Affinity chromatography on 2',5'-ADP-Sepharose 4B for purification of malic enzyme from crustacean muscle.
Shrimp abdomenal muscle NADP-dependent malic enzyme (E.C.1.1.1.40) was purified about 1500-fold to a specific activity of 48 units (mumol/min)/mg at 30 degrees C with good quantitative recovery in three chromatographic steps, including affinity chromatography on 2',5'-ADP-Sepharose 4B, a "substrate activation" method using malate substrate plus manganese chloride. In addition to the malate-manganese chloride substrate pair, succinate or glutamate plus manganese chloride or magnesium chloride could be used in this "substrate activation" method for crustacean NADP-malic enzyme purification on 2',5'-ADP-Sepharose 4B. Affinity chromatography alone purified malic enzyme almost 43 fold, and the overall method resulted in homogeneous enzyme since polyacrylamide gel electrophoresis of the native purified enzyme revealed only a single band staining for protein and enzyme activity.
D000645:Ammonium Sulfate; D000818:Animals; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D033364:Decapoda / Q000378:metabolism*; D004591:Electrophoresis, Polyacrylamide Gel; D007202:Indicators and Reagents; D008291:Malate Dehydrogenase / Q000302:isolation & purification*; D009124:Muscle Proteins / Q000032:analysis; D009132:Muscles / Q000201:enzymology*; D012685:Sepharose / Q000031:analogs & derivatives
C025246:2',5'-ADP-sepharose; D007202:Indicators and Reagents; D009124:Muscle Proteins; D012685:Sepharose; D008291:Malate Dehydrogenase; D000645:Ammonium Sulfate