INTRODUCTION

Phosphatases are ubiquitous enzymes that remove a phosphate group from its substrate by
hydrolysing phosphoric acid monoesters into a phosphate ion and a molecule with a free
hydroxyl group. This action is directly opposite to that of phosphorylases and kinases, which
attach phosphate groups to their substrates by using energetic molecules like ATP. A common
phosphatase in many organisms is alkaline phosphatase. Another large group of proteins present
in archaea, bacteria, and eukaryote exhibits deoxyribonucleotide and ribonucleotide phosphatase
or pyrophosphatase activities that catalyse the decomposition of dNTP/NTP into dNDP/NDP and
a free phosphate ion or dNMP/NMP and a free pyrophosphate ion. The other group of
phosphatase is collectively called as protein phosphatase, which removes a phosphate group
from the phosphorylated amino acid residue of the substrate protein. Protein phosphorylation is a
common posttranslational modification of protein catalyzed by protein kinases, and protein
phosphatases reverse the effect.
Acid phosphatases produced by both prokaryotic and eukaryotic cells and are presumed to
convert organic phosphorus into available Pi (Amlabu et al., 2009). Phosphate is an important
molecule for cellular growth that involved in many different biological reactions (Barret-Lennard
et al., 2012). The hydrolysis of phosphomonoesters by phosphatases in biological systems is an
important process. This process is linked to energy metabolism, metabolic regulation and a wide
variety of cellular signal transduction pathways (Allan et al., 2010). The role of acid phosphatase
in phosphorus metabolism has been extensively studied in prokaryotic and eukaryotic systems.
The physiological function of acid phosphatase is to provide inorganic phosphate for the cellular
growing.
The liberation of phosphate from phosphate ester is mainly affected by phosphatase (Bewley and
Black, 2008). Deficiency of phosphorus in plants leads to metabolic disorders such as a decrease
in photosynthesis, respiration and retardation of growth. Phosphatases have been traditionally
classified as being alkaline or Acid Phosphatase (AP) according to their optimum pH for
catalytic activity above or below pH 7.0 (Barret-Lannard et al., 2012). Several interacellular acid
phosphatases have been suggested to have a role in the hydrolysis of interacellular
polyphosphates (Amlabu et al., 2009).
Acid phosphatase was found to be localized in the cell walls of Pisum sativum (Allan et al.,
2010), phloem of Nicotiana tabacum (Bentwood and Cronshaw, 2006) and xylem of Phaseolus
vulgaris (Bewley and Black, 2008). The mechanism which regulates acid phosphatase
distribution and activity is unclear, although their abundance may be regulated by the level of
phosphate in the environment and their activity is clearly influenced by local polyelectrolytes.
Acid phosphatase expresses its isozymes in many plants such as soybean seeds (Amlabu et al.,
2009); Vigna sinensis (Allan et al., 2010), tea leaves and (Baker and Tadakazu, 2011).
Lastly the purpose of this experiment was to determine the effects of different metal ions on acid
phosphatase activity.

REFERENCES
1. Allan, A.C., M.D. Fricker, J.L. Ward, M.H. Beale and A.J. Trewavas, 2010. Two
Transduction pathways mediate rapid effects of abscisic acid in Commelina guard cells.
Plant Cell, 6: 1319-1328.
2. Amlabu, E., A.J. Nok and A.B. Sallau, 2009. Purification and biochemical
characterization of lysosomal acid phosphatases (EC 3.1.3.2) from blood stream forms,
Trypanosoma brucei brucei. Parasitol. Int., 58: 238-242.
3. Baker, J.E. and T. Tadakazu, 2011. Acid phosphatase in plant tissues. I. Changes in
activity and multiple forms in tea leaves and tomato fruit during maturation and
senescence. Plant Cell Physiol., 14: 459-471.
4. Barret-Lennard, E.D., A.D. Robson and H. Greenway, 2012. Effect of phosphorus
deficiency and water deification phosphatase activity from wheat leaves. J. Exp. Bot., 33:
682-693.
5. Bentwood, B.J. and J. Cronshaw, 2006. Biochemistry and cytochemical localization of
acid phosphatase in the phloem of Nicotiana tabacum. Planta, 130: 97-104.
6. Bewley, J.D. and M. Black, 2008. Seeds: Physiology of Development and Germination.
2nd Edn., Plenum Press, New York, Pages: 444.

MATERIALS AND METHODS

Four germinated seeds of cowpeas (which were soaked in 0.1 % of sodium hypochlorite for 10
minutes, washed 2 successive times with distilled water, soaked in an Erlenmeyer flask closed
with mira cloth or cotton wool, placed on a shaker at 120 rpm for 14 hrs at 28degerres Celsius as
germination pre-treatment) were separated into shoots and cotyledons before pulverizing into
fine powder using pestles and mortars that have been precooled with ice. Pulverized sample was
not allowed to thaw before adding extraction buffer. Extraction buffer was added to the
pulverized sample in 1.5 ml aliquots at a time up to 6- ml total volume of extraction buffer. The
mixture was then incubated at 25 degrees Celsius for 20 minutes prior to centrifuging at 14 000
rpm for 20 minutes. Both the supernatant and the pellet were collected and retained.an enzyme
assay was done using the supernatants with p-nitrophenol as a substrate. 1ml of the substrate was
pipetted into the test tube and pre- incubated at 30 degrees Celsius

RESULTS
Table 1. The Effects of Different Metal Ions on Acid Phosphate
SHOOTS
FEROUS SULPHATE
CONTROL
0.2
MEVEUVIC SULPHATE
CONTROL
0.2
COTYLEDON
FEROUS SUPLHATE
CONTROL
0.2
MEVEUVIC SULPHATE
CONTROL
0.2

EXTRACT
1.4
EXTRACT
0.6
EXTRACT
0.1
EXTRACT
0.8

DISCUSSION
The effects of molybdate, Mn2+, Zn2+, Cu2+ and Fe 3+ on Vigna aconitifolia acid phosphatase were
studied using a saturated concentration of p-NPP (6.0 mM) as substrate (. All metal ions had an
inhibitory effect on the enzyme. Molybdate was found to be relatively the most potent inhibitor
of enzyme. Acid phosphatase from Arabidopsis, soybean, and tomato is reported to be sensitive
to molybdate. Amongst the metal ion effectors, next to molybdate, Fe 3+ was the second most
potent inhibitor for acid phosphatase. An inhibitory effect of Fe 3+ has been observed for acid
phosphatase from germinating seeds ofVigna sinensis. In addition, Cu2 also showed significant
inhibitory effect on acid phosphatase activity, while Mn2+ and Zn2+ were found to be mild
inhibitors.
Other than metal ions, fluoride and phosphate are two most common effectors of acid
phosphatase. The two have been used in elucidation of catalytic mechanism of this enzyme
through inhibition kinetics and structural analysis. Similar to other reports, in our study, both
anions acted as an inhibitor also for Vigna aconitifolia acid phosphatase affinity of molybdate for
the enzyme. Usually, transition metal oxides are reported to be inhibitors for acid phosphatases
due to their similarity with the transition state, or they coordinate with amino acids on the active
site and cause conformational changes in it. However, here we have observed that molybdate
inhibited the enzyme activity in a mixed pattern, which indidates that either it may be acting as
structural analog or it may be involved in complex formation with E-S.
Ferric ion also showed mixed-type inhibition on the enzyme, with Ki and KI values 0.4 and1.0
mM, respectively. No detailed study on inhibition kinetics of ferric ion regarding this enzyme is
available. Cu 2+ exhibited noncompetitive inhibition on acid phosphatase, which reveals that this
ion binds to the no-catalytic site of the enzyme, no matter whether the substrate is bound or not.
Among the three metal ions used in our experiment, Cu2 demonstrated low affinity toward
Vigna aconitifoliaacid phosphatase as its Ki value (2.6 mM) was much higher as compared to
other effectors (Table 1). Huang and Shindo also found that copper inhibition on free and soilbound acid phosphatase activity was of mixed type.
Fluoride and phosphate are well-known inhibitors of acid phosphatase. Also in our study, they
acted as inhibitors for enzyme activity. These effectors exhibited mixed and competitive type of
inhibition on acid phosphatase activity, respectively (Figures 6 and 7). In agreement with our
results, Turner and Plaxton[4] and Bozzo et al. [23] have also found mixed-type inhibition on
banana and tomato acid phosphatase by fluoride, respectively. However, in other reports,

inhibition of acid phosphatase activity due to fluoride was observed to be uncompetitive or

noncompetitive.
Further detailed inhibition studies have explained that fluoride mimics as a nucleophile (terminal
hydroxide at acid phosphatase catalytic site) and thus prevents the hydrolytic reaction catalyzed
by enzyme. Thus, fluoride inhibition of V. aconitifolia acid phosphatase might be due to fluoride
imitation of a nucleophilic hydroxide, which needs to be studied further in detail. Phosphate
generally causes inhibition on acid phosphatase activity by competing with substrate for the
enzyme active site. Inhibition constants for phosphate were previously reported to be in the range
of 0.092.4 mM.
The Ki value reported in our experiment was also found within this range only (Table 1). Acid
phosphatase hydrolyzes phosphorylated organic compounds and metabolites, producing
phosphate as one of hydrolysis products. When plants are phosphate deprived, this enzyme
becomes more active in order to fulfill the plant phosphate requirement.
In our study, inhibition of V. Aconitifolia acid phosphatase due to phosphate may explain the
regulation of this enzyme through product inhibition. Inhibition studies on various acid
phosphatase effectors have been conducted to examine the catalytic mechanism of the enzyme or
development of therapeutics for bone disorders. This study deals with investigation on the effects
of metal ions and a few nonmetal ions on Vigna aconitifolia seed acid phosphatase and their
inhibition kinetics. Three of the effectors, molybdate, ferric ion, and fluoride, exhibited a mixed
pattern of inhibition, while Cu2+ and phosphate showed noncompetitive and competitive
inhibition on the enzyme, respectively. In our experiment, we have found that the enzyme was
strongly inhibited by the two most important acid phosphatase inhibitors, molybdate and
fluoride. Most acid phosphatases follow a similar catalytic mechanism due to their active-site
structural similarity. Thus, further comprehensive study on inhibition kinetics of these two
inhibitors and their derivatives in relation toVigna aconitifolia seed acid phosphatase inhibition
may give ideas to develop other more specific inhibitors for the enzyme, which might be useful
in treatment of osteoporosis and other bone-related disorders where the role of acid phosphatase
is critical.
REFERENCES