Download Molecular characteristics of sucrose synthase

Molecular characteristics of sucrose synthase isolated from bird cherry leaves
41
Molecular characteristics of sucrose synthase isolated from bird cherry
leaves
Hubert Sytykiewicz*, Paweł Czerniewicz, Bogumił Leszczyński
Department of Biochemistry and Molecular Biology
University of Podlasie
Prusa 12
08-110 Siedlce, Poland
*corrresponding author: phone/fax: +4825 6431367, e-mail: [email protected]
Summary
The molecular characteristics of partly purified sucrose synthase (NDP-glucose: D-fructose
2-α-D-glucosyltransferase, EC 2.4.1.13), extracted from the bird cherry (Prunus padus L.)
leaves was elucidated. The sucrose synthase was successfully purified by using a four-step
protocol including ammonium sulfate precipitation, dialysis, gel filtration on Sephadex
G-150 and ion-exchange chromatography with the use of DEAE-cellulose. The analysed enzyme occurred in two isoforms (SuSyI and SuSyII). The relative molecular weight of native
isoenzymes was estimated to be 200 and 180 kDa, respectively. Isoform SuSyI contained
two different subunits of 57.5 and 52.8 kDa, whereas the structure of SuSyII was consisted
of identical 63 kDa subunits. Experimental data indicated that the structure of both SuSy
isoforms was composed of three subunits.
Key words: sucrose synthase, Prunus padus, molecular mass, subunit composition
introduction
The purpose of presented studies was to elucidate molecular characteristics
of partly purified sucrose synthase extracted from the bird cherry leaves (Prunus
padus L., Rosaceae). Sucrose is a major transport form of reduced carbon within
the most higher plants [1, 2]. Sucrose synthase (SuSy, NDP-glucose: D-fructose
2-α-D-glucosyltransferase, EC 2.4.1.13) plays a substantial role in regulation of
intracellular homoestasis and ontogenetic development of plants. Its subcellular
localisation is not restricted to cytosolic or vacuol compartments, but also may
occur as membrane-bound and associated to actin cytoskeleton forms [3-5].
Vol. 54 No 3 2008
H. Sytykiewicz, P. Czerniewicz, B. Leszczyński
42
Biochemical and physiological importance of the analysed enzyme is connected
with its unique functions. The reaction catalysed by SuSy is completely reversible
and depends on metabolic requirements of the cells or tissues. The equilibrium
state in vivo may be displaced into direction of sucrose synthesis or degradation
[6, 7]. The hypothesis that a broad spectrum of SuSy isoforms fulfill distinct metabolic functions is supported by the existence of multiple isoenzymes in many
plant species that possess differentiated structure properties. On the other hand,
level of expression and catalytic activity of those isoforms are influenced by tissue
and subcellular localisation, phase of plant development and environmental factors (especially the presence of abiotic or biotic stressors) [7, 8].
MATERIAL AND METHODS
Plant material
Fresh leaves of the bird cherry grown in Aleksandria Park, Siedlce (central-eastern Poland) were used in the experiments. Tested organs were at sixth flushing
stage (completely mature and fully expanded) in the Carter’s scale [9]. The plant
material was placed in solid carbon dioxide, immediately transferred to laboratory and subjected to biochemical analyses.
Enzyme isolation
Extraction of the sucrose synthase from P. padus leaves was carried out with the
use of Röhring et al. method [10]. Analysed organs were cut and homogenized at
4°C with Tris-HCl buffer (pH 7.5) containing 10 mM MgCl2, 0.5 mM CaCl2, 1 mM
EDTA, 250 mM sucrose, 5 mM DTT (dithiothreitol) and 1 mM PMSF (phenylmethylsulfonyl fluoride). Obtained homogenate was subsequently filtered through four
layers of cheesecloth and centrifuged for 30 min. at 13,500 g. Pellets were discarded, and supernatants were used for further analyses.
Sucrose synthase purification
a) ammonium sulfate precipitation and dialysis
Solid ammonium sulfate (35.4 g) was added to each portion of the supernatant (100 cm3) to obtain 30% saturation. Precipitated proteins were centrifuged
for 30 min at 13,500 g. Supernatant was decanted and ammonium sulfate was
added to obtain 55% saturation. The mixture was centrifuged as described above.
Supernatant was discarded and the pellet was dissolved in extraction buffer at a
temperature of 4°C. Subsequently, the solution was subjected to dialysis against
Tris-HCl buffer (pH 7.5) for 6 h.
Molecular characteristics of sucrose synthase isolated from bird cherry leaves
43
b) gel filtration
Further phase of purification which consisted of separation of SuSy isoenzymes and determination of molecular mass of native isoforms was accomplished with use of Klotz et al. method [11]. Dialysates (each portion of 10 cm3)
were loaded onto a glass column (2 × 40 cm) filled with Sephadex G-150 bed
and equilibrated with 200 cm3 of extraction buffer. Enzyme elution was performed with the same buffer at the flow-rate of 0.8 cm3 · min-1. The eluates were
collected in portions of 2 cm3 and the fractions of SuSy activity were pooled.
Molecular weight of native SuSy isoforms (in kDa) was calculated with the use
of experimentally outlined calibration curve showing relationship between
Kav (coefficient of partitioning) and log10 molecular mass of the used standard
proteins.
c) ion-exchange chromatography
The pooled gel filtration fractions were applied onto a glass column (1 x 34 cm)
filled with DEAE-cellulose, pre-equilibrated with extraction buffer. Elution of SuSy
isoenzymes were performed by means of a linear NaCl gradient (0-500 mM) at the
flow-rate 0.3 cm3 · min-1. The eluates exhibiting SuSy activity were pooled in two
separate fractions.
Determination of SuSy subunit composition
Evaluation of subunit composition of tested isoenzymes was determined
electrophoretically with the use of SDS-PAGE technique. Separation of SuSy isoforms was carried out on 12.5% polyacrylamide gel by the procedure of Laemmli
[12]. The standard discontinuous system under denaturing conditions (with the
application of sodium dodecyl sulfate) was used. After electrophoresis, protein
subunits were visualized within gel by staining with Coomassie Brilliant Blue
R-250.
Enzyme activity assay
The specific activity of the analysed enzyme towards the cleavage direction
was calculated as μg reducing sugars · min-1 · mg protein, whereas activity in
the direction of sucrose synthesis was expressed as a loss in μg reducing sugars·
min-1 · mg protein. Quantitative analyses of reducing sugars within the enzyme
preparates were conduncted according to the standard method of SomogyiNelson [13]. The protein content was measured by using the method of Lowry
et al. [14].
Vol. 54 No 3 2008
H. Sytykiewicz, P. Czerniewicz, B. Leszczyński
44
RESULTS AND DISCUSSION
The performed purification techniques (ammonium sulfate precipitation, dialysis, gel filtration on Sephadex G-150 and ion-exchange chromatography with
the use of DEAE-cellulose) allowed to obtain near-homogenous preparate of the
sucrose synthase (tab. 1, fig. 1-2). Two different peaks that possessed SuSy activity
were identified in the gel filtration (Sephadex G-150) step, and the two isoforms
were named in order of elution as SuSyI and SuSyII (fig.1). After the ion-exchange
chromatography on DEAE-cellulose, specific activity of the analysed SuSy isoforms
slightly increased (approximately 2-fold increase of activity in relation to the prior
phase of purification, see fig. 2).
Figure 1. Fractions of SuSy obtained from bird cherry leaves, eluted from chromatographic
column filled with Sephadex G-150 (A and B – fractions that possess activity of SuSy, subsequent
peaks represent other biomolecules)
Figure 2. Fractions of sucrose synthase isolated from P. padus leaves eluted from column filled with
DEAE-cellulose (I, II –protein fractions that possess activity of SuSy, III, IV –other protein fractions)
Molecular characteristics of sucrose synthase isolated from bird cherry leaves
45
Ta b l e 1 .
Purification of sucrose synthase isolated from bird cherry leaves
purification phase
crude extract
ammonium sulfate precipitation
dialysis
fraction A
sephadex
G-150
fraction B
fraction I
DEAE-cellulose
fraction II
protein
content
(mg ∙ cm-3)
120.00
98.20
40.02
12.40
10.30
6.20
5.80
specific activity of sucrose
synthase
cleavage
synthesis
reaction
reaction
1.13
0.39
4.70
2.61
6.35
5.55
8.55
8.26
9.02
8.77
20.56
19.40
23.90
22.60
purification efficiency
(n-fold)
cleavage
synthesis
reaction
reaction
1.00
1.00
4.16
6.69
5.62
14.23
7.58
21.17
8.00
22.49
18.20
49.74
21.19
57.95
The relative molecular mass of the native sucrose synthase isoenzymes was
estimated to be 200 kDa (Kav=0.12) and 180 kDa (Kav=0.14), respectively (fig. 3).
Electrophoretic separation of identified SuSy isoenzymes was carried out in discontinuous system under denaturing conditions, using the SDS-PAGE technique.
Analyses of the electropherograms revealed that isoform SuSyI contained two
different subunits of 57.5 kDa (Rf=0.36) and 52.8 kDa (Rf=0.39), whereas the structure of SuSyII was consisted of identical 63 kDa monomers (Rf=0.31) (fig. 4).
Figure 3. Calibration curve for molecular weight estimation of chromatographically separated
proteins on Sephadex G-150 gel
Kav – coefficient of partitioning, A – cytochrome c (12.3 kDa), B – trypsin (23.8 kDa), C –
ovoalbumin (45.0 kDa), D – bovine serum albumin (66.0 kDa), E – myosine (205.0 kDa)
Vol. 54 No 3 2008
H. Sytykiewicz, P. Czerniewicz, B. Leszczyński
46
Figure 4. Electropherograms of sucrose synthase fractions isolated from bird cherry leaves
separated with the use of SDS-PAGE
The sucrose synthase has been isolated and highly purified from several organs
and tissues of different plant species, i.e. wheat, cucumber, Japanese pear fruit,
soybean nodules, sugarcane, tobacco and sycamore cells [5-8, 15]. Many authors
emphasize that number of SuSy isoenzymes occurring within relevant subcellular
compartments or tissues depends on numerous factors [16-18]. One of the most
important variables affecting the sucrose synthase profile within cells is maturing
and physiological modifications of plant organs. According to Klotz et al. [11]
activity of SuSy isoenzymes was correlated with phase of ontogenetic development of sugar beet. SuSyII showed its biochemical activity only in mature organs,
whereas SuSyI catalysed reactions of sucrose synthesis and cleavage mostly within
growing tissues. The DNA of Arabidopsis thaliana (L.) encodes six SuSy-like genes
with distinct expression, down- or up-regulated by various environmental stressors such as anoxia, dehydration, cold treatment and wounding [19-22]. The two
Molecular characteristics of sucrose synthase isolated from bird cherry leaves
47
SuSy isoformes of analysed enzyme which were distinguished in presented studies have also been found in several eudicotyledons, i.e. potato, carrot, Japanese
pear fruit and BY-2 tobacco heterotrophic cells [5, 6, 8]. Matic et al. [6] suggested
that SuSyI is more involved in channeling metabolites into glycolysis, and SuSyII
is more active during cell wall biosynthesis. Differences in net charges or charge
distribution within isoenzymes implicate variable biochemical functions they act.
In maize, the SS1 isoform was involved in starch synthesis and dominated in the
endosperm, whereas the SS2 isoenzyme was active in providing substrate for cell
wall formation and glycolysis [6].
Application of gel filtration with the use of Sephadex G-150 bed for native SuSy
molecular mass estimation allowed to determine that both isoenzymes possessed
comparable molecular weight (SuSyI – 200 kDa, SuSyII – 180 kDa). Review of published experimental data indicate that molecular weight of this enzyme is placed in
the range of 320 kDa (Beta vulgaris L., Daucus carota L.) to 540 kDa (Cucumis sativus L.,
Ipomoea batatas L.) [20]. Depending on ionic strength of buffer solution, the enzyme
may form aggregated structures. Explanation of high molecular mass of SuSy is also
S170 phosphorylation that promotes the formation of complex dimensional forms.
Subunit composition of the sucrose synthase was evaluated using the SDS-PAGE
technique under denaturing conditions. This method allowed to separate and reveal SuSy subunits within polyacrylamide gel. Electropherograms of the analysed
isoenzymes isolated from bird cherry tissues indicated that their structure was probable composed of three subunits. Published data referring to analysed enzyme
extracted from different plant species proved that SuSy may possess monomers
with molecular mass reaching up to 94 kDa and form complex three-dimensional
structures, mostly tetrameric or oligomeric [5, 7, 8, 20]. Klotz et al. showed that
SuSyI isolated from Beta vulgaris L. roots was consisted of two 84 kDa subunits and
two 86 kDa subunits (heterotetramer), whereas sucrose synthase isoform II (SuSyII)
was composed of four subunits of 86 kDa (homotetramer) [11].
The proposed protocol of isolation and purification of two SuSy isoforms extracted from bird cherry leaves enables elucidating their more extensive kinetic
properties (i.e. nature of inhibition in sucrose synthesis and cleavage reactions)
and also evaluating metabolic mechanisms regulating isoenzymes’ activity.
CONCLUSIONS
1.Two isoforms of SuSy have been isolated from bird cherry leaves with high
yield and purified to near-homogeneity state.
2.The relative molecular mass of the native isoenzymes was estimated to be 200
and 180 kDa, respectively.
3.Isoform SuSyI was a heteromer contained two different subunits (57.5 and
52.8 kDa), whereas the structure of SuSyII was consisted of identical 63 kDa
subunits (homomer).
Vol. 54 No 3 2008
H. Sytykiewicz, P. Czerniewicz, B. Leszczyński
48
4.The dimensional structure of the sucrose synthase isoenzymes is probably
composed of three subunits.
REFERENCES
1.Ren G, Healy RA, Horner HT, James MG, Thornburg RW. Expression of starch metabolic genes in the
developing nectarines of ornamental tobacco plants. Plant Sci 2007; 173:621-37.
2. Hirose T, Scofield GN, Terao T. An expression analysis profile for the entire sucrose synthase. Plant Sci
2008; 174:534-43.
3.Rosales MA, Rubio-Wilhelmi MM, Castellano R, Castilla N, Ruiz JM, Romero L. Sucrolytic activities in
cherry tomato fruits in relation to temperature and solar radiation. Sci Horticult 2007; 113:244-49.
4.Etxeberria E, Gonzalez P. Evidence for a tonoplast-associated form of sucrose synthase and its potential
involvement in sucrose mobilization from the vacuole. J Exp Bot 2003; 54:1407-14.
5.Tanase K, Yamaki S. Purification and characterization of two sucrose synthase isoforms from Japanese
pear fruit. Plant Cell Physiol 2000; 41:408-14.
6. Matic S, Åkerlund H-E, Everitt E, Widell S. Sucrose synthase isoforms in cultured tobacco cells. Plant
Physiol Biochem 2004; 42:299-306.
7.Barratt DH, Barber L, Kruger NJ, Smith AM, Wang TL, Martin C. Multiple, distinct isoforms of sucrose
synthase in pea. Plant Physiol 2001; 127:655-64.
8.Römer U, Schrader H, Gunther N, Nettelstroth N, Frommer WB, Elling L. Expression, purification
and characterization of recombinant sucrose synthase 1 from Solanum tuberosum L. for carbohydrate
engineering. J Biotechnol 2004; 107:135-49.
9. Leather SR. Biological flora of the British Isles. Prunus padus L. J Ecology 1996; 84:125-32.
10.Röhring H, John M, Schmidt J. Modification of soybean sucrose synthase by S-thiolation with ENOD40
peptide A. Biochem Bioph Res Co 2004; 325:864-70.
11.Klotz KL, Finger FL, Shelver WL. Characterization of two sucrose synthase isoforms in sugar beet root.
Plant Physiol Biochem 2003; 41:107-15.
12. Laemmli UK. Cleavage of structural proteins during the assembly of the head of bacteriophage T4.
Nature 1970; 227:680-85.
13. Nelson N. A photometric adaptation of the Somogyi method for the determination of glucose. J Biol
Chem 1944; 153:375-80.
14. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ. Protein measurement with the Folin phenol reagent. J
Biol Chem 1951; 193:265-75.
15. Lutfiyya LL, Xu N, D’Ordine RL, Morrell JA, Miller PW, Duff SM. Phylogenetic and expression analysis of
sucrose phosphate synthase isozymes in plants. J Plant Physiol 2007; 164:923-33.
16. MacGregor EA. Possible structure and active site residues of starch, glycogen and sucrose synthases. J
Protein Chem 2002; 21:297-306.
17.Klotz KL, Haagenson DM. Wounding, anoxia and cold induce sugar beet sucrose synthase transcriptional
changes that are unrelated to protein expression and activity. J Plant Physiol 2008; 165:423-34.
18.Watkinson JI, Hendricks L, Sioson AA, Heath LS, Bohnert HJ, Grene R. Tuber development phenotypes
in adapted and acclimated, drought-stressed Solanum tuberosum ssp. andigena have distinct expression
profiles of genes associated with carbon metabolism. Plant Physiol Biochem 2008; 46:34-45.
19.Zabalza A, Gálvez L, Marino D, Royuela M, Arrese-Igor C, González EM. The application of ascorbate
or its immediate precursor, galactono-1,4-lactone, does not affect the response of nitrogen-fixing pea
nodules to water stress. J Plant Physiol 2008; 165:805-12.
20. www.brenda.com
21. Marraccini P, Geromel C, Ferreira LP, Pereira LFP, Vieira LGE, Leroy T, Pot D, Mazzafera P. Sucrose
metabolism during fruit development of Coffea racemosa. Ann Appl Biol 2008; 152:179-87.
22. López M, Herrera-Cervera JA, Iribarne C, Tejera NA, Lluch C. Growth and nitrogen fixation in Lotus
japonicus and Medicago truncatula under NaCl stress: Nodule carbon metabolism. J Plant Physiol 2008;
165:641-50.
Molecular characteristics of sucrose synthase isolated from bird cherry leaves
49
CHARAKTERYSTYKA MOLEKULARNA SYNTAZY SACHAROZY IZOLOWANEJ Z LIŚCI
CZEREMCHY ZWYCZAJNEJ
Hubert Sytykiewicz*, Paweł Czerniewicz, Bogumił Leszczyński
Katedra Biochemii i Biologii Molekularnej
Akademia Podlaska
ul. Prusa 12
08-110 Siedlce
*autor, do którego należy kierować korespondencję: tel./faks: +4825 6431367,
e-mail: [email protected]
Streszczenie
W przeprowadzonych badaniach dokonano charakterystyki molekularnej częściowo
oczyszczonej syntazy sacharozy (NDP-glukoza: D-fruktoza 2-α-D-glukozylotransferaza, EC
2.4.1.13), ekstrahowanej z liści czeremchy zwyczajnej (Prunus padus L.). Syntaza sacharozy
została oczyszczona w wysokim stopniu przy wykorzystaniu czteroetapowej procedury
obejmującej precypitację siarczanem amonu, dializę, filtrację żelową na złożu Sephadex G-150 i chromatografię jonowymienną na złożu DEAE-celuloza. Analizowany enzym
występował w postaci dwóch izoenzymów (SuSyI i SuSyII). Względny ciężar cząsteczkowy
natywnych form analizowanego enzymu wynosił odpowiednio 200 i 180 kDa. Izoforma
SuSyI zawierała w swym składzie dwie różne podjednostki (57,5 i 52,8 kDa), podczas gdy
struktura SuSyII składała się z monomerów o ciężarze cząsteczkowym 63 kDa. Przeprowadzone badania wskazują, że obydwie izoformy SuSy zawierały po trzy podjednostki.
Słowa kluczowe: syntaza sacharozy, Prunus padus, ciężar cząsteczkowy, skład podjednostkowy
Vol. 54 No 3 2008