Purification and characterization of glycerol-3-phosphate dehydrogenase (flavin-linked) from rat liver mitochondria.
We have purified the membrane-intrinsic glycerol-3-phosphate dehydrogenase from both normal and hyperthyroid rat liver mitochondria by extraction with Triton X-100, hydrophobic affinity chromatography, ion exchange chromatography, gel filtration, and FAD-linked Sepharose 4B affinity chromatography. The yields in both cases were over 20%, and purification ranged from 800- to 650-fold in mitochondria from hyperthyroid and normal rats, respectively. The final preparations appeared to be greater than 95% pure by polyacrylamide gel electrophoresis in the presence or absence of sodium dodecyl sulfate. The pure enzyme focused at pH 5.5 and produced a biphasic thermal inactivation plot at 50 degrees C. The holoenzyme was found to have a molecular mass of 250,000 daltons on gel filtration. The subunit molecular mass was found to be 74,000 daltons +/- 3,000 by sodium dodecyl sulfate-gel electrophoresis and high-performance liquid chromatography gel filtration in 0.1% sodium dodecyl sulfate. 1 mol of the holoenzyme preparation contains 1.1 mol of non-heme iron and 0.7-0.9 mol of noncovalently bound FAD. The absorption spectrum has a maximum at 375 nm and a shoulder at 450 nm which is bleached on treatment with sodium dithionite. The enzymatic reaction is competitively inhibited by glyceraldehyde 3-phosphate, dihydroxyacetone phosphate, phosphoenolpyruvate, and phosphoglycolic acid. The apparent Km for DL-alpha-glycerol 3-phosphate and noncovalently bound FAD were found to be 6 mM and 7 microM, respectively.
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D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002846:Chromatography, Affinity; D004591:Electrophoresis, Polyacrylamide Gel; D005182:Flavin-Adenine Dinucleotide / Q000032:analysis / Q000378:metabolism*; D005993:Glycerolphosphate Dehydrogenase / Q000302:isolation & purification* / Q000378:metabolism; D006980:Hyperthyroidism / Q000378:metabolism; D007700:Kinetics; D008930:Mitochondria, Liver / Q000201:enzymology*; D051381:Rats; D013961:Thyroid Gland / Q000502:physiology
D000596:Amino Acids; D005182:Flavin-Adenine Dinucleotide; D005993:Glycerolphosphate Dehydrogenase