PRIMARY STRUCTURE AND SPECIFICITY OF THE MAJOR SERINE PROTEINASE-INHIBITOR OF AMARANTH (AMARANTHUS-CAUDATUS L) SEEDS

A novel member of the potato inhibitor I family of serine proteinase inactivating proteins has been isolated from seeds of grain amaranth (Amaranthus caudatus L.) and characterized. The mature form of the amaranth trypsin/subtilisin inhibitor (ATSI) with pI approximate to 8.3 and molecular mass 7887 Da contains 69 amino acids in a sequence showing 33-51% identity with members of the inhibitor I family from other plant families. A minor form with pI approximate to 7.8 and same inhibitory properties lacked the N-terminal dipeptide Ala-Arg. In accordance with the reactive-site bond Lys(45)-Asp(46), which was identified by specific cleavage on a subtilisin column, ATSI is a potent inhibitor of trypsin (K-i approximate to 0.34 nM) and more weakly of plasmin (K-i approximate to 38 nM) and Factor XII, (K-i approximate to 440 nM). However, ATSI also inactivates chymotrypsin (K-i approximate to 0.41 nM), cathepsin G (K-i approximate to 122 nM) and several alkaline microbial proteinases, including subtilisin NOVO (K-i approximate to 0.37 nM). Interestingly, ATSI contains a Trp residue instead of the highly conserved Arg in position 53 (P'(8)), which is assumed to play a central role in stabilization of the active-site loop during complex formation. ATSI was immediately inactivated by pepsin and hardly represents an antinutritional component in foods or feeds.