A rapid assay for tyrosine hydroxylase activity, an indicator of chronic stress in laboratory and domestic animals.
Tyrosine hydroxylase (TH) (EC 1.14.16.2) activity has been frequently employed as a marker of adrenomedullar catecholamine-synthesizing capacity and, thus, as an indicator of chronic stress exposure in various animal species. We have developed a thin layer chromatography (TLC) procedure for its assay in adrenal glands of rats and large animals that reduces some of the drawbacks of currently employed methods, thereby facilitating routine use. Preparation of tissue samples was adapted for rats and pigs. The activity of the enzyme is expressed as the rate of the TH-catalysed tyrosine hydroxylation to 3,4-dihydroxyphenyl-alanine (DOPA) using tritium-labeled tyrosine, in the presence of cofactors and a DOPA decarboxylase inhibitor. The subsequent separation of the radioactive product (DOPA) from the substrate (tyrosine) is accomplished by TLC on silicagel plates, in a n-butanol/acetic acid/water solvent system (4:1:1). Radioactivity in the scraped zones, in which DOPA has been detected by means of an internal standard, is measured by beta-counting. An advantage of this procedure is its simplicity, reliability, and convenience for routine assays. Levels of endogenous adrenal tyrosine (HPLC assay) are considerably higher in pig (2.5-5 nmol/mg protein) than in rat (0.15 nmol/mg protein); their effects upon assay results being, in both cases, negligible. Michaelis constants estimated by this procedure amounted to 0.9 mmol l(-1) (at 0.7 mM DMPH4) for pig, and 1.1 mmol l(-1) (at 1.5 mM DMPH4) for rat.
Chobotská
K
K
Department of Animal Science, Swiss Federal Institute of Technology, Zürich.
Arnold
M
M
Werner
P
P
Pliska
V
V
D000313:Adrenal Medulla / Q000502:physiology; D000818:Animals; D000829:Animals, Domestic / Q000502:physiology; D000830:Animals, Laboratory / Q000502:physiology; D002395:Catecholamines / Q000032:analysis; D002855:Chromatography, Thin Layer / Q000379:methods; D004295:Dihydroxyphenylalanine / Q000032:analysis; D004298:Dopamine / Q000032:analysis; D004837:Epinephrine / Q000032:analysis; D007700:Kinetics; D009638:Norepinephrine / Q000032:analysis; D051381:Rats; D013312:Stress, Physiological / Q000201:enzymology*; D013552:Swine; D014443:Tyrosine / Q000378:metabolism; D014446:Tyrosine 3-Monooxygenase / Q000378:metabolism*
D002395:Catecholamines; D014443:Tyrosine; D004295:Dihydroxyphenylalanine; D014446:Tyrosine 3-Monooxygenase; D004298:Dopamine; D009638:Norepinephrine; D004837:Epinephrine