CopurifiCation of ChiCken liver soluble thiamine monophosphatase and low moleCular weight aCid phosphatase

Thiamine monophosphatase (ThMPase) is an enzyme of thiamine metabolism in animals whose molecular 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 chromatographic 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.

Thiamine monophosphatase (ThMPase) is an enzyme of thiamine metabolism in animals whose molecular 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 chromatographic 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.
T hiamine (vitamin B 1 ) fulfills vital biochemical functions in all living organisms studied to date.In animals, thiamine diphosphate (ThDP) is well known to serve as a coenzyme for at least five enzyme systems involved in ener gy producing and metabolism of carbohydrates, branchedchain amino acids as well as 3methyl branched fatty acids [1].Usually, ThDP accounts for about 80% of the total thiamine content in the cell.Apart from its coenzyme form, several other phosphorylated deriva tives of vitamin B 1 are present in biological objects , namely thiamine monophosphate (ThMP), thiamine triphosphate (ThTP) and thiamine adenosine triphosphate (AThTP).Quantitatively, the amount of these compounds, with some exceptions in the case of ThTP, varies from less than 1% for AThTP and ThTP to 215% for ThMP in terms of tissue total thiamine content [27].While evidence exists indicating both ThMP and AThTP are likely to be involved in mechanisms of shortterm biochemical adaptation [8,9], a possible biological role of ThMP has not been recog nized yet.In bac-teria, yeast and plants this compound was found to be an intermedia te on thiamine biosynthesis pathway, the enzyme thiamine phosphate synthase (EC 2.5.1.3)joining 4amino5hydroxymethyl 2methylpyrimidine pyrophosphate and 4methyl5 βhydroxyethylthiazole monophosphate to produce ThMP.Some bacterial species, like Escherichia coli and S. typhimurium, are also capable of direct phosphorylation of thiamine to ThMP by the enzyme thiamine kinase (EC 2.7.1.89)[10].In animal cells, ThMP is believed to be produced exclusively by the hydrolysis of ThDP.In addition, ThMP can be transported into the cell from the blood plasma, where it constitutes up to 80% of total thiamine, through reduced folate carrier, RFC1 [11].The hydrolysis to thiamine is then a necessary step for ThMP to serve as a substrate in coenzyme synthesis by thiamine pyrophosphokinase (EC 2.7.6.2) in eukaryo tic organisms [1,12].Little knowledge is available from the literature concer ning the nature of ThMP hydrolyzing enzymes in animal tissues.It is only known that this compound could be a substrate for alkaline phosphatase (ALKP, EC 3.1.3.1)[13,14] and fluorideresistant (prostatic) acid phosphatase (PAP, EC 3.1.3.2) [15,16], both enzymes being membrane bound proteins.Quite recently, we have revealed a soluble ThMPase activity in chicken liver and hypothesized that this activity might belong to a low molecularweight acid phosphatase (LMWAP), also known as a lowmolecularweight protein phosphotyrosine phosphatase (LMWPTP) [17].The present work is to study ThMPase subcellular distribution as well as to purify and characterize its basic properties in chicken liver.
All operations on preparing subcellular fractions were performed at 04 °C (in an ice bath).A sample of fresh liver tissue was forced through a 1mm pore press and homogenized by 10 strokes in a teflon/glass PotterElvehjem device at 600 rpm in 10 volumes of 10 mM TrisHCl buffer, pH 7.4, containing 0.25 M sucrose, 0.5 mM EDTA and 0.1 mM MgCl 2 .The homogenate was passed through four layers of wet gauze and centrifuged at 600 g for 10 min.The pellet was washed by suspending in 10 volumes of the initial buffer and centrifuging (600 g, 10 min) to give a crude nuclear fraction.To prepare mitochondria the postnuclear supernatants were pooled and subjected to further centrifugation at 12 000 g for 10 min.The particles were washed once with the same medium yielding a crude mitochondrial fraction.Postmitochondrial supernatants were then combined and adjusted to pH 7.5 with 0.5 M TrisHCl, pH 8.0, followed by the addition of CaCl 2 to a final concentration of 15 mM.In two hours, the microsomal fraction was obtained as a precipitate (14 000 g, 10 min), while the remaining supernatant represented the fraction of cytosol.The particular fractions were suspended in 50 mM TrisHCl buffer, pH 7.3, containing 0.15 M KCl, 0.2 mM EDTA and frozen at -80 °C along with the cytosol for storage.
The standard mixture for ThMPase activity determination contained 25 mM Trismaleate buffer, pH 6.0, 1 mM ThMP, 20 µg of bovine serum albumin (BSA) and an aliquot of enzyme solution in a total volume of 0.2 ml.The reaction was carried out for 2060 min at 37 °C and stopped by adding 0.1 ml of 10% TCA.Then the mixture was centrifuged (10 min, 2500 rpm), and the amount of inorganic phosphate (P i ) liberated during the reaction was determined in accordance to the method of Lanzetta P. A.et al. [22].Kinetic experiments required some alterations in the assay with respect to pH, substrate concentration or metal composition; it is indicated as necessary.During ionexchange chromatography, when the enzyme elution was carried out with phosphate, ThMPase activity was monitored by HPLC.In this case, TCA was extracted prior to analysis with 9 volumes of diethyl ether.Then a 50 µl aliquot was oxidized with 30 µl of 4.3 mM potassium ferricyanide in 12.5% NaOH, diluted with 0.9 ml of 50 mM Naphosphate buffer, pH 7.2, and adjusted to pH ~8.0 adding 35 µl of 1 M HCl.A 10µl sample was injected into the chromatographic system (Agilent 1200) and the separation was performed at 25 °C at a flow rate of 1 ml/min on a Zorbax SBC18 column (3×100 mm) in 50 mM NaH 2 PO 4 , pH 8.0, contai ning 2.5 % tetrahydrofuran.Thiochrome derivatives of thiamine and ThMP were quantified using a fluorometric detector (λ ex = 365 nm, λ em = 433 nm).
Acid phosphatase (AP) activity was assayed by incubating an aliquot of enzyme solution for 510 min in a total volume of 0.1 ml of 25 mM Tris maleate buffer, pH 6.0, containing 10 µg of BSA and 0.5 mM pNPP.The reaction was terminated by addition of 1.0 ml of 0.1 M NaOH, and the absorbance of the solution was measured at 405 nm after centrifugation (10 min, 2500 rpm).The amount of pnitrophenol formed was calculated from ε 405 = 18 300 M -1 cm -1 .
When studying the enzyme substrate specifici ty, the amounts of Pi liberated were measured under conditions of ThMPase assay.
A unit of activity (U) was defined as the amount catalyzing the formation of 1 µmole of P i per min.Specific activity was expressed in units per mg of protein.
Molecular mass of the purified enzyme was estimated by gel filtration on a Sephacryl S200 column (1.0×27 cm) equilibrated with 20 mM TrisHCl, pH 7.5, containing 0.1 M NaCl, and calibrated with standard size proteins: cytochrome c (12.4 kDa), αchymotrypsinogen (25.7 kDa), ovalbumin (45 kDa), BSA (67 kDa) and LDH (135 kDa).The sample of 0.5 ml was applied to the column and eluted at a flow rate of 5 cm/h.Enzyme activi ty was located in the eluate (V e ) by both ThMPase and acid phosphatase assays; the void volume (V o ) of the column was determined using blue dextran 2000.The value of molecular mass was calculated from the plot of log M r versus the log of V e /V o ratio.
Protein was determined from the absorbance at 280 nm or according to the method of Bradford [23] with BSA as a standard.
Statistical calculations were done with Graph-Pad Prism 5.0.The means ± standard errors are presented for the data.

results and discussion
Though the presence of ThMP in animal tissues was shown more than 50 years ago, not much data concerning its enzymology have appeared since then.Keesling seems to become the first who studied ThMPase activity in rat tissues in 1960 [24], observing two pH optima at 6.0 and 9.0 in the acetone powders of the kidney and brain, but only the acid one in the liver.However, it was not until 1982, when Ogawa K. et al. [14] based on experiments with levamisole inhibition of ThMPase activity in mouse tissues at pH 8.5 concluded that the enzyme appears to be ALKP.It was also the time when ThMPase stained in smalldiameter dorsal root ganglia neurons was recognized as a fluoride resistant AP, wellknown histochemical marker [15].More recently, Zylka M. J. et al. [16] have found this fluorideresistant TMPase to be identical to the transmembrane isoform of PAP.
The above nonspecific enzymes would be expected to participate in ThMP metabolism in animals, but it is evident that neither ALKP nor PAP both being membranebound ectophosphatases concern ThMP cleavage inside the cell.So the molecular nature of TMPase is currently unknown.In the search for an enzyme which would be a constituent of the intracellular thiamine metabolizing system in birds, we started studying some properties of ThMPase activity in chicken liver [17].Two phosphatases were revealed to contribute to ThMP hydrolysis in liver homogenate.One of them possessing a pHoptimum of 9.0 was a membrane bound enzyme inhibited strongly by levamisole in an uncompetitive manner with K i of 53 μM, indica ting the involvement of ALKP.On the other hand, at acidic pH (5.06.0) the activity proved to be due to the presence of a soluble phosphatase.The last one had a molecular mass of 17.8 kDa, as estima ted by gel filtration of liver extract, and seemed to show activity toward pNPP and FMN along with ThMP.Analysing the literature led us to hypothe size that the soluble ThMPase might be identical to LMWAP which is widely expressed in eukaryotic organisms, including chicken liver [25].In an attempt to identify this ThMPase we have conducted some further research.At first, the localization of acid ThMPase activity was examined in subcellular fractions isolated from chicken liver according to a standard protocol of differential centrifugation.As can be seen in Fig. 1, about 69% of total ThMPase activity was recovered in the cytosol.This fraction also contained more than 80% of LDH, while only a few percents of total DNA, succinate dehydrogenase and glucose6phosphatase were measurable as contaminants (data not shown).The mitochondrial and microsomal fractions contained up to 18% of ThMPase activity altogether.A relatively high amount (13%) of TMPase activity measured in the nuclear fraction was probably due to unbroken and damaged cells, which were pelleted with nuclei.It is also possible that PAP, which was shown to be expressed in low amounts in human liver [26], contributes to ThMPase activity in the particular fractions.Thus, based on the overlapping of ThMPase activity profile with the distribution of LDH, one may conclude that soluble acid ThMPase of chicken liver is localized in the cytosol like LMWAP, which has been known for few decades to be a cytosolic enzyme [27].
Further, we carried out the purification of soluble ThMPase from chicken liver tracing both ThMPase and pNPPase activity during chromatographic steps.The purification procedure included 8 steps.
Step 1. Extract preparation.Chicken li vers (100 g) stored at -20 °C were thawed, cut into pieces and homogenized with 250 ml of 50 mM TrisHCl buffer, pH 7.5, containing 0.15 M KCl, 0.2 mM EDTA, 5 mM DTT and 0.1 mM PMSF, in an MWP1 blender at a maximal speed for 2 min.The homogena te was centrifuged at 10 000 g for 60 min.The supernatant was passed through 16 layers of gauze and used as a crude extract.
Step 2. acid treatment.Acetic acid (1 M) was slowly added to the extract under constant stirring to lower pH to 5.0.In 30 min protein aggregates were precipitated by centrifugation (10 000 g, 30 min) and discarded.Cold 1 M TrisHCl, pH 8.9, was then added to the supernatant to give a pH value of 7.5.
Step 3. ammonium sulfate fractionation.The solution from the previous step was brought to 35% saturation by adding solid (NH 4 ) 2 SO 4 .After 30 min of gentle stirring, the precipitate was removed by centrifugation at 10 000 g for 30 min.Then the second portion of ammonium sulfate was added to 60% saturation, the suspension was stirred for 30 min and the precipitate was collected by centrifugation (10 000 g, 30 min).
Step 4. Gel filtration on Sephadex G-75.The precipitate was dissolved in MES buffer (20 mM MES, 50 mM NaCl, 1 mM EDTA, 5 mM DTT, pH 6.0) to give approximately 40 ml volume, and chromatographed on a Sephadex G75 column (5×75 cm) in the same buffer at a flow rate of 5 cm/h.Fractions of 6.5 ml were collected and analyzed for ThMPase and pNPPase activities as well as protein content.The elution profiles for both activities proved to be coincident (data not shown).The frac-tions enhanced in specific activity as compared with the previous step were pooled for further processing.
Step 5. Ion exchange chromatography.The combined fractions were applied to an SP Sephadex C50 column (1.6×25 cm) equilibrated with MES buffer.The column was washed with the same buffer and elution was carried out with a 300 ml linear gradient of increasing phosphate concentration from 0 to 100 mM at a flow rate of 15 cm/h.Like in step 4, both ThMPase and pNPPase emerged from the column in the same fractions (data were not shown).The fractions possessing a high specific activity were combined for the next step.
Step 6. ammonium sulfate precipitation.To prepare the enzyme for further purification, the solution was salted out with solid (NH 4 ) 2 SO 4 at 80% saturation and the aggregated proteins were collec ted by centrifugation for 30 min at 10 000 g.
Step 7. Gel filtration on Sephadex G-50.The precipitate from the previous step was dissolved in MES buffer to give a final volume of 5 ml and loaded on a Sephadex G50 column (2.5×100 cm) equilibra ted with the same buffer.The chromatography was carried out at a flow rate of 5 cm/h and fractions of 6 ml were collected.As one might expect, ThMPase and pNPPase elution profiles matched again (Fig. 2), implying the involvement of the same enzyme.The active fractions were pooled and subjected to further purification.
Step 8. Servacel P-23 chromatography.The fractions from the previous step were applied on a Servacel P23 column (0.7×3 cm) equilibrated with MES buffer and the column was washed with 10 ml of the same buffer.The enzyme did not bind to the resin emerging from the column in the effluent, which was then concentrated with Corning SpinX UF Concentrators and used for analysis.The resul ting preparation had a specific ThMPase activity of 2.5 U/mg exceeding 714fold the activity of the extract.Summary of the purification procedure is shown in Table 1.
Thus, during purification ThMPase and pNPPase activity coeluted on each chromatographic step, indicating they reside on the same protein.
As a comment on the purification procedure, it is to be noted the unexpectedly high effectiveness of ThMPase precipitation from SP Sephadex C50 effluent.This manipulation gave a 3.4fold increase in specific activity with respect to the previous step.Evidently, this was due to much lower ThMPase solu bility as compared to contaminating proteins.After the final ThMPase solution was concentrated, an apparent molecular mass of the enzyme was estimated by gel filtration on a calibrated Sephacryl S200 column (1.0×27 cm).ThMPase was found to emerge from the column as a single activi ty peak.The standard proteins gave a linear plot of log Mr versus log V e /V o , from which the appa rent molecu lar mass of ThMPase was calculated to be 18 kDa (data not shown).This value matches the molecular mass of LMWAP from various sources which is known being a small protein of 1421 kDa as determined by similar techniques [25,2832].
Kinetic experiments revealed that the purified enzyme exhibits a belllike dependence of ThMPase activity on the acidity of the reaction medium in the range of pH values from 4.5 to 7.5, with pH optimum being observed around pH 5.5 (data not shown).None of the divalent cations tested, such as Mg 2+ , Mn 2+ or Ca 2+ , each at 5 mM concentration, affected the rate of ThMP hydrolysis in the presence of the enzyme (data not shown).
The studying of the effect of ThMP concentrations on an initial reaction velocity at pH 5.5 in the absence of metal ions revealed the enzyme to obey a classical MichaelisMenten kinetics giving a hyperbolic saturation curve (Fig. 3), with K m of 0.90 ± 0.07 mM and V max of 8.6 ± 0.3 µmol/min/mg as estimated by nonlinear regression fit.The same values of kinetic parameters (K m = of 0.91 mM, V max = 8.1 µmol/min/mg) were also computed from the Hanes plot.It is to be noted, that the K m of the enzyme for ThMP in the chicken liver extract was evalua ted before to be 0.73 ± 0.02 mM [17].Thus, in gene ral, the kinetic behavior of purified  is very close to the kinetics observed in the crude extracts [17].
Table 2 shows the hydrolysis rates of various phosphate monoesters by the chicken liver enzyme.Among compounds tested only pNPP, FMN, phosphotyrosine and ThMP were efficiently hydrolyzed.No hydrolysis of other substances, including βglycerophosphate, glucose6phosphate, fructose1,6diphosphate, IMP, AMP, NADP and pyridoxal5phosphate, occurred at appreciable rates.Essentially, the same restricted substrate specificity towards pNPP, FMN and phosphotyrosine is charac teristic of LMWAP [28,3235].

Fig. 2 .Fractions
Fig. 2. Elution profiles of protein, ThMPase and low molecular weight acid phosphatase (reduced twofold) from a Sephadex G-50 column.The volume of each fraction is 6 ml Fractions Enzyme activity, U/l 20 40 60 80

Fig. 3 .
Fig. 3. Effect of varying ThMP concentrations on the initial reaction velocity.Inset: The Hanes plot of the data.The reaction was carried out for 30 min at 37 °C in the mixture consisted of 25 mM Tris-maleate, pH 5.5, 20 µg of BSA and 0.19 µg of the purified enzyme preparation in a volume of 0.2 ml [ThMP], µM