Purification and properties of the glycoprotein processing N-acetylglucosaminyltransferase II from plants

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Abstract
The presence of an N-acetylglucosaminyltransferaser (GlcNAc-transferase) capable of adding a GlcNAc residue to GlcNAcMan3GlcNAc was demonstrated in mung bean seedlings. This enzyme was purified about 3400-fold by using (diethylaminoethyl)cellulose and phosphocellulose chromatographies and chromatography on Concanavalin A-Sepharose. The transferase was assayed by following the change in the migration of the [3H]mannose-labeled GlcNAc.beta.1,2Man.alpha.1,3(Man.alpha.1,6)Man.beta.1,4GlcNAc on Bio-Gel P-4, or by incorporation of [3H]GlcNAc from UDP-[3H]GlcNAc into a neutral product, (GlcNAc)2Man3GlcNAc. Thus, the purified enzyme catalyzed the addition of a GlcNAc to that mannose linked in .alpha.1,6 linkage to the .beta.-linked mannose. GlcNAc.beta.1,2Man.alpha.1,3(Man.alpha.1,6)Man.beta.1,4GlcNAc was an excellent acceptor while Man.alpha.1,6(Man.alpha.1,3)Man.beta.1,4GlcNAc, Man.alpha.1,6(Man.alpha.1,3)Man.alpha.1,6-(Man.alpha.1,3)(Man.beta.1,4GlcNAc, and Man.alpha.1,6(Man.alpha.1,3)Man.alpha.1,6[GlcNAcMan.alpha.1,3]Man.beta.1,4GlcNAc were not acceptors. Methylation analysis and enzymatic digestions showed that both terminal GlcNAc residues on (GlcNAc)2Man3GlcNAc were attached to the mannoses in .beta.1,2 linkages. The GlcNAc transferase had an almost absolute requirement for divalent cation, with Mn2+ being best at 2-3 mM. Mn2+ could not be replaced by Mg2+ or Ca2+, but Cd2+ showed some activity. The enzyme was also markedly stimulated by the presence of detergent and showed optimum activity at 0.15% Triton X-100. The Km for UDP-GlcNAc was found to be 18 .mu.M and that for GlcNAcMan3GlcNAc about 16 .mu.M.

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