S-adenosylhomocysteine-hydrolase from bovine kidney: enzymatic and binding properties.
In the present study S-adenosylhomocysteine (SAH) hydrolase from the bovine kidney has been purified to apparent homogeneity by standard chromatographic procedures. The purified enzyme was free from adenosine deaminase activity and showed a one-banded pattern in SDS-PAGE with a monomer molecular mass of 47,500. The molecular mass of the native enzyme estimated by gel filtration was about 190,000. The pI was 5.5. For hydrolysis of SAH we found a Km of 5.0 +/- 1.2 microM and a V of 0.25 mumol/min/mg. In the direction of synthesis the Km for adenosine was 5.6 microM and V 0.53 mumol/min/mg. The enzyme activity was inhibited in the presence of adenosine with a Ki = 3 microM. In a second set of experiments we determined the binding characteristics of [3H]-adenosine to purified enzyme. The enzyme bound [3H]-adenosine with three apparent affinities: Kd1 = 6.8 +/- 0.7 nM and Bmax1 = 0.24 +/- 0.04 nmol/mg protein; Kd2 = 387 +/- 41 nM and Bmax2 = 1.4 nmol/mg protein, and Kd3 = 7.05 +/- 0.9 microM and Bmax3 = 9 nmol/mg protein. Binding of 25 nM [3H]-adenosine obeyed a monophasic reaction with a k+1 value of 0.025 min/nM. Dissociation of [3H]-adenosine-SAH hydrolase complex was markedly temperature dependent. After a 240-min incubation at 0 degrees C only 5-10% and at 20 degrees C 75% were displaceable. A fraction of 25% bound [3H]-adenosine was not displaceable by unlabeled adenosine. Our data show that the renal SAH hydrolase exhibits similar enzyme kinetics as the well-characterized SAH hydrolase from liver. The amount of SAH hydrolase present in renal tissues (1.4 nmol/g wet weight) could account almost entirely for the binding of renal tissue adenosine. Finally, we report for the first time a high affinity binding site of SAH hydrolase for adenosine, which remains unexplained at present.
Kloor
D
D
Department of Pharmacology, University of Tübingen, Germany.
Kurz
J
J
Fuchs
S
S
Faust
B
B
Osswald
H
H
D000241:Adenosine / Q000378:metabolism*; D043383:Adenosylhomocysteinase; D000818:Animals; D002417:Cattle; D004791:Enzyme Inhibitors / Q000494:pharmacology; D005069:Evaluation Studies as Topic; D006867:Hydrolases / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*; D007668:Kidney / Q000201:enzymology*; D007700:Kinetics; D016014:Linear Models; D017711:Nonlinear Dynamics
D004791:Enzyme Inhibitors; D006867:Hydrolases; D043383:Adenosylhomocysteinase; D000241:Adenosine