PURIFICATION AND CHARACTERIZATION OF UDP-GLCNAC-IV(3)-BETA-GAL-GB(4)CER BETA-1,6-GLCNAC TRANSFERASE FROM MOUSE KIDNEY

The Gsl-5 gene mapped on mouse chromosome 19 controls the expression of IV6 beta Gal beta 1-4(Fuc alpha 1-3)GlcNAc, IV3 beta Gal-Gb(4)Cer in mouse kidney through regulation of the activity of UDP-GlcNAc:IV3 beta Gal-Gb(4)Cer beta-1,6-GlcNAc transferase (GNT), which transfers GlcNAc to the C-6 position of GalNAc of IV3 beta Gal-Gb(4)Cer (GL-X). Here me report that GNT has been purified to apparent homogeneity from mouse kidney by means of preparation of a microsomal fraction, solubilization with Triton X-100, and sequential column chromatographies on CM-Sepharose, UDP-hexanolamine-Sepharose, and Gg(4)Ose-Aminocellulo-fine. GNT purified 11,000 fold from the microsomal fraction exhibited a specific activity of 26.15 mu mol/min/mg protein and an apparent molecular mass of 50 kDa on SDS-polyacrylamide gel electrophoresis. The K-m Values for UDP-GlcNAc and GL-X were 0.36 and 0.11 mM, respectively. Among glycolipids tested as substrates, GL-X was the best and Gg(4)Cer the next best, but Lc(3)Cer, Gb(4)Cer, and G(M1) did not act as a substrate. GNT was also able to transfer GlcNAc to Gal beta 1-3GalNAc beta 1-benzyl and Gal beta 1-3GalNAc alpha 1-p-nitrophenyl at C-6 of GalNAc through beta linkage but not to GlcNAc beta 1-3GalNAc alpha 1-p-nitrophenyl. The purified GNT was digested with lysyl endopeptidase, and four peptides generated were sequenced. The sequence of four peptides spanning 35 amino acid residues in total exhibited 80% homology with that of the reported human core 2 GlcNAc transferase.