Purification of uridine diphosphate-galactose:glucosyl ceramide, β1-4 galactosyltransferase from human kidney

S. Chatterjee, N. Ghosh, S. Khurana

Research output: Contribution to journalArticlepeer-review

Abstract

A galactosyltransferase that transfers galactose from UDP-galactose to glucosylceramide was purified 440-fold to apparent homogeneity from normal human kidney 'buffy coat' preparation employing detergent extraction, ultrafiltration, and Sepharose Q column chromatography. On reducing and nonreducing gels, the enzyme resolved into two bands with apparent molecular weights on the order of 60,000 and 58,000, respectively. The activity of the enzyme was also associated with these two bands following separation on polyacrylamide gels. Analytical isoelectric focusing revealed that the pI of this enzyme is approximately 4.55. Product characterization and substrate specificity studies employing chromatography, enzymatic digestion with various glycosidases, and use of a variety of glycosphingolipid substrates revealed that the major product synthesized by this enzyme was Cer1-1βGlc4- 1Gal, and Cer1-1βGlc was the preferred substrate. Digestion of the 60- and 58-kDa proteins with Staphylococcus aureus (V-8) protease revealed at least six peptides having identical electrophoretic migration. This finding suggests that the two proteins may be related to each other. Western immunoblot assays revealed that the antibody against UDP-galactose:GlcCer, β1-4 galactosyltransferase (GalT-2) but not galactosyltransferase UDP-Gal:N- acetyl-D-glucosaminyl-glycopeptide 4-β-D-galactosyltransferase (EC 2.4.1.38) (B-GT) immunoprecipitated (recognized) the kidney GalT-2. In contrast, antibody against B-GT did not immunoprecipitate GalT-2. Thus our data indicate that GalT-2 and B-GT are two distinct enzymes. The availability of the enzyme GalT-2 and corresponding antibody will allow functional studies in the near future.

Original languageEnglish (US)
Pages (from-to)7148-7153
Number of pages6
JournalJournal of Biological Chemistry
Volume267
Issue number10
StatePublished - 1992

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology
  • Cell Biology

Fingerprint

Dive into the research topics of 'Purification of uridine diphosphate-galactose:glucosyl ceramide, β1-4 galactosyltransferase from human kidney'. Together they form a unique fingerprint.

Cite this