全 文 ：广 西 植 物 Guihaia 28(3)：390— 394 2008年 5月
phatas(fm from g~ ainating pigeon[ seede|sotorm t- ernu u l pea o
DONG Deng-Feng1，2，U Yang-RuiH ，YANG Jie2，Ⅵ G Yong-Feng2
(1．Key-Lab of Guangxi Crop Genetic Improvement and Biotechnology，Nanning 530007，China；
2．Colege of Agriculture，Guangxi University，Nanning 530004，China)
Abstract：Using p-nitrophenolphosphate(pNPP)as substrate，one isoform of acid phosphatase from germinating pi-
geonpea seed，encoded as APaseⅡ，was purified to 247 folds and the specific activity 51．8 U／mg protein through am—
monium sulfate fractionation and three sequential DEAE-Sephadex A 25，Hydroxyapatite and Concanavalin A-Sepha-
rose 4B column chromatography．The purified APH was demonstrated by Native-and SDS-PAGE to be electro—
phoretic homogeneity and as a 33 kDa monomer．APaseⅡexhibited optimal pH at 5．0 and optimal temperature at 35
℃ and strong stabilization at the pH ranging from 3．5 to 7．0 and at temperature below 55℃．APase 1 showed a
highest specifdty against pyrophosphate．and was activated by K+ and Mg2+ ，while inhibited by Fez+，Mnz+，
MoT0246 ，F-as wel as by organic acids including as tartrate，malate，isocitrate，oxalate，citrate，glycolate，glyoxylate
and ascorhate．
Key words：pigeonpea；acid phosphatase；purification；kinetic characteristics
CLC Nutuber：Q556．1 Document Code：A Article ID：1000．一3142(2008)O3—0390-‘05
Acid phosphatases(EC 3．1．3．2)，catalyzing the
hydrolysis of a wide range of ortho,phosphate monoes—
ters at pH optima below 6．0，are widely distributed in
plants and localized in nearly all organisms and tissues．
Plant add phosphatases usually present in multiple
forms，display diferent biochemical properties，and ex-
hibit a broad spedfidty towards natural and synthetic
phosphoric esters(Ferreira et a1．，1998)． Plant add
phosphatases are involved in phosphorus mobifizafion
and recycle and also up-regulated during salt or osmot-‘
ic stress，seed germination，flowering，senescence and
fruit ripening(Duf eta1．，1994；Dong eta1．，2005)．
Pigeonpea plays an important role in sustainable
agriculture development in arid district because of its
high economic value and tolerance to extremely drought
and nutrition deficiency(Ae et a1．，2000)，and more and
more researc J：hes have been focusing on its tolerance to
abiotic stress．In our prelim nary experiments，APase ac—
tivity was found to increase sharply during the germina—
ring period and whe n seedling was exposed to salt and
drought stresses． In the present paper，APase protein
was isolated and purified from germinated pigeon seed，
its physiological and biochemi cal properties investigated
and possible fun ction discussed．
Material and methods
1．1 Plant material
300 gram of pigeonpea(Cajanus cajan cv．Guimu
2)seeds were surface-sterihzed with 10 H2 O2 for 5
mi n，rinsed wi th distilled water，and germi nated in 25
℃ incubator．
1．2 APase activity determination
APase activity determination was perform ed by
Received date：2007-05—09 Accepted date：2007-09-21
Foundation item !Supported by Postdoctoral Research Foundation Guangxi Academy Agriculture Sciences；Natural Science Foundation o{
Colege of Agricultural，Guangxi University
mography：DONG Deng-Feng(1971一)，male，Born in Jinshan，Hubei Province，Philosophy Doctor and associate professor，Researching on plant
stress physiology and molecular biology．
t Author for correspo ndence
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3期 董登峰等：萌发木豆种子中酸性磷酸酯酶的纯化和动力学特性 391
determining the concentration of product(Pi)released
as described by Duff nZ．(1989)，and one Unit was
defined as 1 t~mol Pi hydrolyzed from the correspond—
ing substrates per minute． Protein concentration was
determ ined as described by Bradford(1 97 6)，using 13o—
vine serum albumi n as standard．
1．3 Isolation and purification
1．3．1 Extraction of seed protein The germinated
seeds were homogenized in the cold HAc-NaAc buffer
(100 mmol／L，pH8．0，containng 2 mmol／L MgC12，14
mmol／L mercaptoethanol and 2 mmol／L EDTA)in a
ratio of 3 mL per gram．The homogenate was centri—
fuged at 10 000 g for 10 mi n，the pellet was re-extracted
with the same buffer and procedure，and then supema—
tams were pooled and used as crude for purification．
1．3．2 Ammonium sulfate fractionation The crude
was transferred into a beaker，and ammonium sulfate
was added and stired slowly to 20 saturation．After
standing for 30 mi n at 4 ℃ 。the solution was centri-
fuged at 10 000 g for 10 mi n．The pellet was discarded
and the supematant transferred into another beaker，
and then ammonium sulfate was added to the 6O sat—
uration． After standing and centrifuging described as
above，the supernatant was discarded，and the precipi—
tant was suspended in rifs—HCl buffer(25 mmol／L，
pH7．1，containing 2 mmol／L MgC12)．The solution
was dialysised with same buffer overnight until no
SO4。‘detectable wi th Baz reagent，and concentrated
with PEG一6000 to the mi nimum volume．
1．3．3 DEAE-Sepadex A 25 column chromatography
The solution was loaded to DEAE-Sephadex A 25 col—
unin pre-equilibrated with商 s—HC1 buffer(25 retool／
L，pH7．1，containing 2 mmol／L MgC12)，and eluted
with 400 mL of linear gradient buffer(0～0．5 mol／L
KCl in the buffer)at the rate of 0．7 mL／min． Every
3mL of eluate was collected into a tube，and protein
concentration and APase activity were detected，frac—
tions containing higher APase activity were pooled and
concentrated．
1．3．3 Hydroxyapatite column chromatography The
concentrated protein from last step was loaded to
hydroxyapatite column pre-equilibrated with HEPE
KOH buffer(5 mmol／L，pH7．0，containing 2 mmol／L
MgCl2)，and then eluted wi th a linear gradient buffer
(5～100 mmol／L KClin the buffer)at the rate of 0．1
mL／min．Each fraction containing APase activity was
pooled and concentrated．
1．3．4 Concanavalin A-Sepharose 4B column chroma—
tography The solution from last step was loaded to
Con A-Sepharose 4B colun3D．pretreated wi th HAc-
NaAc buffer(0．1 mol／L，pH5．8，containng 1 mmol／L
KC1，1 mmol／L CaC12，1 mmol／L MgC12，1 mmol／L
MnC12，0．5 mol／L NaCl and 1 mmol／L EDTA)，and
eluted with linear gradient buffer(0～0．5 mol／L glu—
cose in the buffer)at the rate of 0．3 mL／min．Every 1
mL of eluate was collected into a tube，and protein con—
centration and APase activity were detected，fractions
containing higher APase activity were pooled．
1．3．5 Identification of purity and estimation of molec—
ular weight(MW ) Proteins were run on native poly—
acrylami de gel electrophoresis(PAGE)(4 stacking
gel，7．5 resolving ge1)and SDS-PAGE(4 stacking
gel，12．5 resolving ge1)respectively，and then stained
wi th coomassie brilliant blue as described by Guo
(1999)．MW was calculated referring to the plot of l g
MW against Rf values of standard protein on SDS-
PAGE ge1．
2 Results
2．1 Purification 0f the APase
After DEAE-Sepadex A 25 column chromatogra—
phy，two peaks wi th Ngher APase activities were de—
tected．The first peak(encoded as APase I)appeared
from the 2nd to the 9th tubes，further purification and
characterization of APase 1 will be reported in another
paper．The second peak(encoded as APase lI)，which
appeared from the 54th to the 64th tubes，was pooled
and further purified through two sequential Hydroxya-
patite and Concanavalin A-Sepharose 4B column chro—
matography．In totally，the enzyme was purified 247
fold，and the specific activity was 5 1．8 U／mg protein．
Among all the procedures，Hydroxyapatite colun3D．was
most effective，through which APase activity was in—
creased about 10 folds(Table 1)．
The purified APaseⅡappeared as a single band in
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392 广 西 植 物 28卷
both non-denaturing PAGE(Fig．1：A)and SDS-PAGE
(Fig．1：B)，indicating our target protein was monomer
and purified to electrophoretic homogeneity．
Table 1 Purification of the APaseⅡfrom pigeonpea seeds
2 3 4 5
Fig．1 Proteins on non-denatured PAGE
gel(A)and SD PAGE(B)
1．proteins in crude；2．proteins after ammonium sulfate fractiona—
tion；3．proteins after DEAE-Sephadex A25 column；4
． proteins after
Hydroxyapatite colUlTln|5．protein after ConA-Sepharose 4B column．
2．2 Biochemical properties of purified enzyme
2．2．1 Molecular weight Referring to plot of logMW
standard proteins versus relative migration distance，
molecular weight of APase I1 was estimated to be
33．1KDa(Fig．2)
2．2．2 Optimal pH and pH stability Using pNPP as
substrate，the enzyme were assayed in various reaction
system with pH arrange from 3．5 to 8．0 to investigate
the optimal pH；and the enzymes，after being stored in
various buffers with pH arrange from 3．5 to 8．0 for 1
h at 4 ℃ ，were assayed in optimal reaction system to
investigate the pH stability．APaseⅡexhibited an op—
timal pH of 5．0 and strong pH tolerance，APaseⅡlost
less than 30 activity even stored in pH 3 or 7 buffer
for 1 h(Fig．3)．
2．2．3 Optimal temperature and thermal stability U一
4-
o
E
工
● 一
L
c口
o
5．2
5
4．8
4 6
4 4
4 2
4
0 0 2 0．4 0．6 0 8 1 1 2
Relative migration distance
Fig．2 Logarithm of MW versus relative migration
distance(R／)of proteins on SDS-PAGE ge1
standard protein a is Phosphorylase b(97．4kDa)；h Bovine serum
albumin(66．2kDa)；c Aldolase(39．2kDa)； Tfiose phosphate
isomerase(26．6kDa)； Trypsin inhibitor(21．5kDa)I f．Lysozyme
(14．4kDa)．
3 3．5 4 4 5 5 5 5 6 6．5 7 7 5 8
pH
Fig．3 Optimal pH and pH stability of APaseⅡ
sing pNPP as substrate，the enzyme was assayed at va—
rious temperatures to investigate the optimal tempera—
ture；and the enz ym es，after being stored at various
temperatures，ware assayed in optimal reaction system
to investigate the thermal stability．APaseⅡexhibited
an optima l temperature of 35 ℃ ，and was stable at
temperatures up to 50℃ ，above which point the activi—
ty decreased sharply(Fig．4)．
2．2．4 Substrate specificity In place of pNPP，a varie—
ty of na tural phosphoric compounds including 3-phos—
phoglycerate(3-PGA)，fructose-6一phosphate(F-6一P)，
phosphoenolpyruvate (PEP)，adenosine diphosphate
(ADP)，phytate，fructose-1，6一diphosphate(F-1，6一P)，
glucose-6一phosphate(G 6一P)，adenosine triphosphate
(ATP)and pyrophosphate，all at a concentration of 2
mmol／L，were used as substrates to determine the
APaseⅡactivities，and relative activities as compared to
pNPP were calculated．
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一 一 一>一 。四 ∞>一 四一∞
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3期 董登峰等：萌发木豆种子中酸性磷酸酯酶的纯化和动力学特性 393
APaseⅡ showed relatively divergent substrate
specificities，the highest specificity activity was recor—
ded with pyrophosphate，followed by ATP，phytate and
G-6一P，as 3．7一，2．3-,0．64一，0．37一fold higher than wi th
pNPP。respectively(Fig．5)．
1
1 2O
1OO
8O
60
40
2O
O
3O 35 4O 45 5O 55 6O 65 7O
Temperature(℃ )
Fig．4 Optimal temperature and heat stability of APase I
500
450
400
350
‘ 300
譬 250
．
200
蚕： 5
5O
O
岛
震
厂]一目叨 图圉
Substrate
Fig．5 Relative activity of APaseⅡ
against various substrates
2．2．5 Effects of ions on enzyme activty In order to
determine the effects of some ions as possible activators
or inhibitors of APase lI，the enzym e was incubated
with these compounds at concentration of 1 mmol／L
and 5 retool／L，respectively，and then the activity was
determined．The activity of APase was activated by
K ，but inhibited by Fez ，Mnz ，Mo7024 and F-．As
to Znz and Md ，activatory efects were observed at
low concentration of 1 mmol／L while inhibitory effects
observed at high concentration of 5 mmol／L(Fig．6)．
2．2．6 Effects of organic acids on enzyme activity In
order to determine the effects of intermediate metabo—
lites on APase I，some organic acids including glyco—
late，glyoxylate，citrate，oxalate,tartrate,malate,ascor—
bate and isocitrate were added，respectively，to the reac—
tion system to the ultimate concentration of 2 mmol／
L，and then APaseⅡactivities were determined．APase
l1 was inhibited by all organic acids used(Fig．7)．
1OO
95
90
85
8O
75
70
重
量 耋
雪 星
雪 ． 雪 雾 1． 量
1 5
Ion concentration(mmol／L)
Fig．6 Effects of ions on APase activity
O rganic acid
Fig．7 Effects of organic acids on APase activity
3 Discussion
Although bodies of studies have been reported on
acid phosphatases from plant tissues such as seeds
(Werreira Pta1．，1998)，leaves(Noel et a1．，2004；Tian，
2004)。roots(Panara et a1．，1990)，tubers(Gellatly et
口1．，1994)，and even exudates(Bozzo eta1．，2002)，and
their pos．sible function have been assigned．Little infor—
mation is available on APase purified from plant germi—
nated seed。neither the possible function as relative to
germination．To our knowledge，the present study re—
presents the first detailed purification and characteriza—
tion of an APase from pigeonpea and germinating seed．
APase generally exist as heterogeneities．In our ex-
periment，two isoforms were isolated from germinated
pigeonpea seed，the possibility could not be excluded that
one or more other isoforrrls existed in the discarded su—
Dernatant after 60 ammonium sulfate fractionatior~
The APaseⅡcould bind with Concanavalin，indi—
cating that it might be a kind of glycosylprotein．The
加 ∞ 如 如 ∞
． 苦喜 a^麦
一 一 一>一 0 >一 一
一 一 l^【1口∞u AI la
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394 广 西 植 物 28卷
APaseⅡwas a 33．1kDa monomeric protein with opti—
mal pH5．0 and temperature 35℃ ，the properties are
like those of the isoform APSB from soybean seed(Fer—
reira et a1．，1998)．The sharp loss of enzyme activity
after treated with temperature above 50 ℃ might be
due to the irreversible denaturation．
Like most of other plant APases reported，the
APase I1 was inhibited by molybdate and fluoride，and
activated by magnesium．The APase I1 was inhibited
by tartrate，indicting that it mi ght not be purple acid
phosphatase，because the purple acid phosphatases are
generally resistant to tartrate (Olczak et a1．，1 9 97；
Bozzo et a1．，2002)．
The APase I had activity against all phosphate
esters tester tested。indicating it was a mult讧unctional
enzyme，but relatively high activity was against pyro—
phosphate，ATP，phytate，and G 6一P．Pyrophosphate is
the byproduct during RNA，protein and saccharide syn—
thesis，can be accumulated during the germinating
process as reservoir of P and energy(Li et a1．，2004)．
Phytate is the major storage form of P in the plant
seed．Therefore，the APase I mi ght play a role in
phosphate mobilization and energy provision during
germi nating，but direct evidence in support of this as—
signment should be necessaries． Cloning and function
identification of the APase I gene mi ght facilitate the
understanding of its explicit physiological functions in
relative with germinating．
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子中酸性磷酸酯酶的纯化和动力学特性
董登峰1，2，李杨瑞1*，杨 杰2，王永峰
(1．广西作物遗传改良和生物技术重点实验室，南宁 530007；2．广西大学 农学院，南宁 530004)
摘 要 ：以对硝基苯磷酸为底物检测酶活性 ，通过 2O ～6O 硫酸铵分部 、DEAE-葡聚糖 A25、羟基磷灰石、伴
刀豆球蛋白一琼脂糖 4B柱层析 ，从木豆萌发的种子中纯化到一个同工酶 APase U，酶最终纯化倍数为 247倍，比
活力达 51．8 U／mg蛋白。非变 性 PAGE和 SDS-PAGE表明所纯化的酶 已经达 到电泳纯，是一个分子量为
33．1kDa的单体蛋白。APII的最适pH为 5．0，最适温度为35℃，在pH3．5～7以及 55℃以下稳定。该酶对焦
磷酸有最大活性 ，受 K+和 MgZ+激活，受 Fe2+，Mn2+，MoT0246一，F-及酒石酸、苹果酸、异柠檬酸、草酸、柠檬酸、
乙醇酸、乙醛酸和抗坏血酸等有机酸抑制。
关键词 ：木豆；酸性磷酸酯酶 ；纯化 ；动力学特性
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