Purification of UDP-N-acetylglucosamine:glycoprotein N-acetylglucosamine-1-phosphotransferase from Acanthamoeba castellanii and identification of a subunit of the enzyme.
UDP-N-acetylglucosamine:glycoprotein N-acetylglucosamine-1-phosphotransferase (GlcNAc-phosphotransferase) from the soil amoeba Acanthamoeba castellanii has been purified over 100,000-fold by means of wheat germ agglutinin-Sepharose affinity chromatography, DEAE-cellulose chromatography, concanavalin A-Sepharose affinity chromatography, orange A-agarose dye chromatography, and gel filtration on Superose 6. The most purified enzyme has an estimated specific activity of at least 5 mumol of GlcNAc-phosphate transferred/min/mg of protein using alpha-methylmannoside as acceptor. The molecular weight of the native enzyme is approximately 250,000, as determined by gel filtration and glycerol gradients in H2O and D2O. A protein with an apparent M(r) of 97,000 in small scale preparations and its putative proteolytic fragment of 43,000 in large scale preparations co-purifies with the enzyme activity. This protein is covalently modified with GlcNAc-[32P]phosphate when the enzyme preparation is incubated with [beta-32P]UDP-GlcNAc in the absence of an acceptor substrate. The labeling of the 97(43)-kDa protein requires active enzyme and is completely inhibited by the addition of the acceptor substrate alpha-methylmannoside. The GlcNAc-[32P]phosphate transferred to the protein is not bound to serine, threonine, tyrosine, or mannose residues. The 97(43)-kDa protein with covalently bound GlcNAc-P does not serve as a kinetically competent enzyme-substrate intermediate. However, preincubation of GlcNAc-phosphotransferase with UDP-GlcNAc does result in a decrease in the Vmax of the enzyme in subsequent assays. Taken together, these data are consistent with the 97(43)-kDa protein being a subunit of GlcNAc-phosphotransferase.
Ketcham
C M
CM
Department of Medicine, Washington University School of Medicine, St. Louis, Missouri 63110.
Kornfeld
S
S
D000048:Acanthamoeba / Q000201:enzymology*; D000818:Animals; D002853:Chromatography, Liquid; D002854:Chromatography, Paper; D004591:Electrophoresis, Polyacrylamide Gel; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007700:Kinetics; D008766:Methylmannosides / Q000494:pharmacology; D010770:Phosphotransferases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D017855:Transferases (Other Substituted Phosphate Groups)
D008766:Methylmannosides; C008466:methylmannoside; D010770:Phosphotransferases; D017855:Transferases (Other Substituted Phosphate Groups); C030012:UDP-N-acetylglucosamine-lysosomal-enzyme-N-acetylglucosaminephosphotransferase