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The complete amino acid sequence of trypsin inhibitor DE-3 from Erythrina latissima seeds.

1985; Elsevier BV; Volume: 260; Issue: 24 Linguagem: Inglês

10.1016/s0021-9258(17)38817-8

1083-351X

François J. Joubert, Christa Heussen, Eugene B. Dowdle,

Plant tissue culture and regeneration

Trypsin inhibitor DE-3 from Erythrina latissima seeds contains 172 amino acids, including 4 half-cystine residues, and resembles the Kunitz-type inhibitors.Limited hydrolysis of DE-3 with trypsin at pH 3 produced two fragments, F1 and F2, containing 63 and 109 amino acids, respectively.Amino-terminal sequence studies revealed that F1 was the N-terminal and that F2 was the C-terminal fragment.The complete amino acid sequence of fragments F1 and F2 was then determined on peptides produced by enzymatic digestion with trypsin and Staphylococcus aureus VS protease.The sequence of trypsin inhibitor DE-3 from E. latissima seeds shows a high degree of homology to those of Kunitz-type trypsin inhibitors from soybeans and winged bean seeds.Protease inhibitors are of widespread occurrence in the animal and plant kingdoms, and they have been isolated from various Leguminosae seeds (1).The trees and shrubs of the genus Erythrina, which belongs to the Leguminosae, are distributed throughout tropical to warm regions of the world (2, 3).Recently, it has been confirmed that seeds from Southern African species of Erythrina, namely E. acanthocarpa (4), E. caffra ( 5 ) , E. humeana (6), E. latissima (7), and E. lysistemon (8), contain large concentrations of trypsin and chymotrypsin inhibitors.The inhibitors contain about 170 amino acids (M, 19,000), including 4 half-cystine residues and resemble the Kunitz-type inhibitors (9), and their N-terminal amino acid sequences show a high degree of homology.From the seeds of E. latissima, two proteinase inhibitors (DE-1 and DE-3) were purified.Inhibitor DE-1 inhibits bovine a-chymotrypsin and not porcine trypsin, whereas DE-3 has the opposite inhibitory properties (7).Besides trypsin, it has been shown that DE-3 also inhibits plasmin and tissue plasminogen activator, but it has no comparable effect on urokinase or thrombin (10).When coupled to agarose, inhibitor DE-3 provided an effective reagent for the one-step affinity purification of tissue plasminogen activator from tissue culture medium that had been conditioned by melanoma cells cultivated in vitro (10).The Portions of this paper (including "Materials and Methods," "Results," Fig. 3, and Tables S-I-S-VIII) are presented in miniprint at the end of this paper.Miniprint is easily read with the aid of a standard magnifying glass.