Tag Archives: kinetic properties
Purification and properties of a catalytically active fragment of soluble nucleoside triphosphatase from bovine kidney
А. F. Makarchikov
Grodno State Agricultural University, Belarus;
e-mail: a_makarchikov@yahoo.com
A catalytic fragment of soluble NTPase has been isolated from bovine kidneys.The 236-fold purification was carried out to obtain the preparation with a specific activity of 37.7 U/mg of protein. The purification scheme included the enzyme extraction followed by four column chromatography steps. The catalytic fragment was activated with divalent metal ions, had a pH optimum of 7.0, and possessed specificity for ITP, GTP, UTP and XTP. The apparent Km for Mg–ITP, Mg–GTP and Mg–UTP complexes were calculated from Hanes plots to be 1.70 mM, 0.93 mM and 0.48 mM, respectively. As estimated by gel filtration and SDS-PAAGE, the catalytic fragment has Mw 54.7 kDa being composed of two identical polypeptide chains. Our results suppose soluble NTPase from bovine kidney to consist of regulatory and catalytic structural units.
Identification of thiamine monophosphate hydrolyzing enzymes in chicken liver
I. K. Kolas, A. F. Makarchikov
Grodno State Agrarian University;
Institute of Biochemistry of Biologically Active Compounds,
National Academy of Sciences of Belarus, Grodno;
e-mail: a_makarchikov@yahoo.com
In animals, thiamine monophosphate (TMP) is an intermediate on the path of thiamine diphosphate, the coenzyme form of vitamin B1, degradation. The enzymes involved in TMP metabolism in animal tissues are not identified hitherto. The aim of this work was to study TMP hydrolysis in chicken liver. Two phosphatases have been found to contribute to TMP hydrolysis in liver homogenate. The first one, possessing a maximal activity at pH 6.0, is soluble, whereas the second one represents a membrane-bound enzyme with a pH optimum of 9.0. Membrane-bound TMPase activity was enhanced 1.7-fold by 5 mM Mg2+ ions and strongly inhibited by levamisole in uncompetitive manner with Ki of 53 μM, indicating the involvement of alkaline phosphatase. An apparent Km of alkaline phosphatase for TMP was calculated from the Hanes plot to be 0.6 mM. The soluble TMPase has an apparent Km of 0.7 mM; this enzyme is Mg2+ independent and insensitive to levamisole. As estimated by gel filtration on a Toyopearl HW-55 column, the soluble enzyme has a molecular mass of 17.8 kDa, TMPase activity being eluted simultaneously with peaks of flavinmononucleotide and p-nitrophenyl phosphatase activity. Thus, TMP appears to be a physiological substrate for a low-molecular weight acid phosphatase, also known as low-molecular-weight protein phosphotyrosine phosphatase.
Properties of chicken liver membrane-associated thiamine triphosphatase
I. K. Kolas, A. F. Makarchikov
Grodno State Agrarian University,
Institute of Biochemistry of Biologically Active Compounds,
National Academy of Sciences of Belarus;
e-mail: a_makarchikov@yahoo.com
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.