Deoxynucleotide-polymerizing enzymes in normal and malignant human cells.

The cytoplasmic (175,000 x g supernatant) and the chromatin fractions from phytohemagglutinin-stimulated normal human lymphocytes, human thymus tissue, lympho cytes 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 polymeruse was found only in the cytoplasmic fraction. In glycerol gradients containing 0.1 MNaCl, the 6 to 7 S DNA-directed DNA polymerase and 6 to 7 S polyribosyladenylic aciddependent DNA polymerase sedimented as 10 S aggregate and the 3 to 4 S DNA-directed DNA polymerase sedi mented 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 polyribosyl adenylic acid-dependent DNA polymerase could give double peaks on diethylaminoethyl cellulose column. In addition to this examination of Chromatographie and glyc erol 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 doexynucleotidepolymerizing 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.