Abstract
The enzymes involved in thiamine triphosphate (ThTP) metabolism in birds are not characterized so far. The aim of the present work was to study some properties of ThTPase in chicken liver. In liver homogenate, ThTPase activity has been found to display a bell-like pH-profile with a maximum of 5.5-6.0. Low activity was observed without divalent metal ions, while the addition of Mg2+ or Ca2+, each at 5 mM concentration, enhanced the rate of ThTP hydrolysis by a factor of 17-20. In the presence of 5 mM Mg2+ an apparent K(m) of the enzyme for ThTP was estimated by the method of non-linear regression as well as from the Hanes plot to be 1.7-2.2 mM. Monovalent anions such as I-, SCN-, NO3-, Br-, Cl- (at 150 mM concentration) showed inhibitory effect decreasing the rate of ThTPase reaction by 20-60%. After the homogenate was centrifuged, more than 85% of ThTPase activity was revealed in the fraction of insoluble particles indicating a membrane localization of the enzyme. The precipitate treatment with 1% sodium deoxycholate caused about 53% solubilization of the activity. During Toyopeal HW-55 chromatography, ThTPase activity was eluted simultaneously with ATPase and ITPase peaks in the void volume of the column. Thus, a non-specific high molecular mass protein complex seems to be involved in ThTP hydrolysis in the chicken liver. The chicken liver phosphatase is clearly distinguishable from all membrane-bound ThTPases reported previously.
Highlights
Т иаминтрифосфат (ТТР) – трифосфорный эфир тиамина – впервые синтезирован в 1948 г
In the presence of 5 mM Mg2+ an apparent Km of the enzyme for thiamine triphosphate (ThTP) was estimated by the method of non-linear regression as well as from the Hanes plot to be 1.7-2.2 mM
Monovalent anions such as I, SCN, NO3, Br, Cl– showed inhibitory effect decreasing the rate of ThTPase reaction by 20-60%
Summary
Участвующие в метаболизме тиаминтрифосфата (ТТР) в тканях птиц, в настоящее время не охарактеризованы. Цель данной работы заключалась в исследовании свойств тиаминтрифосфатазы (ТТРазы) печени кур (Gallus gallus). После центрифугирования гомогената печени более 85% ТТРазной активности обнаруживалось в осадке, что свидетельствует о мембранной локализации энзима. В тканях млекопитающих при обычных физиологических условиях концентрация ТТР поддерживается на низком уровне (порядка 30–400 пмоль/л) благодаря экспрессии растворимой тиаминтрифосфатазы (ТТРаза, 3.6.1.28) – Mg2+-зависимого энзима с Мм 25 кДа, обладающего абсолютной субстратной специ фичностью [11,12,13]. Цель данной работы состояла в изучении основных характеристик энзима, катализирующего гидролиз ТТР в печени кур (Gallus gallus); энзимы с ТТРазной активностью из тканей птиц в настоящее время не описаны
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