Deoxyribonucleic acid nucleotidyltransferase from Landschütz ascites-tumour cells. Partial purification and general properties

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Abstract
A purification procedure for DNA nucleotidyltransferase from Land-schutz ascites-tumor cells is described. The enzyme can be separated from endogenous nucleic acid and from triphosphatase and deoxyribo-nuclease activities measurable at pH 7.5. The basic properties of the nucleotidyltransferase reaction are as follows. The enzyme has optimum activity at pH 7-2-7-4. It displays an absolute requirement for DNA-primer, thermally-denatured DNA serving 3 to 10 times as efficiently in this respect as native DNA. Maximum synthesis of poly-deoxyribonucleotide occurs in the presence of all 4 deoxyribonucleo-side 5[image]-triphosphates, but a limited incorporation of mononucleotide into polynucleotide is observed when the system is provided with only one triphosphate, or with various combinations of mono-, di- and tri-phosphates. The reaction requires the presence of a bivalent cation, and of those tested, Mg2+ ions were by far the most effective. Man-ganous ions promoted synthesis but to a much smaller extent. Ca ions did not support synthesis at all. At the appropriate concentrations, the univalent cations (Na K) stimulated the reaction by 25% and 125% respectively. The presence of EDTA in the reaction mixture stimulates the system 5 to 10-fold. The storage characteristics of the enzvme (as well as the activities of the various fractions) improve markedly if EDTA and 2-mercaptoethanol are included in the enzyme solution and in all preparative buffer solutions. The enzyme loses more than 95% of its activity after heating for 1 min. at 45[degree]. If the heating is conducted in the presence of DNA, the enzyme becomes relatively heat-resistant (presumably as a consequence of complex-formation with the DNA) and may actually display an activation effect. This is discussed in relation to a possible molecular conformation of the enzyme. The product of the nucleotidyltransferase reaction is precipitable by acid or ethanol, and is susceptible to the actions of deoxyribonucleases I and n, snake-venom and spleen phosphodiesterases, and micrococcal nuclease. It forms a band in a density gradient of cesium chloride at a density similar to that of the DNA-primer. By the criteria of nearest-neighbor frequency analyses, the product of the nucleotidyltransferase reaction has the characteristics to be expected of a polynucleotide synthesized in accordance with the template directions of the primer.