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Tyrosine aminotransferase in isolated rat liver cells

Marston, Fiona A. O. (1979) Tyrosine aminotransferase in isolated rat liver cells. Doctor of Philosophy (PhD) thesis, University of Kent. (doi:10.22024/UniKent/01.02.94510) (KAR id:94510)


Tyrosine aminotransferase (TAT; L-tyrosine: 2- oxoglutarate transaminase) catalyses the first reaction of tyrosine catabolism in the liver. Glucagon, hydrocortisone, triamcinolone and dexamethasone each induced a significant increase in TAT activity, in liver cells isolated from fed, adrenalectomized rats. Glucagon and glucocorticoid hormones appear to act by independent mechanisms. In cells isolated from fed, normal rats, glucagon and each glucocorticoid hormone together increased TAT significantly, but alone had no effect on the enzyme activity. Glucocorticoid hormones, but not glucagon, stimulated an increase in TAT activity in cells isolated from fasted rats. Cycloheximide and cordycepin each prevented glucagon- and glucocorticoid-stimulated induction of TAT. In addition cycloheximide decreased the basal activity of TAT but lactate dehydrogenase activity remained unchanged. At a concentration of 2.5mM tryptophan stimulated a significant increase in TAT activity in isolated liver cells from fed and adrenalectomized rats. The increase was small compared with that obtained after in vivo administration of tryptophan. The latter effect must be primarily an indirect one. TAT activity also increased when the level of amino acids in the medium was elevated 8-fold. The effect was not attributable to tryptophan and in combination with hydrocortisone additive increases in TAT activity were stimulated. TAT in crude cell extracts was separated into at least 3 forms by ion-exchange chromatography. A crude particulate liver extract promoted interconversion of the forms, thus they are post-translational modifications of the same enzyme. Polyacrylamide gel electrophoresis separated TAT from crude extracts into 3 bands; these were detected using a specific activity stain. However, the forms of TAT separated by ion-exchange chromatography each migrated to the same position in acrylamide gels. One band was identified as glutamate-oxaloacetate transaminase, which reacts with tyrosine to a limited extent. When TAT activity in cells isolated from adrenalectomized rats was induced by glucagon or glucocorticoid hormones, the forms, separated by ion-exchange chromatography, increased to the same extent. During transamination, the side-chain C-2-H of tyrosine is released. An assay for TAT was developed using L-[side chain 2,3-3H3]tyrosine; 3H2O formed was isolated by adsorption of the other H-compounds onto charcoal. With this assay at least 20µU TAT can be detected. Exchange of both the 2- and 3-C-3H was detected; but the latter occurred subsequent to transamination and required a factor present in crude extracts. By including L-[side chain 2,3-3H] tyrosine in the incubation medium, TAT activity in intact isolated liver cells was measured from the 3H2O formed. The rate of transamination was dependent on the concentration of tyrosine and the enzyme exhibited Michaelis-Menten kinetics. A Km for tyrosine of 2.2mM-2.5mM was estimated. The time course of the changes in TAT activity in vitro during induction by hydrocortisone correlated with changes measured in cells. 2.5mM tryptophan decreased the rate of tyrosine transamination in cells and the results were consistent with competition between tryptophan and tyrosine for uptake or transamination. The increase in the rate of transamination in cells in the presence of elevated amino acid levels was attributed to the increased tyrosine concentration and masked the smaller, significant increase in TAT activity measured in vitro.

Item Type: Thesis (Doctor of Philosophy (PhD))
DOI/Identification number: 10.22024/UniKent/01.02.94510
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Uncontrolled keywords: tyrosine catabolism, liver
Subjects: Q Science > QH Natural history > QH581.2 Cell Biology
Divisions: Divisions > Division of Natural Sciences > Biosciences
SWORD Depositor: SWORD Copy
Depositing User: SWORD Copy
Date Deposited: 23 May 2023 15:38 UTC
Last Modified: 23 May 2023 15:38 UTC
Resource URI: (The current URI for this page, for reference purposes)

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