Effects of nucleotides and nucleotide:analogs on human serum sialyltransferase
Klohs, W.D.; Bernacki, R.J.; Korytnyk, W.
Cancer Research 39(4): 1231-1238
1979
ISSN/ISBN: 0008-5472 PMID: 421206 Document Number: 146917
The properties of human serum 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 sialytransferase were also studied. Cytidine nucleotides, which act as competitive inhibitors, showed the greatest inhibitory activity. Like cytidine nucleotides its analogs (5'-fluorocytidine monophosphoric acid, cytosine-.beta.-D-arabinofuranoside-5'-triphosphate, cytosine-.beta.-D-arabinofuranoside-5'-monophosphoric acid and [cyclic CMP 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 Ki for CMP was 50 .mu.M, and for 5'-fluorocytidine monophosphoric acid it was 70 .mu.M. 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 (Ki = 16, 19 and 50 .mu.M for CTP, CDP 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, since increases in serum sialyltransferase are noted in breast cancer.