Purification and cDNA cloning of human red cell pyrimidine 5'-nucleotidase isozymes

嘧啶5‘-脱氧核糖核酸酶(P5N)是一种遗传性非球形细胞溶血性贫血，在临床上具有重要意义。我们一直在进行P5N同工酶的结构分析，以期阐明P5N缺乏症的主要原因。在第一步，我们尝试了P5N-II同工酶的纯化，使用标准的层析技术，从2000毫升的人红细胞中纯化了90微克的P5N-II，大约是134,000倍。纯化后的酶比活力为44130mU/mg，在聚丙烯酰胺凝胶电泳板上可见一条主带。用纯化的P5N-II进行的动力学研究表明，该酶具有一种独特的性质，即使用糖磷酸酯6-磷酸葡萄糖作为其有效底物之一。这可能对阐明该同工酶的生理功能很重要。虽然我们已经对该酶进行了氨基酸组成分析和部分氨基酸序列测定，但发现N端氨基酸残基被封堵。我们已经从人的网织红细胞中构建了一个cDNA文库，并正在进行氨基酸残基N-末端的解块和进一步测序，以便进行cDNA克隆。

Pyrimidine 5'-uncleotidase (P5N) is important in clinical medicine because the deficiency of the enzyme causes a hereditary nonspherocytic hemolytic anemia. We have been carrying out the structural analysis of P5N isozymes for the purpose of elucidating the primary cause of P5N deficiency. For the first step, we have tried the purification of P5N-II isozyme, Using standard chromatographical techniques, ninety micro grams of P5N-II were purified about 134,000-fold from 2,000 ml of human red blood cells. The purified enzyme had a specific activity of 44,130 mU/mg and gave a main band on SDS polyacry lamide gel electrophoresis. Kinetic studies using the purified P5N-II revealed that the enzyme had a unique property of using a suger phosphate ester 6-phosphogluconate as one of its potent substrates. This might be important in elucidating the physiological function of this isozyme. Although we had performed the amino acid compsition analysis and the partial amino acid sequencing of the enzyme, the N-terminal amino acid residue was found to be blocked. We have already constructed a cDNA library from human reticulocytes, and deblocking and further sequencing of amino acid residues N-terminal region for cDNA cloning is in progress.