{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2023,3,16]],"date-time":"2023-03-16T05:29:14Z","timestamp":1678944554179},"reference-count":0,"publisher":"Portland Press Ltd.","issue":"2","content-domain":{"domain":["portlandpress.com"],"crossmark-restriction":true},"short-container-title":[],"published-print":{"date-parts":[[1990,3,1]]},"abstract":"<jats:p>5\u2032-Nucleotidases play an important role in the metabolism of nucleosides; for example, the hydrolysis of AMP generates adenosine, which can modulate a variety of cellular functions. We have used the membrane-bound AMPase from chicken gizzard and a secreted form of these enzymes to analyse their modification by the substrate analogue 5\u2032-p-fluorosulphonylbenzoyladenosine (5\u2032-FSBA). 5\u2032-FSBA irreversibly inactivates 5\u2032-nucleotidases by means of covalent modification of the proteins. ATP, a competitive inhibitor of chicken gizzard and snake-venom 5\u2032-nucleotidase, abolished the inactivation by 5\u2032-FSBA, demonstrating that the inactivation was due to the modification of amino acid residues essential for AMPase activity. We have synthesized radioactive 5\u2032-FSBA, which was employed for the radiolabelling of chicken gizzard 5\u2032-nucleotidase. Incorporation of radioactivity was completely abolished in the presence of ATP, which showed that 5\u2032-FSBA acted by the selective modification of amino acid residues at the active site whereas other potential reactive residues of the protein were not attacked. Limited proteolysis of affinity-labelled chicken gizzard 5\u2032-nucleotidase permitted the identification of digestion products containing the catalytic centre. Pseudo-first-order kinetics indicate that modification of a minimum of one amino acid side chain at the active centre is sufficient to result in inactivation of both chicken gizzard and snake-venom 5\u2032-nucleotidases. Incorporation of the radioactive p-sulphonylbenzoyladenosine moiety parallels the inactivation of 5\u2032-nucleotidase by 5\u2032-FSBA and further substantiated the idea that modification of one amino acid residue at the active centre results in loss of the AMPase activity.<\/jats:p>","DOI":"10.1042\/bj2660447","type":"journal-article","created":{"date-parts":[[2015,8,10]],"date-time":"2015-08-10T21:15:01Z","timestamp":1439241301000},"page":"447-451","update-policy":"http:\/\/dx.doi.org\/10.1042\/crossmark_policy","source":"Crossref","is-referenced-by-count":4,"title":["Affinity labelling of 5\u2032-nucleotidases with 5\u2032-<i>p<\/i>-fluorosulphonylbenzoyladenosine"],"prefix":"10.1042","volume":"266","author":[{"given":"U","family":"Stochaj","sequence":"first","affiliation":[{"name":"Institut f\u00fcr Anatomie und Zellbiologie, Robert-Koch-Strasse 5, 3550 Marburg, Federal Republic of Germany"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"given":"H G","family":"Mannherz","sequence":"additional","affiliation":[{"name":"Institut f\u00fcr Anatomie und Zellbiologie, Robert-Koch-Strasse 5, 3550 Marburg, Federal Republic of Germany"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"288","container-title":["Biochemical Journal"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/portlandpress.com\/biochemj\/article-pdf\/266\/2\/447\/598323\/bj2660447.pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"syndication"},{"URL":"https:\/\/portlandpress.com\/biochemj\/article-pdf\/266\/2\/447\/598323\/bj2660447.pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2021,11,25]],"date-time":"2021-11-25T18:56:57Z","timestamp":1637866617000},"score":1,"resource":{"primary":{"URL":"https:\/\/portlandpress.com\/biochemj\/article\/266\/2\/447\/35807\/Affinity-labelling-of-5-nucleotidases-with-5-p"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[1990,3,1]]},"references-count":0,"journal-issue":{"issue":"2","published-print":{"date-parts":[[1990,3,1]]}},"URL":"https:\/\/doi.org\/10.1042\/bj2660447","relation":{},"ISSN":["0264-6021","1470-8728"],"issn-type":[{"value":"0264-6021","type":"print"},{"value":"1470-8728","type":"electronic"}],"subject":[],"published":{"date-parts":[[1990,3,1]]}}}