A novel trypsin inhibitor from the hemolymph of the horseshoe crab Limulus polyphemus.
Trypsin inhibitory activity from the hemolymph of Limulus polyphemus was found to co-purify with coagulogen (the clottable protein in blood coagulation) after acidification, ammonium sulfate precipitation, and gel filtration. Limulus trypsin inhibitor (LTI) was separated from coagulogen by ion-exchange chromatography on carboxymethyl-Sephadex. LTI is an inhibitor of trypsin (Ki = 3.3 nM) on both high and low molecular weight substrates. It also inhibits chymotrypsin but has little or no effect on thrombin, thermolysin, pepsin, or papain, nor does LTI inhibit the proteolytic cascade produced in endotoxin-stimulated Limulus amoebocyte lysate coagulation. Electrophoresis under nonreducing conditions on denaturing polyacrylamide gel yields a doublet migrating with an estimated Mr of 20,000. Under reducing conditions, a single broad band migrates with an estimated Mr of 15,000. The native structure is a monomer of moderate asymmetry with a molecular weight of 16,300 and a so20,w = 1.5(5), as determined by analytical ultracentrifugation. The amino acid composition of LTI yields a calculated molecular weight of 15,680 and a calculated partial specific volume of 0.71(7) ml/g. LTI does not contain methionine, tryptophan, or detectable levels of reducing carbohydrate. The NH2-terminal sequence (V-S-P-P-F-I-K-Q-T-K-F-S-T-X-F-L-G-X-S-S) consists primarily of hydrophobic amino acid residues. Comparison of the amino acid composition and amino-terminal sequence of LTI with those of other known protease inhibitors reveals no significant similarity to other trypsin inhibitors. The novel physical characteristics suggest that LTI represents a new type of protease inhibitor.
Donovan
M A
MA
Department of Biochemistry, University of New Hampshire, Durham 03824.
Laue
T M
TM
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001798:Blood Proteins / Q000378:metabolism; D002852:Chromatography, Ion Exchange; D006458:Hemolymph / Q000737:chemistry*; D006737:Horseshoe Crabs / Q000032:analysis*; D008297:Male; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011108:Polymers / Q000378:metabolism; D012689:Sequence Homology, Nucleic Acid; D014361:Trypsin Inhibitors / Q000032:analysis / Q000302:isolation & purification / Q000378:metabolism*
D000596:Amino Acids; D001798:Blood Proteins; D011108:Polymers; D014361:Trypsin Inhibitors; C013445:coagulogen