الفهرس 
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Abstract
One of the natural materials derived from plants are smallproteins known as plant protease inhibitors (PIs) which are
considered among the defensive chemicals in plant tissues that
contribute to their resistance towards insects such as the cotton
leafworm, Spodoptera littoralis (Boisd.), which is a serious
lepidoptern pest in Egypt. The difficulty in controlling this kind
of pests promotes the development of alternatives for pest
control, among them the use of proteins (PIs) with insecticidal
effect, and act by inhibiting gut proteases. So PIs extracted
from tomato leaves, Solanum lycopersicum L., and the attacked
gut proteases from the fourth larval instar of S. littoralis were
partially characterized in some of their physicochemical and
kinetic properties. Besides, in vivo effects of PIs on the
digestive physiology and biology of the larvae were also
studied.
1. Optimum conditions for proteolytic
activity :
Effect of PH, temperature and substrate concentration on the
fourth larval instar of S. littoralis proteases system-catalyzed
reactions were studied to detect some kinetic properties of such
enzyme .Therefore, optimum conditions were determined for
each factor separately, all other factors being at the optimum.
Proteases system activity towards bovine serum albumin was
studied at 7 PH values ranging from 5-11. The results showed
that proteases had a relatively broad range of PH values and
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they exhibit their most activity in alkaline medium. At acidic
medium (PH 5) the activity was only 23.5 % as compared with
that of optimum ,while in strong alkaline (PH 11) it decreased
by 53 % than optimal PH. The optimal PH of gut proteases was
8.5 .
Hydrolysis of albumin by proteases was studied at
temperatures ranged from 25 to 70 ºC. The activity increased
sharply from 25 to 37 ºC reaching to optimal temperature at 55
ºC , however the activity at temperatures from 45-50 ºC was
somewhat equal to the optimal temperature. This indicates that
the enzyme is thermostable. Proteases lost their major activity
at 70 ºC , where the activity decreased by 76 % less than the
activity at 55 ºC .
Thirty minutes were allowed for protease reaction to
determine the suitable time allowed for reaction. A period of
10 minutes for cotton leafworm larval protease activity was
found to fit well within the linear part of the enzyme activity
curve .
Effect of substrate concentration in the reaction mixture on
the proteases activity was studied by measuring the activity at
six concentrations ranged between 10-10 to 10-5 M bovine serum
albumin. The hydrolysis had a linear dependence on the
substrate concentration from 10-7 to 10-6 M, where the peak is
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reached .Concentrations below 10-7 were very low and the
activity was at its lowest level. Also the reaction rate decreased
as the concentration of the substrate was further increased
,indicating substrate inhibition .
2. Michaelis-Menten kinetics of general
proteases:-
The kinetics of proteases from the fourth larval instar of the
cotton leafworm, S. littoralis were detected. Maximum velocity
of the reaction (Vmax), and substrate concentration (Km) were
determined using Lineweaver-Burk plot. When the linear
reciprocal plot is extrapolated , it intersects the negative portion
of the abscissa at -4 µM, which equal to 1/Km. Thus Km of
S.littoralis larval protease catalyzed reaction was 25 ×10-8 M,
and Vmax was 142 n mole D,L-alanine/min/mg protein.
The results address that cotton leafworm, S. littoralis fourth
larval instar gut proteases are thermostable, and exhibit their
most activity in alkaline medium, suggesting that larvae use
serine proteinases to digest proteins. Kinetic studies suggest
that 1 ml of the reaction mixture consists of about 100 µl of gut
extract and 1 µM albumin in 0.1 M Tris-Hcl buffer (pH 8.5) at
55ºC for 10 min represent the optimum conditions for S.
littoralis larval gut proteases activity.
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3. Plant protease inhibitors :
3.1. Stability to pH :
Inhibitors extracted from tomato leaves (S. lycopersicum)
are proteins, so denaturates of proteins such as pH and heat
were expected to affect them. PIs crude extract protein was
incubated for 1 hr in variable degrees of pH (6-11) at 37ºC to
evaluate the effect of pH on the ability of the extract to inhibit
proteases. The results show that antiproteolytic activity % was
non-significantly changed by the change in pH of the buffers
used. It was 100, 97, 98, 105, 100 and 96% at pH 6, 7, 8, 9, 10
and 11, respectively. i.e. pH degree had no effect on the
inhibition ability of PIs.
3.2. Thermal stability :
PIs crude extract protein was subjected, for 1 hr, to heat
treatment (30-80ºC) to evaluate the effect of temperature on PIs
antiproteolytic activity. The results also show, as the case of
pH, that the ability of PIs to inhibit proteases (anti- proteolytic
action) was non- significantly altered either at low temperature
(30ºC) or at high temperature (80ºC). PIs lost 10% of their
inhibition activity at 80ºC. The antiproteolytic activity was 99,
100, 105, 97, 102 and 90% at 30, 40, 50, 60, 70 and 80ºC ,
respectively.
3.3. In vitro PIs inhibitory kinetics of gut
proteases:
The proteolytic digestive activity of S. littoralis larvae was
very sensitive to the inhibition of tomato PIs. The crude extract
protein had an IC50 value of 21×10-9 mole ml-1. On the other
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hand, Dixon plot analysis revealed that tomato PIs crude extract
proteins are relatively strong proteases inhibitors (Ki=19.6×10-6
M protein), and because the lines do not intersect at the x-axis,
inhibition was competitive. This means that the inhibitor
(protein) competes with proteases substrate (albumin) for
binding the same active site on the enzyme (protease).
The high degree of tomato leaves PIs stability either to heat
or pH, and low inhibition kinetic values to pest gut proteases,
might be of a significant value in the field of pest control.
3.4. In vivo studies :
3.4.a. Effect on growth and development:
Feeding on PIs significantly (p<0.05) reduced the growth of
the cotton leafworm different larval instars. However, PIs did
not cause any mortality (=0%) in the treated larvae. Also, they
did not affect the larval duration, but treatment lead to
appearance of larval pupal intermediate in 6.7% of insects.
3.4.b. Effect on gut proteases :
The continuous feeding of larvae on PI treated leaves from
egg hatching led to inhibition of 4thlarval instar gut proteases,
in vivo. The results revealed that gut proteases were
significantly decreased by 18% (p<0.05) less than control.
3.4.c. Effect on protein metabolism :
Continuous feeding on PIs treated leaves from egg hatching
slightly reduced total protein of fourth larval instar larvae
(p<0.05), while free amino acids were more affected than
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proteins (p<0.01). on the other hand, enzymes related to protein
metabolism were also changed due to treatment. Treatment
enhanced glutamic oxaloacetic transaminase (GOT) activity,
while it decreased the titre of glutamic pyruvic transaminase
(GPT) (p<0.01).
It could be suggested that tomato PIs act by in vivo
inhibiting proteases not by resulting in hyperproduction of
proteolytic enzymes due to chronic ingestion of proteinase
inhibitors as reported by some authors. Instead, PIs result in
activation of some enzymes related to protein metabolism such
as GOT to compensate the reduced amount of available
proteins due to treatment.
It is the first time to test PIs activity towards the native pests.
PIs extracts from tomato S. lycopersicum leaves caused
deleterious effects on the digestive proteinases, growth and
development of S. littoralis larvae. Physicochemical analysis
has shown that they are strong and stable inhibitors. Results
presented here demonstrating that the use of PIs from tomato
could be a useful strategy in development of modified plants
with enhanced resistance to the cotton leafworm, S. littoralis.