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

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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.