Characterization of Trypsin-Like Serine Protease from Lethocerus indicus Salivary Venom and its Cytotoxic Effect against Human Epidermoid Carcinoma Cell, A431
Journal Title: Biotechnology Journal International - Year 2014, Vol 4, Issue 9
Abstract
Objective: Lethocerus indicus salivary venom characterization and evaluation of extracellular degradation activity and cytotoxic effect against native human collagen type 1 and epidermoid carcinoma cell, A431. Method: Salivary venom extract was collected from adult insects by injecting 2% pilocarpine of 50 µml. Enzyme presence was detected by the apiZYM assay. The proteolytic activity was tested by the photometric and zymogram methods using specific fluorescent substrates and inhibitors. The cytotoxic activity was determined by the MTT assay and Trypan blue exclusion method. Apoptosis induction was observed using AO/EB staining solution. Digestion of extracellular matrix protein was detected against native human type I collagen. Result: L. indicus salivary venom presents amylases, proteases, carbohydrases, phosphatases and lipases. Among them, protease enzyme showed highest composition. The highest rate of proteolytic activity observed at pH 8 in 35ºC (100 %). Serine proteases present predominantly in salivary venom. Cysteine and metalloproteases are also detected. The activation energy of salivary venom is 49.86 kJ. Use of serine inhibitor, PMSF inhibited 92.77% which indicated that the maximum activity was due to serine protease. Detection of trypsin-like protease was confirmed by using PMSF and TLCK with specific substrate, BApNA. It shows significant inhibitions, 82% and 78% respectively suggesting maximum influence in salivary venom. Degradation of the fibrillar native state collagen Type I into 8 smaller peptide bands showed it importance in medical application. IC50 concentration of venom that induces cytotoxicity in epidermoid carcinoma cells, A431was 2.3 µg/ml only. It gives prominent apoptotic features such as cytoplasmic membrane blebbing, nuclear contraction, nuclear fragmentation and contact inhibition. Conclusion: We suggest that further investigation of the venom will lead to identification of active compound in L. indicus salivary venom for its potential use in therapeutic application.
Authors and Affiliations
T. Shantibala, H. Debaraj, R. K. Lokeshwari, Sarbani Giri
Keywords
Related Articles
- EP ID EP237724
- DOI 10.9734/BBJ/2014/12217
- Views 91
- Downloads 0
Morphological and SSR Assessment of Putative Drought Tolerant M3 and F3 Families of Wheat (Triticum aestivum L.)
In an attempt to develop drought tolerant genotypes of bread wheat, two procedures, i.e., mutation and hybridization were used to induce new genetic variation. Selection for high grain yield/plant (GYPP) and other desira...
Insights on Pharmacological Properties of Combretum leprosum Mart.
Combretum leprosum Mart. is a plant widely used in folk medicine; several studies revealed anti-inflammatory, antinociceptive, toxic, antiproliferative, antibacterial, antiparasitic, neuroprotective and gastroprotective...
Use of Gene Specific Universal Primers for Isolation of DNA Sequences Encoding Laccase Enzyme from a Wild Isolate of Schizophyllum commune
Laccase enzymes plays a vital role in innumerable biotechnological applications and hence their large scale production has stimulated considerable research. In the present study, degenerate universal primer pairs were em...
In vitro and in silico Approach to Evaluate the Urease and Collagenase Inhibitory Activity of Embilica officinalis Gaertn Fruit
Aim: The key virulent factors of bacteria are enzymes. Urease and collagenase enzyme play a vital role in pathogenesis of wide array of bacterial strains and cause numerous diseases. So the aim of present study was to fi...
Effect of Abscisic Acid on Soluble Sugar Contents in Tomato Fruits under Condition of Short-Term Night Sub-Low Temperature
Aims: This research explored the effect of ABA treatment before short-term night sub-low temperature on the sugar composition and contents, and provided theoretical basis for temperature management in tomato protected cu...