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FRACTIONATION OF THYMIDINE PHOSPHOKINASE, THYMIDINE 5'-MONOPHOSPHATE PHOSPHOKINASE AND THYMIDINE 5'-DIPHOSPHATE PHOSPHOKINASE IN EXTRACTS OF LANDSCHUTZ ASCITES-TUMOUR CELLS.

ABSTRACT: 1. Extracts of Landschutz ascites-tumour cells have been fractionated by treatment with acid, alumina C(gamma) gel and Sephadex G-100 to yield purified preparations of thymidine phosphokinase, thymidine 5'-monophosphate phosphokinase and thymidine 5'-diphosphate phosphokinase. 2. These results clearly demonstrate the existence in Landschutz ascites tumour of three phosphokinases each of which catalyses one step in the reaction sequence: thymidineright harpoon over left harpoonthymidine 5'-monophosphateright harpoon over left harpoonthymidine 5'-diphosphateright harpoon over left harpoonthymidine 5'-triphosphate. Though these results do not preclude the participation of other enzymes in the formation of thymidine 5'-triphosphate from thymidine by Landschutz ascites-tumour cells, they provide strong support for the view that thymidine 5'-diphosphate is an intermediate in the formation of thymidine 5'-triphosphate from thymidine 5'-monophosphate by this system.

SUBMITTER: GRAV HJ

PROVIDER: S-EPMC1206583 | biostudies-other | 1965 Mar

REPOSITORIES: biostudies-other

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Project description:1. A purification procedure for DNA nucleotidyltransferase from Landschütz ascites-tumour cells is described. The enzyme can be separated from endogenous nucleic acid and from triphosphatase and deoxyribonuclease activities measurable at pH7.5. 2. The basic properties of the nucleotidyltransferase reaction are as follows. The enzyme has optimum activity at pH7.2-7.4. It displays an absolute requirement for DNA-primer, thermally-denatured DNA serving three to ten times as efficiently in this respect as native DNA. Maximum synthesis of polydeoxyribonucleotide occurs in the presence of all four deoxyribonucleoside 5'-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, Mg(2+) ions were by far the most effective. Manganous ions promoted synthesis but to a much smaller extent. Calcium ions did not support synthesis at all. At the appropriate concentrations, the univalent cations (sodium and potassium) stimulated the reaction by 25% and 125% respectively. The presence of EDTA in the reaction mixture stimulates the system five- to ten-fold. 3. The storage characteristics of the enzyme (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. 4. The enzyme loses more than 95% of its activity after heating for 1min. at 45 degrees . 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. 5. The product of the nucleotidyltransferase reaction is precipitable by acid or ethanol, and is susceptible to the actions of deoxyribonucleases I and II, snake-venom and spleen phosphodiesterases, and micrococcal nuclease. It forms a band in a density gradient of caesium chloride at a density similar to that of the DNA-primer. 6. By the criteria of nearest-neighbour 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.

Dataset Information

FRACTIONATION OF THYMIDINE PHOSPHOKINASE, THYMIDINE 5'-MONOPHOSPHATE PHOSPHOKINASE AND THYMIDINE 5'-DIPHOSPHATE PHOSPHOKINASE IN EXTRACTS OF LANDSCHUTZ ASCITES-TUMOUR CELLS.

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