METABOLISM OF N-ACETYLNEURAMINIC ACID IN MAMMALS - ISOLATION AND CHARACTERIZATION OF CMP-N-ACETYLNEURAMINIC ACID

Radiolabeled sialoglycoproteins were detected upon analysis of liver and kidneys of mice that had been injected with N-acetyl[4,5,6,7,8,9-3H]neuraminic acid. Similar results were also found upon incubation of this radiolabel with liver slices. CMP-N-acetylneuraminic acid (CMP-NeuNAc), a postulated intermediate in the conversion of free NeuNAc to NeuNAc bound to glycoproteins and glycolipids, was detected and characterized for the first time from a mammalian tissue; neither the nucleotide moiety nor the biological activity of this compound, when derived from a mammalian tissue, has been previously reported. Identification of CMP-NeuNAc, labeled in both the nucleotide and sugar moieties, was based on (1) comigration with authentic standard in several chromatographic systems, (2) detection of labeled CMP and NeuNAc after mild acid hydrolysis, (3) determination of its susceptibility to NaBH4 reduction and NeuNAc aldolase cleavage before and after mild acid hydrolysis, and (4) demonstration of its ability to transfer NeuNAc in an assay in vitro. The concentration of CMP-NeuNAc in mouse liver is 37 ± 10 nmol/g of wet tissue. After labeling with a mixture of,N-acetyl[l-l4C]-and -[4,5,6,7,8,9-3H]neuraminic acid, we found that the differences in isotope ratio between free and covalent-bound NeuNAc (in CMP-NeuNAc and proteins) were less than 12%. This suggests that NeuNAc had remained intact during the above experiments and that aldolase-catalyzed cleavage to pyruvate and N-acetyl[3H]mannosamine and subsequent condensation of this latter compound with cellular phos-phoenolpyruvate had not occurred. © 1979, American Chemical Society. All rights reserved.