A KAZAL-TYPE INHIBITOR WITH THROMBIN SPECIFICITY FROM RHODNIUS-PROLIXUS

A thrombin-specific inhibitor with an apparent molecular mass of 11 kD a has been purified from the insect Rhodnius prolixus. Amino-terminal protein sequence analysis allowed the molecular cloning of the corresp onding cDNA. The open reading frame codes for a protein of about 103 a mino acid residues and displays an internal sequence homology of resid ues 6-48 with residues 57-101 indicating a two-domain structure. Based on the amino acid sequence the two domains exhibit high homology to p rotease inhibitors belonging to the Kazal-type family. Model building suggests that the first domain binds to the active site with residue H is10 pointing into the specificity pocket. From gel filtration and tig ht-binding inhibition experiments the inhibitor appears to form 1:1 co mplexes with thrombin. Periplasma-directed heterologous expression of the rhodniin cDNA in Escherichia coli yields the intact thrombin inhib itor. Natural and recombinant rhodniin both display inhibition constan ts of about 2 x 10(-13) M.