Printer Friendly

PARTIAL PURIFICATIONCHARACTERIZATION AND SOME KINETIC PROPERTIES OF LOW MOLECULAR WEIGHT ACID PHOSPHATASE FROM LEAVES OF GERMINATING VIGNA RADIATA SEEDS.

Byline: A. Saeed M. Salim R. Naz U. Zaman A. L. Baloch S. Nadir and A. Saeed

: Abstract

Acid phosphatase isoenzyme (AcP-II) from leaves of germinating seeds of vigna radiata (mung beans) was partially purified by CM-Cellulose chromatoghraphy gelfiltration on Ultrogel AcA 44 and Con A-Sepharose affinity chromatoghraphy. The specific activity of 25U/ mg of protein was obtained with recovery of 4 %. The enzyme showed a purification by a factor of 86. Gel filtration experiment and sodium dodecyl sulphate polyacrylamide gel electrophoresis indicated that the isoenzyme had a molecular weight of 29 kDa. The Km value of the isoenzyme was 0.5 mM with p-nitrophenyl phosphate as substrate. The enzyme had pH optimum of 5.5 and optimum temperature of 60oC. The enzyme was inhibited by phosphate vanadate fluoride and molybdate. It was also strongly inhibited by Cu++ Hg++ Zn++ and Al+++. The enzyme had very little effect of inhibition by thiol specific reagents such as iodoacetamide N-ethyl- maleimide etc. suggesting that no -SH groups are involved in the enzyme catalysis.

Dithiothreitol and AY-mercaptoethanol had small activating effect at low concentrations indicating their properties as reducing agents but at their high concentrations the activation was replaced by inhibition suggesting that these thiols may cause a conformational change in the enzyme at a place other than the active site. Variation of Km values with pH alteration study showed that a histidine may constitute a part of an active site. This was confirmed by inhibitory effect of high concentration of iodoacetate at pH 7.2

Key words: Acid phosphatase; Mung beans; Vigna radiata; purification; characterization.

INTRODUCTION

Acid phosphatases (EC 3.1.3.2) are enzymes that catalyze the removal of inorganic phosphate from phosphomonoesters in acid media (Anand and Srivastava 2012). These are ubiquitous in nature and found in bacteria fungi animals and plants (Guo and Pesacreta1997; Leitao et al. 2010; Naz et al. 2006; Siddiqua et al. 2008; Al-Omair 2010). The studying acid phosphatases is difficult due to their occurrence in multiple forms and their small quantity (Park and Van Etten 1986; Waymack and Van Etten 1991). Their study is even more difficult because of wide variations in the activity and variations of multiple forms between species and between different organs during various stages of plant development (Alves et al. 1994; Baes and Van Cutasem 1993).

Four isoforms in v. sinensis (Biswas and Cundiff 1991) and six isoforms in v. mungo ( Haraguchi et al. 1990) rise in activity in the axes during the germinating of soybean seeds at the early stage (Okuda et al. 1987) and also the increase of acid phosphatase activity of 10-fold in soybean leaves on seed pod removal after flowering season (Staswick et al.1994) are the examples of the facts. These factors together with occurrence of their small quantity and instability in dilute solution makes the isolation of highly purified acid phosphatase difficult.

A number of acid phosphatases have been purified to homogeneity or near to homogeneity from the different plant sources such as sweet potato (Durmus et al.1999) aleurone particles of rice grain (Yamagata et al. 1980) cotton seed (Bhargava and Sachar1987) lupin seed (Olczak et al. 1997) cotyledons of germinating soybean seeds (Ullah and Gibson 1988) axes and cotyledons of germinating soybeans (Kaneko et al. 1990) in order to study their structures and functions in the cells. The physiological roles of acid phosphatase are not well understood because of the heterogeneity and lack of substrate specificity (Duff et al. 1994). Generally acid phosphatases are believed to function in the production transport and recycling of inorganic phosphate (Pi) (Yoneyama et al. 2004).

In plant roots acid phosphatases seem to be involved in the solubilization of macromolecular organic phosphates in soils by its catalytic action to release Pi which can then be absorbed by plants (Panara et al. 1990) for growth and development. In tubers Kouadio (2004) described the important role acid phosphatase concerning the transport of Pi in metabolic processes during the preservation of cocoyam. In seeds and seedlings the physiological function of the acid phosphatase is to provide inorganic phosphate to the growing plant during the germination and many different phosphate esters of sugars and phosphorylated compounds stored in seeds and seedlings are hydrolyzed to release Pi through the catalytic action of increased enzyme activity which is either due to de novo synthesis of enzyme protein or activation of the enzyme by imbibition (Gahan and Mc Lean 1969; Schultz and Jensen1981; Akiyama and Suzuki 1981; Hoehamer et al. 2005).

Most of the purified plant acid phosphatases have been found to contain molecular weights from 50 kDa to 200 kDa (Gonnety et al. 2006) and very few reports on low molecular weight acid phosphatases (18 kDa -31 kDa) have been cited in the literature.

In our preceding report (Nadir et al. 2012) we attempted to purify 29 kDa acid phosphatase to homogeneity from germinating v. radiata seeds ( whole plants axes and cotyledons) and characterized with respect to molecular weight pH optimum Km Vmax and Ki values with various inhibitors etc. This paper describes the presence of multiple forms of acid phosphatase in the leaves of germinating v. radiata seeds and presents a simple procedure of purification of low molecular weight form of acid phosphatase along with some kinetic properties. This kinetic data provides a basic knowledge about the structure at the active site.

MATERIAL AND METHODS

Chemicals: Ion-exchanger CM-Cellulose from Whatman

Biosystem gel media Sephadex G-100 and Ultrogel AcA 44 affinity gel Concanavalin A-Sepharose 4B (Con A- Sepharose) and marker proteins were obtained from Sigma Chemical Chemical Co. The chemicals for SDS- PAGE were supplied by Sigma-Aldrich Chemical Co. Substrates and other chemicals were purchased from Fluka and BDH Chemical Company.

Enzyme assays: Acid phosphatase activity was determined as described by Panara et al. (1990). To 900l solution of substrate containing 4mM p- nitrophenyl phosphate in 0.1M acetate buffer pH 5.5 100l of enzyme solution was added and incubated at 37C for 5 min. The reaction was terminated by adding 1- 4 ml of 0.1N KOH and the intensity of the yellow color (phenolate ions) produced was measured at 405nm (e = 18000 M-1 cm-1). One unit of enzyme was defined as amount of the enzyme that produces 1 mol of p- nitrophenol / min. Specific activity was expressed as enzyme units / mg of proteins.

The pH dependence of enzyme activity was determined by measuring the hydrolysis of p-nitrophenyl phosphate at 37C in a series of buffer at various pH values ranging from 3.6 to 9.0. Buffers used were 0.1 M sodium acetate buffer from pH 3.1 to 6.0 and Tris-HCl buffer (0.1M) from pH 7.0 to 9.0.

The temperature optimum was determined by measuring the activities at temperatures between 40-80C at intervals of 5C. To determine the temperature stability the enzyme was first pre-incubated in 0.1 M acetate buffer pH 5.5 at different temperatures ranging from 50oC to 80oC for 30 min. Following cooling at 4C the enzyme assay was done as usual to estimate residual activity.

Substrate specificity studies were carried out by determining the release of inorganic phosphate as the result of hydrolysis of various substrates. Inorganic phosphate was determined by Black and Jones method (1983). The incubation mixture consisted of 450 l of 0.1 M acetate buffer pH 5.5 containing 4 mM of substrate and 50 l enzyme solution was incubated at 37o C for 5 minutes to release Pi from enzymatic reaction. This reaction was stopped by addition of 200 l of 10 % trichloroacetic acid. The blue color was developed with molybdic acid reaction which was as follows: The 500 l mixture (composed of 200 l of 2 % ammonium molybdate and 300 l of 14 % ascorbic acid in 50 % trichloroacetic acid) was added to the above mixture (700 l) followed by the addition of 1 ml solution containing 2 % trisodium citrate and 2 % sodium arsinate in 2 % acetic acid to make the total volume of 2200 l. The color was developed for 30 minutes and absorption was determined at 700 nm.

The enzyme activity was expressed as a percent of p-nitro phenyl phosphate.

Inhibitions by metal ions: The mixture consisting of 100 l of the 0.1M cation solution under test 600 l of 1M acetate buffer pH 5.5 and 100 l of enzyme solution was pre-incubated for 10 min at 37C. After pre- incubation 200 l of 20 mM p-nitrophenyl phosphate was added to determine the activity as usual. Simultaneously control and blank experiments were run in which the cation and enzyme solutions were replaced by water respectively in the pre-incubation mixtures.

Similarly the effect of some compounds reacting with SH-groups of the enzyme on the enzyme activity at pH values varying from 3 to 9 was determined as described above.

Kinetic parameters: The Km Vmax and Ki values were determined using p-nitrophenyl phosphate as the substrate in concentrations of 0.06 - 4 mM in the absence or presence of two or three fixed concentrations of inhibitors. These kinetic parameters were determined from Line-weaver-Burk plots. Straight lines were drawn by applying least square rule.

The pH dependence studies of Km Vmax and specificity constants were carried out as described by Pasqualini et al.(1997).

Protein determination: Protein concentration was determined by the Biuret method. In chromatographic procedures the relative protein concentration was estimated from the absorbance at 280 nm.

Electrophoresis: SDS-polyacrylamide gel electrophoresis was carried out by the method of Laemmli (1970) under reduced conditions. The sample was prepared in sample buffer with AY-mercaptoethanol and heated at 95C for 2-3 min. The enzyme purity was checked in 12% acrylamide mini-slab gel. After the run the proteins in gel were stained with coomassie blue and molecular weight estimates were made using standard size marker proteins as indicated in the respective figure.

Molecular weight determination: Two different acid phosphatase isoenzymes were placed on Ultrogel AcA 44 column (1.8x85 cm) separately and eluted with 0.01 M Tris-HCl buffer pH 7.0 containing 0.1M NaCl. The operating flow rate was 25 ml/h and 5 ml fractions were monitored. The molecular weights of the isoenzymes were estimated on calibrated Ultrogel AcA 44 column by comparing its elution volumes with those of standard protein markers.

Germination of seeds extraction and purification of enzyme: Seeds of v. radiata (mung beans) were washed with water three times and soaked in water for 3-4 h. After hydration germination of seeds was performed on moist sand trays during at least 7 days in a room at ambient temperature of 28-35C. The enzyme was purified by a procedure of Nadir et al. (2012) with slight modifications. The leaves obtained after 4-7th day of germinating seeds were homogenized in Warring Blender with five volumes of 0.1M acetate buffer pH 5.5. The homogenate was centrifuged at 45000xg for30 min and supernatant was collected. To the supernatant solid ammonium sulfate was added and brought to 80 % saturation. The mixture was stirred well and centrifuged. The precipitate obtained was dissolved in 0.01 M acetate buffer pH 5.9 and dialyzed against the same buffer over night. After centrifugation the clear dialysate was applied to CM-Cellulose column (2.2x17.5 cm).

The column was washed with buffer and some of the acid phosphatase activity peak (P-1) was eluted as unbound protein. After extensive washing the bound acid phosphatase activity peak (P-II) was eluted by linear gradient of 0-0.5M NaCl in the same buffer (50ml) with flow rate of 40 ml/h. Fractions of approximate 10ml each were collected (Fig.1A). The enzymes from both peaks (P-1 and P-II) were pooled separately and concentrated to 5 ml each by ultrafiltration using Amicon YM2 membrane at 20 psi pressure. The enzyme sample P-II was placed on Ultrigel AcA 44 column (1.8x85 cm) previously equilibrated and eluted with 0.05 M Tris-HCl buffer pH 7.0 containing 0.1M NaCl. Fractions of 5ml were collected with flow rate of 25 ml/h (Fig. 1B).

The highest activity containing fractions were pooled and dialysed against 0.01 M Tris-HCl buffer pH 7.0 containing 1mM Ca++and 1mM Mn++. The dialysed enzyme was applied to a Con A-Sepharose column (2x11cm) which had been previously equilibrated with dialyzing buffer and washed with same buffer. The column was then eluted with 10 % a (+) D- methylglucopyranside in buffer. Fractions of 5 ml were collected at flow rate of 25 ml/h (Fig.1C). The most active fractions were pooled concentrated by ultrafiltration and used for further study.

RESULTS AND DISCUSSION

Enzyme purification: Acid phosphatase isoenzyme (AcP-II) from leaves of germinating v. radiata seeds was partially purified. A summary of the purification is presented in table 1. About 86- fold purification was achieved with a specific activity of 25 U/mg of protein and a recovery of 4 %. The specific activity was higher in comparison with acid phosphatase purified from Phaseolus vulgaris to specific activity of 18.1 U/ mg of protein (Cabello-Diaz et al. 2012). However it was much lower than the specific activity (598 U/ mg of protein) reported for acid phosphatase from Euphorbia latex ( Pintus et al. 2011). The isoenzyme AcP-I could not be purified. The SDS-PAGE of AcP-II showed a major band with molecular weight at 29 kDa (Fig. 2). Very faint bands corresponding to 18 kDa and 14 kDa were also observed. These two bands are probably NH2 terminus truncated fragments originating from proteolysis during purification.

Gel filtrations of enzymes P-I and P-II obtained from CM-Cellulose chromatography on Ultrogel AcA 44 column have shown their elution profiles in figs.3A and 1B. The elution volumes of 100 ml and 115 ml for the peaks AcP-1 and AcP-II were obtained. The molecular weights of these two native isoenzymes were found to be 58 kDa and 29 kDa (Fig. 3B) indicating that AcP-II is a monomeric protein. The 58 kDa acid phosphatase in v. aconitifolia seeds was also reported (Anand and Srivastava 2013). These results showed the presence of multiple forms of acid phosphatase as reported in other plant sources (Panara et al. 1990; Pasqualini et al.1997). Our results were consistent with acid phsphatases isolated from germinating blackgram (v. mungo)(Asaduzzman et al. 2011) and wheat seedlings (Chen and Tao 1989) which had molecular weights of 25 kDa and 35 kDa respectively.

Effect of pH and temperature: Acid phosphatases isolated from leaves of v. radiata showed a pH optimum of 5.5 (Fig. 4). The same results were obtained for enzymes from seedlings of v. radiata (Nadir et al. 2012) caster bean seeds (Granjeiro et al. 1999) and leaf of p. vulgaris (Tejera-Garcia et al. 2004). But this optimum pH value was higher than optimum pH (pH 4.75) of acid phosphatase from Agaricus bisporus (Wannet et al. 2000).

The enzyme had optimum temperature of 60C (Fig. 5) which was almost similar to the values reported for acid phosphatases purified from germinating soybean seeds (Ullah and Gibson 1988) and axes of v. radiata seedlings (Kundu and Banerjee1990). Optimum temperature of 60C was higher than for barley roots (35C) (Panara et al.1990) cotton seeds and zea mays seeds (37C) (Bhargava and Sacchar1987; Senna et al. 2006) caster beans seeds (45C)(Granjeiro et al. 1999) and garlic seedlings (50C)(Yenigun and Guvenilir 2003). But was lower than optimum temperature (80C) for isoenzymes from soybean seeds (Ferreira et al. 1998).

The enzyme was found to be stable at 50C. The same value was reported for the enzyme purified from seedlings of v. radiata (Nadir et al. 2012) but it had lost 14 % of its activity at 55C and 50 % of activity at 60C after pre-incubation for 30 min. The enzyme was inactivated completely at 70C.

This broad range of substrate specificity was similar to those of other plant acid phosphatases (Tejera- Garcia et al. 2004; Turner and Plaxton 2001; Koffi et al. 2010). A non-specificity of this enzyme seems to exhibit the metabolic significance in utilizing extracellular as well as intracellular phosphorylated compounds in release of Pi.

Action of modifiers and inhibitors: The action of various compounds as possible activators or inhibitors of acid phosphatase was determined. Alcohols such as methanol ethanol and glycerol at concentrations of 10 % showed no activating effect on the activity suggesting that acid phosphatase was not involved in the transphosphorylation reaction. The lack of effect of 4mM EDTA showed that divalent ions were not necessary for the activity. 1 % Triton X-100 activated the enzyme to 142 %. This activation may be due to its interaction with hydrophobic portions of the enzyme. Shekar et al. (2002) had also reported the same effect on acid phosphatase from developing pea nut cotyledons. Two known acid phosphatase inhibitors tartrate and citrate at concentration of 5 mM did not inhibit this enzyme.

Tartrate resistance was also observed in many other acid phosphatases (Olczak et al. 1997; Pan 1987; Rossi et al. 1981; Ching et al. 1987; Doi et al. 1987). Phosphate fluoride vanadate and molybdate inhibited the enzyme as was the case for other phosphatases (Tabaldi et al. 2008) and their inhibition pattern seemed to be very similar to our enzyme already reported (Nadir et al. 2012). The comparison of their Ki values is shown in table 3. Phosphate acted as competitive inhibitor. Competitive inhibition was observed for acid phosphatase from artemecia vulgaris pollen extract (Cirkovic et al. 2002) and from other plant sources. Vanadate was also competitive inhibitor of this enzyme. This result was in accord with the findings of purple acid phosphatase in the walls of tobacco cells (Kaida et al. 2008).

Fluoride inhibited non-competitively. A similar type of inhibition was reported for acid phosphatase from rice plants (Tso and Chen 1997) whereas the molybadate showed a very strong inhibition of mixed type (Ki 3 M). Such type of inhibition was obtained in enzymes from axes and cotyledons of germinating soybeans (Kaneko et al. 1990).

Effect of metal ions: Metal ions showed different effects on the acid phosphatase activity. The activity was reduced by Fe Cu Hg and Zn which was consistent with the results reported (Tso and Chen 1997; Bozzo et al. 2004) while other divalent ions such as Ca Mg and Mn had no significant effect on activity.

The Zn and Hg inhibited the enzyme non- competitively with Ki values of 4 mM and 13 M respectively (Table 3). It was observed from the Lineweaver-Burk plot while calculating the kinetic parameters both Km and Vmax decreased with elevation in Hg++ concentration. The data demonstrated that HgCl2 concentrations ranging from 0.01-0.02 mM decreased the apparent Km values from 28 % to 37 % of real Km value while the enzymatic activity apparent Vmax values decreased from 40 % to 57 % of real Vmax. By using a Cornish-Bowden plot the Ki was found to be 13M ( Table 3). The effects of EDTA and AY-mercaptoethanol on the inhibition of enzyme activity by some metal ions are shown in Table 4. At 12.5 mM concentrations Al+++ Zn++ and Hg++ showed around 80 % 78 % and 100 % inhibitions respectively (Table 4a and b). The addition of 20 mM EDTA to portions of enzyme solutions which had been inhibited by metal ions showed that inhibition was substantially reversed.

The activity recovery was 54 % 66 % and 12 % respectively (Table 4a). But addition of AY-mercaptoethanol at 10 mM concentration was found without effect on the inhibition by these metal ions. By this treatment enzyme activity recovery was 0 % (Table 4b). In general inhibition caused by these oxidizing agents (metal ions) was not reversed by reducing agent (AY-mercaptoethanol).

Effect of some SH-reacting compounds: Pre-incubating the enzyme with SH-reacting compounds such as iodoacetic acid (5mM) iodoacetamide (10mM) N-ethyl- maleimide (10mM) and p-hydroxymercuri-benzoate (0.5mM) for 10 min at various pH ranging from 3 to 8 before the addition of p-nitrophenyl phosphate showed that these compounds inhibited the enzyme to lesser extent (10-20%) revealing that SH-group containing amino acids in the enzyme may not be significant for its catalytic activity. The suitable controls indicated that enzyme was almost stable over pH range 3-8 (Table 5). Similar to our observation Panara et al. (1990) found no substantial role of free SH- groups in the barley root acid phosphatase but Granjeiro et al.(2003) demonstrated the importance of SH- groups in the catalytic mechanism of caster bean seed acid phosphatase.

Effect of some SH-protecting compounds: Under above stated conditions the pre-incubation of enzyme with dithiothreitol (DTT) or AY-mercaptoethanol at 12.5 mM for 10 min at pH 5.5 showed activation by nearly 5 %. The effect of these two compounds on the enzyme activity was further studied at their different concentrations when added to the enzyme at the same time as the p-nitro phenyl phosphate solution at pH 5.5. The results are shown in Table 6. A small activation (6- 10%) was observed with concentrations up to 100 mM of DTT while AY-mercaptoethanol at these concentrations displayed very little or no activation. Thus DTT or AY- mercaptoethanol at low concentrations behaves as reducing agent. At high concentrations of DTT or AY- mercaptoethanol (200-500 mM) activation was replaced by inhibition (Table 6).

Prolonged incubation of enzyme with 50 mM of DTT or AY-mercaptoethanol at 4oC a very little activation was observed but ascorbic acid caused strong inhibition and complete inhibition was obtained in 17 days (Table 7). Thus DTT or AY-mercaptoethanol has a stabilizing and protective effect on the enzyme activity.

From the above discussion it may be concluded that this enzyme was not susceptible to inactivation by some SH- protecting reagents ( DTT AY-mercaptoethanol or ascorbic acid) or SH-blocking reagents (iodoacetic acid iodoacetamide N-ethyl-maleimide or p- hydroxymercuri-benzoate) and thus the SH-groups seemed to have no catalytic role in the mechanism of enzyme action. The inhibition by DTT or AY- mercaptoethanol at very high concentrations may cause influence on the catalytic process perhaps by producing conformational changes in regions other than the active site.

The effect of pH on the Km and Vmax of acid phosphatase is shown in Table 8. At lower pH values there seemed to be a trend of decrease in Vmax but Km was almost the same at each pH. It may be suggested that some ionizable groups were protonated resulting in a slow rate of hydrolysis. The protonation of ionizable group was not affecting the substrate affinity for the enzyme as the Km values in range of pH 4.6-5.8 were the same. The similar finding was also reported by Anand and Srivastava (2013) confirming that this ionizable group may not be located in the enzyme active site.

The pH dependence of the Km curve showed (Fig. 6) two inflections one at pH 5.8 and the other at pH 7.6. These inflections are due to ionization of groups on the enzyme or substrate which may possibly correspond to the pKa2 of p-nitophenyl phosphate substrate (5.3-5.5) and pKa of histidine group on the enzyme (Andrews and Pallavicini 1973) respectively. Saini and Van Etten (1978) described that monoanionic form of p-nitophenyl phosphate was hydrolyzed by the enzymes from wheat germ human prostate and potato with pKa2 value of 5.2 found for second ionization constant of p-nitophenyl phosphate and pKa of 7.8 for the ionization of phosphohistidine covalent intermediate. Our conclusion from kinetic data of pH dependence suggests that p- nitophenyl phosphate is hydrolyzed by enzyme involving two groups with pKas of 5.8 and 7.6.

The participation of histidine as a part of an active site was supported by enzyme-iodoacetate reaction in time dependent manner. As previously shown in table 5 the reaction of enzyme with 5 mM iodoacetate was slow and had little inhibitory effect (10-20 % at all pH values) during 10 min pre-incubation period but prolonged pre-incubation of enzyme at pH 7.2 in the presence of 100 mM iodoacetate was accompanied by complete inactivation (Table 9) and this was consistent with the presence of an active site histidine residue.

Table 1 Purification of acid phosphatase from 5 g leaves of germinating of v. radiate seeds

Steps###Vol.###T. Act.###T. Prot.###S.A###P.F.###Rec.

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

Extract###15.5###65###224.75###0.29###1###100

Ammonium sulphate (80 %saturation)###16.5###64.35###108.9###0.59###2###99

Dialysis###16.5###63###146.85###0.43###1.48###97

CM-Cellulose Chromatography. (P-II)###38###31.5###45.6###0.69###2.38###48

Concentration by ultrafiltration###5###29.3###40###0.73###2.5###45

Ultrogel AcA 44 Chromatography of P-II.###35###4.2###2.45###1.714###5.91###6.5

(AcP-II )

Con-A Sepharose chromatography (AcP-II )###5###2.5###0.1###25###86###3.8

Table 2. Substrate specificity of acid phosphatase from the leaves of v. radiata

Substrates###% activity

p-Nitrophenyl phsphate###100

Phenyl phopshate###64.7

- Naphthyl phosphate###11.3

- Naphthyl phosphate###63

-Glycerophosphate###17.8

- Glycerophosphate###35

Phosphotyrosine###80

Phosphoserine###18

Phosphothreonine###15.4

Glucose-1-phosphate###15

Glucose-6-phosphate###136

cAMP###11.5

cGMP###22.4

GMP###27.2

ADP###1.5

ATP###48

FMN###19.7

Table 3. Effect of inhibitors on the acid phosphatase activities

Inhibitors###Type of inhibition###( Ki)###(Ki)

###29 kDa enzyme from leaves of###29 kDa enzyme from seedlings of

###v.radiata###v.radiata

Phosphate###Competitive###5 mM###3.5 mM

Vanadate###Competitive###5 M###11.5 M

Fluoride###Non- competitive###0.3 mM###0.6 mM

Molybdate###Mixed type###6-10 M###3 M

Zn++###Non- competitive###4 mM###16 mM

Hg++###Non- competitive###13 M###30 M

Table 4a. Inhibition by metal ions in the presence or absence of EDTA

###Metal ions###without EDTA treatment###with EDTA (20 mM)###Recovery

###(12.5 mM)###Act.###% Act.###Act.###% Act.###(% Act.)

No metal ions (control)###1.264###100###1.302###100###-

###Al+3###0.25###19.7###0.962###73.8###54.1

###Zn+2###0.273###21.6###1.138###87.4###65.8

###Hg+2###0.00###0.00###0.158###12.1###12.1

Table 4b Inhibition by metal ions in the presence or absence of -merceaptoethanol

###with -merceaptoethanol###Recovery

###Metal ions###without -merceaptoethanol

###(10 mM)###(% Act.)

###(12.5 mM)

###Act.###% Act.###Act.###% Act.

No metal ions (control)###0.73###100###0.77###100###-

###Al+3###0.12###16.4###0.12###15.6###no

###Zn+2###0.17###23.3###0.13###16.9###no

###Hg+2###0.00###0.00###0.00###0.00###no

Table 5: Effect of some SH- reacting compounds on the acid phosphatase activity

###Iodoacetic acid###Iodoacetamide###N-ethylmaleimide###hydroxymercuroben-

3.14###0.92###1.1###16###1.174###1.328###11.6###-###1.407###-###-###-###-

4.06###1.029###1.26###18###1.279###1.508###15###1.144###1.459###21.5###-###-###-

4.65###1.075###1.296###17###1.171###-###-###1.248###1.042###19.8###-###-###-

5.4###1.18###1.416###16.6###1.41###1.64###14###1.383###1.843###25###-###-###-

5.5###1.193###1.48###20###1.446###1.608###10###1.456###1.852###21.4###1.102###1.343###18

5.85###1.31###1.5###13###1.431###1.85###22.6###1.522###1.944###21.7###-###-###-

6.2###1.41###1.731###18###1.52###1.713###11.2###1.52###1.872###14.7###-###-###-

6.44###1.595###1.67###-###1.521###1.637###7###1.573###1.763###10.8###-###-###-

7.25###1.511###1.788###16###1.66###1.758###5.5###1.511###1.85###11.3###-###-###-

8.34###1.49###1.69###12###1.525###1.764###13.5###1.49###1.73###14###-###-###-

Table 6. Effect of different concentrations of SH-protecting or reducing agents on the enzyme activity

###Concentration###DTT###-mercaptoethanol

###(mM)###Activity###Activity###Activity###Activity

###(A405)###(%)###(A405)###(%)

###0###1.44###100###1.20###100

###10###1.492###106.6###1.26###105

###20###1.513###107.8###1.226###102

###50###1.548###110.5###1.209###102

###100###1.539###110.0###1.225###102

###200###1.375###95.5###1.08###90

###500###1.278###91.3###1.02###85

Table 7. Effect of prolonged exposure to 50 mM SH-protecting/reducing agents on the acid phosphatase enzyme

###Days###0###5###11###14###17

###(% Activity)###(% Activity)###(% Activity)###(% Activity)###(% Activity)

###H2O###100###100###100###100###100

###DTT###105###105###103###101###96

###-mercaptoethanol###102###105###101###102###99

###Ascorbic acid###95###17###20###14###0

Table 8. pH dependence of the hydrolysis of p-nitrophenyl phosphate by acid phosphatase from leaves of germinating v. radiata

###pH###Vmax (U/mg)###Km (mM)###Vmax / Km

###3.0###3.7###1.11###3.33

###3.5###12.5###0.20###62.5

###4.0###19.4###0.18###107.77

###4.2###22###0.133###165.41

###4.4###22###0.200###110.0

###4.6###28.6###0.281###101.78

###4.8###29.4###0.333###88.29

###5.0###30.1###0.303###99.34

###5.2###31.14###0.315###98.86

###5.4###33.02###0.295###111.93

###5.6###35.52###0.357###99.49

###5.8###25.36###0.333###76.16

###6.0###29.58###0.526###56.36

###7.0###35.52###1.670###21.27

###7.2###14.8###2.000###7.4

###7.4###12.6###2.500###5.04

###7.6###8.88###3.330###2.67

###7.8###11.1###1.428###7.77

###8.0###7.8###2.500###3.12

###8.2###8.88###2.500###3.55

###8.4###5.0###3.330###1.50

###8.6###8.0###1.666###4.80

###8.8###6.56###1.666###3.94

Table 9. Inhibition of enzyme with 0.1M iodoacetate at pH 7.2 in function of time

###Time###Activity (%)

###0 min###100

###2min###55

###1h###15

###2h###9.3

###4h###4

###24h###1.1

###96h###0

Conclusions: In this study low molecular acd phosphatase isoenzyme of (29 kDa) from the leaves of germinating v. radiata seeds has been purified and biochemically characterized. The enzyme purification electrophoretic pattern biochemical properties and some other kinetic study reveal that AcP-II from leaves and enzyme isolated from germinating seeds of v. radiata are very similar. However the sequencing data of both 29 kDa enzymes needs to be resolved. No -SH groups are involved in enzyme catalysis but histidine may constitute a part of an active site. Since the enzyme was found insensitive to tartrate inhibition it may be recognized as a tartrate resistant acid phosphatase class.

Acknowledgements: This research was carried out under M.Phil/Ph.D Scheme program in the Department of Biological Sciences Gomal University Dera Ismail Khan Pakistan in collaboration with Department of Chemistry University of Science and Technology Bannu Pakistan.

REFERENCES

AL- Omair M.A. (2011). Purification and biochemical characterization of acid phosphatase from vigna aconitifolia. Am. J. Plant Physiol. 5(6): 361- 370.Anand A. and P.K. Srivastava (2012). A molecular description of acid phosphatase. Appl. Biochem.Biotechnol. 167: 2174-2197.Alves J.M. C.D. Siachakr. M. Allot. S. Tizroutine. I.Mussio and A. Servaes (1994). Isoenzyme modification and plant regeneration through somatic embryogenesis in sweet potato (Ipomoea batatas L. Lam.). Plant Cell Rep. 13: 437-441.Akiyama T. and H. Suzuki (1981). Localization of acid phosphatase in aleurone layers of wheat seeds. Pfanzenphysiol. 101:131.Anand A. and P.K. Srivastava (2013). Isolation and enzymatic properties of a non-specific acid phosphatase from Vigna aconitifolia seeds. Biotechnol. Appl. Biochem. DOI:10.1002/bab.1131.Asaduzzaman AK.M. M.H. Rahman and T.Yeasmin (2011). Purification and characterization of acid phosphatase from a germinating black gram (Vigna Mungo L.) seedlings. Arch. Biol. Sci. Belgrade 63(3): 747-756.Andrews A.T. and Pallavicini C. (1973). Biochim.Biophys. Acta. 321 197-209.Baes P.G. and P.J. Van Cutasem (1993). Isoenzyme polymorphism in three gene pools of cultivated chicory (Cichorium intybus L.). Euphytica. 71: 143-150.Biswas T.K. and C. Cundiff (1991). Multiple forms of acid phosphatase in germinating seeds of Vigna sinensis. Phytochem. Phytochemistry. 30(7): 2119-2125.Bhargava R. and R.C. Sachar (1987). Induction of acid phosphatase in cotton seedlings: Purification sub unit structure and kinetic properties. Phytochemistry. 26: 1293-1297.Black M.J. and M.E. Jones (1983). Inorganic phosphate determination in the presence of a labile organic phosphate: assay for carbonyl phosphatase activity. Anal. Biochem. 135: 233- 238.Bozzo G.G. K.G. Raghothama and W.C. Plaxton (2004). Structural and kinetic properties of a novel purple acid phosphatase from phosphatestarved tomato (Lycopersicon esculentum) cell cultures. Biochem. J. 377: 419- 428.Cabello-Diaz J.M. F.A. Quiles. R. Lambert. M. Pineda and P. Piedras (2012). Idetification of a novel phosphatase with high affinity for nucleotides monophosphate from common bean (Phaseolus vulgaris). Plant Physiol.Biochem. 53: 54-60.Chen H.E. and M.Tao (1989). Purificaion and characterization of a phosphotyrosylprotein phosphatasefrom wheat seedlings. Biochim. Biophys. Acta. 19: 271-276.Ching T.M. T.P. Lin and R.J. Metzger (1987).Purification and properties of acid phosphatase from plump and shriveled seeds of Triticale. Plant physiol. 84: 789-795.Cirkovic T.D. M.D. Gavrovic-Jankulovic. M.N.Bukilica. L. Mandic. S.Z. Petrovic and R.M. Jankov (2002). Isolation and partial characterization of an acid phosphatase from Artemisia vulgaris pollen extract. J. Serb. Chem. Soc. 67: 567-572.Duff S.M.G. G. Sarath and W.C. Plaxton (1994). The role of acid phosphatase in plant phosphorus metabolism. Plant Physiology. 90: 791-800.Durmus A. C. Eicken. B.H. Sift. A. Cratel. R. Kappl. J. Huttermann and B. Krebs (1999). The active site of Purple acid phosphatase from sweet potatoes (Ipomoea batatus). Eur.J. Biochem. 260: 709-716.Doi K. B.L. Antanaitis and P. Aisen (1988). The binuclear iron centers of uteroferrin and the purple acid phosphatase. Structur Bond. 70: 1- 26.Ferreira C.V. P.A. Granjeiro. E.M. Taga and H.Aoyama (1998). Purification and characterization of multiple forms of soybean seeds acid phosphatase. Plant Physiol Biochem. 36: 487-494.Guo J. and T.C. Pesacreta (1997). Purification and characterization of an acid phosphatase from the bulb of Allium cepa L.var.sweet spanish. J. Plant Physiol. 151: 520-527.Gahan P.B. and J. McLean (1969). Subcellular localization and possible functions of acid AY- glycerophosphatase and naphthol esterases in plant cells. Planta 89: 126-136.Gonnety J.T. S. Niamke. B.M. Faulet. E.J.P.N.Kouadio and L.P. Kouame (2006) .Purification and characterization of three low molecular weight acid phosphatase from peanut (Arachis hypogaea) seedlings. Afr. J. Biotechnol. 5: 35- 44.Granjeiro P.A. C.V. Ferreira. E.M Taga and H.Aoyama (1999). Purification and kinetic properties of of a caster bean seed acid phosphatase containing sulfhydryl groups. Plant Physiol. 107: 151-158.Granjeiro P.A. C.V. Ferreira. A. Donizeti. M. Cavagis. J.M. Granjeiro and H. Aoyama (2003). Essential sulfhydryl groups in the active site of castor bean (Ricinus communis) seed acid phosphatase. Plant Sci. 164: 529-633.Haraguchi H. D. Yamauchi and T. Minamikawa (1990).Multiple forms of acid phosphatase in cotyledons of Vigna mungo seedlings: immunological detection and quatitation. Plant Cell Physiol. 31: 917-923.Hoehamer C.F. C.S. Mazur and N.L. Wolfe (2005).Purification and partial characterization of an acid phosphatase from spirodela oligorrhiza and its affinity for selected organophosphate pesticide. J. Agric. Food Chem. 53: 90-97.Kaneko J. M. Kuroiwa. K. Aoki. S. Okuda. Y. Kamio and K. Izaki (1990). Purification and properties of acid phosphatase from axes and cotyledons of germinating soybeans. Agric. Biol.Chem. 54: 745-751.Kouadio N.E.J.P. (2004).Contribution a letude tubercule de Taro Xanthosoma sp variete atoumbou orono: evolution de quelques parameters biochimiques au cours de la conservation et purification et caracterisation de deux phosphatases acides. These de doctorat 3eme cycle de IUFR des science et technologie des aliments de IUniversite dAbobo-Adjame (Cote dIvoire) pp.10.Kundu P.D. and A.C. Banerjee (1990). Multiple forms of acid phosphatase from seedling axes of Vigna radiata. Phytochemistry. 29(9): 2825-2828.Koffi D.M. J.T. Gonnety. B.M. Faulet. M.E. Bedikou. L.P. Kouame. I.A. Zoro Bi and S.L. Niamke (2010). Biochemical characterization of two non-specific acid phosphatases from cucurbitaceae ( Lagenaria siceraria) edible seeds exhibiting phytasic activity. J. Anim.Plant. Sc. 7: 860-875.Kaida R. T. Hayashi and T.S. Kaneko (2008). Purple acid phosphatase in the walls of tobacco cells. Phytochemistry. 69: 2546-2551.Leitao V.O. R.C. de Melo Lima. M.H. Vainstein and C.J. Ulhoa (2010). Purification and characterization of an acid phosphatase from Trichoderma harzianum Biotechnol. Lett. 10.1007/s10529-010-0264-2.Laemmli U.K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 277: 680-685.Nadir S. Asma Saeed. R. Naz. A. Siddiqua. M..Sherazi. S. M. Wazir and A. Saeed (2012). Isolation purification and characterization of acid phosphatase from germinating Vigna radiata seeds. J. Chem. Soc. Pak. 34: 717-727.Naz R. Asma Saeed. and A. Saeed (2006). An 18 k Da acid phostphatase from chicken heart possessing phosphotyrosine phosphatase activity. The Protein Journal. 25: 135-146.Okuda S. J. Kaneko. T. Ogawa. T. Yamaguchi. K.Izaki and H. Takahashi (1987) Increase in enzyme activities in embryonic axes of soybean seeds during germination. Agric. Biol. Chem. 51: 109-113.Olczak M. W. Watorek and B. Morawiecka (1997).Purification and charactertization of acid phosphatase from yellow Lupin (Lupinus luteus) seeds. Biochim. Biophys. Acta. 134: 14 -25.Pintus F. D. Spano. S. Corongiu. G. Floris and R.Medda ( 2011). Purification primary structure and properties of Euphorbia characias latex purple acid phosphatase. Biochem. (Moscow) 76: 694-701.Park H.C. and R.L. Van Etten. (1986). Purification and characterization of a homogeneous sunflower acid phosphatase. Phytochemistry. 25: 351-357.Panara S. S. Pasqualini and M. Antonielli (1990).Multiple forms of barley root acid phosphatase: Purification and some characterization of the major cytoplasmic isoenzyme. Biochim. Biophys. Acta. 1037: 73-80.Pasqualini S. F. Panara. L. Ederli. P. Batini and M.Antonielli (1997). Multiple acid phosphatase in barley coleoptiles: Isolation and partialcharacterization of the 63 kDa soluble enzyme form. Plant Physiol. Biochem. 35(2): 95-101.Pan S. (1987). Characterization of multiple acid phosphatase in salt-stressed spinach leaves.Aust. J. Plant Physiol. 14: 117-124.Rossi A. M.S. Palma. F.A. Leone and M.A. Brigliador (1981). Properties of acid phosphatase from scutella of germinating maize seeds. Phytochemistry. 20: 1823-1826.Staswick P.E. C. Papa. J. Huang and Y. Rhee ((1994).Purification of major soybean leaf acidphosphatase that is increased by seed-pod removal. Plant Physiol. 104: 49-57.Schultz P. and W.A. Jensen (1981). Pre-fertilization ovule development in capsella: ultrstructure and ultracytochemical localization of acid phosphatase in the meiocyte. Protoplasma. 107: 27-45.Senna R. V. Simonin. M.A.C. Silva-Neto and E. Fialho (2006). Induction of acid phosphatase activity during germination of maize (Zea mays) seeds. Plant Physiol. Biochem. 44: 467-473.Siddiqua A. M. Rehmat Asma Saeed. S. Amin. R.Naz. M. Sherazi. G. M. Khan and A. Saeed (2008). Acid phosphatase from the liver of Labeo rohita: Purification and characterization. Biol. Pharm. Bull. 31: 802-808.Shekar S. A.W. Tumaney. T.J. Rao and R.Rajasekharan (2002). Isolation of lysophosphatidic acid phosphatase from developing peanut cotyledons. Plant Physiol. 128: 988-996.Saini M.S. and R.L. Van Etten (1978). A homogeneous isoenzyme of human liver acid phosphatase. Arch. Biochem. Biophys. 191(2): 613-624.Tejera-Garcia N.A. M. Olivera. C. Iribarne and C.Lluch (2004). Partial purification and characterization of a non-specific acid phosphatase in leaves and root nodules of Phaseolus vulgaris. Plant Physiol. Biochem. 42: 585-591.Tabaldi L.A. R. Ruppenthal. I.B.Pereira. D.Cargnelutti. J.F. Goncalves. V.M. Morsch and M.R.C. Schetinger (2008). Presence of multiple acid phosphatase activity in seedlings of cucumber raddish and rocket salad. Ciencia Rural. Santa Maria. 38: 650-657.Tso S.C. and Y.R. Chen (1997). Isolation and characterization of a group III isoenzyme of acid phosphatase from rice plants. Bot. Bull. Acad. Sin. 38: 245-250.Turner W. and W.C. Plaxton (2001). Purification and charactertization of banana fruit acid phosphatase. Planta. 214: 243-249.Ullah A.H. and D.M Gibson (1988). Purification and charactertization of acid phosphatase from cotyledons of germinating soybean seeds. Arch. Biochem. Biophys. 260: 514-520.Waymack P.P. and R.L Van Etten (1991). Isolation and characterization of a homogeneous isoenzyme of wheat germ acid phosphatase. Arch. Biochem. Biophys. 288: 621-633.Wannet W.J.B. R.W Wassenaar. H.J.M.M Jorissen. C.Van der Drift and H.J.M. Opden Camp (2000). Purification and characterization of an acid phosphatase from commercial mushroom Agarcus bisporus. Antonic van Leeuwenhoek 77: 215-220.Yamagata H. K. Tanaka and Z. Kasai (1980).Purification and characterization of acid phosphatase in aleuron particles of rice grains. Plant Cell Physiol. 21: 1449-1460.Yoneyama T. M. Shiozawa. M. Nakamura. T. Suzuki. Y. Sagane. Y. Katoh. T. Watanabe and T. Ohyama (2004). Characterization of a novel acid phosphatase from embryonic axes of kidney bean exhibiting vanadate dependent chloroperoxidase activity. J. Biol. Chem. 279: 37477-37484.Yenigun B. and Y. Guvenilir (2003). Partial purification and kinetic characterization of acid phosphatase from garlic seedlings. Appl. Biochem. Biotechnol. 105: 677-687.
COPYRIGHT 2014 Asianet-Pakistan
No portion of this article can be reproduced without the express written permission from the copyright holder.
Copyright 2014 Gale, Cengage Learning. All rights reserved.

Article Details
Printer friendly Cite/link Email Feedback
Publication:Journal of Animal and Plant Sciences
Date:Oct 31, 2014
Words:6841
Previous Article:ROLE OF POTASSIUM IN PHYSIOLOGICAL FUNCTIONS OF SPRING MAIZE (Zea mays L.) GROWN UNDER DROUGHT STRESS.
Next Article:SEQUENCE-RELATED AMPLIFIED POLYMORPHISM (SRAP) FOR STUDYING GENETIC DIVERSITY AND POPULATION STRUCTURE OF PLANTS AND OTHER LIVING ORGANISMS: A...
Topics:

Terms of use | Privacy policy | Copyright © 2020 Farlex, Inc. | Feedback | For webmasters