BOVINE UDP-N-ACETYLGLUCOSAMINE-LYSOSOMAL-ENZYME N-ACETYLGLUCOSAMINE-1-PHOSPHOTRANSFERASE .1. PURIFICATION AND SUBUNIT STRUCTURE

UDP-N-acetylglucosamine:lysosomal-enzyme N-acetylglucosamine-1-phospho transferase (GlcNAc-phosphotransferase) catalyzes the initial step in the synthesis of the mannose 6-phosphate determinant required for effi cient intracellular targeting of newly synthesized lysosomal hydrolase s to the lysosome, The enzyme was partially purified similar to 30,000 -fold by chromatography of solubilized membrane proteins from lactatin g bovine mammary glands on DEAE-Sepharose, reactive green 19-agarose, and Superose 6, The partially purified enzyme was used to generate a p anel of murine monoclonal antibodies, The anti-GlcNAc-phosphotransfera se monoclonal antibody PT18 was coupled to a solid support and used to immunopurify the enzyme similar to 480,000-fold to apparent homogenei ty with an overall yield of 29%, The purified enzyme has a specific ac tivity of 10-12 mu mol 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 filtratio n chromatography, sodium dodecyl sulfate-polyacrylamide gel electropho resis, and amino-terminal sequencing. The data indicate that bovine Gl cNAc-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.