The synthesis of substrates and two assays for the detection of N-acetylglucosamine-1-phosphodiester α- N-acetylglucosaminidase (uncovering enzyme)

A method for the synthesis and purification of large quantities of four radiolabeled substrates for quantitation of uncovering enzyme is described. Four substrates, [ 3H]GlcNAc - α- P - ManαMe, [ 3H]GlcNAc-α-P-uteroferrin, [ 3H]GlcNAcα-P-Manα1-2Man- O-Me, and [ 3H]GlcNAcα-P-Man 9GlcNAc, were enzymat...

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Published inAnalytical biochemistry Vol. 205; no. 2; pp. 200 - 207
Main Authors Mullis, Karen Gheesling, Ketcham, Catherine M.
Format Journal Article
LanguageEnglish
Published San Diego, CA Elsevier Inc 01.09.1992
Elsevier
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Summary:A method for the synthesis and purification of large quantities of four radiolabeled substrates for quantitation of uncovering enzyme is described. Four substrates, [ 3H]GlcNAc - α- P - ManαMe, [ 3H]GlcNAc-α-P-uteroferrin, [ 3H]GlcNAcα-P-Manα1-2Man- O-Me, and [ 3H]GlcNAcα-P-Man 9GlcNAc, were enzymatically synthesized using GlcNAc-phosphotransferase from Acanthamoeba castellanii and uridine diphosphate N-acetyl-[ 3H]glucosamine and, as acceptor, methyl-α- d-mannopyranoside (ManαMe), uteroferrin, Manα1-2Man- O-methyl, or Man 9GlcNAc. The isolation of the [ 3H]GlcNAc-P-modified product of each reaction is detailed. Two assays for the detection of uncovering enzyme activity using [ 3H]GlcNAc-α-P-uteroferrin and [ 3H]GlcNAc-α-P-ManαMe are outlined. The ability to easily synthesize four relevant substrates for uncovering enzyme offers flexibility in assaying uncovering enzyme.
Bibliography:ObjectType-Article-1
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ISSN:0003-2697
1096-0309
DOI:10.1016/0003-2697(92)90424-6