Boophilus microplus: its saliva contains microphilin, a small thrombin inhibitor.
Saliva of the cattle tick Boophilus microplus contains two thrombin inhibitors, BmAP and microphilin. This work presents the purification and characterization of microphilin. It was purified from the saliva by gel filtration, ultrafiltration through a 3 kDa cut-off membrane and affinity chromatography in a thrombin-Sepharose column. Analysis by mass spectrometry showed a molecular mass of 1770 Da. Microphilin is the smallest salivary thrombin inhibitor peptide known to date. It inhibits fibrinocoagulation and thrombin-induced platelet aggregation with an IC(50) of 5.5 microM, is temperature resistant and its inhibitory activity was abolished by protease K treatment. Microphilin did not inhibit the amidolytic activity of the enzyme upon a small chromogenic substrate, but inhibited the hydrolysis of a substrate that binds both catalytic site and exosite I. Therefore, we propose that microphilin blocks thrombin at exosite I.
Ciprandi
Alessandra
A
Centro de Biotecnologia, Universidade Federal do Rio Grande do Sul, P.O. Box 15005, 91501-970 Porto Alegre, RS, Brazil.
de Oliveira
Simone Kobe
SK
Masuda
Aoi
A
Horn
Fabiana
F
Termignoni
Carlos
C
D000577:Amides / Q000378:metabolism; D000818:Animals; D000925:Anticoagulants / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism / Q000494:pharmacology*; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D004305:Dose-Response Relationship, Drug; D004355:Drug Stability; D019286:Endopeptidase K / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism / Q000494:pharmacology*; D005260:Female; D006358:Hot Temperature; D026863:Ixodidae / Q000737:chemistry*; D008970:Molecular Weight; D010974:Platelet Aggregation / Q000187:drug effects; D011506:Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism / Q000494:pharmacology*; D012463:Saliva / Q000737:chemistry; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013917:Thrombin / Q000037:antagonists & inhibitors*; D014462:Ultrafiltration
D000577:Amides; D000925:Anticoagulants; D004791:Enzyme Inhibitors; D011506:Proteins; D013917:Thrombin; D019286:Endopeptidase K