Purification and characterization of glycerol-3-phosphate dehydrogenase (flavin-linked) from rat liver mitochondria

• Published in: The Journal of biological chemistry 1986-06, Vol.261 (17), p.8042-8048
• Main Authors: Garrib, A, McMurray, W C
• Format: Article
• Language: English
• Subjects: Amino Acids - analysis; Analytical, structural and metabolic biochemistry; Animals; Biological and medical sciences; Chromatography, Affinity; Electrophoresis, Polyacrylamide Gel; Enzymes and enzyme inhibitors; Flavin-Adenine Dinucleotide - analysis; Flavin-Adenine Dinucleotide - metabolism; Fundamental and applied biological sciences. Psychology; Glycerolphosphate Dehydrogenase - isolation & purification; Glycerolphosphate Dehydrogenase - metabolism; Hyperthyroidism - metabolism; Kinetics; Mitochondria, Liver - enzymology; Oxidoreductases; Rats; Thyroid Gland - physiology
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1016/S0021-9258(19)57509-3

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.