R. Kornfeld et al., PURIFICATION AND MULTIMERIC STRUCTURE OF BOVINE N-ACETYLGLUCOSAMINE-1-PHOSPHODIESTER ALPHA-N-ACETYLGLUCOSAMINIDASE, The Journal of biological chemistry, 273(36), 1998, pp. 23203-23210
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.