Role of isovaleryl-CoA dehydrogenase and short branched-chain acyl-CoA dehydrogenase in the metabolism of valproic acid: implications for the branched-chain amino acid oxidation pathway.
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Effect of an oxygen-tolerant bifurcating butyryl coenzyme A dehydrogenase/electron-transferring flavoprotein complex from Clostridium difficile on butyrate production in Escherichia coli.
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Short chain acyl-coenzyme A dehydrogenase (SCAD) deficiency. Immunochemical demonstration of molecular heterogeneity due to variant SCAD with differing stability.
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Using a [35S]methionine labeling/immunoprecipitation technique, we have previously shown that cultured skin fibroblast from three patients with short chain acyl-CoA dehydrogenase (SCAD) deficiency each synthesize a normal-sized (41 kD) variant SCAD in an amount comparable to that of normal cells. In the current study, these same cell lines were reexamined with immunoblot analysis. In one cell line (YH2065) no SCAD protein was detectable. In the other two deficient cell lines, the amount of variant SCAD was similar to, or only slightly less than, normal. These results suggested that SCAD-YH2065 is labile. In the pulse-labeling experiments, labeled SCAD was readily detectable for at least 30 h in a normal control and two other SCAD-deficient cell lines. In contrast, the labeled SCAD band in YH2065 cells was barely detectable at 6 h and undetectable at 20 h. [35S]Methionine-labeling in the presence of rhodamine 6G demonstrated that SCAD-YH2065 was synthesized as a 44-kD precursor and imported normally into mitochondria, as were the normal SCAD and two other variant SCADs, excluding the possibility that SCAD-YH2065 is a truncated precursor that cannot be imported into mitochondria. These results indicate that the mutations responsible for SCAD deficiency are heterogeneous, and emphasize the importance of using both radiolabeling and immunoblotting when evaluating such genetic defects at the protein level. (+info)
Large-scale preparation and reconstitution of apo-flavoproteins with special reference to butyryl-CoA dehydrogenase from Megasphaera elsdenii. Hydrophobic-interaction chromatography.
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A new method is described for the large-scale reversible dissociation of flavoproteins into apoprotein and prosthetic group using hydrophobic-interaction chromatography. Lipoamide dehydrogenase from Azotobacter vinelandii and butyryl-CoA dehydrogenase from Megasphaera elsdenii are selected to demonstrate the usefulness of the method. In contrast to conventional methods, homogeneous preparations of apoproteins in high yields are obtained. The apoproteins show high reconstitutability. The holoenzymes are bound to phenyl-Sepharose CL-4B at neutral pH in the presence of ammonium sulfate. FAD is subsequently removed at pH 3.5-4.0 by addition of high concentrations of KBr. Large amounts of apoenzymes (200-500 mg), showing negligible residual activity, are eluted at neutral pH in the presence of 50% ethylene glycol. The holoenzyme of lipoamide dehydrogenase can be reconstituted while the apoprotein is still bound to the column or the apoenzyme can be isolated in the free state. In both cases the yield and degree of reconstitution of holoenzyme is more than 90% of starting material. Apo-lipoamide-dehydrogenase exists mainly as a monomer in solution and reassociates to the native dimeric structure in the presence of FAD. The apoenzyme is stable for a long period of time when kept in 50% ethylene glycol at -18 degrees C. Steady-state fluorescence-polarization measurements of protein-bound FAD indicate that reconstituted lipoamide dehydrogenase possesses a high stability which is governed by the low dissociation rate constant of the apoenzyme-FAD complex. The holoenzyme of butyryl-CoA dehydrogenase cannot be reconstituted when the apoenzyme is bound to the column. However, stable apoprotein can be isolated in the free state yielding 50-80% of starting material, depending on the immobilization conditions. The coenzyme A ligand present in native holoenzyme is removed during apoprotein preparation. The apoenzyme is relatively stable when kept in 50% ethylene glycol at -18 degrees C. From kinetic and gel filtration experiments it is concluded that the reconstitution reaction of butyryl-CoA dehydrogenase is governed by both the pH-dependent hydrodynamic properties of apoenzyme and the pH-dependent stability of reconstituted enzyme. At pH 7, the apoenzyme is in equilibrium between dimeric and tetrameric forms and reassociates to a native-like tetrameric structure in the presence of FAD. The stability of reconstituted enzyme is strongly influenced by the presence of CoA ligands as shown by fluorescence-polarization measurements. The degree of reconstitution of butyryl-CoA dehydrogenase is more than 80% of the original specific activity under certain conditions.(ABSTRACT TRUNCATED AT 400 WORDS) (+info)