生化学と分析生化学

概要

Time Course Studies on Impact of Low Temperature Exposure on the Levels of Protein and Enzymes in Fifth Instar Larvae of Eri Silkworm, Philosamia ricini (Lepidoptera: satuniidae)

Anita Singh, Vivek Kumar Gupta, Nikhat Jamal Siddiqi, Shaily Tiwari, Anita Gopesh and Bechan Sharma

Lactate dehydrogenase (LDH; EC 1.1.1.27) and malate dehydrogenase (MDH; EC 1.1.1.37) are the enzymes involved in energy metabolism of Eri silkworm, Philosamia ricini. However, no previous study has been reported about effect of low temperature exposure on their levels in different tissues of Eri silkworm. The present study was aimed to the time-course effects of low temperature (~10°C) exposure of 5th instar P. ricini on the levels of protein and energy metabolism enzymes of three major tissues (haemolymph, silk gland and fat body). The Eri silkworm larvae, reared on fresh leaves of castor-oil-plant (Ricinus communis), were divided into 4 groups: a control group reared at 25 ± 2°C along with three experimental groups reared at 10 ± 1°C containing 50 larvae in each, for varying durations (2, 4 and 7 days). The cell free extract was prepared by centrifuging the tissue homogenate at 9000 g and used for biochemical estimations (total protein content, lactate dehydrogenase and malate dehydrogenase activities). For isozyme analysis, another set of homogenates (20% w/v) was prepared in buffer (0.2 M Tris HCl, pH 7.0) containing 0.2 M sucrose and 10 mM EDTA, and analyzed by native-PAGE followed by activity staining. The activities of lactate dehydrogenase and malate dehydrogenase showed significant decrease in haemolymph, whereas in fat bodies both enzymes showed increased activity. In silk gland, Lactate dehydrogenase activity decreased uniformly, whereas malate dehydrogenase activity increased at all exposure durations. The isozyme analysis revealed significant perturbations in their expression profiles. The low temperature exposure resulted into accumulation of protein content in haemolymph and depletion in silk gland and fat body tissues. Fat bodies emerged as the main energy producing organ under this condition. Lactate dehydrogenase and malate dehydrogenase displayed presence of only one isozyme in all the tissues tested. The isozyme behaviour of lactate dehydrogenase and malate dehydrogenase towards low temperature varied in different tissues. These results suggested that alterations in expression and functions of these enzymes might be associated to the acclimation of larvae at low temperature.