Biosynthesis of chondroitin sulfate. Purification of glucuronosyl transferase II and use of photoaffinity labeling for characterization of the enzyme as an 80-kDa protein.

The Journal of Biological Chemistry
G SugumaranR R Drake

Abstract

A photoaffinity analogue, [beta-32P]5-azido-UDP-GlcA, was used to photolabel the enzymes that utilize UDP-GlcA in cartilage microsomes and rat liver microsomes. SDS-polyacrylamide gel electrophoresis analysis of photolabeled cartilage microsomes, which are specialized in chondroitin sulfate synthesis, showed a major radiolabeled band at 80 kDa and other minor radiolabeled bands near 40 and 60 kDa. Rat liver microsomes, which are enriched for enzymes of detoxification by glucuronidation, had a different pattern with multiple major labeled bands near 50-60 and 35 kDa. To determine that the photolabeled 80-kDa protein is the GlcA transferase II, we have purified the enzyme from cartilage microsomes. This membrane-bound enzyme, involved in the transfer of GlcA residues to non-reducing terminal GalNAc residues of the chondroitin polymer, has now been solubilized, stabilized, and then purified greater than 1350-fold by sequential chromatography on Q-Sepharose, heparin-Sepharose, and WGA-agarose. The purified enzyme exhibited a conspicuous silver-stained protein band on SDS-polyacrylamide gel electrophoresis that coincided with the major radiolabeled band of 80 kDa. SDS-polyacrylamide gel analysis of photoaffinity-labeled active fract...Continue Reading

References

Sep 1, 1969·Proceedings of the National Academy of Sciences of the United States of America·A E BrandtG W Jourdian
Dec 20, 1995·Biochimica Et Biophysica Acta·J E Silbert, G Sugumaran
Mar 22, 1996·The Journal of Biological Chemistry·H KitagawaK Sugahara

❮ Previous
Next ❯

Citations

Nov 6, 2002·Biochimica Et Biophysica Acta·Dorota A Bulik, Phillips W Robbins
Oct 24, 2000·Current Opinion in Structural Biology·K Sugahara, H Kitagawa
Mar 7, 2007·Organic & Biomolecular Chemistry·Hai Yu, Xi Chen
Jul 26, 2002·Yakugaku zasshi : Journal of the Pharmaceutical Society of Japan·Hiroshi Kitagawa
May 20, 2000·The Journal of Biological Chemistry·P L DeAngelis, A J Padgett-McCue
Aug 22, 2001·The Journal of Biological Chemistry·H KitagawaK Sugahara

❮ Previous
Next ❯

Related Concepts

Related Feeds

ASBMB Publications

The American Society for Biochemistry and Molecular Biology (ASBMB) includes the Journal of Biological Chemistry, Molecular & Cellular Proteomics, and the Journal of Lipid Research. Discover the latest research from ASBMB here.