A simple preparation method for apoaspartate aminotransferase from Escherichia coli B, and its application for the assay of pyridoxal and pyridoxamine 5'-phosphate.
A simple and rapid preparation method for apoaspartate aminotransferase from Escherichia coli B was developed. A crude extract of the bacterial cells was treated batchwise with DEAE-cellulose. The enzyme fraction obtained was then applied to a pyridoxamine-Sepharose column. Apoaspartate aminotransferase was eluted with 50 mM potassium phosphate buffer (pH 7.0), and found to be electrophoretically homogeneous. The apoenzyme preparation thus obtained showed very low holoenzyme activity (only 0.4% of the activity seen in the fully saturated condition with pyridoxal 5'-phosphate) and was successfully used for assaying pyridoxal and pyridoxamine 5'-phosphate.
D001051:Apoenzymes / Q000302:isolation & purification*; D001059:Apoproteins / Q000302:isolation & purification*; D001219:Aspartate Aminotransferases / Q000302:isolation & purification*; D002846:Chromatography, Affinity; D004926:Escherichia coli / Q000201:enzymology*; D011732:Pyridoxal Phosphate / Q000032:analysis*; D011733:Pyridoxamine / Q000031:analogs & derivatives* / Q000032:analysis
D001051:Apoenzymes; D001059:Apoproteins; D011732:Pyridoxal Phosphate; D011733:Pyridoxamine; D001219:Aspartate Aminotransferases; C010627:pyridoxamine phosphate