Characterization and partial purification of two enzymes transferring N-acetylglucosamine to dolichyl monophosphate and ribonuclease A.
Two N-acetylglucosamine (GlcNAc) transferases which catalyze the incorporation of GlcNAc into GlcNAc-P-P-dolichol (dolichol enzyme) and into bovine pancreatic ribonuclease A (RNAseA enzyme) were solubilized from the rat liver microsomes in a non-ionic detergent, Triton X-100. Both enzyme activities were adsorbed on activated CH-Sepharose 4B, and could be eluted with a linear KCl gradient. Two enzyme activities were separated by this column with the dolichol enzyme eluting before the RNAseA enzyme. A 49-fold and 136-fold purification was achieved for the dolichol and the RNAseA enzyme, respectively. The addition of exogeneous dolichyl phosphate resulted in a 3-5-fold stimulation of the purified dolichol enzyme, but did not affect the purified RNAseA enzyme. The addition of RNAseA stimulated only the RNAseA enzyme. Whereas, tunicamycin could inhibit only the dolichol enzyme. The purified dolichol enzyme had a Km of 14 X 10(-6) M for UDP-GlcNAc and the reaction was saturated with about 0.25 M dolichyl phosphate. The purified RNAseA enzyme had a Km of 4.55 X 10(-6) M for UDP-GlcNAc and was saturated with about 0.36 mM RNAseA. The pH optima and the metal ion requirement for the two enzymes were different. These results suggest that because of the different properties of these two enzymes they may have distinct functions regarding the core glycosylation of N-linked glycoproteins. It is well established that the dolichol enzyme catalyzes the formation of the first dolichol-linked intermediate GlcNAc-P-P-dolichol, whereas according to the present finding, the RNAseA enzyme may catalyze the transfer of GlcNAc directly from UDP-GlcNAc into acceptor protein.
D000818:Animals; D004288:Dolichol Phosphates / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D005964:Glucosyltransferases / Q000302:isolation & purification / Q000378:metabolism*; D008297:Male; D008862:Microsomes, Liver / Q000201:enzymology*; D017351:N-Acetylglucosaminyltransferases; D011106:Polyisoprenyl Phosphates / Q000378:metabolism*; D051381:Rats; D011919:Rats, Inbred Strains; D012259:Ribonuclease, Pancreatic / Q000378:metabolism*; D012995:Solubility
D004288:Dolichol Phosphates; D011106:Polyisoprenyl Phosphates; C028306:dolichol monophosphate; D005964:Glucosyltransferases; D017351:N-Acetylglucosaminyltransferases; C037649:N-acetyllactosaminide beta-1,6-N-acetylglucosaminyltransferase; D012259:Ribonuclease, Pancreatic