Purification of a novel enzyme, 5'-nucleotide phosphodiesterase, and studies on its physiological role and relation to tumor.

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We have found that many tumor cell lines express a 5'-nucleotide phosphodiesterase (5'-NPDase) with properties clearly distinguishable from enzymes of normal tissues. Such an enzyme with 5'-NPDase activity was purified from Ehrlich ascites carcinoma after several steps of column chromatography to show a single band on SDS-polyacrylamide gel electrophoresis. Unlike the normal cell type enzyme, the tumor cell type enzyme is a soluble protein, has a pH optimum of 7.5, and the molecular mass estimated by SDS-PAGE is 6.7 kDa. The enzyme is activated by dithiothreitol and inhibited by N-ethylmaleimide, whereas the normal cell type enzyme was inhibited by dithiothreitol. The enzyme cleaves thymidine 5'-mono- phosphate p-nitrophenyl ester (TMP-NP) but does not hydrolyze other chromogenic substrate for phosphodiesterases, nor pyrophosphate bond of various nucleotides which are cleaved by 5' of normal tissues. But, it hydrolyzes dinucleotides to from 5'-phosphates, and is more active on 2', 5'-than on 3', 5'-phosphodiester bonds. These results indicate that the TMP-NP splitting enzyme in Ehrlich ascites carcinoma cell is a 2', 5'-phosphodiesterase. We are trying to get a monoclonal antibody against this enzyme from Ehrlich ascites tumor cells to know whether this enzyme could be used for diagnosis of cancer. However, we have not succeeded in it so far.