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Effects of Nucleotides and Nucleotide:Analogs on Human Serum Sialyltransferase¹

Department of Experimental Therapeutics, Grace Cancer Drug Center, Roswell Park Memorial Institute, State Department of Health, Buffalo, New York 14263

The properties of human serum cytidine 5'-monophosphate (CMP)-N-acetylneuraminic acid:glycoprotein sialyltransferase (EC 2.4.99.1) were studied. Optimal enzyme activity was observed in an assay medium buffered with 0.1 M cacodylate at pH 6.5 and using desialyzed fetuin as an acceptor. Neither Mn²⁺ nor Mg²⁺ were required for optimal enzyme activity, and when present, they inhibited the enzyme activity at concentrations greater than 2 and 5 mM, respectively. Enzyme inhibition was also observed in the presence of detergents such as Triton X-100, deoxycholate, and sodium dodecyl sulfate. Two sulfhydryl agents, N-ethylmaleimide and p-chloromercuriphenylsulfonic acid, showed inhibitory activity toward sialyltransferase. The effects of nucleotides and nucleotide:analogs on sialyltransferase were also studied. It was found that cytidine nucleotides, which act as competitive inhibitors, showed the greatest inhibitory activity. Like cytidine nucleotides its analogs (5'-fluorocytidine monophosphoric acid, cytosine-ß-D-arabinofuranoside-5'-triphosphate, cytosine-ß-D-arabinofuranoside-5'-monophosphoric acid, and cytidine 3':5'-cyclic monophosphoric acid) acted as competitive inhibitors but were not as effective in enzyme inhibition, although 5'-fluorocytidine monophosphoric acid was nearly as potent in its inhibitory capacity as CMP. The apparent K₁ for CMP was 50 µM, and for 5'-fluorocytidine monophosphoric acid it was 70 µM. Enzyme inhibition was also observed with ribodialdehyde CMP, and to a lesser extent with 5'-(trans-4-N-acetylcyclohexyl)cytidylic acid hydrochloride and its cis counterpart which were synthesized as analogs of CMP:N-acetylneuraminic acid. A noncompetitive inhibition was observed for both adenosine and uridine nucleotides. The extent of inhibition for all nucleotides and their analogs increased with an increasing number of phosphate groups in the compound (K₁= 16, 19, and 50 µM for cytidine 5'-triphosphate, cytidine 5'-diphosphate, and CMP, respectively). These studies are important in assessing the activity of potential inhibitors of glycoconjugate biosynthesis and should aid in the design of more active and specific agents which may be useful in the chemotherapy of cancer.

¹ Supported in part by USPHS Grants CA-15757, CA-19814, CA-13038, CA-09072, and CA-08793 from NIH, Department of Health, Education, and Welfare.

² Present address: Gastrointestinal Laboratory, Department of Medicine, Clinical Center CC 186, State University of N. Y. at Buffalo, 462 Grider Street, Buffalo, N. Y. 14215.

³ To whom requests for reprints should be addressed.

Received 9/28/78. Accepted 12/27/78.

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