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 K-m of 5.0 +/-1.2 mu M and a V of 0.25 gamma mu mol/min/mg. In the direction of synthesis the K-m for adenosine was 5.6 mu M and V 0.53 mu mol/min/mg. The enzyme activity was inhibited in the presence of adenosine with a K-i = 3 mu M. In a second set of experiments we determined the binding characteristics of [H-3]-adenosine to purified enzyme. The enzyme bound [H-3]-adenosine with three apparent affinities: K-d1 = 6.8+/-0.7 nM and B-max1 = 0.24+/-0.04 nmol/mg protein; K-d2 = 387+/-41 nM and B-max2 = 1.4 nmol/mg protein, and K-d3 = 7.05+/-0.9 mu M and B-max3 = 9 nmol/mg protein. Binding of 25 nM [H-3]-adenosine obeyed a monophasic reaction with a k(+1) value of 0.025 min/nM. Dissociation of [H-3]-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 [H-3]-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.