Measurement of UDP-N-acetylglucosaminyl transferase (OGT) in brain cytosol and characterization of anti-OGT antibodies.
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.
Marshall
Stephen
S
Hexos, Inc., 18304 NE 153rd Street, Woodinville, WA 98072, USA. Hexos@comcast.net
Duong
Trung
T
Orbus
Ryan J
RJ
Rumberger
John M
JM
Okuyama
Ryo
R
D000818:Animals; D000906:Antibodies / Q000276:immunology / Q000494:pharmacology; D015153:Blotting, Western; D001921:Brain / Q000201:enzymology*; D011232:Chemical Precipitation; D003600:Cytosol / Q000187:drug effects / Q000201:enzymology; D004591:Electrophoresis, Polyacrylamide Gel; D005944:Glucosamine / Q000031:analogs & derivatives* / Q000494:pharmacology; D008297:Male; D017351:N-Acetylglucosaminyltransferases / Q000187:drug effects / Q000276:immunology / Q000378:metabolism*; D011092:Polyethylene Glycols / Q000737:chemistry; D011189:Potassium Chloride / Q000494:pharmacology; D051381:Rats; D017207:Rats, Sprague-Dawley; D012965:Sodium Chloride / Q000494:pharmacology; D013696:Temperature; D013997:Time Factors; D014530:Uridine Diphosphate / Q000494:pharmacology
D000906:Antibodies; C026712:UDP-glucosamine; D011092:Polyethylene Glycols; D012965:Sodium Chloride; D014530:Uridine Diphosphate; D011189:Potassium Chloride; D017351:N-Acetylglucosaminyltransferases; D005944:Glucosamine