Biotechnology


https://sites.google.com/a/gauhati.ac.in/research/biotechnology#hks27122018 Insect immunological response
Insect immunological response

Hridip Kumar Sarma and his fellow researchers have characterised a key enzyme in insect immunological response. This research work is published in the journal Archives of Insect Biochemistry and Physiology.



Authors
Gayatri Sarma Baruah1, Hridip Kumar Sarma, Sunayan Bardoloi1, and Dipsikha Bora1
1 from other institutions

Abstract
This is the first report on the characterization of phenoloxidase in Antheraea Assamensis Helfer, the key enzyme in insect immunological responses against pathogen infestation. In the current study, a dimeric phenoloxidase (PO) from the hemolymph of healthy and diseased (pebrine infected) larvae of the insect was extracted and purified. The enzyme comprised of two subunits of ~76.8 and 76 kDa that showed PO activity in 6 mM L‐3,4‐dihydroxyphenylalanine (L‐DOPA) and 8 mM catechol but not in hydroquinone. Specific activity of the purified PO from healthy larvae was 53.9 µM/min per mg of protein per ml in L‐DOPA and 50.77 µM/min per mg of protein per ml in catechol while that of diseased larvae was 30.0 µM/min per mg of protein per ml in L‐DOPA and 28.55 µM/min per mg of protein per ml in catechol. The enzyme showed the Michaelis constant (Km) of 2.46–2.85 mM for healthy and diseased fractions in L‐DOPA. In catechol Km of 9.23–17.71 mM was observed. Peptidoglycan appeared as the best activator of purified PO from both healthy and diseased fractions. Interactions between controls and activators appeared statistically significant (F = 767.5; df = 3; P < 0.0001). Na+, K+, and Cu2+ increased, whereas Ca2+, Zn2+, Mg2+, and Co2+ decreased PO activity. The overall interactions appeared highly significant (F = 217.0; df = 27; P < 0.0001). Kojic acid, dithiothreitol, thiourea, phenylthiourea, carbendazim, N‐bromosuccinimide, N,N,N′,N′‐tetraacetic acid, and diethyldithiocarbamate inhibited PO activity. The importance of POs in arthropod immunity has been consistently debated and all arthropod genomes explored till date are reported to contain the genes necessary for PO synthesis (Cerenius et al., 2008). This report emphasizes the characteristics of PO from A. assamensis Helfer during healthy and diseased states. Observations revealed distinct differences in enzymatic activity during different states of the insect and propose that highly contagious diseases like pebrine significantly impair cellular reaction cascade in the silkworm, concomitant to their physiological states. The purified PO appeared as a dimeric protein with decreased activity compared to its crude equivalent. Biological components from microbial sources elicited PO activity in crude hemolymph, whereas diminished activity in purified PO was probably due to the absence of endogenous activators in the purified fractions. A better understanding of the role of endogenous activators and inhibitors need to be evaluated to understand the mechanism of PO activation in A assamensis Helfer. The enzyme was sensitive to certain chemicals and inhibitors at minute concentrations in both crude and purified forms. This suggests that the effect of chelators and hazardous substances are critical towards the survival of the species in the wild. It is proposed that further investigation of the insect PO is vital toward the restoration of the species which calls for immediate attention.

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