title: Purification, characterization, and cloning of a serine proteinase inhibitor from the ectoparasite Haematobia irritans irritans (Diptera: Muscidae).
abstract: The fly Haematobia irritans irritans is one of the most important ectoparasites in cattle production, due to its ability to suck large amounts of blood. This report describes the purification and characterization of a serine proteinase inhibitor (HiTI) present in H. i. irritans head and thorax extracts. The HiTI purified by affinity chromatography on trypsin-Sepharose has a molecular mass of 7029Da by MALDI-TOF mass spectrometry. HiTI inhibited bovine trypsin, human neutrophil elastase, and a trypsin-like enzyme purified from H. i. irritans abdomen with dissociation constants of 0.57, 1.30, and 0.20nM, respectively. The HiTI partial amino acid sequence allowed its classification into the BPTI-Kunitz-type family. An HiTI cDNA fragment was cloned in the pGEMT vector using RT-PCR. The translated amino acid sequence of HiTI cDNA confirmed a unique Kunitz-type-domain protein. Our results suggest that HiTI could control some endogenous enzyme, e.g., the H. i. irritans trypsin-like protein.
authors.affiliation: Departamento de Bioquímica, UNIFESP-EPM, Rua Três de Maio 100, 04044-020, São Paulo, SP, Brazil.
authors.forename: Simone S
authors.initials: SS
authors.lastname: Azzolini
authors.forename: Joyce M C
authors.initials: JM
authors.lastname: Santos
authors.forename: Adriana F
authors.initials: AF
authors.lastname: Souza
authors.forename: Ricardo J S
authors.initials: RJ
authors.lastname: Torquato
authors.forename: Izaura Y
authors.initials: IY
authors.lastname: Hirata
authors.forename: Renato
authors.initials: R
authors.lastname: Andreotti
authors.forename: Aparecida S
authors.initials: AS
authors.lastname: Tanaka
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002417:Cattle; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000737:chemistry; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009117:Muscidae / Q000737:chemistry* / Q000201:enzymology / Q000235:genetics; D020539:Sequence Analysis, Protein; D015842:Serine Proteinase Inhibitors / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D018076:DNA, Complementary; D015842:Serine Proteinase Inhibitors