M. Bao et al., BOVINE UDP-N-ACETYLGLUCOSAMINE-LYSOSOMAL-ENZYME N-ACETYLGLUCOSAMINE-1-PHOSPHOTRANSFERASE .1. PURIFICATION AND SUBUNIT STRUCTURE, The Journal of biological chemistry, 271(49), 1996, pp. 31437-31445
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.