Solid-phase assembly of cow colostrum trypsin inhibitor gene.

A gene for cow colostrum trypsin inhibitor (CTI) was constructed from synthetic oligonucleotides using a novel method of solid-phase gene assembly. In the first step an anchor oligonucleotide was covalently bound to the CNBr-activated Sephacryl S-500 support. Next, triads or tetrads of separately annealed oligonucleotides were stepwise hybridized to the immobilized complementary sequence, with washing after each step. In the last step a linearized vector molecule was ligated to the assembled gene. The whole construct was released from the solid support with a restriction enzyme, circularized, and used for transformation, with a high yield of recombinant clones being obtained. The method represents a generally applicable approach to rapid and efficient assembly of extended DNA duplexes.     

On the carbohydrate composition of bovine colostrum trypsin inhibitor.

The trypsin inhibitor from bovine colostrum has been separated into several forms by CM-Sephadex and DEAE-cellulose chromatography. These forms differ in the amount and composition of the carbohydrate they contain, which has been quantitated for four components by gas-liquid chromatography and standrad colorimetric procedures. The monosaccharides fucose, mannose, galactose, galactosamine, glucosamine and sialic acids have been determined. A microheterogeneity was establish ed in the carbohydrate moiety, which amounts to about 40% of the total molecular weight (Mr 11 000 - 14 000) of bovine colostrum inhibitor.     

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.     

The reactive site of eggplant trypsin inhibitor.

The reactive site peptide bond of the eggplant inhibitor against trypsin [EC 3.4.21.4] was identified by chemical modifications with 1,2-cyclohexanedione, 2,4,6-trinitrobenzenesulfonic acid, acetic anhydride and glyoxal, and by sequential treatments with trypsin and carboxypeptidase B [EC 3.4.12.3]. The inhibitor was significantly inactivated by chemical modifications of arginine residues, but was not affected by lysine modifications. Free arginine was released from the trypsin-modified inhibitor by carboxypeptidase B digestion, accompanied by a marked loss of inhibitory activity. A serine residue was newly exposed at the N-terminal amino acid of the inhibitor after modification with trypsin. The reactive site of the inhibitor against trypsin was concluded to be an arginylseryl bond. The inhibitor was completely inactivated by full reduction of its disulfide bonds.     

Secretion and immunochemical properties of the trypsin inhibitor from bovine colostrum.

A trypsin inhibitor was isolated from bovine colostrum by affinity chromatography. Immunoelectrophoresis detected two immunogenic components in the isolated inhibitor, but only one of these was specific for the inhibitor; the other one was identical with an antigen present in liver, kidney, spleen, adrenal, thyroid, thymus, brain, ovarian, testicular and udder tissue and in bull seminal plasma. Using immunoabsorption and immunofluorescence it was shown that the antigens specific for the trypsin inhibitor of colostrum could be demonstrated only in the tissue of an udder that is secreting colostrum. The inhibitor is secreted by the secretory epithelium of the milk alveoli of the udder, during the period when the latter secretes colostrum. This inhibitor was not detected in the milk. Cross-reaction between antisera to colostral inhibitor and basic pancreatic inhibitor or seminal plasma inhibitors yielded negative results. Antiserum to bovine colostral inhibitor showed a positive reaction with inhibitor isolated from porcine colostrum.     

The amino acid sequence and reactive site of a single-headed trypsin inhibitor from wheat endosperm

The sequence of a trypsin inhibitor, isolated from wheat endosperm, is reported. The primary structure was obtained by automatic sequence analysis of the S-alkylated protein and of purified peptides derived from chemical cleavage by cyanogen bromide and digestion withStaphylococcus aureus V8 protease. This protein, named wheat trypsin inhibitor (WTI), which is comprised of a total of 71 amino acid residues, has 12 cysteines, all involved in disulfide bridges. The primary site of interaction (reactive site) with bovine trypsin has been identified as the dipeptide arginyl-methionyl at positions 19 and 20. WTI has a high degree of sequence identity with a number of serine proteinase inhibitors isolated from both cereal and leguminous plants. On the basis of the findings presented, this protein has been classified as a single-headed trypsin inhibitor of Bowman-Birk type.     

The catalytic active site of thioredoxin: conformation and homology with bovine pancreatic trypsin inhibitor

Rabbit polyclonal antibody was raised to a chemically synthesized nonapeptide (Trp-Ala-Glu-Trp-Cys-Gly-Pro-Cys-Lys) corresponding to the active-site sequence of Escherichia coli thioredoxin. The antiserum efficiently inhibited thioredoxin activity in the standard thioredoxin reductase/NADPH coupled assay. This inhibition was blocked by preincubation of the antiserum with the nonapeptide. Tight association of the E. coli thioredoxin to the active-site antibody required SDS denaturation. These results suggest that thioredoxin reductase (NADPH: oxidized-thioredoxin oxidoreductase, EC 1.6.4.5) alters the conformation of thioredoxin sufficiently to permit binding to the antibody. The antiserum bound to plant and liver thioredoxins. Bovine pancreatic trypsin inhibitor, whose active site (Gly-Pro-Cys-Lys) is homologous to that of thioredoxin, also competes for the active-site antibody. This result led to experiments showing that thioredoxin can inhibit the digestion of cytochrome c by trypsin. The ability of thioredoxin to act as a trypsin inhibitor analogue provides a rationale for thioredoxin's resistance to digestion by trypsin.     

Hydrogen exchange kinetics of bovine pancreatic trypsin inhibitor beta-sheet protons in trypsin-bovine pancreatic trypsin inhibitor, trypsinogen-bovine pancreatic trypsin inhibitor, and trypsinogen-isoleucylvaline-bovine pancreatic trypsin inhibitor

Hydrogen exchange rates of six beta-sheet peptide amide protons in bovine pancreatic trypsin inhibitor (BPTI) have been measured in free BPTI and in the complexes trypsinogen-BPTI, trypsinogen-Ile-Val-BPTI, bovine trypsin-BPTI, and porcine trypsin-BPTI. Exchange rates in the complexes are slower for Ile-18, Arg-20, Gln-31, Phe-33, Tyr-35, and Phe-45 NH, but the magnitude of the effect is highly variable. The ratio of the exchange rate constant in free BPTI to the exchange rate constant in the complex, k/kcpIx, ranges from 3 to much greater than 10(3). Gln-31, Phe-45, and Phe-33 NH exchange rate constants are the same in each of the complexes. For Ile-18 and Tyr-35, k/kcpIx is much greater than 10(3) for the trypsin complexes but is in the range 14-43 for the trypsinogen complexes. Only the Arg-20 NH exchange rate shows significant differences between trypsinogen-BPTI and trypsinogen-Ile-Val-BPTI and between porcine and bovine trypsin-BPTI.     

Effect of modification of reactive lysine on antitryptic and antichymotryptic activity of proteinase inhibitor from cow colostrum.

40% of the primary structure of the cow colostrum proteinase inhibitor (CTI) is homologous with the structure of the trypsin kallikrein inhibitor (TKI) from bovine organs; the positions of the reactive lysine residues are also the same in both inhibitors. Both CTI and TKI were modified by carbamoylation and the fully labeled derivatives were isolated by ion-exchange chromatography. The effect of the modification on the antitryptic and antichymotryptic activity of both inhibitors was investigated. The antichymotryptic activity of both inhibitors is not decreased after the modification. The antitryptic activity of modified TKI is retained, yet the dissociation constant of the complex of the modified inhibitor with trypsin is considerably increased; nevertheless, modified TKI is a good trypsin inhibitor. The antitryptic activity of modified CTI is hardly detectable. We explain this difference in the behaviour of both inhibitors by a replacement of basic residues Arg-17 and Arg-39 in TKI by neutral amino acids Ala-20 and Gln-42 in CTI.     

Genetic linkage of a leucine aminopeptidase locus with the Kunitz trypsin inhibitor locus in soybeans

The soybean (Glycine max (L.) Merrill) seed leucine aminopeptidase locus (Lap1) was found to be linked to the Kunitz trypsin inhibitor locus (Ti) with a recombination frequency of 15.3 percent +/- 0.9 percent. The two loci are in linkage group 9. Both Lap1 and Ti loci are inherited independently of the flower color locus (W1) in linkage group 8.