Enzymatic measurement of saccharopine with saccharopine dehydrogenase.
We have developed an enzymatic method for measuring saccharopine, a key intermediate in lysine metabolism. With the enzyme saccharopine dehydrogenase, saccharopine can be oxidized to lysine and alpha-ketoglutarate with the corresponding conversion of NAD to NADH. The natural equilibrium favors saccharopine formation, but using hydrazine to trap one of the products, alpha-ketoglutarate, shifts the reaction toward quantitative oxidation of saccharopine. A stable endpoint is reached in 15-20 min, and although high concentrations of alpha-ketoglutarate slow the reaction, the end product is fully recovered. Unlike previous assays this technique is specific, convenient, and capable of measuring saccharopine directly in protein-free biological fluids or extracts.
D000818:Animals; D004590:Electrophoresis, Paper; D008099:Liver / Q000378:metabolism; D008239:Lysine / Q000031:analogs & derivatives* / Q000032:analysis* / Q000096:biosynthesis; D008297:Male; D009243:NAD / Q000096:biosynthesis / Q000378:metabolism; D010084:Oxidation-Reduction; D000587:Oxidoreductases Acting on CH-NH Group Donors; D051381:Rats; D011919:Rats, Inbred Strains; D012443:Saccharopine Dehydrogenases
D009243:NAD; D000587:Oxidoreductases Acting on CH-NH Group Donors; D012443:Saccharopine Dehydrogenases; D008239:Lysine; C100169:saccharopine