Purification and characterization of UDP-GlcNAc:IV3 beta Gal-Gb4Cer 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-Gb4Cer in mouse kidney through regulation of the activity of UDP-GlcNAc:IV3 beta Gal-Gb4Cer beta-1,6-Glc-NAc transferase (GNT), which transfers GlcNAc to the C-6 position of GalNAc of IV3 beta Gal-Gb4Cer (GL-X). Here we 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 Gg4Ose-Aminocellulo-fine. GNT purified 11,000-fold from the microsomal fraction exhibited a specific activity of 26.15 mumol/min/mg protein and an apparent molecular mass of 50 kDa on SDS-polyacrylamide gel electrophoresis. The Km 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 Gg4Cer the next best, but Lc3Cer, Gb4Cer, and GM1 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-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.
Sekine
M
M
Department of Membrane Biochemistry, Tokyo Metropolitan Institute of Medical Science, Japan.
Hashimoto
Y
Y
Suzuki
M
M
Inagaki
F
F
Takio
K
K
Suzuki
A
A
D000595:Amino Acid Sequence; D000818:Animals; D002240:Carbohydrate Sequence; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D015180:Electrophoresis, Gel, Two-Dimensional; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007668:Kidney / Q000201:enzymology*; D051379:Mice; D008861:Microsomes / Q000201:enzymology; D008969:Molecular Sequence Data; D017351:N-Acetylglucosaminyltransferases / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D011110:Polymorphism, Genetic; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
D017351:N-Acetylglucosaminyltransferases; C066287:UDP-GlcNAc-Gb(5)Cer beta1-6N-acetylglucosaminyltransferase