low abundance and instability. Using oligonucleotides based on primary amino acid sequence derived from purified dCyd kinase, we have screened T-lymphoblast cDNA libraries and identified a cDNA sequence that encodes a 30.5-kDa protein corresponding to the subunit molecular mass of the purified protein. Expression of the cDNA in Escherichia coli results in a 40-fold increase in dCyd kinase activity over control levels. In dCyd kinase-deficient murine L cells, transfection with dCyd kinase cDNA in a mammalian expression vector produces a 400-fold increase over control in dCyd phosphorylating activity. The expressed enzyme has an apparent K(m) of 1.0-mu-M for dCyd and is also capable of phosphorylating dAdo and dGuo. Northern blot analysis reveals a single 2.8-kilobase mRNA expressed in T lymphoblasts at 5- to 10-fold higher levels than in B lymphoblasts, and decreased dCyd kinase mRNA levels are present in T-lymphoblast cell lines resistant to arabinofuranosylcytosine and dideoxycytidine. These findings document that this cDNA encodes the T-lymphoblast dCyd kinase responsible for the phosphorylation of dAdo and dGuo as well as dCyd and arabinofuranosylcytosine.