Ammonium containing buffers should be avoided during enzymatic release of glycans from glycoproteins when followed by reducing terminal derivatization

the different specificity of the enzymes. In addition, the similarity at the HCA level of the SialylmotifS extends beyond the region defined by linear alignments. We did not find any sequence similarity in the NH2~ te il region, but analysis of the COOH-terminal region showed other conserved regions (see Figure 1). Firstly, considering the same enzyme obtained from various mammalian species, we observed that this COOH-terminal region is highly conserved. Secondly, within this region one Glu (E) residue is always found among all cloned sialyltransferases (Table I) separated by four amino acid residues from an His (H) residue present in all the sialyltransferases sequences with the only exception of the rat ST6Gal I sequence (GenBank accession Ml8769). (The difference between the two codons of His (CAU, CAC) and Asp (GAU, GAC) resides in the first nucleotide. A rereading of the rat ST6Gal I sequence would probably show the presence of the missing Glu residue.) We have named this region Sialylmotif VS (Very Short). Due to the intrinsic physicochemical properties of Glu, and the lack of other conserved amino acid residue that may be involved in catalysis, it is reasonable to suppose that this conserved Glu residue is involved in the catalytic process. Site directed mutagenesis will confirm the crucial role of this amino acid residue in the transfer of sialic acid residues.