Characterization and isolation of oligosaccharyl transferase
Oligosaccharyltransferases (OT), the enzyme catalyzing the transfer of oligosaccharride from dolichol-PP-GlcNAc/sub 2/Man/sub 9/Glc/sub 3/ to asparagine residues of -Asn-X-Thr/Ser-sequences within nascent polypeptides, was labeled with a new active site-directed photoaffinity probe, N/sup ..cap alpha../-(/sup 125/I)-3-(4-hydroxyphenylpropionyl)-Asn-Lys(N/sup epsilon/-p-azidobenzoyl)-Thr-NH/sub 2/. Using this sensitive selective labelling probe, the authors have characterized and isolated the enzyme from hen oviduct. Analysis by 2-D gel electrophoresis revealed that the major photoaffinity-labeled OT has a pI = 5.2; minor forms exist at pI = 6.0-6.2. Kinetic experiments demonstrated that the probe interacts with a 57 kDa Coomassie Blue stainable protein at short incubation times, with the radioactive signal appearing at 60 kDa after longer incubations. The 60 kDa /sup 125/I-photoaffinity labeled OT decreased in molecular mass to 57 kDa following endoglucosaminidase H digestion with no shift in pI. The 57 dKa Coomassie Blue stainable protein is not endoglucosaminidase H sensitive. When microsomal dolichol-P-P-oligosaccharide was depleted by preincubating with excess competing peptide, only the 57 kDa /sup 125/I-labeled OT formed. These data suggest that (1) OT is a 57 kDa protein that does not contain polymannose chains, and (2) the formation of the 60 kDa /sup 125/I-labeled OT is likely the result of the glycosylation of the covalently linked tripeptide probe. The enzyme has been isolated to homogeneity by preparative 2-D gel electrophoresis and amino acid sequence analysis and polyclonal antibody production is being performed.
- Research Organization:
- M.D. Anderson Hospital and Tumor Institute, Houston, TX
- OSTI ID:
- 5020131
- Report Number(s):
- CONF-8606151-; TRN: 86-034841
- Journal Information:
- Fed. Proc., Fed. Am. Soc. Exp. Biol.; (United States), Vol. 45:6; Conference: 76. annual meeting of the Federation of American Society for Experimental Biology, Washington, DC, USA, 8 Jun 1986
- Country of Publication:
- United States
- Language:
- English
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Related Subjects
TRANSFERASES
LABELLING
PURIFICATION
AFFINITY
AMINO ACID SEQUENCE
ANTIBODIES
BIOCHEMICAL REACTION KINETICS
CHICKENS
ELECTROPHORESIS
IODINE 125
OVARIES
TRACER TECHNIQUES
ANIMALS
BETA DECAY RADIOISOTOPES
BIRDS
BODY
DAYS LIVING RADIOISOTOPES
ELECTRON CAPTURE RADIOISOTOPES
ENZYMES
FEMALE GENITALS
FOWL
GONADS
INTERMEDIATE MASS NUCLEI
IODINE ISOTOPES
ISOTOPE APPLICATIONS
ISOTOPES
KINETICS
MOLECULAR STRUCTURE
NUCLEI
ODD-EVEN NUCLEI
ORGANS
RADIOISOTOPES
REACTION KINETICS
VERTEBRATES
550201* - Biochemistry- Tracer Techniques