TRYPSIN AND ELASTASE INHIBITORS FROM BITTER GOURD (MOMORDICA-CHARANTIA LINN) SEEDS - PURIFICATION, AMINO-ACID-SEQUENCES, AND INHIBITORY ACTIVITIES OF 4 NEW INHIBITORS

Serine proteinase inhibitors of the squash family were isolated from b itter gourd (Momordica charantia LINN.) seeds by the conventional puri fication method. Heat treatment of the extract of the seeds allowed re moval of large amounts of protein without loss of trypsin and elastase inhibitory activities. From the supernatants thus obtained, the inhib itors were isolated to homogeneity by ion-exchange chromatography, gel filtration, and reversed phase chromatography. One trypsin inhibitor (Momordica charantia trypsin inhibitor-III; MCTI-III) and three elasta se inhibitors (Momordica charantia elastase inhibitor-II, -III, and -I V; MCEI-II, -III, and -IV) were newly isolated in addition to trypsin inhibitors MCTI-I and -II and elastase inhibitor MCEI-I previously rep orted [Hare, S. et al. (1989) J. Biochem. 105, 88-92]. The primary str uctures of the four new inhibitors were determined as follows. [GRAPHI CS] The dissociation constants, K-1, of MCTI-III complex with bovine b eta-trypsin, and of MCEI-II, -III, -IV with porcine elastase were dete rmined to be 1.9 x 10(-7) M, 9.4 x 10(-9) M, 4.0 x 10(-9) M, and 4.7 x 10(-9) M, respectively. Although MCTI-III differed from MCTI-I in onl y two amino acids, having Gly(3) and Gln(13) in place of Arg(3) and Ar g(13), the K-1 value of MCTI-III was 20-fold larger than that of MCTI- I. Addition of an amino terminal Grin residue, a dipeptide (Glu-Glu-), and a tripeptide (Glu-Glu-Glu-) to MCEI-I strengthened its elastase i nhibitory activity by 200-fold.