ISSN 2409-4943. Ukr. Biochem. J., 2017, Vol. 89, N 6 UDC 577.164.11:577.152.3 doi: https://doi.org/10.15407/ubj89.06.013 COPURIFICATION OF CHICKEN LIVER SOLUBLE THIAMINE MONOPHOSPHATASE AND LOW MOLECULAR WEIGHT ACID PHOSPHATASE 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: [email protected] Thiamine monophosphatase (ThMPase) is an enzyme of thiamine metabolism in animals whose mo- lecular nature has still to be elucidated. In this study we have achieved a 714-fold purification of a soluble enzyme possessing ThMPase activity from a chicken liver extract. In addition to ThMPase, acid phosphatase activity was traced during purification. Both activities proved to have coincident elution profiles at all chro- matographic steps implying the same enzyme involved. The molecular weight of the enzyme was 18 kDa as estimated by gel filtration. Along with ThMP and p-nitrophenyl phosphate, the purified enzyme was capable of hydrolyzing flavin mononucleotide as well as phosphotyrosine. Subcellular distribution of ThMPase activity was also explored indicating its cytosolic localization. The results of the present work imply the involvement of low molecular weight acid phosphatase in thiamine metabolism in the chicken liver. K e y w o r d s: thiamine monophosphatase, low molecular weight acid phosphatase, HPLC, chicken liver. hiamine (vitamin B1) fulfills vital biochemi‑ teria, yeast and plants this compound was found to cal functions in all living organisms studied be an intermedia te on thiamine biosynthesis path‑ T to date. In animals, thiamine diphosphate way, the enzyme thiamine phosphate synthase (ThDP) is well known to serve as a coenzyme (EC 2.5.1.3) joining 4­amino­5­hydroxymethyl­ for at least five enzyme systems involved in ener­ 2­methylpyrimidine pyrophosphate and 4­methyl­5­ gy producing and metabolism of carbohydrates, β­hydroxyethylthiazole monophosphate to produce branched­chain amino acids as well as 3­methyl­ ThMP. Some bacterial species, like Escherichia coli branched fatty acids [1]. Usually, ThDP accounts for and S. typhimurium, are also capable of direct phos‑ about 80% of the total thiamine content in the cell. phorylation of thiamine to ThMP by the enzyme Apart from its coenzyme form, several other phos‑ thiamine kinase (EC 2.7.1.89) [10]. In animal cells, phorylated deriva tives of vitamin B1 are present in ThMP is believed to be produced exclusively by the biological objects , namely thiamine monophosphate hydrolysis of ThDP. In addition, ThMP can be trans‑ (ThMP), thiamine triphosphate (ThTP) and thiamine ported into the cell from the blood plasma, where adenosine triphosphate (AThTP). Quantitatively, it constitutes up to 80% of total thiamine, through the amount of these compounds, with some excep‑ reduced folate carrier, RFC1 [11]. The hydrolysis tions in the case of ThTP, varies from less than 1% to thiamine is then a necessary step for ThMP to for AThTP and ThTP to 2­15% for ThMP in terms serve as a substrate in coenzyme synthesis by thia‑ of tissue total thiamine content [2­7]. While evi‑ mine pyrophosphokinase (EC 2.7.6.2) in eukaryo­ dence exists indicating both ThMP and AThTP are tic organisms [1, 12]. Little knowledge is available likely to be involved in mechanisms of short­term from the literature concer ning the nature of ThMP biochemical adaptation [8, 9], a possible biological hydrolyzing enzymes in animal tissues. It is only role of ThMP has not been recog nized yet. In bac‑ known that this compound could be a substrate for © 2017 Kolas I. K., Makarchikov A. F. This is an open­access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. 13 ISSN 2409-4943. Ukr. Biochem. J., 2017, Vol. 89, N 6 alkaline phosphatase (ALKP, EC 3.1.3.1) [13, 14] and to a final concentration of 15 mM. In two hours, the fluoride­resistant (prostatic) acid phosphatase (PAP, microsomal fraction was obtained as a precipitate EC 3.1.3.2) [15, 16], both enzymes being membrane­ (14 000 g, 10 min), while the remaining supernatant bound proteins. Quite recently, we have revealed a represented the fraction of cytosol. The particular soluble ThMPase activity in chicken liver and hy‑ fractions were suspended in 50 mM Tris­HCl buffer, pothesized that this activity might belong to a low­ pH 7.3, containing 0.15 M KCl, 0.2 mM EDTA and molecular­weight acid phosphatase (LMW­AP), also frozen at –80 °C along with the cytosol for storage. known as a low­molecular­weight protein phospho‑ Lactate dehydrogenase (LDH, EC 1.1.1.27), tyrosine phosphatase (LMW­PTP) [17]. The present succinate dehydrogenase (EC 1.3.5.1) and glucose­ work is to study ThMPase subcellular distribution as 6­phosphatase (EC 3.1.3.9) activities, as well as well as to purify and characterize its basic properties DNA content, were assayed in subcellular fractions in chicken liver. by conventional methods [18­21]. The standard mixture for ThMPase activity de‑ Materials and Methods termination contained 25 mM Tris­maleate buffer, Fresh livers of broiler chicken ROSS 308 were pH 6.0, 1 mM ThMP, 20 µg of bovine serum albu‑ obtained from local poultry farm, placed on ice min (BSA) and an aliquot of enzyme solution in a and frozen during about 1 h after the animals were total volume of 0.2 ml. The reaction was carried out killed. The tissues were stored at –20 °C before use. for 20­60 min at 37 °C and stopped by adding 0.1 ml Sephadex G­50, Sephadex G­75, SP Sephadex C­50 of 10% TCA. Then the mixture was centrifuged and Sephacryl S­200 were purchased from Phar‑ (10 min, 2500 rpm), and the amount of inorganic macia (Uppsala, Sweden), Servacel P­23, ThMP, phosphate (Pi) liberated during the reaction was phenylmethylsulfonyl fluoride (PMSF), pyridoxal­ determined in accordance to the method of Lanzet‑ 5­phosphate and protein molecular weight markers ta P. A.et al. [22]. Kinetic experiments required some were from Serva (Heidelberg, Germany), dithio­ alterations in the assay with respect to pH, substrate threitol (DTT), p­nitrophenyl phosphate (p­NPP), concentration or metal composition; it is indicated flavin mononucleotide (FMN), phosphotyrosine, as necessary. During ion­exchange chromatography, β­glycerophosphate, glucose­6­phosphate, fruc‑ when the enzyme elution was carried out with phos‑ tose­1,6­diphosphate, inosine 5′­monophosphate, phate, ThMPase activity was monitored by HPLC. In adenosine 5′­monophosphate, 2­morpholinoethane‑ this case, TCA was extracted prior to analysis with sulfonic acid (MES) and trichloroacetic acid (TCA) 9 volumes of diethyl ether. Then a 50 µl aliquot was were from Sigma (St. Louis, Mo., USA). All other oxidized with 30 µl of 4.3 mM potassium ferricya‑ chemicals were of analytical grade. nide in 12.5% NaOH, diluted with 0.9 ml of 50 mM All operations on preparing subcellular frac‑ Na­phosphate buffer, pH 7.2, and adjusted to pH ~8.0 tions were performed at 0­4 °C (in an ice bath). A adding 35 µl of 1 M HCl. A 10­µl sample was in‑ sample of fresh liver tissue was forced through a jected into the chromatographic system (Agilent 1­mm pore press and homogenized by 10 strokes in 1200) and the separation was performed at 25 °C at a teflon/glass Potter­Elvehjem device at 600 rpm in a flow rate of 1 ml/min on a Zorbax SB­C18 column 10 volumes of 10 mM Tris­HCl buffer, pH 7.4, con‑ (3×100 mm) in 50 mM NaH2PO4, pH 8.0, contai ning taining 0.25 M sucrose, 0.5 mM EDTA and 0.1 mM 2.5 % tetrahydrofuran. Thiochrome derivatives of thiamine and ThMP were quantified using a fluoro‑ MgCl2. The homogenate was passed through four layers of wet gauze and centrifuged at 600 g for metric detector (λex = 365 nm, λem = 433 nm). 10 min. The pellet was washed by suspending in 10 Acid phosphatase (AP) activity was assayed volumes of the initial buffer and centrifuging (600 g, by incubating an aliquot of enzyme solution for 10 min) to give a crude nuclear fraction. To prepare 5­10 min in a total volume of 0.1 ml of 25 mM Tris­ mitochondria the post­nuclear supernatants were maleate buffer, pH 6.0, containing 10 µg of BSA pooled and subjected to further centrifugation at and 0.5 mM p­NPP. The reaction was terminated 12 000 g for 10 min. The particles were washed once by addition of 1.0 ml of 0.1 M NaOH, and the ab‑ with the same medium yielding a crude mitochon‑ sorbance of the solution was measured at 405 nm drial fraction. Post­mitochondrial supernatants were after centrifugation (10 min, 2500 rpm). The amount then combined and adjusted to pH 7.5 with 0.5 M of p­nitrophenol formed was calculated from ε = 18 300 M‑1cm–1. Tris­HCl, pH 8.0, followed by the addition of CaCl2 405 14 I. K. Kolas, A. F. Makarchikov When studying the enzyme substrate specifici­ concern ThMP cleavage inside the cell. So the mo‑ ty, the amounts of Pi liberated were measured under lecular nature of TMPase is currently unknown. In conditions of ThMPase assay. the search for an enzyme which would be a con‑ A unit of activity (U) was defined as the stituent of the intracellular thiamine metabolizing system in birds, we started studying some proper‑ amount catalyzing the formation of 1 µmole of Pi per min.