Aromatic Aminotransferase Research Articles

Studies of brain aminotransferase and psychotropic agents—I: Interaction of β-β'-iminodipropionitrile and rat brain aromatic aminotransferases

Rat cerebral phenyl (tyrosine and phenylalanine) and indolyl (tryptophan and 5-hydroxytryptophan) aminotransferases used in this study were separated and partially purified from acetone precipitation extracts on hydroxyapatite chromatography and Sephadex G-200 gel filtration columns eluted with phosphate buffers of increasing molarity. The substrate specificity and kinetics of these aromatic aminotransferases have been determined. The effect of various concentrations of pyridoxal phosphate on both the enzymic activity and the stability were investigated. The administration in vivo of β-β'-iminodipropionitrile (IDPN) was found to markedly decrease the specific activity of L-5-hydroxytryptophan:2-oxoglutarate aminotransferase, whereas phenylalanine, tyrosine and tryptophan aminotransferases remained unaltered. Studies in vitro showed that IDPN inhibits the aminotransferases in the following order—5-HTP-T > Tyr-T > Phe-T ⩾ Tryp-T. On the basis of kinetic studies, IDPN was found to inhibit the Tyr-T and 5-HTP-T in competition with its substrate, the K i for these reactions being 4.3 × 10 −3M (Tyr at pH 8.35) and 3.8 × 10 −3M (5-HTP at pH 8.30). The possibility of IDPN competing with the amino group acceptors (2-oxoglutarate or oxalacetate) or with its coenzyme, pyridoxal phosphate, has been eliminated. In addition, it was shown that IDPN produced a greater inhibitory effect on these aminotransferases than a structurally similar compound, β-aminopropionitrile (BAPN). It is suggested therefore that this observation may account for their dissimilar physiological role; IDPN is a psychotropic agent, whereas BAPN is an osteolathyrogen.

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

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.

A new radioactive assay procedure of aromatic aminotransferase

A rapid and sensitive assay of aromatic aminotransferase activity based on semimicro cation-exchange paper chromatography to separate 14C- l-glutamic acid from 14C-α-ketoglutaric acid is described. Its sensitivity, specificity, and broad flexibility constitute some of the principal advantages over the existing procedure. The stoichiometry of the reaction indicated a 1:1 relationship between the amount of phenylpyruvic and glutamic acid formed over a 2 hr interval.