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Deoxynucleotide-polymerizing Enzymes in Normal and Malignant Human Cells¹

Department of Experimental Therapeutics, Roswell Park Memorial Institute, New York State Department of Health, Buffalo, New York 14203

The cytoplasmic (175,000 x g supernatant) and the chromatin fractions from phytohemagglutinin-stimulated normal human lymphocytes, human thymus tissue, lymphocytes from chronic lymphocytic leukemia and acute lymphoblastic leukemia patients and cultured cells of normal (RPMI 1788), multiple myeloma (RPMI 8226), Burkitt lymphoma (HR1K), and acute lymphoblastic leukemia (Molt-4) origin were examined for deoxynucleotide-polymerizing enzymes by diethylaminoethyl cellulose and phosphocellulose chromatography, glycerol gradient centrifugation, and other properties.

In all the cells examined, 6 to 7 S (polyribosyladenyl acid-dependent polymerase, 3 to 4 S DNA-directed DNA polymerase, and 3 to 4 S terminal deoxynucleotidyltransferase were found both in the cytoplasmic (latter two enzymes only in small amounts) and in the chromatin fraction; whereas 6 to 7 S DNA-directed DNA polymerase was found only in the cytoplasmic fraction. In glycerol gradients containing 0.1 M NaCl, the 6 to 7 S DNA-directed DNA polymerase and 6 to 7 S polyribosyladenylic acid-dependent DNA polymerase sedimented as 10 S aggregate and the 3 to 4 S DNA-directed DNA polymerase sedimented as the 7 S aggregate, whereas at 0.2 to 0.4 M NaCl they sedimented in unaggregated form. Both 3 to 4 S DNA-directed DNA polymerase and 6 to 7 S polyribosyladenylic acid-dependent DNA polymerase could give double peaks on diethylaminoethyl cellulose column. In addition to this examination of chromatographic and glycerol gradient behavior of enzymes, the template-primer and metal requirements and the effect of sulfhydryl inhibitors and monovalent cations were also studied. These results, in addition to the demonstration of four doexynucleotide-polymerizing enzymes in the two fractions examined, showed that terminal deoxynucleotidyltransferase was not specific for thymus or thymocytes. The results also suggest that several analytical steps as used here may be necessary to avoid reporting of artifact peaks as new enzymes.

¹ This work was supported in part by Core Grant CA-13038 from the National Cancer Institute, USPHS. A brief report dealing with this work was presented at the 1973 meeting of the American Association for Cancer Research.

Received 10/11/73. Accepted 1/25/74.