Purification of phosphoenolpyruvate carboxykinase (GTP) by affinity chromatography on agarose-hydrazide-GTP.
The cytosolic form of phosphoenolpyruvate carboxykinase (GTP; EC 4.1.1.32) from rat liver was purified by a procedure involving affinity chromatography on agarose-hydrazide-GTP. Phosphoenolpyruvate carboxykinase is retained quantitatively by the affinity medium in the presence of manganese and can be specifically eluted by a pulse of GTP. On the contrary, no binding to agarose-hydrazide-GTP occurs in the absence of manganese. This suggests that the affinity of the enzyme for GTP is enhanced by prior interaction with manganese. A combination of several conventional purification steps followed by affinity chromatography provides pure phosphoenolpyruvate carboxykinase in good yields. The final specific activity is 19 U/mg protein. The enzyme migrates as a single polypeptide of molecular weight 70,600 during electrophoresis on sodium dodecyl sulfate polyacrylamide gels.
D000818:Animals; D002846:Chromatography, Affinity / Q000379:methods; D003600:Cytosol / Q000201:enzymology; D004591:Electrophoresis, Polyacrylamide Gel; D006160:Guanosine Triphosphate; D006834:Hydrazines; D008099:Liver; D008297:Male; D008345:Manganese / Q000378:metabolism; D008970:Molecular Weight; D010729:Phosphoenolpyruvate Carboxykinase (GTP) / Q000302:isolation & purification*; D051381:Rats; D012685:Sepharose
D006834:Hydrazines; D008345:Manganese; D006160:Guanosine Triphosphate; D012685:Sepharose; D010729:Phosphoenolpyruvate Carboxykinase (GTP)