Boguslavskaya L.V, Shupranova L.V.
Researche institute of biology, Oles Honchar Dnepropetrovsk National
University
Peroxidase activity and its isozyme composition of extensibility zone of maize root
cells under xenobiotics effect
It is
known, that peroxidase is sensibility enzyme to anthropogenic factors effect.
Investigation of isozyme composition of plants gives possibility to understand
and explain the cause of steadiness or sensibility of plants to unfavourable
conditions of environmental [1, 2]. The simultaneous presence of several
molecular forms of the same enzyme, that differ in kinetics and other
properties, side by side with others mechanisms of regulation, conductives to
co-ordination of substances exchange in cells and rapid adaptation to surroundings,
that continuelly changes. The isozymes differ between itself in
electrophoretical mobile, that gives possibility to analyse its in detail. In
this connection, the purpose of this paper was to study the combined influence of ions cadmium and
soil herbicide metolachlor on component composition of soluble peroxidase in
extensibility zone of maize roots. The
objects of investigation were the two-day roots of maize shoots of three-linear
average-early hybrid Biloserskyj 295 CB, that were growed in medium with cadmium nitrate Cd(NO3)2 in concentration of
50 µM, herbicide of chlorazetanilide groupe – metolachlor (Mch) - in
concentration 1 mg/l as well as its combination. The selection of root samples
carried out after 48 h of treatment. Lightsoluble proteins were obtained by the
water extraction from extensibility zone of maize roots. Protein concentration in homogenates was
determined by Bradford method [4]. For isoelectrical focusing (IEF) of
peroxidase system was used 5% PAAG (polyacrilamide gel) containing 10%
glycerine and 1.5% ampholines, pH 3,5-6,0 (“LKB”, Sweden). Isozymes of
peroxidase revealed by 0.01% solution of 3,3-diamino-benzidine of
hydrochloro-sour in presence of 0.001% H2O2 in
Na-phosphate buffer, pH 6.2. Densitograms of isoelectrophoregram obtained by
computer program “TCX-manager 2.1” (2005).
The
investigation results showed, that the changes in peroxidase system under
xenobiotics effect have been accompanied by qualitative and quantitative
alterations in enzyme spectrum. General changes apply to quantitative
redistribution between available isoenzymes of peroxidase, that depended on the
type of xenobiotic (table 1).
Table 1
Effect of Cd(NO3)2,
herbicide metolachlor and its combination
(Cd2+, 50 µM + Mch, 1 mg/l) on the content of peroxidase isoforms in
maize roots extensibility zone after 48 h of treatment
|
Values of pI |
Relative content, % |
|||
|
control |
Cd(NO3)2,
50µM |
Mch,1 mg/l |
Cd, 50 µM + Mch, 1 mg/l |
|
|
4.00 |
- |
- |
0,04 |
0,06 |
|
4.05 |
0,06 |
0,08 |
0,41 |
0,65 |
|
4.10 |
0,35 |
0,15 |
5,18 |
0,11 |
|
4.15 |
9,87 |
5,95 |
5,01 |
6,51 |
|
4.20 |
2,24 |
6,44 |
6,29 |
9,22 |
|
4.21 |
10,6 |
8,54 |
8,61 |
7,35 |
|
4.25 |
1,37 |
2,21 |
2,82 |
2,10 |
|
4.28 |
1,95 |
2,14 |
1,98 |
2,10 |
|
4.30 |
0,21 |
2,48 |
0,47 |
1,98 |
|
4.35 |
18,8 |
20,0 |
20,0 |
20,6 |
|
4.40 |
21,2 |
21,3 |
20,9 |
21,4 |
|
4.45 |
4,67 |
5,21 |
6,00 |
6,71 |
|
4.50 |
7,08 |
7,72 |
5,44 |
5,98 |
|
4.60 |
0,11 |
0,23 |
0,36 |
0,34 |
|
4.70 |
3,79 |
3,85 |
3,25 |
3,63 |
|
4.75 |
1,23 |
1,57 |
0,75 |
0,21 |
|
4.80 |
6,70 |
1,26 |
1,22 |
1,21 |
|
4.90 |
0,64 |
1,03 |
1,00 |
1,00 |
|
5.00 |
0,34 |
0,25 |
0,30 |
0,28 |
|
5.15 |
0,39 |
0,98 |
0,47 |
0,85 |
|
5.20 |
2,65 |
1,91 |
2,00 |
1,53 |
|
5.45 |
2,00 |
2,34 |
3,14 |
2,10 |
|
5.60 |
1,24 |
1,51 |
1,35 |
1,67 |
|
5.80 |
0,58 |
0,61 |
0,64 |
0,64 |
|
6.00 |
0,69 |
0,97 |
1,55 |
0,82 |
|
6.25 |
0,87 |
1,25 |
0,85 |
0,97 |
|
6.45 |
0,06 |
0,08 |
0,04 |
0,06 |
|
6.70 |
0,35 |
0,15 |
0,41 |
0,65 |
Cytosol
peroxidase was presented in control sample 28 components in pH diapason
4.05-6.70. General activity of peroxidase system falled on isoenzymes with pI
4.35 and 4.40 (18.8-21.3% from total isoperoxidases). Isoform with pI 4.00
appears under herbicide effect. The main reorganizations in peroxidase system
of extensibility zone
in conditions of toxical stress occurred for account quantitative adaptation
strategy. Four isoenzymes with pI 4.35, 4.40, 4.70 and 5.80 are posessed the
high stability under Cd effect, while in conditions of herbicide expousure it
was found seven: 4.28, 4.35, 4.40, 5.00, 5.15, 5.60 and 6.25. Under cadmium action
observed essential content increasing of isoform with pI 4.20, 4.25, 4.30, 4.60
and 5.15 and under metolachlor effect – with pI 4.05, 4.10, 4.20, 4.25, 4.30,
4.60 and 6.00. Essential content level lowering of isoperoxidases with pI 4.15
(at an average on 44%) and 4.80 (on
79%) was typical for all xenobiotics. Cadmium reduced the level content of peroxidase component with pI 6.70 (in 2.3
times as compared with control). Typical line of xenobiotic combined effect was
increasing of isoenzymes content with pI 4.05 (in 10.5 times), 4.20 (in 4.1
times), 4.30 (in 9.4 times), 4.60 (in 3 times) and 5.15 (in 2.2 times) as
compared with control.
In
conclusion, both cadmium and soil
herbicide metolachlor have essential influence on functioning of lightsoluble
peroxidase system in extensibility zone of maize roots, that
expresses in change of separate isoform activity and it is unspecific
reaction on unfavourable environment.
Literature:
1. Газарян И.Г. Пероксидазы растений // Итоги науки и техники. Серия
Биотехнология. М.: ВИНИТИ.- 1992. - Т. 36. - С. 4-28
2. Серегин И.В., Иванов В.Б.
Физиологические аспекты токсического действия кадмия и свинца на высшие
растения // Физиология растений. - 2001. -Т. 48, №4. -С. 606-630
3. Bradford M.M. A rapid and
sensitive method for quantitation of microgram quantities of protein utilizing
the principle of protein dye binding // Anal. Biochem. – 1976. – P. 248–254