Shahbaz M, Hoch J A, Trach K A, Hural J A, Webber S, Whiteley J M
Department of Basic and Clinical Research, Scripps Clinic and Research Foundation, La Jolla, California 92037.
J Biol Chem. 1987 Dec 5;262(34):16412-6.
The cleavage of reductively alkylated rat liver dihydropteridine reductase with cyanogen bromide afforded a mixture of peptides, six of which (CB-1 to CB-6) were isolated and purified by C8 reverse-phase high performance liquid chromatography. Portions of peptides CB-1, CB-4, and CB-6 were sequenced by automated Edman degradation and high performance liquid chromatography and the carboxyl-terminal region by conventional procedures. Further proteolytic digestion of CB-6 and isolation of the products afforded a seven-amino acid peptide. A low degeneracy probe comprising 20 nucleotides was synthesized from the sequence of this peptide and was used to screen a rat liver cDNA expression library constructed in the vector lambda gt 10. Positive clones were isolated, and detailed examination of five of these by restriction endonucleases and dideoxy sequence analyses allowed identification of the entire coding region for dihydropteridine reductase. The gene was found to code for a protein of 240 amino acids (excluding the methionine initiator) of Mr = 25,420. Each of the sequences corresponding to the peptides CB-1, CB-4, CB-6, and the carboxyl terminus were identified in the deduced protein sequence. The rat enzyme is highly homologous to the human dihydropteridine reductase; the two proteins differ in only 10 amino acids, and all are conservative substitutions. In contrast, the sequence shows little homology with that of mammalian dihydrofolate reductase: reduced pyridine nucleotide-requiring enzymes with superficial mechanistic similarities.
用溴化氰裂解经还原烷基化的大鼠肝脏二氢蝶啶还原酶，得到了一个肽混合物，其中6个肽段（CB - 1至CB - 6）通过C8反相高效液相色谱法进行了分离和纯化。肽段CB - 1、CB - 4和CB - 6的部分序列通过自动Edman降解和高效液相色谱法进行了测序，其羧基末端区域则通过常规方法进行了测序。对CB - 6进行进一步的蛋白酶消化并分离产物，得到了一个七肽。根据该肽段的序列合成了一个由20个核苷酸组成的低简并度探针，并用于筛选构建于λgt 10载体中的大鼠肝脏cDNA表达文库。分离出了阳性克隆，通过限制性内切酶和双脱氧序列分析对其中5个进行了详细检查，从而确定了二氢蝶啶还原酶的整个编码区。发现该基因编码一个含240个氨基酸（不包括甲硫氨酸起始密码子）的蛋白质，其相对分子质量为25420。在推导的蛋白质序列中鉴定出了与肽段CB - 1、CB - 4、CB - 6以及羧基末端相对应的各个序列。大鼠的这种酶与人二氢蝶啶还原酶高度同源；这两种蛋白质仅在10个氨基酸上有所不同，且均为保守性替换。相比之下，该序列与哺乳动物二氢叶酸还原酶的序列几乎没有同源性：后者是需要还原型吡啶核苷酸的酶，在表面机制上有相似之处。
