PURIFICATION AND MULTIMERIC STRUCTURE OF BOVINE N-ACETYLGLUCOSAMINE-1-PHOSPHODIESTER ALPHA-N-ACETYLGLUCOSAMINIDASE

N-Acetylglucosamine-1-phosphodiester alpha-N-Acetylglucosaminidase (EC 3.1.4.45; phosphodiester alpha-GlcNAcase) catalyzes the second step i n the synthesis of the mannose 6-phosphate determinant required for ef ficient intracellular targeting of newly synthesized lysosomal hydrola ses to the lysosome, A partially purified preparation of phosphodieste r alpha-GlcNAcase from bovine pancreas was used to generate a panel of murine monoclonal antibodies. The anti-phosphodiester alpha-GlcNAcase monoclonal antibody UC1 was coupled to a solid support and used to im munopurify the bovine liver enzyme 670,000-fold in two steps to appare nt homogeneity with an overall yield of 14%. The purified phosphodiest er alpha-GlcNAcase has a specific activity of 498 mu mol of [H-3]GlcNA c-alpha-phosphomannose-alpha-methyl cleaved per h per mg of protein us ing 0.5 mM [H-3]GlcNAc-alpha-phosphomannose-alpha-methyl as substrate. The subunit structure of the enzyme was determined using a combinatio n of analytical gel filtration chromatography, SDS-polyacrylamide gel electrophoresis, and amino-terminal sequencing. The data indicate that bovine phosphodiester alpha-GlcNAcase is a 272,000-Da complex of four identical 68,000-Da glycoprotein subunits arranged as two disulfide-l inked homodimers. A soluble form of the enzyme, isolated from fetal bo vine serum, showed the same subunit structure. Both forms of the enzym e reacted with a rabbit antibody raised to the amino-terminal peptide of the liver enzyme, suggesting that phosphodiester alpha-GlcNAcase is a type I membrane-spanning glycoprotein with its amino terminus in th e lumen of the Golgi apparatus.