Possibilities of enzymic synthesis of nucleoside-.5'-triphosphates- 14CC(U) of adenine, guanine, cytosine and uracil of high spec@ activity and radiochemical purity by means of a purijied enzyme isolatedfrom Escherichia coli B were studied. The efect of enzyme concentration in the reaction mixture in dependence on the time of the enzyme reaction is decisive for an optimal production of radioactive nucleoside-5'-triphosphates. A simple and rapid method of preparation of radioactive nucleoside-5'-triphosphates, including preparative paper chromatography as the single isolation step, is described and information on the radiolysis of the isolated nucleotides is presented.INTRODUCTION.

SUMMARY The e n z y m a t i c e x t r a c t p t e p a r e d f r o m 48h. r e g e n e r at i n g r a t l i v e r s t a b i l i z e d w i t h d i e t a r y L -t r y p t o p h a n p o s s e s s e s t h e enhanced t h y m i d i n e and t h y m i d y l a t e k inaae a c t i v i t y . The o p t i m a l c o n d i t i o n 8 f o r t h e phoap h o r y l a t i o n o f t h y m i d i n e in vitro were f o u n d and the p O 8 8 i b i Z i t y f o r t h e p r e p a r a t i v e u s e 6 was c o n s i d e r e d . The amount of newly formed t h y m i d

The catalytic properties of an unpurijied enzyme preparation from cells of Brevibacterium ammoniagenes toward phosphoribosylation of the individual nucleic acid bases are pronouncedly specijic, display a high efficiency toward phosphoribosylation of uracil where the conversion to uridine-5'-phosphate is quantitative, phosphoribosylation of adenine takes place with lower eflciency and guanine and cytosine are practically not phosphoribosylated at all. Using orotic acid-14C and a complete inhibition of orotidine 5'-phosphate decarboxylase by means of 6-azauridine-5'-phosphate, a 60 % production of orotidine-5'-phosphate-14C has been achieved.The catalytic efficiency of the enzyme preparation remains preserved even upon substitution of 5-phosphoribosyl-I-pyrophosphate by its direct metabolic precursors.The possibility of direct application of the unpurijied cell-free extract of the bacteria, the simple composition of the jinal reaction mixtures and the high degree of production of biologically important compounds makes it possible to employ the method studied for an enzyme synthesis of radioactive uridine-5'-phosphate, orotidine-5'-phosphate and adenosine-5'-phosphate.

SUMMARY A K R leukemic mouse cells possess a considerable uridine X-inase activity. The ability of unpurijied cell-fvee extracts prepared .from these cells to phosphorylate nucleosides-ldC was investigated.The substrate specijicity of the enzyme preparation and the possibility to regulate the course of the enzymatic reactions were examined. The method is convenient for the preparative synthesis of uracil, cytosine and hypoxanthine S-n~cleotides-~~C.

Z d e n e k N e j e d l y a n d K a r e l Hybs. I n s t i t u t e f o r R e s e a r c h , P r o d u c t i o n a n d U s e s o f R a d i o i s o t o p e s , P f i s t a v n i 2 4 , P r a g u e 7, Czechoslovakia . R e c e i v e d on A u g u s t 6, 1971