Effect of copper on the activation of the acid phosphatase from the green algae Pseudokirchneriella subcapitata

Article abstract of DOI:10.1007/s10534-009-9270-z

The presence of copper in water environment may have detrimental effects on aquatic organisms, including algae, where different enzymatic systems can be affected. Algae acid phosphatase plays important roles in metabolic processes such as decomposition of

Full text of DOI:10.1007/s10534-009-9270-z

Biometals (2010) 23:93–98
DOI 10.1007/s10534-009-9270-z
Effect of copper on the activation of the acid phosphatase
from the green algae Pseudokirchneriella subcapitata
´
Claudio Martın Jonsson Æ Hiroshi Aoyama
Received: 13 March 2009 / Accepted: 9 September 2009 / Published online: 19 September 2009
Ó Springer Science+Business Media, LLC. 2009
Abstract The presence of copper in water environ-
ment may have detrimental effects on aquatic organ-
isms, including algae, where different enzymatic
systems can be affected. Algae acid phosphatase
plays important roles in metabolic processes such as
decomposition of organic phosphate, autophagic
digestive process, recycling cellular materials and
zygote formation during reproduction. This work
describes an in vitro activation effect of copper on the
acid phosphatase of the green algae Pseudokirch-
neriella subcapitata (formely Selenastrum capricor-
nutum) under preincubation condition. Apparent
Michaelis constant values of 1.21 and 0.37 mM,
and activation energy values of 26.8 and
13.6 kJ mol-1 were determined in the absence and
in the presence of 0.2 mM Cu2?, respectively. The
dissociation constant value for Cu2? binding to
the enzyme was determined to be 22.04 lM. The
decrease of the apparent Michaelis constant (Km) and
activation energy values in the presence of Cu2?
correlates well with its activating effect on the acid
phosphatase activity. This propriety could be used as
a sensitive bioindicator for copper in environmental
samples.
Keywords Enzyme Á Phytoplankton Á
Toxicity Á Fungicide Á Metal Á
Selenastrum capricornutum
Introduction
There has been an increasing interest in studying the
action of metal on enzyme activity and in using their
biochemical changes in the aquatic biota as an index
of metal toxicity (Lan et al. 1995). In this context,
copper, an essential metal ion, has been intensively
studied as a potential toxic compound (Gill et al.
1992, Guasch et al. 2002). This element is widely
used in agriculture to control fungal diseases and its
increasing concentration in the environment has been
attributed to mining operations, sewage sludge
disposal and industrial discharges (WHO 1998).
Copper sulphate has also been used in the control
of natural phytoplanktonic communities (Larsen et al.
2003; Le Jeune et al. 2006). The increase of
availability and potential toxicity of copper in water
may have detrimental effects on aquatic organisms
including algae, an important component of primary
production and thus the entire aquatic food chain.
Algae are able to bioconcentrate and to metabolize
C. M. Jonsson (&)
Embrapa Meio Ambiente, CP 69, Rod. SP 340, Km 127,5,
´
13820-000 Jaguariuna, Sa˜o Paulo, Brazil
e-mail: jonsson@cnpma.embrapa.br
H. Aoyama
Departamento de Bioquımica, Instituto de Biologia,
´
Universidade Estadual de Campinas, 13083-970
Campinas, Sa˜o Paulo, Brazil
123

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Biometals (2010) 23:93–98
several aquatic pollutants like insecticides and poly-
cyclic aromatic hydrocarbons (Warshawsky et al.
1995; Jonsson et al. 2001).
The activities of algal enzymes like esterase
(Franklin et al. 2001), b-D-galactosidase (Peterson
and Stauber 1996), nitrate redutase, urease, ATPase
(Rai and Rai 1997) and antioxidants enzymes (Li
et al. 2006) were affected in presence of the metal.
Previously, we reported the activator effect of
copper on the activity of acid phosphatase from P.
subcapitata. Also, we demonstrated that the metal
behaved as a slight antagonist for the inhibitor effect
of mercury on the enzyme activity (Jonsson and
Aoyama 2007). In the present work, we studied the
effect of copper ions on the enzyme activity from the
algae. We analyzed the stability and determined dose
effects and kinetic parameters in order to understand
the magnitude and mechanism of metal ion–phos-
phatase interaction.
Pseudokirchneriella subcapitata (formely Selena-
strum capricornutum), an unicellular Chlorophyceae
(green) algae present in the freshwater aquatic and
terrestrial compartments (Keddy et al. 1995), has been
widely used in studies of pollutants effects (Jonsson
et al. 1998; Okamura et al. 2002) and is recommended
by regulatory national (Gherardi-Goldstein et al.
1990; Jonsson and Maia 1999) and international
(OECD 1984) agencies as a test organism.
Acid phosphatase (EC 3.1.3.2) plays important
roles in the metabolism such as decomposing organic
phosphates into free phosphates and organic com-
pounds. Several functions have been attributed to
algae acid phosphatases, such as autophagic digestive
processes, hydrolysis of phospholipid materials (Coo-
per et al. 1974), fertilization (breakdown of plasma-
lemma and absorption of flagella) (Braten 1975),
availability of inorganic phosphate from the extra-
cellular medium (Sommer and Blum 1965), recycling
of inorganic phosphate for its reassimilation (Theod-
orou et al. 1991), endomembrane recycling (Domo-
zych 1989) and spore differentiation (Tsekos and
Schnepf 1991). In the freshwater algae S. tenue, the
enzyme was found in the cell wall, plasmalemma and
vacuole (Michetti et al. 2006). The induction of acid
phosphatase activity in unicellular green algae at low
N and P concentrations has been observed by
Kruskopf and Du Plessis (2004).
Materials and methods
Materials
p-Nitrophenylphosphate (pNPP) was obtained from
Sigma Chemical Co. Stock solutions of CuSO45H2O
(Merck) were prepared in Milli-Q water or in liquid
medium (OECD 1984). All the other reagents were
AR grade.
Organisms and growth conditions
Unicellular green algae Pseudokirchneriella subcap-
itata was maintained and subcultured in an inorganic
liquid medium prepared as recommended by OECD
(OECD 1984). Cultures were grown in 250 ml flasks
sealed with cotton bungs and containing 200 ml of
sterilized medium. The flasks were incubated in a
controlled temperature chamber (20 2°C) under a
continuous white fluorescent light of 3,000–4,000 lux
and manually shaken twice a day. Every 40–60 days,
a new stock culture maintained at 4°C (in dark) was
prepared by inoculating approximately 5 9 104
cells ml-1 (Jonsson and Aoyama 2007).
It has been reported that copper or other metal ion
are needed for the catalytic action of different acid
phosphatases. Vincent and Averill (1990) have
observed that the oxidized form of some mammalian
acid phosphatases exhibited a iron–phosphorus inter-
action indicating that phosphate, and presumably the
phosphate esters, bound to one of both irons present
in the protein. Reilly et al. (1999) demonstrated that
the addition of copper restored nearly all the enzy-
matic activity of an acid phosphatase from Hae-
mophilus influenzae. The acid phosphatases from the
microorganisms Mycoplasma fermentans, Pseudomo-
nas aeruginosa and Yarrowia lipolytica are enhanced
in the presence of copper (Domenich et al. 1992;
Noda et al. 1994; Ito et al. 2007). This last effect was
also observed by Bounias et al. (1996) in the kinetic
studies for alkaline and acid phosphatases present in a
gut crude extract obtained from honeybees .
Harvesting and preparation of extracts
All centrifugation procedures were carried out at 4°C.
Exponential phase organisms were harvested by
centrifugation at 4,000 r.p.m. for 5 min in a Beckman
J2-21 refrigerated centrifuge (rotor SER# 7644,
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Biometals (2010) 23:93–98
95
Apparent Michaelis constant (Km) and maximum
velocity (Vmax) determination
JA-20) and washed twice with 0.1 M sodium acetate
buffer, pH 5.0. The algae pellet was suspended in
0.1 M sodium acetate buffer (1:4 w/v) and the cell
suspension was submitted to the cell disruption
procedure for phosphatase extraction as described
previously (Jonsson and Aoyama 2007): The sample
was frozen at -20°C, thawed at room temperature
and submitted to a probe sonication at 0°C (ice bath)
for 50 s followed by 20 s interval (1 cycle) with an
amplitude of 70 (Vibra Cell, Sonics Materials Inc.,
45 mm tipped probe). This procedure was repetead
twice. The resultant cell disrupted suspension was
centrifuged at 10,000 r.p.m. for 20 min and the
supernatant fluid (extract) was used for acid phos-
phatase activity determination.
The enzyme was preincubated in the absence or in the
presence of 0.2 mM Cu2?, and after that the enzyme
activity was determined, using pNPP as substrate, in a
concentration range of 0.1–10.0 mM. The apparent
Km and Vmax values were calculated from Linewe-
aver–Burk plot.
Activation energy determination
The enzyme activity was determined after preincu-
bation at different temperatures (27, 32, 37, 42 and
47°C) in the absence or in the presence of 0.2 mM
Cu2?. The activation energy values were determined
from the slopes of the Arrhenius plots (log V versus
1/T (Kelvin)), according to Dixon and Webb (1979).
Assay of phosphatase activity
Acid phosphatase activity wasroutinelyassayed atleast
in duplicate by incubating the enzyme with pNPP as
substrate and measuring the p-nitrophenol (pNP)
produced as previously described (Prazeres et al.
2004). The enzyme activity was determined in a final
volume of 1 ml containing 0.1 M sodium acetate buffer
(pH 5.0) and 10 mM substrate. After incubation for
40 min at 37°C, the reaction was terminated by the
addition of 1 ml of 1 M NaOH. The pNP released was
measured at 405 nm in a UNICAM 8625 UV/VIS
spectrophotometer. For the initial velocity (V) deter-
mination, the amount of pNP produced was calculated
using a molar extintion coefficient of 18,300 M-1cm-1
(Chaimovich and Nome 1970). Units (U) of enzymatic
activity are defined as lmoles of pNP released per min.
Data analysis
The results were analyzed by a Simples Regression
program with a StatgraphicsÒ Plus Version 2 soft-
ware package.
Results
In vitro effect of copper on P. subcapitata
acid phosphatase activity
A remarkable increase in pNPP dephosphorylation
was observed in the presence of Cu2? up to a
concentration of 0.05 mM, when this metal was
previously incubated with the enzyme (Fig. 1). The
dissociation constant (KdCu2?), for the complex
Cu2?-enzyme was calculated from the double
reciprocal plot to be 22.04 lM (Fig. 1, inset). In
contrast to the results described in the Fig. 1, no
significant enzyme activity changes were observed in
the presence of Cu2? without preincubation (results
not shown).
Effect of Cu2?concentration
The enzyme activity was determined in the absence
(control) or in the presence of several copper concen-
trations in a range of 0.1–200 lM Cu2?. Lineweaver–
Burk plot was used to determine the Cu2?-enzyme
dissociation constant by plotting the velocity change
(delta V) versus the reciprocal of copper concentration
(1/[Cu2?]), as described by Dixon and Webb (1979).
Stability of acid phosphatase in the presence
of copper
Enzyme stability
The enzyme was stable for at least 60 min at 37°C
The enzyme activity was determined after preincu-
when preincubated in the presence of 0.2 mM Cu2?
.
bation at 37°C for 0, 20, 40 and 60 min in the absence
or in the presence of 0.2 mM Cu2?
.
In contrast, a loss of acid phosphatase activity of
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Biometals (2010) 23:93–98
300
250
200
150
100
50
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
10
8
6
4
2
0
-100 -50
0
50
100 150 200
-1
1/ [Cu2+] (mM )
0
20
40
60
0
0
0.05
0.1
0.15
0.2
0.25
Time (min)
[Cu2+] (mM)
Fig. 2 Preincubation time course of acid phosphatase activity.
The enzyme activity was determined at the indicated times by
preincubation at 37°C in the absence (d) or in the presence (j)
of 0.2 mM Cu2?. Bars represent the standard deviations of the
averages of two replicates
Fig. 1 Effect of copper concentration on acid phosphatase
activity. The preincubation of the enzyme and Cu2?(at
different concentrations) was performed for 20 min at 37°C.
At the end of this period, the reaction was initiated by the
addition of pNPP 10 mM as substrate, as described in Methods.
The activity in the absence of Cu2? was considered as 100%
(control) and the other values were determined as percentages
of this value. Determination of dissociation constant for the
interaction Cu2?-acid phosphatase (inset). Bars represent the
standard deviations of the averages of two replicates
Table 1 Effect of Cu2?on the kinetic parameters for P. sub-
capitata acid phosphatasea
Parameter
[Cu2?] (mM)
0.0
0.2
about 60% was observed when the enzyme was
preincubated in the absence of the metal ion (Fig. 2).
Km (mM)
Vmax (mU ml-1
Activation energy (kJ mol-1
1.21 0.42
0.55 0.16
26.8 1.5
0.37 0.05
0.75 0.06
13.6 3.9
)
)
Effect of Cu2?on the acid phosphatase
kinetic parameters
a
The Km and Vmax values were determined from
Lineweaver–Burk plot and the activation energy from the
Arrhenius plot. The standard deviations ( ) represent the
averages of two replicates
The values of apparent Km and Vmax were deter-
mined from the double reciprocal plot of Linewe-
aver–Burk, and the activation energy from the
Arrhenius plot (not shown). These results are shown
in the Table 1. In the presence of 0.2 mM Cu2?, the
apparent Km value decreased from 1.21 to 0.37 mM
castor bean seed (Granjeiro et al. 1999) were
inhibited 97 and 93% in the presence of 10 mM
and 1 mM Cu2?, respectively.
In the present work we demonstrated that Cu2?
when preincubated with P. subcapitata acid phos-
phatase activated the pNPP-directed reaction. Li-
neweaver–Burk plot was used to determine the
dissociation constant value for Cu2? binding to the
enzyme (KdCu2?), as described by Dixon and Webb
(1979) (Fig. 1, inset). Youngs et al. (2000) have also
applied this method to determine the dissociation
constant for the Cd-Mn-peroxidase complex. The low
magnitude of the KdCu2? value (22.04 lM) obtained
for the complex Cu2?-acid phosphatase indicated a
strong binding capacity of this metal to the enzyme.
Even after preincubation of Cu2?, and enzyme, no
activation effect was observed for the acid
and the activation energy from 26.8 to 13.6 kJ mol-1
;
no significant difference was observed for the Vmax
values (0.55 and 0.75 mU ml-1).
Discussion
The metallic ion Cu2? has been generally used in
enzymatic systems in order to inactivate the reactions
by binding to essential sulfhydryl groups located at or
near the active site. In the case of acid phosphatases,
we have previously reported that the enzyme purified
from bovine kidney (Granjeiro et al. 1997) and from
123

Biometals (2010) 23:93–98
97
phosphatase obtained from the algae Ochromonas
danica (Patni and Aaronson 1974). However, others
in vitro studies showed that acid phosphatases from
crude extracts of Rizopus delemar (Tsekova and
Galabova 2003) and honeybee guts (Bounias et al.
sound basis for comparison with enzymatic processes
affected by other pollutants.
Acknowledgements This work was supported by grants from
`
Fundac¸a˜o de Amparo a Pesquisa do Estado de Sa˜o Paulo
(FAPESP), Conselho Nacional de Desenvolvimento Cientıfico
´
1996) were activated by Cu2?
.
´
e Tecnologico (CNPq) and Empresa Brasileira de Pesquisa
´
Agropecuaria (Embrapa). The authors are grateful to Dr. Fred
In order to explain the reasons for the activation of
the P. subcapitata acid phosphatase by Cu2?, we
performed three different experiments: enzyme sta-
bility study, determination of apparent Km, and
determination of activation energy, in the absence
and in the presence of the metal.
´
Y. Fujiwara (Instituto de Quımica, UNICAMP) for critically
reading the manuscript.
References
The presence of Cu2?promoted a protection of the
P. subcapitata acid phosphatase from thermal inacti-
vation at 37°C for at least 1 h (Fig. 2), which supports
an enzyme–Cu2? interaction. Protection of enzymes
by metal ions in stability studies was also described for
fungal laccase by Cu2? (Baldrian and Gabriel 2002),
fungal peroxidase by Cd2?and Mn2? (Youngs et al.
2000), and E. coli alkaline phosphatase by Cu2?and
other metals (Trotman and Greenwood 1971).
Baldrian P, Gabriel J (2002) Copper and cadmium increase
laccase activity in Pleurotus ostreatus. FEMS Microbiol
Lett 206:69–74. doi:10.1111/j.1574-6968.2002.tb10988.x
Bennum A, Blum JJ (1966) Properties of the induced acid
phosphatase and of the constitutive acid phosphatase of
Euglena. Biochim Biophys Acta 128:106–123
Bounias M, Kruk I, Nectoux M, Popeskovic D (1996) Toxicol-
ogy of cupric salts on honeybees. V. Gluconate and sulfate
action on gut alkaline and acid phosphatases. Ecotoxicol
Environ Saf 35:67–76. doi:10.1006/eesa.1996.0082
Braten T (1975) Ultrastructural localization of phosphohydro-
lases in gametes, zygotes and zoospores of Ulva mutabilis
FØYN. J Cell Sci 17:647–653
Chaimovich H, Nome F (1970) Purification and properties of
an acid phosphatase from bovine brain. Arch Biochem
Biophys 139:9–16. doi:10.1016/0003-9861(70)90039-1
Cooper A, Bowen ID, Lloyd D (1974) The properties and
subcellular localization of acid phosphatases in the col-
ourless alga Polytomella caeca. J Cell Sci 15:605–618
Dixon M, Webb EC (1979) Enzymes, 3rd edn. Longmans
Green and Co Ltd., London
Domenich CEA, Teresita AL, Salvano MA et al (1992)
Pseudomonas aeruginosa acid phosphatase: activation by
divalent cations and inhibition by aluminium ions. FEBS
Lett 299:96–98. doi:10.1016/0014-5793(92)80108-S
Domozych DS (1989) The endomembrane system and mech-
anism of membrane flow in the green alga, Gloeomonas
kupfferi (Volvocales, Chlorophyta) II. A cytochemical
analysis. Protoplasma 149:108–119. doi:10.1007/BF0132
2983
The kinetic parameter apparent Km for pNPP
decreased (3.3 fold) in the presence of Cu2?
(Table 1), suggesting an enhancement on the affinity
enzyme-substrate by copper.
Bounias et al. (1996) also described a decrease of
the Km value for honeybee gut acid phosphatase
contained in the crude extract in the presence of
copper. The apparent Km value of 0.37 mM, obtained
in the presence of Cu2?is similar to those reported for
other algae species when the enzyme was assayed in a
non-preincubated system and in the absence of the
metal (Bennum and Blum 1966; Patni and Aaronson
1974). The decrease of the activation energy value
from 26.8 to 13.6 kJ mol-1 in the presence of Cu2?
(Table 1), also contributed to the activating effect of
Cu2?on the P. subcapitata acid phosphatase activity.
Summarizing, different factors contributed to the
increase in the acid phosphatase activity in the
presence of Cu2?: protection of the enzyme against
thermal inactivation, increase in the enzyme-substrate
affinity, and reduction of the activation energy. The
high sensitivity of the enzyme crude extract to copper
may be useful as a tool to detect this metal in natural
waters and other environmental samples.
Franklin NM, Stauber JL, Lim RP (2001) Development of flow
cytometry—based algal bioassays for assessing toxicity of
copper in natural waters. Environ Toxicol Chem 20:160–
170. doi:10.1897/1551-5028(2001)020\0160:DOFCBA[
2.0.CO;2
Gherardi-Goldstein E, Bertoletti E, Zagatto PA et al (1990)
Procedimentos para a utilizac¸a˜o de testes de toxicidade no
´
controle de efluentes lıquidos, Serie Manuais. CETESB,
Sa˜o Paulo
Gill TS, Tewari H, Pande J (1992) Short and long term effects
of copper on the rosby barb (Puntius conchonius Ham).
Ecotoxicol Environ Saf 23:294–306. doi:10.1016/0147-
6513(92)90079-I
Granjeiro JM, Ferreira CV, Juca MB et al (1997) Bovine
kidney low molecular weight acid phosphatase: FMN-
dependent kinetics. Biochem Mol Biol Int 41:1201–1208
We hope that the results presented in this work can
improve the understanding of the basic events of the
impact of copper at biochemical levels in primary
producer organisms. Moreover, they could provide a
123

98
Biometals (2010) 23:93–98
Granjeiro PA, Ferreira CV, Granjeiro JM et al (1999) Purifi-
cation and kinetic properties of a castor bean seed acid
phosphatase. Physiol Plant 107:151–158. doi:10.1034/
j.1399-3054.1999.100201.x
Guasch H, Paulsson M, Sabater S (2002) Effect of copper on algal
communitiesfromoligotrophiccalcareousstreams. J Phycol
38:241–248. doi:10.1046/j.1529-8817.2002.01114.x
Ito H, Inouhe M, Tohoyama H, Joho M (2007) Effect of copper
on acid phosphatase activity in yeast Yarrowia lipolytica.
Z Naturforsch [C] 62:70–76
OECD (1984) Guidelines for Testing of Chemicals. Alga,
Growth Inhibition Test. Organization for Economic
Cooperation and Development. OECD, Paris
Okamura H, Mingyu P, Aoyama I et al (2002) Algal growth
inhibition by river water pollutants in the agricultural area
around lake Biwa, Japan. Environ Pollut 117:411–419.
doi:10.1016/S0269-7491(01)00196-8
Patni NJ, Aaronson S (1974) Partial characterization of the
intra- and extracellular acid phosphatase of an alga,
Ochromonas danica. J Gen Microbiol 83:9–20
Jonsson CM, Aoyama H (2007) In vitro effect of agriculture
pollutants and their joint action on Pseudokirchneriella
subcapitata acid phosphatase. Chemosphere 69:849–855.
doi:10.1016/j.chemosphere.2007.06.024
Peterson SM, Stauber JL (1996) New algal enzyme bioassay for
the rapid assessment of aquatic toxicity. Bull Environ
Contam Toxicol 56:750–757. doi:10.1007/s001289900110
Prazeres JN, Ferreira CV, Aoyama H (2004) Acid phosphatase
activities during the germination of Glycine max seeds.
Plant Physiol Biochem 42:15–20. doi:10.1016/j.plaphy.
2003.10.009
Jonsson CM, Maia AHN (1999) Protocolo de avaliac¸a˜o de
´
agentes microbianos de controle biologico de pragas para
registro como biopesticidas. III. Testes em organismos
´
na˜o alvo do ambiente aquatico. Serie Documentos.
Embrapa Meio Ambiente, Jaguariuna
Rai PK, Rai LC (1997) Interactive effects of UV-B and Cu on
photosynthesis, uptake and metabolism of nutrients in a
green alga Chlorella vulgaris under simulated ozone
column. J Gen Appl Microbiol 43:281–288. doi:10.2323/
jgam.43.281
´
Jonsson CM, Maia AHN, Ferreira CJA et al (1998) Risk
assessment of the herbicide clomazone in the aquatic life.
Verh Int Verein Limnol 26:1724–1726
Jonsson CM, Paraiba LC, Mendoza MT et al (2001) Biocon-
centration of the insecticide pyridaphenthion by the green
algae Chlorella saccharophila. Chemosphere 43:321–325.
doi:10.1016/S0045-6535(00)00145-4
Keddy CJ, Greene JC, Bonnel MA (1995) Review of whole-
organism bioassay: soil, freshwater, sediment, and fresh-
water assessment in Canada. Ecotoxicol Environ Saf
30:221–251. doi:10.1006/eesa.1995.1027
Kruskopf MM, Du Plessis S (2004) Induction of both acid
and alkaline phosphatase activity in two green-algae
(Chlorophyceae) in low N and P concentrations. Hyd-
robiologia 513:59–70. doi:10.1023/B:hydr.0000018166.
15764.b0
Reilly TJ, Chance DL, Smith AL (1999) Outer membrane
lipoprotein e (P4) of Haemophilus influenzae is a novel
phosphomonoesterase. J Bacteriol 181:6797–6805
Sommer JR, Blum JJ (1965) Cytochemical localization of acid
phosphatase in Euglena gracilis. J Cell Biol 24:235–251.
doi:10.1083/jcb.24.2.235
Theodorou ME, Elrifi IR, Turpin DH et al (1991) Effect of
phosphorus limitation on respiratory metabolism in the
green algae Selenastrum minutum. Plant Physiol 95:1089–
1095. doi:10.1104/pp.95.4.1089
Trotman NA, Greenwood C (1971) Effects of zinc and other
metal ions on the stability and activity of Escherichia coli
alkaline phosphatase. Biochem J 124:25–30
Lan WG, Wong MK, Chen N et al (1995) Effect of combined
copper, zinc, chromium and selenium by orthogonal array
design on alkaline phosphatase activity in liver of the red
sea bream, Chrysophrys major. Aquaculture 131:219–
230. doi:10.1016/0044-8486(94)00326-J
Larsen DK, Wagner I, Gustavson K et al (2003) Long-term
effect of sea-nine on natural coastal phytoplanktonic
communities assessed by pollution induced community
tolerance. Aquat Toxicol 62:35–44. doi:10.1016/S0166-
445X(02)00065-6
Le Jeune AH, Charpin M, Deluchat V et al (2006) Effect of
copper sulphate treatment on natural phytoplanktonic
communities. Aquat Toxicol 80:267–280. doi:10.1016/
j.aquatox.2006.09.004
Li M, Hu C, Zhu Q, Chen L et al (2006) Copper and zinc
induction of lipid peroxidation and effects on antioxidant
enzyme activities in the microalga Pavlova viridis
(Prymnesiophyceae). Chemosphere 62:565–572. doi:
10.1016/j.chemosphere.2005.06.029
Tsekos I, Schnepf E (1991) Acid phosphatase activity during
spore differentiation of the red algae Gigartina teedii and
Chondria tenuissima. Plant Syst Evol 176:35–51. doi:
10.1007/BF00937944
Tsekova K, Galabova D (2003) Phosphatase production and
activity in copper (II) accumulating Rizopus delemar.
Enzyme Microb Technol 33:926–931. doi:10.1016/
j.enzmictec.2003.06.001
Vincent JB, Averill BA (1990) An enzyme with double iden-
tity: purple acid phosphatase and tartrate-resistant acid
phosphatase. FASEB J 4:3009–3013
Warshawsky D, Cody T, Radike M et al (1995) Biotransfor-
mation of benzo[a]pyrene and other polycyclic aromatic
hydrocarbons and heterocyclic analogs by several green
algae and other algal species under gold and white light.
Chem Biol Interact 97:131–148. doi:10.1016/0009-2797
(95)03610-X
WHO (1998) Copper. Environmental health criteria 200.
International Programme on chemical safety. World
Health Organization, Geneva
´
Michetti KM, Leonardi PI, Caceres EJ (2006) Cytochemical
localization of acid phosphatase in Stigeoclonium tenue
(Chaetophorales, Chlorophyceae). Biocell 30:491–496
Noda M, Shibata K, Sawa Y et al (1994) Purification and
characterization of an acid phosphatase from Mycoplasma
fermentans. Microbiol Immunol 38:103–107
Youngs HL, Sundaramoorthy M, Gold MH (2000) Effects of
cadmium on manganese peroxidase. Competitive inhibi-
tion of MnII oxidation and thermal stabilization of the
enzyme. Eur J Biochem 27:1761–1769. doi:10.1046/
j.1432-1327.2000.01173.x
123