Abstract
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.
Highlights
3-phosphate dehydrogenase from both normal and hy- from rabbit skeletal muscle and brain (15)
Very little is known of FAD-linked Sepharose 4B affinity chromatography. the structure and properties of this enzyme because of its The yields in bothcases were over 20‘70,and purifica- extreme insolubility and thedifficulty of purifying it in good tfpfeirinuonocrnameelrabopnhyrrgyepaeppobdaelsryfreartahontciycmroeyrnolosaifdma8psi0aodp0ndeed-iagurtemneoldo6edr5ltmeoo0cd-atrefrlbaocoetlypsdlh,gisnorrueresleafpsatmeietscerit.ttioivThnceahhlneyoth.np9edTu5rhrip%aeeresy-a(usieenIlnmdftroahondimsidfinepiodnar)pmFeaarAnul nDwamne-lodidndhekifyseicpedredigrbftleohyryctmrheor.eiodlp-3rua-rptifhliicovasetpirohmnatietoodfcehthhoyenddrarocigatievtnoeenzyme focused at pH 5.5 and produced a biphasic over 95% homogeneity and from 650- to 800-foldpurification, thermal inactivation plot at 50 ‘C. The holoenzyme respectively.The holoenzyme has been characterized in terms was found to have a molecular mass of 250,000 daltons of pH stability and temperature inactivation
We report here the purification to homogeneity of the FADlinked glycerol-3-phosphatedehydrogenase from rat liver mitochondria
Summary
3-phosphate dehydrogenase from both normal and hy- from rabbit skeletal muscle and brain (15). It has a single perthyroid rat liver mitochondria by extraction with polypeptide of molecular weight000 when analyzed by Triton X-100, hydrophobic affinity chromatography, SDS-polyacrylamide gel electrophoresis andhas variable ion exchangechromatography, gel filtration,and amounts of FAD and non-heme iron. The holoenzyme respectively.The holoenzyme has been characterized in terms was found to have a molecular mass of 250,000 daltons of pH stability and temperature inactivation. The subunmitolecular mass was found for its substrate andcofactor have been determined, and the to be 74,000 daltons f 3,000 by sodium dodecyl sul- effects of various substrate analogs have been studied. A fate-gel electrophoresis and high-performance liquid preliminary report of this study has been presented (16). Hyperthyroidism was induced by the method of Lee and Lardy (4)
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