Characterization of the cAMP binding site of purified S-adenosyl-homocysteine hydrolase from bovine kidney.
The enzyme S-adenosyl-homocysteine hydrolase (AdoHcyase) which catalyzes the reversible hydrolysis of AdoHcy to adenosine and homocysteine is an adenosine binding protein. In the present study we examined the characteristics of [(3)H]cAMP binding to purified AdoHcyase from bovine kidney in comparison with the high affinity adenosine binding site of AdoHcyase. AdoHcyase exhibits one [(3)H]cAMP binding site with an affinity of K(d)=23.1+/-1.1nM and a B(max) of 116.6+/-3.8pmol/mg protein. Binding of [(3)H]cAMP obeyed a monophasic reaction with a k(+1) value of 0.035min/M. The dissociation of AdoHcyase-[(3)H]cAMP complex exhibited a time- and temperature-dependent character. After a 240min incubation at 0 degrees only 5-10%, however, at 20 degrees 90% were displaceable. Adenosine and cAMP displace each other with similar affinities of EC(50) 57nM vs. EC(50) 65nM. 2'-Deoxyadenosine, N(6)-methyladenosine, and NECA displace 25nM [(3)H]cAMP and 10nM [(3)H]adenosine with EC(50) values of 94, 90 and 80nM, respectively. All other nucleosides studied, adenine, inosine, adenosine-2',3'-dialdehyde, 2-chloroadenosine, aristeromycin, and adenine nucleotides were only week competitors for [(3)H]cAMP and [(3)H]adenosine. These compounds displace [(3)H]cAMP and [(3)H]adenosine with equal potencies. Our data indicate that the binding site for nanomolar concentrations of cAMP and adenosine at the AdoHcyase appears to be identical. The physiological implications of a cAMP binding site at the AdoHcyase remain to be established.
Kloor
Doris
D
Department of Pharmacology, Faculty of Medicine, University of Tübingen, Wilhelmstrasse 56, D-72074 Tübingen, Germany. doris.kloor@uni-tuebingen.de
Danielyan
Lusine
L
Osswald
Hartmut
H
D043383:Adenosylhomocysteinase; D000818:Animals; D001665:Binding Sites; D002417:Cattle; D000242:Cyclic AMP / Q000378:metabolism*; D006867:Hydrolases / Q000187:drug effects / Q000378:metabolism*; D019271:Hypoxanthine / Q000378:metabolism; D007668:Kidney / Q000201:enzymology*; D013806:Theophylline / Q000494:pharmacology; D014316:Tritium; D014970:Xanthines / Q000494:pharmacology
D014970:Xanthines; D014316:Tritium; D019271:Hypoxanthine; C051360:1,3-dipropyl-8-cyclopentylxanthine; D013806:Theophylline; D000242:Cyclic AMP; D006867:Hydrolases; D043383:Adenosylhomocysteinase