S-Adenosylhomocysteine-Hydrolase from Bovine Kidney: Enzymatic and Binding Properties

Abstract

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.

S-Adenosyl-l-homocysteine Hydrolase Regulates Aldosterone-induced Na+ Transport

“…1) consistent with that reported previously for SAHHase (11,19). Overexpression of SAHHase increased enzyme activity in both HEK293 (Fig.…”

“…4A) was similar to hydrolytic activity reported in crude extract of guinea pig cardiac muscle (18) and bovine kidney (19); however, activity was greater compared with crude extract of cat, rabbit, rat, and dog heart (18). Aldosterone repletion for 4 h to A6 cell monolayers doubled SAHHase activity to a level similar to that observed in rat liver extract (20).…”

Effect of Vitamin B6 Deficiency on S-Adenosylhomocysteine Hydrolase Activity as a Target Point for Methionine Metabolic Regulation

“…There were two explanations for the mechanism of SAH accumulation. One explanation is the inhibition of SAH hydrolysis by adenosine, because SAH hydrolase is an adenosine binding protein and bound adenosine was reported to inhibit the activity (20). Another explanation is due to enhancement of the SAH-synthetic rate from adenosine and Hcy.…”

Localization of S-adenosylhomocysteine Hydrolase in the Rat Kidney

“…SAH hydrolase was purified from bovine kidney using chromatographical techniques as described previously (Kloor et al 1996). The purified enzyme was frozen at Ϫ 20C until use.…”

“…SAH hydrolase activity controls the cytosolic concentration of SAH, which is the most potent endogenous inhibitor of trans methylations (Schatz et al 1981). In addition, SAH hydrolase binds adenosine with high affinity and with a capacity of 4 moles adenosine/mol SAH hydrolase, and is one of the adenosine binding proteins in the cytosol of the kidney (Hershfield and Kredich 1978;Kloor et al 1996). This adenosine binding could be responsible for the relatively high adenosine tissue content of the kidney under normoxic conditions (Osswald et al 1977).…”