全 文 ：萱草花粉类动力蛋白生物物理学及药理学性质 ?
廖俊杰1 , 2 , 吴英杰3
??
, 阎隆飞1
( 1 中国农业大学生物学院植物生理生化国家开放重点实验室 , 北京 100094; 2 广东轻工职业技术学院食品与生物
工程系 , 广东 广州 510300 ; 3 Department of Medicine, Mount Sinai School of Medicine, New York, NY 10029)
摘要 : 利用 FPLC 技术从萱草花粉中鉴定并纯化了动力蛋白 , 研究了它的酶学性质及部分生物化学性质。
结果如下 : 纯化的类动力蛋白分子量为 100 kD, 等电点 pI = 6.15 和 6 .80。在 280 nm波长激发下 , 最大的荧
光发射波长是 346 nm。荧光光谱分析结合紫外吸收光谱及导数光谱分析推断它含有色氨酸和酪氨酸残基。
药理学性质研究表明巯基可能在酶的活性中心起重要作用。
关键词 : 类动力蛋白 ; 分子马达 ; 微管 ; 花粉
中图分类号 : Q 946.1 文献标识码 : A 文章编号 : 0253 - 2700 (2007) 02 - 247 - 04
Biophysical and Pharmacological Characterization of a Dynamin-like
Protein from Day-lily ( Hemerocallis fulva, Liliaceae) Pollens
LIAO Jun-J ie
1 , 2
, WU Ying-Jie
3 * *
, YAN Lung-Fei
1
( 1 Collegeof Biological Sciences, China Agricultural University, State Key Labratory of Plant Physiology and Biochemistry, Beijing
100094 , China; 2 Department of Food and Bio-technology, Guangdong Light Industry Technologic College, Guangzhou 510300 ,
China; 3 Department of Medicine, Mount Sinai School of Medicine, New York, NY 10029 , America)
Abstract : A novel motor protein, dynamin- likeprotein fromday lily, was purified and identified by FPLC . Herewereport
its biochemical characterization . The molecular weight of the dynamin-like protein is 100 kD on SDS-PAGE . Isoelectric
points are about 6.15 and 6 .80 . The fluorescence emission wave lengthof dynamin-like protein is 346 nmby excitation at
280 nm . Through fluorescence spectra analysis, ultraviolet absorption spectrum and derivative spectrum, we conclude that
it contains tryptophan and tyrosine residues . Pharmacological study indicates that mercapto may play an important role in
enzyme activity of dynamin-like protein .
Key words: Dynamin-like protein; Molecular motor; Microtubule; Pollen
Movement is an essential function of all the cells
and the basisof all life activities . Long before, cell bi-
ologists have speculated that all activities in the cell
were the result of the functions of so-called mecha-
noenzymes . However, none of these mechanoenzymes
has been identified . It iswell known thatmyosin is the
movement motor of the microfilament system . Kinesin
and cytoplasmic dynein were identified as motor pro-
teins (Vale et al . 1985) , which were regarded as the
enzymes to couple the hydrolysis of nucleotide and
movements of themicrotubules . Recently, the study on
motor protein, which plays a significant role in eukary-
otic cells, has been one of the focuses in modern cell
biology and biochemistry (Skoutias and Scholey, 1993;
Wu and Yan, 2002 ) . The protein that was found in
bovine brain tissue with the molecular weight 100 kD
was named dynamin-likeprotein (Shpetner and Vallee,
1989) , which GTPase activity was shown to be high,
while ATPase activity to be low . However they could
be also stimulated 1 .6-fold by microtubules . This was
云 南 植 物 研 究 2007 , 29 (2) : 247～250
Acta Botanica Yunnanica

?
?? ?Author for correspondence; E-mail : yyjjwu@yahoo. com
Received date: 2006 - 05 - 11 , Accepted date: 2006 - 12 - 04
作者简介 : 廖俊杰 (1965 - ) 男 , 副教授 , 主要从事植物细胞研究工作。 E-mail : junjie33@gdqy. edu. cn ?
Foundation item: This work was supported by the National Science Fund ( No . 39730280) .
the third microtubulemotor protein that was importance
to microtubule slippage and transport of the vesicles,
succeeding to kinesin and cytoplasmic dynein which
were found in bovine brain tissue .
Previously, all the reports on dynamin-like pro-
tein were from animal materials . While Wu and Yan
( 2002 ) recently have firstly isolated a dynamin-like
protein fromplant day-lilywhich is similar to nervecell
protein . If like this, it may prove that the plant cell
and animal cell has the same mechanism in the energy
transmission and the information transmission . The re-
search proved that this dynamin-like protein, which
can take the immuno-cross reaction with bovine brain
protein antibody, has both GTPase and ATPase activi-
ty, but the former one is far higher than the latter one .
And the investigations on thebiophysical and the phar-
macological properties of day-lily′s dynamin-like pro-
tein are reported here .
1 Materials and Methods
1 .1 Materials
Day-lily ( Hemerocallis fulva L .) pollenswereobtainedfrom
Institute of Medicinal Plant, Chinese Academy of Medical Sci-
ences . The anthers were irradiated in 60W lamp to dehisce .
Then pollenswere selected by shakesieve . Thepollenswere dried
and stored at - 80℃ .
1 . 2 Separation and purification
25 g day-lily pollensweretriturated in ice water with 60 ml
- 100 ml extract buffer (100 mmol?L PEPES, pH 6 .9 including1
mmol?L MgCl2 , 1 mmol?L EGTA , 1 mmol?L DTT, 1 mmol?L
PMSF , 50μg?ml TAME , 50μg?ml TPCK 和 0 .5 % PVP) The
extracted liquid was isolated in30%- 80% fraction of ammonium
sulfate precipitation . Then supernatant was treated with DEAE-
Sephadex A50 column chromatography with 0 .5 mol?L KCl as
eluent . The liquid was collected at 280 nm peak . After centrifu-
gation at 15 000 r?min for 15 min at 4℃ , the supernatant was
purified by FPLC-Mono Q ion exchange chromatography using 0
- 600 mol?L continuous concentration gradient KCl as eluent .
1 . 3 kDetermination of the molecular weight of the dynamin-
like protein
SDS-PAGE was performed according to the Laemmli (1970)
method, withaconcentration of 4.8% for the separatinggel anda
concentration of 10% for the concentratinggel . Gelswere stained
with silver nitrate . Themolecular weight of the dynamin- like pro-
tein was determined by therelationship betweenthe logarithmmo-
lecular weight and the electrophoretic mobility .
1 . 4 Determination of isoelectric point
Themeasurement of isoelectric point of the protein was de-
termined with phast system IEF ( Pharmacia) . The sample was
loaded0 .4μl . PhastGelTM (5%T , 3% C) , pI 3 .0 - 9 .0 , were
used as prepared gels and stained with silver nitrate . The stan-
dard was fromPharmacia Company .
1 . 5 Fluorescence spectra analysis
Thefluorescence emission wave length of dynamin-like pro-
tein is measured with a F-4010 fluorescence spectrophotometer
(Hitachi ) .
1 . 6 2Ultraviolet absorption spectrum and derivative spec-
trum analysis
The Ultraviolet absorption spectrumand derivative spectrum
aremeasured with a Beckman DU-7000 spectrophotometer .
1 . 7 Determination by the Pharmacology
In thedetermination of Pharmacological character of day-lily
dynamin- like protein, various inhibitors or activatingagent, such
as 100μmol?L Oligomycin, 100μmol?L Ouabain, 2 . 4 mmol?L
NaF , 2 .4 mmol?L NaF+ and 0 .1 mmol?L AlCl3 , 0. 25% Tri-
tonX-100 , 1 mmol?L PCMS, 0 .1 mmol?L NEM , 1 mmol?L
NEM , wereadded inrespectively, compared with theblank sam-
ple in which no inhibitor was added . The method given by
Gonzalez-Romo ( 1992 ) was used to determine ATPase activity .
The enzyme activity unit is nmol Pi·min- 1·mg- 1 Pr .
1 . 8 Determination of protein concentration
Theproteinconcentration was measured by UV-240 spectropho-
tometry (Shimadzu) , BSA as a standard protein (Bradford, 1976) .
2 Results
2 .1 Separation and purification
Ammoniumsulphate fractional precipitation, DE-
AE-Sephadex A50 column chromatography and FPLC-
Mono Q ion exchange chromatography could purify the
dynamin-likeprotein fromthe extractof day-lily pollens
and increase its purity by 86 times (Table 1 ) .
2 . 2 Determination of the molecular weight
Results of SDS-PAGE that stainedby silver nitrate
arepresented in Fig . 1 . The molecular weight of the
dynamin-like protein is 100 kD estimated by the rela-
tionshipbetween the logarithmmolecular weight and the
electrophoretic mobility, which is similar to the animal
dynamin-like protein (Shpetner and Scholy, 1993) .
2 . 3 Determination of isoelectric point
Isolectric points of the plant dynamin-like protein
are 6 .80 and 6 .15 ( Fig . 2 ) which may be resulted
fromthe composition and structure of the amino acids .
It shows that the dynamin-like protein is electrically
neutral in both buffer solutions pH 6 .80 andpH 6 .15 .
2 .4 Fluorescence spectra analysis
Table 1 Purification of dynamin- like protein from day-lily pollen
Purification Total Specific activity Purified
steps protein ( nmol Pi . mg - 1 Vfold
min - 1 ZPr .)
Crude extracts 669 6. 6 6 :. 8 1 t
( NH4 ?)2 SO4 precipitate 238 6. 4 17 M. 6 2 O. 5
DEAE-Sephadex A50 ?8 ?. 7 272 _. 4 40 b. 1
FPLC-Mono Q 0 ?. 84 585 _. 1 86
842 云 南 植 物 研 究 29 卷
Fig . 1 SDS-PAGE of plant dynamin- like protein
by FPLC-Mono column
lane l : Sigma Mr . Standard
lane 2: Crude dynamin- like protein from ( NH4 )2 SO4 precipitate
lane 3: Purified dynamin- like protein fromDEAE A50
lane 4 - 11: Different fractions fromMono Q column
Fig . 2 Determination of PI volue of dynamin- like protein
from day lily pollen by IEF-PAGE
lane 1: Purified dynamin-likeprotein; lane 2: Standard Marker proteins .
The fluorescence analysis method is usually used
to investigate the structure of the protein that based on
its self-fluorescence . The fluorescence emission wave
length of dynamin-like protein is 346 nm by excitation
at 280 nm ( Fig . 3 ) , indicating it may contain the
tryptophan residue .
2 . 5 ?Analysis of ultraviolet absorption spectrum
and derivative spectrum
The ultraviolet absorption spectrum of dynamin-
likeprotein was shown in Fig . 4 . Its maximal absorb-
ing wave is 276 .2 nm (Fig . 4A) , contributed by in-
dole cycle in tryptophan and hydroxybenzene in ty-
rosine . Derivative spectrum is sensitive to the intensity
transformed by length of the wave, especially producing
strong signals in the slopeor bent site so as to provide
more message and complicated structure, which is
meaningful to the inspection of spectrumcurve obliter-
ated by broad band spectrum or non-structure back-
ground spectrum . We have inspected the first, second
and third rank derivative spectrumof dynamin-like pro-
tein, shown in Fig . 4B, C , D . It represents an ab-
sorption peak in ultraviolet absorption spectrum ( Fig .
4A ) , while it represents four absorption peak in the
first rank of derivativespectrum ( Fig . 4B) , which pr-
esents more complicated structure (Fig . 4C , D) .
2.6 ?Pharmacological character of day-lily dynamin-
like protein
The effect of somekinds of inhibitor and the acti-
vating agent on dynamin-like protein ATPase enzymatic
activity is listed in Table 2 , indicating that Triton X-
100 has the activity function slightly but Oligomycin is
insensitive to it . NaF、Ouabain presents a certain kind
of restraint feature, whileAlCl3 + NaF andPCMS strongly
suppress its activity . NEM can suppress59% of them,
Fig . 3 Fluorescence spectrum of dynamin-like protein
which is similar to thestudy result onbovinebrainprotein
(Shpetner and Vallee, 1992) . Wu and Yan (2002) also
obtained thesametrend in theinspectionof GTPase activ-
ity of dynamin-likeprotein . NEM can only inhibit kine-
sin as concentrations above 2 mM, but suppress dy-
namin-like protein at concentration as low as 0 .1 mM .
The inhibition becomes stronger when the concentration
of NEM becomes higher . Themercapto inhibitor, NEM
and PCMS, which can strongly inhibit the enzyme ac-
tivity, indicates that mercaptowas presumed to play an
important role in the enzyme′s active site .
3 Discussions
In all plant microtubemotor proteins, only kinesin
was identified (Tiezzi et al . 1992) , so we anticipated
that all the movement of cell would have the corre-
sponding motor . If microtubes exist in the cell , there
must be molecular motors reacting with the microtubes
in it . For microtubes participate in many life move-
ments, the quantity of the corresponding motor protein
should be enough to show adequately the function of
9422 期 LIAO Jun-J ie et al .: Biophysical and Pharmacological Characterization of a Dynamin-like Protein . . .
Fig . 4 Ultraviolet absorption and derivative spectrum
of dynamin- like protem trom day lily pollen
A . Ultraviolet absorption spectrum; B . 1st derivative spectrum;
C . 2nd derivative spectrum; D . 3nd derivative spectrum
Table 2 Pharmacological characterization of dynamin- like protein
Inhibitor ATPase activity % control
( bnol Pi .min - 1 ?.mg - 1 .Pr .)
100 (μmol?L Oligomycin 189 ?. 55 92 . 96
100 (μmol?L Ouabain 99 ?. 08 48 . 59
2 ?. 4 mmol?L NaF 101 .11 49 . 59
2 ?. 4 mmol?L Naf + 30 .09 17 . 70
0 ?. 1 mmol?L AlCl3
0 ?. 25 % Triton X-100 220 .21 108 .00
1 ?mmol?L PCMS 30 .08 14 . 75
0 ?. 1 mmol?L NEM 85 .50 41 . 90
1 ?mmol?L NEM 113 .82 55 . 82
No inhibitor 203 ?. 90 100
microtube system . In the investigation of Yan and Wu
( 1993 ) , dynamin-likeproteinwas found inmyxomyce-
te and towel gourdpollen . Wu and Yan (2002) report-
ed several properties of day-lily pollen dynamin-like
protein, it isobviously proved that its properties is very
similar to those of animal dynamin on some aspects
such as molecular weight, substrate pharmacological
character on GTPase . Here we continue to reveal the
biophysical and the pharmacological properties of dy-
namin-like protein . The molecular weight of dynamin-
like protein of purified day-lily pollen is 100 kD (Fig .
1) . The result is consistent with that of bovine dy-
namin (Shpetner and Vallee, 1989 ) . Pharmacological
character on ATPase (Table 2) is very silimar to phar-
macological character on GTPase ( Wu and Yan,
2002) . Through fluorescence spectra analysis and ul-
traviolet absorption spectrum, we can conclude that it
contains tryptophan base and tyrosine base ( Fig . 3 ,
Fig . 4A ) . And derivative spectrum gives the finer
structureof dynamin-like protein ( Fig . 4B, C, D) .
The results arenot referred in animal dynamin . On our
study Oligomycin, inhibiting the ATPase in mitochon-
drion membrane, was not the inhibitor to dynamin-like
protein ( Table 2) , indicating it is not only energy sup-
ply but also can join the cell life movement involving
particulate transportation, endocytic activity and signal
transudation . The further study on the orientation and
physical function of plant dynamin-like protein is ex-
pected earnestly . Whatever it will be molecular motor
or one member of G-protein, the significance of it in
cell is undoubted .
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