Control of glycoprotein synthesis. Purification of UDP-N-acetylglucosamine:alpha-D-mannoside beta 1-2 N-acetylglucosaminyltransferase II from rat liver.
A new affinity chromatography adsorbant, in which UDP-GlcNAc has been linked to thiopropyl-Sepharose at the 5 position of the uracil via a 5-mercuri mercaptide bond, was utilized to purify UDP-GlcNAc:alpha-D-mannoside beta 1-2 N-acetylglucosaminyltransferase II 60,000-fold from rat liver. After extraction of rat liver membranes with Triton X-100, the enzyme was found to exist in two molecular weight forms of markedly differing size, separable on Sephadex G-200. The low Mr form was separated from the high Mr form on columns of CM-Sephadex and hydroxylapatite, and was further purified by sequential elutions with NaCl, UDP-GlcNAc, and EDTA from the 5-mercuri-UDP-GlcNAc affinity adsorbant. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified low Mr form under reducing conditions revealed two protein bands of Mr 48,000 and 43,000. The purified enzyme catalyzes the transfer of N-acetylglucosamine from UDP-GlcNAc to the compound: (Formula: see text) The high Mr form of the enzyme, which eluted in the void volume of Sephadex G-200, was resistant to a number of treatments in attempts to reduce its molecular weight. These results suggest that the high Mr form of the enzyme may represent either a complex which normally exists in Golgi membranes as a result of strong protein-protein interactions or a protein with one or more "anchor" segments.
D000818:Animals; D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D002462:Cell Membrane / Q000201:enzymology; D002846:Chromatography, Affinity; D005964:Glucosyltransferases / Q000302:isolation & purification* / Q000378:metabolism; D006023:Glycoproteins / Q000096:biosynthesis*; D008099:Liver / Q000201:enzymology*; D046911:Macromolecular Substances; D008970:Molecular Weight; D017351:N-Acetylglucosaminyltransferases; D051381:Rats
D006023:Glycoproteins; D046911:Macromolecular Substances; D005964:Glucosyltransferases; D017351:N-Acetylglucosaminyltransferases; C063313:alpha-1,6-mannosyl-glycoprotein beta-1,2-N-acetylglucosaminyltransferase