Brain aromatic aminotransferase: I. Purification and some properties of pig brain L-phenylalanine-2-oxoglutarate aminotransferase

Abstract

1. 1. Pig brain l-phenylalanine-2-oxoglutarate aminotransferase was purified approximately 900-fold. The purification procedure included preparation of cell-free extract by sonication, (NH 4) 2SO 4 fractionation, and adsorption on and elution from calcium phosphate gel chromatography. 2. 2. The assay used throughout the experiments described consisted of two procedures devised in this laboratory. One of these, a spectrophotometric assay employs a borate-arsenate enolizing mixture; the other, a radiometric procedure, makes use of ion-exchange paper and permits a direct determination of the percent of substrate converted to product. 3. 3. On polyacrylamide gel electrophoresis, the enzyme appears to be made up of one slow-moving component. Maximal activity was observed in the pH range 8.0–9.0 in both phosphate and arsenate buffer. The apparent Michaelis constants for l-phenylalanine and 2-oxoglutarate are 5·10 −2 M and 7.4·10 −4 M, respectively. 4. 4. Various studies concerning substrate, apoenzyme and coenzyme specificities, the effects of pyridoxal phosphate analogues, sulfhydryl reagents and metal ion chelators were carried out.