5′-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′-p-fluorosulphonylbenzoyladenosine (5′-FSBA). 5′-FSBA irreversibly inactivates 5′-nucleotidases by means of covalent modification of the proteins. ATP, a competitive inhibitor of chicken gizzard and snake-venom 5′-nucleotidase, abolished the inactivation by 5′-FSBA, demonstrating that the inactivation was due to the modification of amino acid residues essential for AMPase activity. We have synthesized radioactive 5′-FSBA, which was employed for the radiolabelling of chicken gizzard 5′-nucleotidase. Incorporation of radioactivity was completely abolished in the presence of ATP, which showed that 5′-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′-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′-nucleotidases. Incorporation of the radioactive p-sulphonylbenzoyladenosine moiety parallels the inactivation of 5′-nucleotidase by 5′-FSBA and further substantiated the idea that modification of one amino acid residue at the active centre results in loss of the AMPase activity.
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Research Article|
March 01 1990
Affinity labelling of 5′-nucleotidases with 5′-p-fluorosulphonylbenzoyladenosine
U Stochaj;
U Stochaj
1Institut für Anatomie und Zellbiologie, Robert-Koch-Strasse 5, 3550 Marburg, Federal Republic of Germany
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H G Mannherz
H G Mannherz
1Institut für Anatomie und Zellbiologie, Robert-Koch-Strasse 5, 3550 Marburg, Federal Republic of Germany
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Publisher: Portland Press Ltd
Online ISSN: 1470-8728
Print ISSN: 0264-6021
© 1990 London: The Biochemical Society
1990
Biochem J (1990) 266 (2): 447–451.
Citation
U Stochaj, H G Mannherz; Affinity labelling of 5′-nucleotidases with 5′-p-fluorosulphonylbenzoyladenosine. Biochem J 1 March 1990; 266 (2): 447–451. doi: https://doi.org/10.1042/bj2660447
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