Large scale preparation of PA-oligosaccharides from glycoproteins using an improved extraction method.
We have developed a new method for the large scale preparation of pyridylaminated (PA-) oligosaccharides from glycoproteins. Phenol/chloroform extraction was adapted for the removal of protein and excess 2-aminopyridine, improving the efficiency of preparation. From a 2.5 g sample of human apo-transferrin, 25-30 mu mol of agalacto biantennary PA-oligosaccharide could be obtained. By increasing the concentration of PA-oligosaccharide substrate, we were able to detect a very low level of N-acetylglucosaminlytransferase IV activity in CHO cell extracts.
Tokugawa
K
K
Central Laboratories for Key Technology, Kirin Brewery Co., Ltd. Fukuura, Japan.
Oguri
S
S
Takeuchi
M
M
D000631:Aminopyridines; D000818:Animals; D001059:Apoproteins / Q000737:chemistry; D016466:CHO Cells; D002240:Carbohydrate Sequence; D002725:Chloroform; D002851:Chromatography, High Pressure Liquid; D006224:Cricetinae; D005456:Fluorescent Dyes; D006023:Glycoproteins / Q000737:chemistry*; D006801:Humans; D008722:Methods; D008969:Molecular Sequence Data; D017351:N-Acetylglucosaminyltransferases / Q000378:metabolism; D009844:Oligosaccharides / Q000737:chemistry / Q000302:isolation & purification*; D019800:Phenol; D010636:Phenols; D013379:Substrate Specificity; D014168:Transferrin / Q000737:chemistry
D000631:Aminopyridines; D001059:Apoproteins; D005456:Fluorescent Dyes; D006023:Glycoproteins; D009844:Oligosaccharides; D010636:Phenols; D014168:Transferrin; C028284:apotransferrin; D019800:Phenol; D002725:Chloroform; D017351:N-Acetylglucosaminyltransferases; C038111:alpha-1,3-mannosylglycoprotein beta-1,4-N-acetylglucosaminyltransferase; C032439:alpha-aminopyridine