Interaction between alpha-2 macroglobulin and trypsin with synthetic trypsin inhibitors]

The complex formed between trypsin (Tn) and alpha 2 Macroglobulin (alpha 2 M) retains the whole hydrolytic activity of the enzyme for synthetic substrates. Moreover synthetic inhibitors of low molecular weight stiel inhibit this activity. A comparative study of three inhibitors (Benzylamine, Butylamine, Benzamidine) has been carried out and shows that their behavior is similar. These inhibitors bind trypsin when it is bound to alpha 2 M and reciprocally alpha 2 M can bind Tn-inhibitor complex. Nevertheless the dissociation constant of the enzyme-inhibitor complex (Ki) is increased by alpha 2 M. In the case of Benzamidine the value of Ki is 2.22.10(-5) M for native enzyme and 13.4.10(-5) M for Tn-alpha 2 M and in the case of Butylamine this value increases from 0.5.10(-3) M to 2.95.10(-3) M. These variations of the Ki values are due to the modification of the accessibility of the inhibitor to the active site. Unpublished results show that the alpha 2 M molecule undergoes a deep structural modification in the course of the complex formation, which must lead to an increase of the value of Ki. This structural modification is probably irreversible so that the alpha 2 M complex has never been dissociated without altering the alpha 2 M molecule. The increase of the values of Ki cannot therefore result in an effective decrease of the association constant of the Tn-alpha 2 M complex.     

Inhibition of lymphocyte transformation: effect of soy bean trypsin inhibitor and synthetic anti-proteases.

Soybean trypsin inhibitor (SBI) was found to inhibit transformation of human lymphocytes induced by mitogens (leucoagglutinin, concanavalin A, NaIO4) or in mixed lymphocyte reaction (MLR). SBI covalently cross-linked to Sepharose beads inhibited the MLR and mitogen stimulation virtually completely. We have confirmed the work of others which showed that the synthetic anti-proteases epilson-aminocaproic acid and tosyl-L-lysyl-chloromethane (TLCK) also inhibited mitogen-induced blastogenesis and we have shown that phenylmethylsulfonylfluride was effective also; all of these agents were found to inhibit the MLR as well. SBI and TLCK were most inhibitory when added along with mitogen or when mixing allogeneic cells in a MLR; significant decrease in inhibition was noted when TLCK was added 1 h after mitogen. These data support the hypothesis that protease action at a cell surface is an essential early event common to all types of lymphocyte transformation.     

A synthetic operon containing 14 bovine pancreatic trypsin inhibitor genes is expressed in E. coli.

A synthetic gene encoding the protein sequence of mature bovine pancreatic trypsin inhibitor (BPTI) has been cloned into a novel E. coli expression vector. After in vitro gene amplification by successive DNA duplications, more than 600 000 mostly inactive inhibitor molecules may be recovered from a single cell. After purification the inhibitory activity can be reconstituted almost completely. The specificity of BPTI for trypsin is abolished by a single amino acid exchange from lysine to isoleucine at position 15. The altered protein is shown to be an efficient inhibitor of human leukocyte elastase.     

A synthetic 13-residue peptide designed to resemble the primary binding site of the basic pancreatic trypsin inhibitor

A peptide, AC-Pro-Cys-Lys-Ala-Arg-Ile-DPhe-Pro-Tyr-Gly-Gly-Cys-Arg-NH2, which resembles the binding site of the basic pancreatic trypsin inhibitor, has been prepared by solid-phase peptide synthesis. A partially protected peptide was first obtained from the solid-phase product by removal of all side-chain protecting groups except the acetamidomethyl (Acm) groups on the cysteines. This di-Acm-peptide was deprotected, with concomitant formation of the cyclic product, by treatment with I2 in AcOH. The cyclic 13-residue peptide is a reversible, competitive inhibitor of trypsin with a Ki (app) of 2 . 10(-6) M, but loses its inhibitory activity upon incubation with trypsin. The di-Acm-peptide precursor has a Ki (app) of 5 . 10(-5) M and is deactivated more rapidly by trypsin. The effectiveness of the 13-residue peptides as inhibitors is in part attributed to the conformation induced by the beta-turn directing the -DPhe-Pro portion of the sequence.     

New synthetic inhibitors of C1r, C1 esterase, thrombin, plasmin, kallikrein and trypsin

p-Guanidinobenzoate derivates were prepared and their inhibitory effects on trypsin, plasmin, pancreatic kallikrein, plasma kallikrein, thrombin, C1r and C1 esterase were examined. Among the various inhibitors tested, 6'-amidino-2-naphthyl-4-guanidinobenzoate dihydrochloride, 4-(beta-amidinoethenyl)phenyl-4-guanidinobenzoate dimethanesulfonate and 4-amidino-2-benzoylphenyl-4-guanidinobenzoate dimethanesulfonate were the most effective inhibitors of trypsin, plasmin, pancreatic kallikrein. plasma kallikrein and thrombin and they strongly inhibited the esterolytic activities of C1r and C1 esterase, and then strongly inhibited complement-mediated hemolysis.     

Inhibition of alpha 2-macroglobulin-bound trypsin by soybean trypsin inhibitor

Soybean trypsin inhibitor, a protein of Mr = 20,000, has been used to assess the degree of inaccessibility of porcine trypsin within the alpha 2-macroglobulin-trypsin complex. The interaction between alpha 2-macroglobulin-bound trypsin and the inhibitor was demonstrated by affinity chromatography and trypsin inhibition. Whereas the free trypsin-inhibitor association is very fast (k = 1.2 X 10(7) M-1 s-1), the reaction between complexed trypsin and inhibitor takes 10 h to reach equilibrium. In addition, alpha 2-macroglobulin reduces, by several orders of magnitude, the affinity of trypsin for the inhibitor. Only one of the two trypsin molecules of the ternary (trypsin)2-alpha 2-macroglobulin complex is readily accessible to soybean inhibitor. It is postulated that the recently discovered proximity of the alpha 2-macroglobulin binding sites (Pochon, F., Favaudon, V., Tourbez-Perrin, M., and Bieth, J. (1981) J. Biol. Chem. 256, 547-550) accounts for this behavior. In the light of these results it is concluded that the proteinase binding sites are localized on the alpha 2-macroglobulin surface and that the two subunits of this protein are either not identical or not symmetrically arranged.     

Interaction of trypsin with a trypsin inhibitor from bovine colostrum]

Kinetics of trypsin association with trypsin inhibitor from colostrum (IC) was studied. The association rate constant is 3-10-5 M- minus 1 sec- minus 1 at pH 7,8, 25 degrees C. The rate constant for the complex dissociation was determined from the kinetics of the IC displacement from the complex with trypsin by a specific substrate and was found to be 5-10- minus 6 sec- minus 1 (pH 7,8; 25 degrees C). The equilibrium constant (Ki) was measured in a special experiment and was equal to 4-10- minus 12 M (p H 7,8; 25 degrees C). The similarity of this reaction and the association of trypsin with other protein inhibitors was discussed.