ENZYMATIC SULFATION OF GANGLIOTRIAOSYLCERAMIDE IN HUMAN RENAL-CANCER CELLS

Biosynthesis of sulfoglycolipid is markedly increased in a human renal cancer cell line, SMKT-R3, We investigated the sulfotransferase catal yzing the transfer of sulfate from 3'-phosphoadenosine 5'-phosphosulfa te (PAPS) to gangliotriaosylceramide (Gg3Cer) in SMKT-R3 cells, On thi n-layer chromatography, the reaction product comigrated with Gg3Cer II I3-sulfate (SM2b), not with Gg3Cer II3-sulfate (SM2a), To examine whic h monosaccharide of Gg3Cer was sulfated, the product was treated with beta-hexosaminidase A, Unlike authentic SM2a, of which the non-reducin g terminal N-acetylgalactosamine was cleaved off, the product was not hydrolyzed, These results suggest that sulfate was transferred to the non-reducing terminal N-acetylgalactosamine. Gg3Cer sulfotransferase a ctivity was independent of divalent cations but stimulated by Fe2+, an d the optimal pH was approximately 6.5, The apparent K-m values were 1 02 mu M for PAPS and 348 mu mM for Gg3Cer, Gg3Cer II3,III3-bis-sulfate (SB2) and a sulfotransferase activity synthesizing SB2 from SM2a were also detected in SMKT-R3 cells.