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Volume 271, Number 49, Issue of December 6, 1996 pp. 31437-31445
©1996 by The American Society for Biochemistry and Molecular Biology, Inc.

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Bovine UDP-N-acetylglucosamine:Lysosomal-enzyme N-Acetylglucosamine-1-phosphotransferase
I. PURIFICATION AND SUBUNIT STRUCTURE

(Received for publication, May 28, 1996, and in revised form, September 4, 1996)

From the W. K. Warren Medical Research Institute and the Department of Medicine, University of Oklahoma Health Sciences Center, Oklahoma City, Oklahoma 73104

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 ~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 ~480,000-fold to apparent homogeneity with an overall yield of 29%. The purified enzyme has a specific activity of 10-12 µmol of GlcNAc phosphate transferred per h/mg using 100 mM -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.

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