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 µM and a V of 0.25 µmol/min/mg. In the direction of synthesis the K_m for adenosine was 5.6 µM and V 0.53 µmol/min/mg. The enzyme activity was inhibited in the presence of adenosine with a K_i = 3 µM. 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: K_d1 = 6.8 ± 0.7nM 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 = 705+0.9 µM and B_max3 = 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°C only 5-10% and at 20°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.

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