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Exogenous asialo-glycoproteins and endogenous acceptors are both sialylated by incubating cytidine 5'-mono-phosphate N-[14C]acetylneuraminic acid (CMP[14C]NeuAc) with a lysate of human platelets but their respective incorporation levels vary with the divalent cation concentrations. P-Nitrophenyl-β-D-galactoside has also been demonstrated to be an acceptor of sialyl residues, and two different sialyl derivatives are synthesized according to the concentration of divalent cations. P-Nitrophenyl-β-D-[6-³H]galactoside has been prepared by reduction with tritiated borohydride of the compound previously oxidized by galactose oxidase. Using this labelled p-nitrophenyl-β-D-galactoside as acceptor and unlabelled CMP-NeuAc as donor, the two sialyl derivatives have been identified by methylation analysis as α-sialosyl-(2 - 3)-p-nitrophenyl-β-D-galactoside and α-sialosyl-(2 - 6)-p-nitrophenyl-β-D-galactoside. In addition to their different responses to divalent cation requirements, the sialyltransferase activities responsible for the synthesis of the two sialylgalactoside isomers have been clearly distinguished by their temperature and pH optimal values. They also exhibit different susceptibilities to dithioerythritol and different stabilities. These results demonstrate the presence in human platelets of two sialyltransferases: a CMP-NeuAc: galactoside (α2 - 3)-sialyltransferase and a CMP-NeuAc galactoside (α2 - 6)-sialyltransferase. [ABSTRACT FROM AUTHOR]

Interleukin-6 (IL-6)and insulin-like growth-factor-1 (IGF-I) are cytokines produced by a variety of cells that act on a wide range of tissues, influencing cell growth and differentiation. Purified plasma membranes from human U937 monoblastic cells produced in vitro dimeric species of IL-6- and IGF-I-derived peptides through the sequential actions of surface-associated enzymes cathepsin G and transpeptidase activities. Cathepsin G degraded native unglycosylated IL-6 and IGF-I molecules into 8-kDa and 7-kDa peptides respectively. Subsequent dimerisation of these intermediate forms into 16-kDa IL-6- and 14-kDa IGF-I-derived peptides was inhibited by acivicin and glutathione which are specific inhibitors of the standard cell-surface γ-glutamyl transpeptidase (γ-GT). However U937 plasma membranes, cleared of γ-GT activity by immunoprecipitation with anti-γGT and adsorption on protein-G-Sepharose, were still able to convert the intermediate forms of IL-6 and IGF-I into dimers. Together, these observations indicate that the transpeptidase involved in the formation of the dimeric species of IL-6 and IGF-I was related to, but distinct from standard cell-surface γ-GT. Cells of all hematopoietic lineages expressed γ-GT-related activity. In contrast to the 16-kDa IL-6-derived peptide that did not retain growth-stimulating activity, the 14-kDa IGF-1 peptide was at least equipotent with native IGF-I in the BALB/c 3T3 fibroblast DNA synthesis response. The N/O-glycosylated IL-6 was clearly as sensitive to cathepsin-G- and γ-GT-related activities as the unglycosylated IL-6 from Escherichia coli, thus indicating that the sugar chains did not protect the cleavage sites of the two proteases on the IL-6 molecule. Our in vitro findings raise the possibility that similar proteases participate in the regulation of the catabolism of IL-6 and IGF-1 in vivo. [ABSTRACT FROM AUTHOR]