Purification of the bovine lens isozymes which reduce fructose diphosphate to sorbitol diphosphate.
Three isozymes of an enzyme which reduce fructose 1, 6-diphosphate (FDP) to sorbitol 1, 6-diphosphate (SDP) in the presence of NADH have been purified from bovine lens. The isozymes were fractionated by acid precipitation of the lens homogenate followed by DE-52 column chromatography. This step separated the FDP reducing activity into three major peaks, peak 1, peak 2, and peak 3. Each of these peaks were further purified by affinity chromatography using Reactive blue-2-agarose, Sephadex G-150 gel filtration, and DE-52 column chromatography. Polyacrylamide disc gel electrophoresis demonstrated the presence of one major isozyme and one minor isozyme in each of the three peaks. The Km values for FDP were 8.0, 5.7, and 4.7 mM for peaks 1, 2, and 3 respectively. The reaction product SDP was characterized by nuclear magnetic resonance spectroscopy. All the isozymes utilized pyruvate as substrate with the Km for peaks 1, 2, and 3 being 0.63, 0.20, and 0.09 mM respectively. These studies therefore indicate that FDP reducing activity and lactate dehydrogenase activity co-purify and may be expressed by the same enzyme protein.
D000818:Animals; D002237:Carbohydrate Dehydrogenases; D002417:Cattle; D004591:Electrophoresis, Polyacrylamide Gel; D005635:Fructosediphosphates / Q000378:metabolism*; D006598:Hexosediphosphates / Q000378:metabolism*; D006600:Hexosephosphates / Q000378:metabolism; D007527:Isoenzymes / Q000302:isolation & purification*; D007770:L-Lactate Dehydrogenase / Q000302:isolation & purification*; D007908:Lens, Crystalline / Q000201:enzymology*; D009682:Magnetic Resonance Spectroscopy; D009243:NAD / Q000378:metabolism; D011773:Pyruvates / Q000378:metabolism
D005635:Fructosediphosphates; D006598:Hexosediphosphates; D006600:Hexosephosphates; D007527:Isoenzymes; D011773:Pyruvates; D009243:NAD; C035752:sorbitol 6-phosphate; D002237:Carbohydrate Dehydrogenases; C051169:fructose-1,6-diphosphate reductase; D007770:L-Lactate Dehydrogenase