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J Biol Chem, Vol. 273, Issue 42, 27625-27632, October 16, 1998

Purification and Characterization of UDP-GlcNAc:Gal1-4GlcNAc1-3*Gal1-4Glc(NAc)-R(GlcNAc to *Gal) 1,6N-Acetylglucosaminyltransferase from Hog Small Intestine

Yoshihiro Sakamoto^§, Tomohiko Taguchi^¶, Yasuo Tano^§, Tomoya Ogawa^¶, Anne Leppänen, Marjo Kinnunen, Olli Aitio, Pinja Parmanne, Ossi Renkonen, and Naoyuki Taniguchi

From the Department of  Biochemistry and ^§ Ophthalmology, Osaka University Medical School, Suita, Osaka 565-0871, Japan, ^¶ RIKEN (The Institute of Physical and Chemical Research), Wako, Saitama 351-0198, Japan, and  Institute of Biotechnology and Department of Biosciences, University of Helsinki, Biocenter 1, P. O. Box 56, FIN-00014 Helsinki, Finland

A 1,6N-acetylglucosaminyltransferase (1-6GnT) responsible for the formation of the 1,6-branched poly-N-acetyllactosamine structure has been purified 210,000-fold in 2.4% yield from a homogenate of hog small intestine by successive column chromatographies involving CM-Sepharose FF, Ni²⁺-chelating Sepharose FF, and UDP-hexanolamine-agarose, using an assay wherein pyridylaminated lacto-N-neotetraose (Gal1-4GlcNAc1-3Gal1-4Glc-PA) was used as an acceptor substrate, and the reaction product was Gal1-4GlcNAc1-3(GlcNAc1-6)Gal1-4Glc-PA. The apparent molecular weight of the purified 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 were 0.96 and 2.59 mM, respectively. For its activity, this enzyme was shown to have an absolute requirement of at least a complete LacNAc (LacNAc = Gal1-4GlcNAc) 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 could be involved in generating branches to central positions of preformed as well as growing polylactosamine chains, but not in synthesizing the distal branches to growing polylactosamine chains.

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