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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 Summary 79 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 Summary 80 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. Summary 81 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 Summary 82 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 Summary 83 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. |