Measurement of UDP-N-acetylglucosaminyl Transferase (OGT) in Brain Cytosol and Characterization of anti-OGT Antibodies

Anal Biochem. 2003 Mar 15;314(2):169-79. doi: 10.1016/s0003-2697(02)00686-3.

Abstract

UDP-N-acetylglucosaminyl transferase (OGT) catalyzes O-linked glycosylation of cytosolic and nuclear proteins, but enzyme studies have been hampered by the lack of a rapid, sensitive, and economical OGT assay. Employed assay methods typically involved the use of HPLC, formic acid, and large amounts of expensive radiolabeled [3H]UDP-N-acetylglucosaminyl ([3H]UDP-GlcNAc). In the current study, we have developed an OGT assay that circumvents many of these problems through four critical assay improvements: (1) identification of an abundant and enriched source of OGT enzyme (rat brain tissue), (2) utilization of a rapid method for efficiently removing salts and sugar nucleotides from cytosol (polyethylene glycol precipitation of active enzyme), (3) expression of a recombinant p62 acceptor substrate designed to facilitate purification (polyhistidine metal-chelation site), and (4) development of two alternative methods to rapidly separate free [3H]UDP-GlcNAc from 3H-p62ST acceptor peptide (trichloroacetic acid precipitation and metal-chelation affinity purification). To study the enzymology of OGT, independent of potential regulatory proteins within cytosol, we also developed and characterized an alternate OGT assay that uses antibody-purified OGT as the enzyme source. The major advantage of this assay lies in the ability to measure OGT in the absence of other cytosolic proteins.

Publication types

  • Comparative Study

MeSH terms

  • Animals
  • Antibodies / immunology
  • Antibodies / pharmacology
  • Blotting, Western
  • Brain / enzymology*
  • Chemical Precipitation
  • Cytosol / drug effects
  • Cytosol / enzymology
  • Electrophoresis, Polyacrylamide Gel
  • Glucosamine / analogs & derivatives*
  • Glucosamine / pharmacology
  • Male
  • N-Acetylglucosaminyltransferases / drug effects
  • N-Acetylglucosaminyltransferases / immunology
  • N-Acetylglucosaminyltransferases / metabolism*
  • Polyethylene Glycols / chemistry
  • Potassium Chloride / pharmacology
  • Rats
  • Rats, Sprague-Dawley
  • Sodium Chloride / pharmacology
  • Temperature
  • Time Factors
  • Uridine Diphosphate / pharmacology

Substances

  • Antibodies
  • UDP-glucosamine
  • Polyethylene Glycols
  • Sodium Chloride
  • Uridine Diphosphate
  • Potassium Chloride
  • N-Acetylglucosaminyltransferases
  • Glucosamine