Quantitation of S-adenosylhomocysteine hydrolase in mouse L929 cells using the inhibitor neplanocin A.
S-Adenosylhomocysteine (AdoHcy) hydrolase (EC 3.3.1.1) catalyzes the reversible hydrolysis of AdoHcy to adenosine and homocysteine. Neplanocin A, a cyclopentyl analog of adenosine, has previously been shown to act as a tight-binding inhibitor of the purified bovine liver enzyme, binding with a stoichiometry of one molecule per tetramer of enzyme (R.T. Borchardt, B.T. Keller, and U.G. Patel-Thombre, 1984, J. Biol. Chem. 259, 4353-4358). In the current study neplanocin A was also shown to act as a stoichiometric inhibitor of the L929 cell enzyme having Ki = 0.2 nM. Using this inhibitor to titrate the AdoHcy hydrolase, the concentration of the enzyme in intact L929 cells was calculated to be 0.8 microM, assuming a 1:1 inhibitor:protein stoichiometry. It was observed that the specific activity of AdoHcy hydrolase as measured in the hydrolytic direction increased 270% over a 12-h period after L929 cells were given fresh serum-free medium or when the cell extract was dialyzed first against phosphate buffer. Using the neplanocin A titration technique, it was found that the enzyme concentration in L929 cells remained constant over a 48-h period after refeeding the cultures. These results suggest the presence of an endogenous inhibitor or a readily reversible-type enzyme modification which is responsible for regulating AdoHcy hydrolase in vivo.
D000241:Adenosine / Q000031:analogs & derivatives / Q000494:pharmacology; D043383:Adenosylhomocysteinase; D000818:Animals; D002021:Buffers; D002851:Chromatography, High Pressure Liquid; D003956:Dialysis; D006863:Hydrogen-Ion Concentration; D006867:Hydrolases / Q000032:analysis* / Q000037:antagonists & inhibitors; D007739:L Cells / Q000201:enzymology*; D051379:Mice; D010710:Phosphates; D011485:Protein Binding
D002021:Buffers; D010710:Phosphates; C031096:neplanocin A; D006867:Hydrolases; D043383:Adenosylhomocysteinase; D000241:Adenosine