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 in | Analytical biochemistry Vol. 205; no. 2; pp. 200 - 207 |
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Main Authors | , |
Format | Journal Article |
Language | English |
Published |
San Diego, CA
Elsevier Inc
01.09.1992
Elsevier |
Subjects | |
Online Access | Get full text |
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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. |
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Bibliography: | ObjectType-Article-1 SourceType-Scholarly Journals-1 ObjectType-Feature-2 content type line 23 |
ISSN: | 0003-2697 1096-0309 |
DOI: | 10.1016/0003-2697(92)90424-6 |