Characterization of sialyltransferase activity from human placenta

Crude soluble sialyltransferase (CMP-N-acetylneuraminate:d-galactosylgylcoproteinN-acetylneuraminyltransferase, EC 2.4.99.1) activity from human placenta is characterized with acid-treated fetuin and ceruloplasmin as acceptors. Two forms of sialyltransferase in human placental extract can be demonstrated by using either Sephadex G-200 or concanavalin-A-Sepharose column chromatography. The large species (apparent molecular weight larger than 200 000) does not bind to concanavalin-A Sepharose, while the small one (apparent molecular weight about 80 000) does bind to concanavalin-A-Sepharose, and can be eluted with 5% α-methylmannoside. The K_m values of the CMP-sialic acid substrate are different for these two forms of sialyltransferase. No divalent cation requirement can be demonstrated, and 10 mM EDTA stimulates the reaction 2-3-fold. Folic acid and its derivatives, including methotrexate, are inhibitory at concentrations of millimolar range. Kinetic studies indicate that folic acid acts as a competitive inhibitor with a K₁ of 1.1 mM. While heparin inhibits the transferase reaction, protamine acts as a stimulator in this crude preparation. Sialyltransferase activity is inhibited by 0.1 M iodoacetate and 1 mM p-chloromercuribenzoyl sulfonate, suggesting that an intact sulfhydryl group of a methionine residue is involved in the active site of the enzyme.