The Ukrainian Biochemical Journal (Jun 2015)

Properties of chicken liver membrane-associated thiamine triphosphatase

  • I. K. Kolas,
  • A. F. Makarchikov

DOI
https://doi.org/10.15407/ubj87.03.037
Journal volume & issue
Vol. 87, no. 3
pp. 37 – 46

Abstract

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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 Km 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.

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