SUBCELLULAR DISTRIBUTION OF TYROSINE AMINOTRANSFERASE IN RAT BRAIN

Tyrosine aminotransferase activity was found in all regions of the adult rat brain. Enzymatic activity was determined in brain subcellular fractions prepared from homogenates by differential centrifugation or by continuous sucrose gradients. More than 95% of the total tyrosine aminotransferase activity was found to be associated with the particulate fractions and 65 to 70% of the total activity was found rather tightly bound in the mitochondrial fraction. The mitochondrial fraction was purified further by fractionation on discontinuous sucrose gradients (0.8 to 1.4 m). The resultant four mitochondrial subfractions were analyzed for tyrosine aminotransferase and monoamine oxidase activities and were examined for morphological structure with the electron microscope. The crude mitochondrial fraction contained mitochondria, some nerve endings, myelin, and membranous fragments. About two thirds of the total tyrosine aminotransferase activity was found in the highly purified mitochondrial fraction. Monoamine oxidase and tyrosine aminotransferase activities paralleled each other for the different mitochondrial fractions. Tyrosine aminotransferase from rat brain was most active with α-ketoglutarate (100%) as an amino group acceptor but also used oxaloacetic acid (75%) and pyruvate (25%). The apparent Km for tyrosine was 6.3 mm which made direct measurement of maximal velocities impractical due to limited solubility of tyrosine. With crude extracts various aromatic amino acids (tyrosine, monoiodotyrosine, tryptophan, phenylalanine, and dihydroxyphenylalanine) were found to undergo transamination. On the basis of initial velocities, tyrosine and monoiodotyrosine were the best substrates. The solubilized enzyme, partially purified 160-fold over the brain homogenate, catalyzed the transamination reaction equally well with oxaloacetate or α-ketoglutarate at 1 mm; however, oxaloacetate at 10 mm inhibited the reaction whereas α-ketoglutarate did not. © 1969.