title: Isolation and characterization of novel inducible serine protease inhibitors from larval hemolymph of the greater wax moth Galleria mellonella.
abstract: Three inducible serine protease inhibitors (ISPI-1, 2, 3) have been purified from larval hemolymph of greater wax moth larvae, Galleria mellonella, and characterized at a molecular level. These inhibitors were synthesized after larvae were injected with a yeast polysaccharide, zymosan preparation. ISPI-1,2,3 were active against various serine proteases including trypsin and toxic proteases released by the entomopathogenic fungus Metarhizium anisopliae. Precipitation by trichloroacetic acid and heat, followed by FPLC and HPLC separation steps were used for purification of the protease inhibitors from cell-free hemolymph samples. The molecular masses of purified proteins were determined by MS to be 9.2 kDa (ISPI-1), 6.3 kDa (ISPI-2) and 8.2 kDa (ISPI-3) with isoelectric points ranging between 7.2 and 8.3. The N-terminal amino-acid sequences of ISPI-1 and ISPI-3 are not similar to other known proteins, whereas that of ISPI-2 exhibits extensive similarity to known Kunitz-type protease inhibitors.
authors.affiliation: Institute of Zoology, Free University of Berlin, Germany. andreasvi@t-online.de
authors.forename: A C
authors.initials: AC
authors.lastname: Fröbius
authors.forename: M R
authors.initials: MR
authors.lastname: Kanost
authors.forename: P
authors.initials: P
authors.lastname: Götz
authors.forename: A
authors.initials: A
authors.lastname: Vilcinskas
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001798:Blood Proteins / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D006458:Hemolymph / Q000737:chemistry*; D019476:Insect Proteins / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D007814:Larva / Q000737:chemistry*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009036:Moths; D017386:Sequence Homology, Amino Acid; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D001798:Blood Proteins; D019476:Insect Proteins