Studies on properties of membrane-associated oligosaccharyltransferase using an active site-directed photoaffinity probe.
Previous attempts in several laboratories, including ours, to purify oligosaccharyl-transferase have met with limited success because of the lability of the membrane-associated enzyme after solubilization with detergents. In an effort to identify the enzyme in face of this lability, we recently developed a photoaffinity reagent to label the active site [J. K. Welply, P. Shenbagamurthi, F. Naider, H. R. Park, and W. J. Lennarz (1985) J. Biol. Chem. 260, 6459-6465]. In this report, the preparations of a more sensitive selective labeling probe, 125I-labeled N alpha-3-(4-hydroxyphenylpropionyl)-Asn-Lys-(N epsilon-p-azidobenzoyl)-Thr-NH2, is described. Using this new probe, we have confirmed, independently of catalytic activity, that hen oviduct oligosaccharyltransferase is tightly associated with the endoplasmic reticulum membrane. The 125I-labeled oligosaccharyltransferase was released from the membrane by detergent and strong alkali treatments but not by sonication, high salt, or hypotonic shock. However, all procedures that released the enzyme from the membrane resulted in a dramatic loss of enzyme activity. Treatment of sealed microsomal membrane vesicles with phospholipase A resulted in nearly complete enzyme inactivation; in contrast, phospholipase C or D had moderate or little effect, respectively. Taken together, these results suggest that the hydrophobic environment of the membrane is required for oligosaccharyltransferase activity. Trypsin treatment of intact vesicles diminished enzyme activity by nearly 70%, but it had no effect on the binding affinity of the enzyme for the 125I-labeled photoaffinity probe. This result suggests that the polypeptide acceptor portion of oligosaccharyltransferase is lumenally disposed, and that a trypsin-sensitive, cytoplasmically oriented domain or another subunit binds the carbohydrate donor, dolichol-PP-oligosaccharide.
Welply
J K
JK
Kaplan
H A
HA
Shenbagamurthi
P
P
Naider
F
F
Lennarz
W J
WJ
D000345:Affinity Labels / Q000138:chemical synthesis*; D000818:Animals; D001386:Azides / Q000138:chemical synthesis*; D001665:Binding Sites; D002645:Chickens; D004721:Endoplasmic Reticulum / Q000201:enzymology; D005260:Female; D006602:Hexosyltransferases; D007425:Intracellular Membranes / Q000201:enzymology; D008565:Membrane Proteins; D009842:Oligopeptides / Q000138:chemical synthesis*; D010057:Oviducts / Q000201:enzymology; D010777:Photochemistry; D012995:Solubility; D014166:Transferases / Q000378:metabolism*
D000345:Affinity Labels; D001386:Azides; D008565:Membrane Proteins; D009842:Oligopeptides; C048719:N(alpha)-3-(4-hydroxyphenylpropionyl)asparaginyl-lysyl-(N(epsilon)-4-azidobenzoyl)threoninamide; D014166:Transferases; D006602:Hexosyltransferases; C025620:dolichyl-diphosphooligosaccharide - protein glycotransferase