A Kazal-type inhibitor with thrombin specificity from Rhodnius prolixus.
A thrombin-specific inhibitor with an apparent molecular mass of 11 kDa has been purified from the insect Rhodnius prolixus. Amino-terminal protein sequence analysis allowed the molecular cloning of the corresponding cDNA. The open reading frame codes for a protein of about 103 amino acid residues and displays an internal sequence homology of residues 6-48 with residues 57-101 indicating a two-domain structure. Based on the amino acid sequence the two domains exhibit high homology to protease inhibitors belonging to the Kazal-type family. Model building suggests that the first domain binds to the active site with residue His10 pointing into the specificity pocket. From gel filtration and tight-binding inhibition experiments the inhibitor appears to form 1:1 complexes with thrombin. Periplasma-directed heterologous expression of the rhodniin cDNA in Escherichia coli yields the intact thrombin inhibitor. Natural and recombinant rhodniin both display inhibition constants of about 2 x 10(-13) M.
Friedrich
T
T
Department of Biotechnology, BASF Aktiengesellschaft, Ludwigshafen, Germany.
Kröger
B
B
Bialojan
S
S
Lemaire
H G
HG
Höffken
H W
HW
Reuschenbach
P
P
Otte
M
M
Dodt
J
J
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003001:Cloning, Molecular; D004247:DNA; D007301:Insect Hormones / Q000235:genetics* / Q000378:metabolism; D019476:Insect Proteins; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011480:Protease Inhibitors / Q000302:isolation & purification / Q000378:metabolism; D012239:Rhodnius / Q000737:chemistry*; D017386:Sequence Homology, Amino Acid; D013917:Thrombin / Q000037:antagonists & inhibitors*; D014359:Trypsin Inhibitor, Kazal Pancreatic / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism
D007301:Insect Hormones; D019476:Insect Proteins; D011480:Protease Inhibitors; C082506:rhodniin protein, Rhodnius; D014359:Trypsin Inhibitor, Kazal Pancreatic; D004247:DNA; D013917:Thrombin