Purification, characterization and immunolocalization of a novel protease inhibitor from hemolymph of tasar silkworm, Antheraea mylitta

Rai, Shruti ; Aggarwal, K. K. ; Mitra, B. ; Das, T. K. ; Babu, C. R. (2010) Purification, characterization and immunolocalization of a novel protease inhibitor from hemolymph of tasar silkworm, Antheraea mylitta Peptides, 31 (3). pp. 474-481. ISSN 0196-9781

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Official URL: http://www.sciencedirect.com/science/article/pii/S...

Related URL: http://dx.doi.org/10.1016/j.peptides.2009.08.021

Abstract

A novel serine protease inhibitor (AmPI) was purified from larval hemolymph of tasar silkworm, Antheraea mylitta by two-step process of trypsin-affinity and gel-filtration (FPLC) chromatography. AmPI was active against larval midgut and commercial bovine trypsin and chymotrypsin. The extent of purification was determined by SDS and Native PAGE. The protease inhibitor had an apparent molecular weight of approximately 14.5 kDa as determined by SDS-PAGE. Its activity was stable over a pH range of 4.5–9 and temperatures range of 4-65 °C. Molecular weight as determined by MALDITOF-MS was between 13241.63 and 13261.66 Da. MS profile of AmPI also suggests two isoforms of AmPI because of glycosylation by heptose (C7H14O7). This confirmed the result of Native PAGE showing two bands. N-terminal amino acid sequence of this protein did not show similarity to any known protease inhibitor. To study the functional implications of AmPI in insect, it was localized in insect body tissue of different larval instars by immunogold labeling technique using GAR-gold conjugate as secondary antibody. The pattern of localization suggests constitutive nature of AmPI, which may have role in insect's defense mechanism.

Item Type:Article
Source:Copyright of this article belongs to Elsevier Science.
Keywords:Antheraea mylitta; Cuticle; Epidermis; Immunogold; Localization; Mass Spectrum; Protease Inhibitor
ID Code:69289
Deposited On:22 Nov 2011 11:23
Last Modified:22 Nov 2011 11:23

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