Characterization and partial purification of two enzymes transferring N-acetylglucosamine to dolichyl monophosphate and ribonuclease A

Publisher Website
Google Scholar
Abstract
Two N-acetylglucosamine (GlcNAc) transferases which catalyze the incorporation of GlcNAc into GlcNAc-P-P-dolichol (dolichol enzyme) and into bovine pancreatic RNase A were solubilized from the rat liver microsomes in a nonionic 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- to 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 .times. 10-6 M for UDP-GlcNAc and the reaction was saturated with .apprx. 0.25 M dolichyl phosphate. The purified RNase enzyme had a Km of 4.55 .times. 10-6 M for UDP-GlcNAc and was saturated with .apprx. 0.36 mM RNaseA. The pH optima and the metal ion requirement for the 2 enzymes were different. These results suggest that because of the different properties of these 2 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 1st 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.

This publication has 26 references indexed in Scilit: