Polyol-pathway enzymes of human brain. Partial purification and properties of sorbitol dehydrogenase.
Sorbitol dehydrogenase was isolated from human brain and purified 690-fold, giving a final specific activity of 11.1 units/mg of protein. The enzyme preparation was nearly homogeneous, but was unstable at most temperatures. It exhibited a broad pH optimum of 7.5-9.0 in the forward reaction (i.e. sorbitol leads to fructose), and of 7.0 in the reverse reaction (i.e. fructose leads to sorbitol). Substrate-specificity studies demonstrated that the enzyme had the capability to oxidize a wide range of polyols and that the enzyme had a higher affinity for substrates in the forward reaction than in the reverse reaction, e.g. Km for sorbitol was 0.45 mM, and that for fructose was 480 mM. However, the Vmax. was 10 times greater in the reverse reaction. At high concentrations of fructose (500 mM) the enzyme exhibited substrate inhibition in the reverse reaction. The enzyme mechanism was sequential, as determined by the kinetic patterns arising from varying the substrate concentrations. In addition, both fructose and NADH protected the enzyme against thermal inactivation. These findings, together with product-inhibition data, suggested that the mechanism is random rapid equilibrium with two dead-end complexes.
D001921:Brain / Q000201:enzymology*; D001923:Brain Chemistry; D004591:Electrophoresis, Polyacrylamide Gel; D004376:Galactitol / Q000378:metabolism; D006358:Hot Temperature; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D007064:L-Iditol 2-Dehydrogenase / Q000037:antagonists & inhibitors / Q000302:isolation & purification / Q000378:metabolism*; D009243:NAD / Q000494:pharmacology; D011794:Quercetin / Q000494:pharmacology; D013012:Sorbitol / Q000378:metabolism; D013379:Substrate Specificity; D013401:Sugar Alcohol Dehydrogenases / Q000378:metabolism*; D013439:Sulfhydryl Reagents / Q000494:pharmacology
D013439:Sulfhydryl Reagents; D009243:NAD; D004376:Galactitol; D013012:Sorbitol; D011794:Quercetin; D013401:Sugar Alcohol Dehydrogenases; D007064:L-Iditol 2-Dehydrogenase