The domesticated insect Bombyx mori respond well to environmental fluctuations that govern the success of cocoon production due to its piokilothermic nature. Since, the interaction between heat shock proteins and proteases in the whole organism that complete larval life in natural environment remain obscure, we have employed zymography technique to examine different classes of proteases and its activity under imposed temperature stress (heat shock) condition. The total protein extracted from the whole larvae was analyzed in SDS-polyacrylamide gel copolymerized with gelatin, casein and BSA. Discrete proteolytic bands aptly visualized in the gel contained gelatin as substrate revealed strong activity of five proteases with molecular mass of 116 (P1), 83 (P2), 57 (P3), 35 (P4) and 24 kDa (P5) and the only protease that exhibits activity in all the substrates was P5. Various protease inhibitors like leupeptine, aprotonin, pepstatin, EDTA and PMSF were used for identification and most of the proteases activity eventually was inhibited by PMSF while other inhibitors had no effect. With this we could determine the presence of large group of serine proteases in the whole body of B. mori. Further, for the first time, we have employed zymographic technique to evaluate protease activity in the heat shock (40°C) induced larvae. Interestingly, the activity of three proteases (P3 to P5) found inhibited on gelatin substrate while high molecular weight proteases (P1 and P2) were not affected. The protease assay further confirmed decreased protease activity in the heat shock induced larvae compared to control. The differential and altered proteases activities in normal and heat-shocked larvae respectively envisaged its significance in stress physiology and adaptation of whole organism to elevated temperature.
Key words: Bombyx mori, heat shock, lepidoptera, serine-protease, zymography.
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