Abstract
A simple purification for the membrane-associated, flavin-linked, glycerol-3-phosphate dehydrogenase of Escherichia coli has been developed which yields homogeneous enzyme in a detergent-solubilized state. 1. The dissociated form of the enzyme has a molecular weight of 58,000 and contains 0.5 mol of FAD/mol of protein monomer. 2. The solubilized enzyme-catalyzed reaction has a pH profile and temperature dependence similar to that observed for the membrane-bound enzyme. 3. The most efficient electron acceptor is potassium ferricyanide but phenazine methosulfate, methylene blue, menadione, and dichlorophenolindophenol can also be utilized. 4. The reaction is competitively inhibited by dihydroxyacetone phosphate, phosphoenolpyruvate, phosphoglycolic acid, glyceraldehyde-3-phosphate, and D-2- and D-3-phosphoglyceric acid. 5. The activity of the enzyme is regulated in a complex manner by ATP and GTP. 6. Detergent-depleted enzyme can be functionally reconstituted with Escherichia coli membrane vesicles to support glycerol-3-phosphate-dependent active transport of L-proline. 7. Detergent-depleted enzyme requires exogenous phospholipid or nondenaturing detergent for electron transfer activity.
Highlights
A simple purification for the membrane-associated, flavin-linked, glycerol-3-phosphate dehydrogenase of Escherichia coli has been developed which yields homogeneous enzyme in a detergent-solubilized state
(10) was found to contain a low molecular weight protein contaminant which migrated with the bromphenol blue tracking dye in 10% SDS-polyacrylamide gel electrophoresis
A simple purification procedure has been developed for the isolation of large quantities of the enzyme
Summary
Cavalli, (glpRc, phoA, relA, ton A,,) and Strain 8 (glpR”, glpD, phoA, rel A, ton A,,) were kindly provided by Dr E. Enzyme activity was eluted with 20 mM potassium phosphate, pH 6.6, containing 20% ethylene glycol and. For phospholipid activation studies and sedimentation equilibrium ultracentrifugation, the column eluate was dialyzed for 48 h against column elution buffer This resulted in a fraction with a specific activity of 1.5 unitslmg. 0.43 ml of membrane vesicles (0.4 mg/ml in 0.1 M potassium phosphate buffer, pH 6.61 was added detergent-depleted enzyme (0.05 to 0.20 units) and the volume was brought to 0.5 ml with buffer. The vesicle pellet was resuspended in 10 pl of 0.1 M potassium uhosuhate buffer, PH 6.6 To this was added 10 ~1 of 40 mM M&O,, i0 pi of 160 mM ni-glycerol-3-P or 10 ~1 of 20 mr.r n(-)-lactate, and 10 ~1 of 40 pM L-[‘*Clproline (25.4 mCi/mmol). When Brij 58 was present, standard bovine serum albumin solutions were prepared with the same concentration of Brij
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