Determination of sialidase activities in HeLa cells using gangliosides specifically labeled in N-acetylneuraminic acid

Abstract

Radioactive gangliosides, N-[ 14C]-acetylneuraminylgalactosylglucosylceramide ([ 14C]G M3) and N- [ 14C]-acetylneuraminylgalactosyl- N-acetylgalactosaminyl- [ N-acetylneuraminyl]-galactosylglucosylceramide ([ 14C]G D1a), were synthesized from CMP-[ 14C]sialic acid and the appropriate precursor glycolipid using specific sialyltransferase activities. These compounds were isolated and used as substrates to assay sialidase activity in HeLa cells. Although sodium butyrate added to the culture medium increased G M3 biosynthesis in HeLa cells, sialidase activity, as well as that of other glycohydrolases, was the same in control and butyrate-treated HeLa cells. The same sialidase activity appeared to hydrolyze both [ 14C]G M3 and [ 14C]G D1a, but not fetuin; the enzyme had a pH optimum of 5.0 and a K m of 75 μm for the ganglioside substrates. Although the cells contained a high sialidase activity (4–7 nmol/mg of protein/h) and could bind exogenously added [ 14C]G M3, no “ecto”-sialidase activity would be detected in intact cells under conditions where a close to physiological pH is maintained. The results indicate that ganglioside sialidase is not involved directly in the morphological and biochemical differentiation induced in HeLa cells by exposure to sodium butyrate.