A highly sensitive fluorometric assay for sialyltransferase activity using CMP-9-fluoresceinyl-NeuAc as donor
This paper presents a very sensitive fluorometric assay for sialyltransferase activity based on the transfer of 5-acetamido-9-deoxy-9-fluoresceinylthioureidoneuraminic acid onto distinct glycoproteins, thus allowing determination of acceptor specificities. Acceptor protein-bound fluorescence was qua...
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| Veröffentlicht in: | Analytical biochemistry 1990-04, Vol.186 (1), p.127-134 |
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| creator | Gross, H.J. Sticher, U. Brossmer, R. |
| description | This paper presents a very sensitive fluorometric assay for sialyltransferase activity based on the transfer of 5-acetamido-9-deoxy-9-fluoresceinylthioureidoneuraminic acid onto distinct glycoproteins, thus allowing determination of acceptor specificities. Acceptor protein-bound fluorescence was quantified after gel filtration which separated fluorescent sialoglycoprotein from the fluorescence-labeled CMP-glycoside donor. Kinetic constants obtained for five different purified sialyltransferases indicated that CMP-9-fluoresceinyl-NeuAc was a suitable donor substrate for each enzyme, affording low
K
m
values and
V
max values comparable in magnitude (15–100%) to that obtained with the parent CMP-NeuAc. Sensitivity was enhanced 200- to 1000-fold compared to the radiometric sialyltransferase assay as it is used routinely. The method was applied to determination of the kinetic properties of purified rat liver α2,6-sialyltransferase with four separate glycoprotein acceptors differing in glycan structure, employing very small amounts of donor, acceptor, and enzyme, and to the study of sialyltransferase activity of the human promyelocytic cell line HL-60 toward three different acceptors. |
| doi_str_mv | 10.1016/0003-2697(90)90585-W |
| format | Article |
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K
m
values and
V
max values comparable in magnitude (15–100%) to that obtained with the parent CMP-NeuAc. Sensitivity was enhanced 200- to 1000-fold compared to the radiometric sialyltransferase assay as it is used routinely. The method was applied to determination of the kinetic properties of purified rat liver α2,6-sialyltransferase with four separate glycoprotein acceptors differing in glycan structure, employing very small amounts of donor, acceptor, and enzyme, and to the study of sialyltransferase activity of the human promyelocytic cell line HL-60 toward three different acceptors.</description><identifier>ISSN: 0003-2697</identifier><identifier>EISSN: 1096-0309</identifier><identifier>DOI: 10.1016/0003-2697(90)90585-W</identifier><identifier>PMID: 2192578</identifier><identifier>CODEN: ANBCA2</identifier><language>eng</language><publisher>San Diego, CA: Elsevier Inc</publisher><subject>Analytical, structural and metabolic biochemistry ; Animals ; Biological and medical sciences ; Cell Line ; Chromatography, High Pressure Liquid ; Chromatography, Liquid ; Cytidine Monophosphate N-Acetylneuraminic Acid ; Enzymes and enzyme inhibitors ; Fluorometry ; Fundamental and applied biological sciences. Psychology ; Galactose - metabolism ; Humans ; Kinetics ; Liver - enzymology ; Liver - metabolism ; Rats ; Sensitivity and Specificity ; Sialic Acids ; Sialyltransferases - analysis ; Substrate Specificity ; Transferases</subject><ispartof>Analytical biochemistry, 1990-04, Vol.186 (1), p.127-134</ispartof><rights>1990</rights><rights>1990 INIST-CNRS</rights><lds50>peer_reviewed</lds50><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c386t-fea2b69d518981c15e13614e3d63c224cb5f75cf7d1aab22a0b4ceab2c405ff13</citedby><cites>FETCH-LOGICAL-c386t-fea2b69d518981c15e13614e3d63c224cb5f75cf7d1aab22a0b4ceab2c405ff13</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktohtml>$$Uhttps://dx.doi.org/10.1016/0003-2697(90)90585-W$$EHTML$$P50$$Gelsevier$$H</linktohtml><link.rule.ids>314,780,784,3550,27924,27925,45995</link.rule.ids><backlink>$$Uhttp://pascal-francis.inist.fr/vibad/index.php?action=getRecordDetail&idt=6854318$$DView record in Pascal Francis$$Hfree_for_read</backlink><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/2192578$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><creatorcontrib>Gross, H.J.</creatorcontrib><creatorcontrib>Sticher, U.</creatorcontrib><creatorcontrib>Brossmer, R.</creatorcontrib><title>A highly sensitive fluorometric assay for sialyltransferase activity using CMP-9-fluoresceinyl-NeuAc as donor</title><title>Analytical biochemistry</title><addtitle>Anal Biochem</addtitle><description>This paper presents a very sensitive fluorometric assay for sialyltransferase activity based on the transfer of 5-acetamido-9-deoxy-9-fluoresceinylthioureidoneuraminic acid onto distinct glycoproteins, thus allowing determination of acceptor specificities. Acceptor protein-bound fluorescence was quantified after gel filtration which separated fluorescent sialoglycoprotein from the fluorescence-labeled CMP-glycoside donor. Kinetic constants obtained for five different purified sialyltransferases indicated that CMP-9-fluoresceinyl-NeuAc was a suitable donor substrate for each enzyme, affording low
K
m
values and
V
max values comparable in magnitude (15–100%) to that obtained with the parent CMP-NeuAc. Sensitivity was enhanced 200- to 1000-fold compared to the radiometric sialyltransferase assay as it is used routinely. The method was applied to determination of the kinetic properties of purified rat liver α2,6-sialyltransferase with four separate glycoprotein acceptors differing in glycan structure, employing very small amounts of donor, acceptor, and enzyme, and to the study of sialyltransferase activity of the human promyelocytic cell line HL-60 toward three different acceptors.</description><subject>Analytical, structural and metabolic biochemistry</subject><subject>Animals</subject><subject>Biological and medical sciences</subject><subject>Cell Line</subject><subject>Chromatography, High Pressure Liquid</subject><subject>Chromatography, Liquid</subject><subject>Cytidine Monophosphate N-Acetylneuraminic Acid</subject><subject>Enzymes and enzyme inhibitors</subject><subject>Fluorometry</subject><subject>Fundamental and applied biological sciences. Psychology</subject><subject>Galactose - metabolism</subject><subject>Humans</subject><subject>Kinetics</subject><subject>Liver - enzymology</subject><subject>Liver - metabolism</subject><subject>Rats</subject><subject>Sensitivity and Specificity</subject><subject>Sialic Acids</subject><subject>Sialyltransferases - analysis</subject><subject>Substrate Specificity</subject><subject>Transferases</subject><issn>0003-2697</issn><issn>1096-0309</issn><fulltext>true</fulltext><rsrctype>article</rsrctype><creationdate>1990</creationdate><recordtype>article</recordtype><sourceid>EIF</sourceid><recordid>eNp9kMFu1DAQhi1EVbaFNwDJB4TgkGInthNfkFYrCpVa4ADq0XKccWvkxK0nqZS3J-mu9shpRprv_zX6CHnL2QVnXH1mjFVFqXT9UbNPmslGFrcvyIYzrQpWMf2SbI7IK3KG-JcxzoVUp-S05LqUdbMh_Zbeh7v7OFOEAcMYnoD6OKWcehhzcNQi2pn6lCkGG-c4Zjugh2wRqHULH8aZThiGO7q7-VXo4jkN6CAMcyx-wLRdS2iXhpRfkxNvI8Kbwzwnfy6__t59L65_frvaba8LVzVqLDzYslW6k7zRDXdcAq8UF1B1qnJlKVwrfS2drztubVuWlrXCwbI5waT3vDonH_a9Dzk9ToCj6cPyUYx2gDShqXUjGyH0Aoo96HJCzODNQw69zbPhzKyWzarQrAqNZubZsrldYu8O_VPbQ3cMHbQu9_eHu0Vno1-cuYBHTDVSVHzFvuwxWFw8BcgGXYDBQRcyuNF0Kfz_j3-9e5sJ</recordid><startdate>19900401</startdate><enddate>19900401</enddate><creator>Gross, H.J.</creator><creator>Sticher, U.</creator><creator>Brossmer, R.</creator><general>Elsevier Inc</general><general>Elsevier</general><scope>IQODW</scope><scope>CGR</scope><scope>CUY</scope><scope>CVF</scope><scope>ECM</scope><scope>EIF</scope><scope>NPM</scope><scope>AAYXX</scope><scope>CITATION</scope><scope>7X8</scope></search><sort><creationdate>19900401</creationdate><title>A highly sensitive fluorometric assay for sialyltransferase activity using CMP-9-fluoresceinyl-NeuAc as donor</title><author>Gross, H.J. ; Sticher, U. ; Brossmer, R.</author></sort><facets><frbrtype>5</frbrtype><frbrgroupid>cdi_FETCH-LOGICAL-c386t-fea2b69d518981c15e13614e3d63c224cb5f75cf7d1aab22a0b4ceab2c405ff13</frbrgroupid><rsrctype>articles</rsrctype><prefilter>articles</prefilter><language>eng</language><creationdate>1990</creationdate><topic>Analytical, structural and metabolic biochemistry</topic><topic>Animals</topic><topic>Biological and medical sciences</topic><topic>Cell Line</topic><topic>Chromatography, High Pressure Liquid</topic><topic>Chromatography, Liquid</topic><topic>Cytidine Monophosphate N-Acetylneuraminic Acid</topic><topic>Enzymes and enzyme inhibitors</topic><topic>Fluorometry</topic><topic>Fundamental and applied biological sciences. Psychology</topic><topic>Galactose - metabolism</topic><topic>Humans</topic><topic>Kinetics</topic><topic>Liver - enzymology</topic><topic>Liver - metabolism</topic><topic>Rats</topic><topic>Sensitivity and Specificity</topic><topic>Sialic Acids</topic><topic>Sialyltransferases - analysis</topic><topic>Substrate Specificity</topic><topic>Transferases</topic><toplevel>peer_reviewed</toplevel><toplevel>online_resources</toplevel><creatorcontrib>Gross, H.J.</creatorcontrib><creatorcontrib>Sticher, U.</creatorcontrib><creatorcontrib>Brossmer, R.</creatorcontrib><collection>Pascal-Francis</collection><collection>Medline</collection><collection>MEDLINE</collection><collection>MEDLINE (Ovid)</collection><collection>MEDLINE</collection><collection>MEDLINE</collection><collection>PubMed</collection><collection>CrossRef</collection><collection>MEDLINE - Academic</collection><jtitle>Analytical biochemistry</jtitle></facets><delivery><delcategory>Remote Search Resource</delcategory><fulltext>fulltext</fulltext></delivery><addata><au>Gross, H.J.</au><au>Sticher, U.</au><au>Brossmer, R.</au><format>journal</format><genre>article</genre><ristype>JOUR</ristype><atitle>A highly sensitive fluorometric assay for sialyltransferase activity using CMP-9-fluoresceinyl-NeuAc as donor</atitle><jtitle>Analytical biochemistry</jtitle><addtitle>Anal Biochem</addtitle><date>1990-04-01</date><risdate>1990</risdate><volume>186</volume><issue>1</issue><spage>127</spage><epage>134</epage><pages>127-134</pages><issn>0003-2697</issn><eissn>1096-0309</eissn><coden>ANBCA2</coden><abstract>This paper presents a very sensitive fluorometric assay for sialyltransferase activity based on the transfer of 5-acetamido-9-deoxy-9-fluoresceinylthioureidoneuraminic acid onto distinct glycoproteins, thus allowing determination of acceptor specificities. Acceptor protein-bound fluorescence was quantified after gel filtration which separated fluorescent sialoglycoprotein from the fluorescence-labeled CMP-glycoside donor. Kinetic constants obtained for five different purified sialyltransferases indicated that CMP-9-fluoresceinyl-NeuAc was a suitable donor substrate for each enzyme, affording low
K
m
values and
V
max values comparable in magnitude (15–100%) to that obtained with the parent CMP-NeuAc. Sensitivity was enhanced 200- to 1000-fold compared to the radiometric sialyltransferase assay as it is used routinely. The method was applied to determination of the kinetic properties of purified rat liver α2,6-sialyltransferase with four separate glycoprotein acceptors differing in glycan structure, employing very small amounts of donor, acceptor, and enzyme, and to the study of sialyltransferase activity of the human promyelocytic cell line HL-60 toward three different acceptors.</abstract><cop>San Diego, CA</cop><pub>Elsevier Inc</pub><pmid>2192578</pmid><doi>10.1016/0003-2697(90)90585-W</doi><tpages>8</tpages></addata></record> |
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| ispartof | Analytical biochemistry, 1990-04, Vol.186 (1), p.127-134 |
| issn | 0003-2697 1096-0309 |
| language | eng |
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| source | MEDLINE; Access via ScienceDirect (Elsevier) |
| subjects | Analytical, structural and metabolic biochemistry Animals Biological and medical sciences Cell Line Chromatography, High Pressure Liquid Chromatography, Liquid Cytidine Monophosphate N-Acetylneuraminic Acid Enzymes and enzyme inhibitors Fluorometry Fundamental and applied biological sciences. Psychology Galactose - metabolism Humans Kinetics Liver - enzymology Liver - metabolism Rats Sensitivity and Specificity Sialic Acids Sialyltransferases - analysis Substrate Specificity Transferases |
| title | A highly sensitive fluorometric assay for sialyltransferase activity using CMP-9-fluoresceinyl-NeuAc as donor |
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