Phosphoenolpyruvate carboxylase of Escherichia coli. Hydrophobic chromatography using specific elution with allosteric inhibitor.
The adsorption of Escherichia coli phosphoenolpyruvate carboxylase [EC 4.1.1.31] to butyl-, hexyl-, and octyl-Sepharose gels was investigated. The enzyme was nearly completely adsorbed to the latter two gels both in the absence and presence of high concentrations of ammonium sulfate. At intermediate concentrations--0.1 M in the case of hexyl-Sepharose--virtually no adsorption was observed. Upon application of an increasing or decreasing concentration gradient of the salt, the enzyme was eluted at various concentrations of the salt depending on chain length of the immobilized alkyl groups. The adsorption to hexyl-Sepharose at 0.7 M ammonium sulfate was markedly decreased by L-aspartate, the allosteric inhibitor, whereas it was increased by acetyl-CoA, one of the allosteric activators. Evidence was obtained suggesting that these changes in adsorption were due to conformational alterations of the enzyme elicited by these effectors. The enzyme seemed to have been adsorbed at its hydrophobic regions which were distinct from the allosteric site for long-chain fatty acids. The specific elution with L-aspartate in the presence of 0.82 M ammonium sulfate could successfully be applied to purification of the enzyme. By this hydrophobic interaction chromatography, the enzyme was purified about 55-fold over its partially purified preparation with a recovery of 73%. The obtained enzyme preparation was almost homogeneous as judged from sodium dodecylsulfate-polyacrylamide gel electrophoresis.
D000327:Adsorption; D000645:Ammonium Sulfate; D002262:Carboxy-Lyases / Q000302:isolation & purification*; D002846:Chromatography, Affinity / Q000379:methods; D004926:Escherichia coli / Q000201:enzymology*; D010730:Phosphoenolpyruvate Carboxylase / Q000037:antagonists & inhibitors / Q000302:isolation & purification*; D011506:Proteins / Q000032:analysis
D011506:Proteins; D002262:Carboxy-Lyases; D010730:Phosphoenolpyruvate Carboxylase; D000645:Ammonium Sulfate