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Phosphotransferase Activity in Acid Phosphatase in Camel Heart.

Byline: Shakil Abbas, Asma Saeed, Mehrin Sherazi, Umber Zaman, Ahmad Saeed and Rubina Naz

Summary: Two forms of low molecular weight acid phosphatase (LMW-ACPase) from camel heart were purified by salt fractionation, ion exchange chromatography, gel filtration and affinity chromatography. These were designated as Peak-1 and Peak-2. The peak-1 was purified 500-fold with specific activity of 28 U/ mg of protein and a yield of 3 %, while peak-2 was purified 850 times with specific activity of 44 U/ mg. The recovery was 4 %. The homogeneity of the enzymes was checked on SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Both forms showed similar patterns on SDS-PAGE and had molecular weight of 18 kDa. The LMW-ACPase activity was increased in the presence of alcohols. This activation reflected phosphotransferase activity.

The presence of various alcohols in the incubation mixture which acted as phosphate acceptors increased the overall rate of breakdown of substrate p-nitrophenyl phosphate as judged by the amount of p- nitrophenol released and the competition between hydrolysis and phosphate transfer reaction was started. The rate of transfer of phosphate group to phosphate acceptors was increased while the amount of inorganic phosphate (Pi) produced remained same and was equal to the amount of Pi released in water when no alcohol was added. As the alcohol concentrations increased, the rate of formation of p-nitrophenol and the rate of Pi transfer were greatly increased while the level of inorganic phosphate produced was found constant. Thus, smaller than 1 ratios of Pi / p-nitrophenol and greater ratios of phosphate transfer to hydrolysis showed the presence of transphosphorylation reaction.

Key Words: Low molecular weight acid phosphatase; Isoenzymes; Phosphotransferase activity; Phospho- enzyme covalent intermediate.

Introduction

Acid phosphatase (EC 3.1.1.2) belongs to hydrolase class which catalyzes the hydrolysis of phosphomonoesterases in acidic pH [1]. At least two classes of acid phosphatases have been reported in vertebrates [2]. These enzymes are distinguished from each other by localization in cell organelle, molecular mass, substrate specificity and sensitivity to inhibitors [3]. High molecular weight acid phosphatase class (Mr greater than 100 kDa) is lysosomal and low molecular weight acid phosphatase (Mr less than 20 kDa) enzymes are cytosolic in nature[4]. These phosphatases do not require metal ions for the activity [5]. Some acid phosphatases need Zn++ ions during its hydrolytic actions. These are called Zn dependent acid phosphatases which are found in animal tissues [6].

The low molecular weight acid phosphatases have been recognized as low Mr cytosolic phosphotyrosine protein phosphatases (PTPases) because these act on phosphorylated biomolecules such as immunoglobulins, casein and epidermal growth factor receptors [7, 8]. Beside its hydrolytic action, the low Mr PTPases demonstrate phosphotransferase activity [9-11].

Two distinct isoforms of LMW-ACPase exist in rat liver (AcP1 and AcP2), human erythrocytes (Bfast and Bslow) and human placenta (HCPTP-A and HCPTP-B) and these have been isolated, characterized and sequenced [12-14]. The AcP1, Bfast and HCPTP-A have been classified as isoform, IF-1 while AcP2, Bslow, HCPTP-B, Bovine heart PTPase and bovine liver PTPase as IF-2. The IF-1 and IF-2 differ from each other in type specific amino acid sequence of 40-73 regions, in substrate affinity and in the sensitivity to activators and inhibitors [15].

In our laboratory, we have purified and characterized these enzyme couples from liver of chicken and uromastix [16-17]. These differ in pyridoxal-5/-PO4 inhibition, cGMP activation and kinetic parameters. Both isoenzyme couples possess phosphotyrosine protein phosphatase activity also.

This paper describes the purification of low molecular weight acid phosphatase from camel heart and the resolution of two forms possessing phosphotransferase activity and some kinetic properties.

Experimental

Materials

The camel heart was purchased from local market. The cation exchanger SP-Sephadex C-50, the material for gel chromatography- Sephadex G-75, p- nitrophenyl phosphate (pNPP) and standard proteins for the determination of molecular weight by SDS- PAGE were obtained from Sigma while p-amino benzyl phosphonic acid-agarose gel was supplied from Pierce Chemical Co. The substrates and other chemicals related to gel-electrophoresis were arranged from local reputed firms.

Method

Enzyme Assays

Acid phosphatase activity was measured at 37C as described by Ramponi et al. [18] using 4 mM pNPP in 0.1M acetate buffer pH 5.5. The reaction was started by addition of enzyme solution in a final volume of 1 ml. After 5 min, the reaction was stopped by adding 4 ml of 1N KOH. The yellow color (p-nitrophenol ions) appeared, was measured at 405 nm. One unit of activity was defined as the amount of enzyme that was required to produce 1mol of p-nitrophenol from its substrate per min at 37C (405 =18,000 M-1 cm-1). Specific activity was expressed as number enzyme units per milligram of protein.

Phosphotransferase Activity

For phosphotransferase activity, the hydrolysis of pNPP was carried out under above conditions in the presence of various alcohols. The reaction mixture (0.5ml) containing 4mM pNPP, alcohol (10%), 0.1M acetate buffer pH 5.5 and catalytic amount of enzyme, was incubated at 37C for 5 min. For the determination of p-nitrophenol, the reaction was stopped by the addition of 0.5mL of 1N KOH and the absorbance at 405 nm was measured. The amount of p-nitrophenol was determined from the absorbance using p-nitrophenol standard curve.

For inorganic phosphate determination, the reaction was stopped by the addition of 0.2 mL of 10 % trichloroacetic acid and the molybdate color reaction was performed according to Black and Jones method [19] which was started by the addition of 0.5mL mixture (composed of 0.2ml of 2 % ammonium molybdate and 0.3mL of 14 % ascorbic acid in 50 % trichloroacetic acid). After 1-2.5 min, 1 ml solution containing 2 % trisodium citrate and 2 % sodium arsinate in 2 % acetic acid, was added. The color was developed for 30 min and absorption at 700 nm was measured. The amount of inorganic phosphate produced was calculated from the standard curve using KH2PO4 as a standard. The amount of phosphate transferred to alcohol was the difference between the amount of p-nitrophenol produced and inorganic phosphate produced.

Protein Determination

Protein concentration was determined by the Lowery method [20]. For column effluents, the relative protein concentration was estimated from absorbance at 280 nm.

SDS- Polyacrylamide Gel Electrophoreses

The SDS- polyacrylamide gel electrophoresis was performed according to method of Laemmli [21]. The samples of enzymes were prepared in sample buffer with and without reduction by AY-mercaptoethanol and heated at 95C for 5 min. The enzyme purity was checked in 12.5 % acrylamide mini-slab gel. The proteins in the gel were stained with coomassie blue and molecular weight estimate was made using standard size marker protein as indicated in respective Fig.

Enzyme Purification

Two forms of LMW-ACPase from camel heart were purified by a method described by Manao et al. [12] and Saeed et al. [22] The camel heart (1 Kg) was minced and then strirred with 3 volumes of 0.01M acetate buffer pH 5.0, containing 1mM EDTA, 1mM PMSF and 1mM AY-mercaptoethanol for 30 min. The mixture was homogenized in a Waring blender for 4 min with 0.5 min interval, then centrifuged at 2000xg for 60 min, The supernatants were combined for further process. Solid ammonium sulphate was added to it at 30% saturation level, then centrifuged at 2000xg for 60 min. The supernatant was brought to 60% saturation with ammonium sulphate and centrifuged at 2000xg for 60 min.

The resulting precipitate was dissolved in 300 ml of 0.01M acetate buffer pH 4.9, containing 1mM EDTA, 1mM PMSF and 1mM AY-mercaptoethanol and dialyzed against 5L of the same buffer for 36 h. The dialysed supernatant solution was centrifuged at 10,000xg for 30 min and clear solution was applied on a SP-Sephadex C-50 column (6.5x50cm) previously equilibrated with 0.01M acetate buffer pH 5.1, containing 1mM EDTA, 1mM PMSF and 1mM AY-mercaptoethanol. The column was washed with 2.5L of the same buffer to remove the unbound protein. Acid phosphatase activities were eluted with 0.3M sodium hydrogen phosphate that had been dissolved in above buffer. The most active fractions from peak were pooled and concentrated by adding 70% saturation ammonium sulphate.

The precipitate thus obtained, was collected by centrifugation at 3000xg for 30 min and dissolved in 25ml of 0.01M acetate buffer pH 5.1, containing 1mM EDTA, 1mM PMSF and 1mM AY-mercaptoethanol. Then enzyme was chromatographed in a Sephadex G-75 column (4.5x 85 cm) that was equilibrated and eluted with 0.01M acetate buffer pH 5.1, containing 0.1M NaCl, 1mM EDTA, 1mM PMSF and 1mM AY- mercaptoethanol. The active fractions were combined and the enzyme was concentrated by Amicon ultrafiltration using Ym 5 membrane to 7 mL. The enzyme from Sephadex G-75 chromatography was dialysed overnight against 1L of 0.2M citrate buffer pH6.5 containing 1mM EDTA and 1mM dithiothreitol.

Then it was applied to an affinity column (1x5 cm) of p-aminobenzyl phosphonic acid - Agarose that was equilibrated in the same buffer. The column was washed extensively with the said buffer to eliminate unbound proteins. During an early washing, some acid phosphatase form (Peak-1) began to elute together with contaminating proteins but at later stage pure acid phosphatase form was eluted. The acid phosphatase form (Peak-2) was eluted with 0.1M sodium phosphate in the same buffer. The most active fractions from both peaks were pooled separately and concentrated by ultrafiltration.

Results and Discussion

LMW-ACPases were purified to homogeneity. The purification steps are presented in Table-1 and their elution profiles by various chromatographic techniques are shown in Fig. 1(A- C). Ammonium sulphate fractionation step did not purify the enzyme but 50% of the activity was lost and a lot of unwanted proteins were removed. Thirty four folds purification was achieved by SP-Sephadex C-50 chromatography with recovery of 12 % with respect to starting material. Further, 10-folds purification was obtained with Sephadex G-75 column showing 10 % of total recovery.

The enzyme was further purified to almost homogeneity by affinity chromatography on p-aminobenzyl phosphonic acid -Agarose column. A total of 4.5 mg of both forms of LMW-ACPase was obtained. The form (Peak-1) was purified 500-folds while the form (Peak-2) was purified 850-folds. The overall recovery was 3 % and 4 % respectively. The form, peak-1 showed specific activity of 27 U/mg of protein while the form, peak-2 had a specific activity of 44 U/mg of protein. These results are comparable to isoenzymes AcP1 and AcP2 isolated from rat liver [12] and isoenzymes from other sources [23-24].

The homogeneity of the enzymes was checked on 12.5 % SDS-polyacrylamide gel electrophoresis. Both forms showed similar patterns: a main band corresponding to molecular weight of 18 kDa while a small band at 14 kDa was obtained (Fig. 2). Early reports on bovine liver and uromastix liver low Mr PTPases showed that the 14 kDa band is actually a NH2- terminus truncated fragment probably originated by proteolysis during purification [25-26]. The electrophoretic mobility was the same for both reduced and non- reduced enzymes, indicating that these forms of LMW-ACPase were monomeric proteins as reported earlier [27].

Many authors reported that LMW-ACPases have phosphotransferase activity [7,10,28] which had been helpful for identifying the rate limiting step in the hydrolysis of substrate reaction mechanism. LMW-ACPase activity (Peak-2) from camel heart was stimulated in the presence of different concentrations of monofunctional and polyfunctional alcohols (Table-2). Methanol, ethylene glycol and glycerol, at 20% concentrations activated the enzyme activity about two times). The polyfunctional alcohols (glycerol and ethylene glycol) were found better modifiers than methanol and ethanol. This behaviour is a reflection of phosphotransferase activity. Similar activations also appeared in many other LMW-ACPases [29].

Table-1: Purification of acid phosphatase from 1 kg of camel heart.

###Volume###Total activity###Total protein###Specific activity###Purification###Recovery

###Steps

###(ml)###(U)###(mg)###(U/mg)###factor###%

###Extract###2300###2093###39928###0.052###1###100

###30% (NH4)2 SO4 saturation###2450###1979###34545###0.057###1###94.6

###60% (NH4)2 SO4 saturation###300###1159###23460###0.049###1###55.3

###Dialysis and centrifugation###360###1062###21132###0.050###1###50.7

###SP-Sephadex C-50

###25###250###140.5###1.779###34###11.9

###Sephadex G-75

###7###208###11.4###18.24###351###9.9

###Affinity chromatography

###7.5###66.4###2.4###27.66###532###3.2

###Peak-1

###20###87.4###1.98###44.14###849###4.2

###Peak-2

Table-2: Effect of different concentrations of alcohols on the LMW-ACPase (Peak -2) activity.

###Modifiers/concentration###5%###10 %###15 %###20 %

###% activity###% activity###% activity###% activity

###Glycerol###140###186.2###230###250

###Ethylene glycol###130###165.5###185###195

###Methanol###125###144.8###170###180

###Ethanol###120###131###132###135

###Acetone###112###131###137###140

When enzymatic reaction was performed in the presence of any alcohol as phosphate acceptor, there was partitioning of phosphoenzyme intermediate between water and the acceptor as shown below.

Equation

where E represents enzyme, RO-P p-nitro phenyl phosphate, E.RO-P Enzyme-substrate complex, E-P phosphoenzyme intermediate, ROH p-nitrophenol, A the acceptor molecule and A-P phosphorylated acceptor.

According to the concept of Zhang and Van Etten [30] if the rate limiting step is the formation of phosphoenzyme intermediate, then the acceptor molecule (A) reacts with phosphoenzyme, it will not increase the overall rate of breakdown of substrate. So, the addition of a phosphate acceptor in the enzymatic reaction would not change the formation of p-nitrophenol but would decrease the rate of formation of inorganic phosphate because the new pathway competes to decrease the level of E-P. On the other hand, if the rate limiting step is the hydrolysis of phosphoenzyme intermediate, then the acceptor will increase the overall rate of breakdown of substrate. Thus, the addition of a phosphate acceptor in the reaction would increase the rate of formation of p-nitrophenol, the level of E-P would not fall and the rate of formation of inorganic phosphate would remain unchanged. The latter case seems to be the hydrolysis of p-nitro phenyl phosphate catalyzed by LMW-ACPase from camel heart.

Phosphotransferase activity was shown by phospho transfer experiments using various alcohols as phosphate acceptors (Table-3). The glycerol and ethylene glycol were found better phosphate acceptors than methanol ethanol and acetone. The presence of these alcohols in the incubation mixture increased the overall rate of breakdown of substrate, p-nitrophenyl phosphate as determined by the amount of p-nitrophenol produced. The rate of transfer of phosphate group to phosphate acceptors was also increased while the amount of inorganic phosphate (Pi) produced, remained same and was equal to the amount of Pi released in water when no alcohol was added (Table-3). This indicates the formation of phosphoenzyme covalent intermediate.

Thus the determination of p-nitrophenol and Pi produced in the reaction with phosphate acceptor which competes with water in the hydrolytic reaction of phosphoenzyme covalent intermediate demonstrated transphosphorylation reaction as indicated by Pi / p- nitrophenol ratio (less than 1). When concentration of glycerol was increased from 5 % to 20 %, the overall rate of breakdown of p-nitrophenyl phosphate was increased (Table-4). The competition between hydrolysis and phosphate transfer reaction was started. The rate of hydrolytic reaction remained almost constant (Pi produced same) and rate of phosphate transfer reaction was increased with increasing concentration of glycerol. Thus the ratio of phosphate transfer reaction to hydrolysis was also increased. The fact supported by smaller than 1 ratio of Pi / p-nitrophenol and greater ratio of phosphate transfer to hydrolysis showed the presence of transphosphorylation reaction (compare Table-3 and 4).

Table-3: Acid phosphatase catalysed phosphate transfer reaction in the presence of various alcohols as phosphate acceptors

###Concentration###p-Nitro- phenol produced###Inorganic phosphate (Pi)###Phosphate transferred

###Acceptors###Pi /p-nitrophenol

###(%)###(n mol/ml)###Produced (n mol/ml)###(n mol/ml

###Water###29###30###-###1.034

###Acetone###10###38###27###11###0.710

###Ethanol###10###38###31###7###0.815

###Methanol###10###42###26###16###0.619

###Ethylene-

###10###48###28###20###0.583

###glycol

###Glycerol###10###54###30###24###0.555

Table-4: Acid phosphatase catalysed phosphate transfer reaction in the presence of various concentrations of glycerol.

###p-Nitro-###Inorganic phosphate (Pi)

###Concentration of###Phosphate transferred###Ratio of transfer to###Pi /p-

###phenol produced###produced (hydrolysis)

###glycerol (%)###(n mol/ml###hydrolysis###nitrophenol

###(n mol/ml)###(n mol/ml)

###0###29###30###-###-###1.034

###5###49###29###20###0.689###0.591

###10###54###30###24###0.8###0.555

###15###67###28###39###1.392###0.417

###20###72.5###28.5###44###1.54###0.393

Conclusion

In this study two low molecular weight acid phosphatases were purified. The ability to catalyse phosphate transfer and partitioning of an intermediate between competing acceptors has been useful method for demonstrating the formation of phosphoenzyme intermediate and for identifying the rate limiting step in the reaction mechanism.

Acknowledgements

This research work was carried out under PhD program in the Department of Chemistry, Gomal University, D.I.Khan, Pakistan.

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