PURIFICATION AND CHARACTERIZATION OF -BETA-1-4-GLCNAC-BETA-1-3-ASTERISK-GAL-BETA-1-4GLC (NAC)-R(GLCNAC TO ASTERISK-GAL) BETA-1, 6N-ACETYLGLUCOSAMINYLTRANSFERASE FROM HOG SMALL-INTESTINE

A beta 1,6N-acetylglucosaminyltransferase (beta 1-6GnT) responsible fo r the formation of the beta 1,6-branched poly-N-acetyllactosamine stru cture has been purified 210,000-fold in 2.4% yield from a homogenate o f hog small intestine by successive column chromatographies involving CM-Sepharose FF, Ni2+-chelating Sepharose FF, and UDP-hexanolamine-aga rose, using an assay wherein pyridylaminated lacto-N-neotetraose (Gal beta 1-4GlcNAc beta 1-3Galp1-4Glc-PA) was used as an acceptor substrat e, and the reaction product was Gal beta 1-4GlcNAc beta 1-3(GlcNAc bet a 1-6)Gal beta 1-4Glc-PA. The apparent molecular weight of the purifie d enzyme was 76,000 under nonreducing conditions. The enzyme has a pH optimum at 7.0 and has no requirement for any divalent metal ions. The K-m values for pyridylaminated lacto-N-neotetraose and UDP-GlcNAc wer e 0.96 and 2.59 mM, respectively, For its activity, this enzyme was sh own to have an absolute requirement of at least a complete LacNAc (Lac NAc = Gal beta 1-4GlcNAc) residue bound to position 3 of the acceptor Gal residues, i.e. it is capable of acting only on the Gal residues of internal LacNAc units. The data strongly suggest that this enzyme cou ld be involved in generating branches to central positions of preforme d as well as growing polylactosamine chains, but not in synthesizing t he distal branches to growing polylactosamine chains.