Purification, characterization and immunolocalization of a novel protease inhibitor from hemolymph of tasar silkworm, Antheraea mylitta.
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 degrees 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 (C(7)H(14)O(7)). 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.
Rai
Shruti
S
School of Biotechnology, GGS Indraprastha University, Kashmere Gate, Delhi 110006, India. shrutisrai@rediffmail.com
Aggarwal
K K
KK
Mitra
B
B
Das
T K
TK
Babu
C R
CR
D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D002846:Chromatography, Affinity; D002918:Chymotrypsin / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation / Q000187:drug effects; D041981:Gastrointestinal Tract / Q000187:drug effects; D006458:Hemolymph / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D066298:In Vitro Techniques; D007814:Larva / Q000235:genetics / Q000378:metabolism; D046529:Microscopy, Electron, Transmission; D008969:Molecular Sequence Data; D009036:Moths / Q000235:genetics / Q000378:metabolism*; D011480:Protease Inhibitors / Q000302:isolation & purification* / Q000378:metabolism* / Q000494:pharmacology; D017386:Sequence Homology, Amino Acid; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013696:Temperature; D014357:Trypsin / Q000378:metabolism
D011480:Protease Inhibitors; D002918:Chymotrypsin; D014357:Trypsin