Bovine UDP-N-acetylglucosamine:lysosomal-enzyme N-acetylglucosamine-1-phosphotransferase. I. Purification and subunit structure.
UDP-N-acetylglucosamine:lysosomal-enzyme N-acetylglucosamine-1-phosphotransferase (GlcNAc-phosphotransferase) catalyzes the initial step in the synthesis of the mannose 6-phosphate determinant required for efficient intracellular targeting of newly synthesized lysosomal hydrolases to the lysosome. The enzyme was partially purified approximately 30,000-fold by chromatography of solubilized membrane proteins from lactating bovine mammary glands on DEAE-Sepharose, reactive green 19-agarose, and Superose 6. The partially purified enzyme was used to generate a panel of murine monoclonal antibodies. The anti-GlcNAc-phosphotransferase monoclonal antibody PT18 was coupled to a solid support and used to immunopurify the enzyme approximately 480,000-fold to apparent homogeneity with an overall yield of 29%. The purified enzyme has a specific activity of 10-12 micromol of GlcNAc phosphate transferred per h/mg using 100 mM alpha-methylmannoside as acceptor. The subunit structure of the enzyme was determined using a combination of analytical gel filtration chromatography, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and amino-terminal sequencing. The data indicate that bovine GlcNAc-phosphotransferase is a 540,000-Da complex composed of disulfide-linked homodimers of 166,000- and 51,000-Da subunits and two identical, noncovalently associated 56,000-Da subunits.
Bao
M
M
W. K. Warren Medical Research Institute and the Department of Medicine, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 73104, USA. Bill-canfield@uokhsc.edu
Booth
J L
JL
Elmendorf
B J
BJ
Canfield
W M
WM
D000818:Animals; D002417:Cattle; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D005260:Female; D008321:Mammary Glands, Animal / Q000737:chemistry; D008956:Models, Chemical; D008970:Molecular Weight; D011487:Protein Conformation; D017855:Transferases (Other Substituted Phosphate Groups) / Q000737:chemistry / Q000302:isolation & purification*
D017855:Transferases (Other Substituted Phosphate Groups); C030012:UDP-N-acetylglucosamine-lysosomal-enzyme-N-acetylglucosaminephosphotransferase