Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:3.4.21.4 (trypsin)
42,187 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A trypsin inhibitor (ACTI) was isolated and purified from the seeds of Acacia confusa by gel filtration, and trypsin-Sepharose 4B column affinity chromatography. The molecular weight of ACTI was found to be 21,000 +/- 1,000 by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and amino acid composition analysis. ACTI contained four half-cystine and no methionine residues, and was rich in aspartic acid, glutamic acid, glycine, leucine, and lysine residues. The native trypsin inhibitor was composed of two polypeptide chains, and it inhibited trypsin and alpha-chymotrypsin stoichiometrically at the molar ratio of 1:1 and 2:1, respectively. The amino-terminal sequence analysis of the A. confusa trypsin inhibitor A and B chains revealed a more extensive homology with Acacia elata and silk tree trypsin inhibitors, and a less extensive homology with Kunitz soybean trypsin inhibitor.
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PMID:Trypsin inhibitor from the seeds of Acacia confusa. 179 77

1. Of the nineteen plants screened, six were found to contain large quantities of condensed tannins. Black locust (Robinia pseudo-Acacia), bush clover (Lespedeza bicolor), wistaria (Wistaria floribunda) and Japanese knotgrass (Reynoutria japonica) were used for the present experiment. Tannins of the investigated plants were fractionated into three or four molecular forms, according to the degree of polymerization, by chromatography on a column of Sephadex LH-20. 2. The protein-precipitating capacity of the fractionated tannins increased with the increase in degree of polymerization. The inhibitory effect of tannins on trypsin (EC 3.4.21.4), alpha-amylase (EC 3.2.1.1) and lipase (EC 3.1.1.3) activities in vitro also increased with the increase in degree of polymerization. The digestion of tannin-bovine serum albumin complex by trypsin was related to the degree of polymerization of tannins complexed. 3. Inclusion of black locust tannins in the diet (10 g/kg) depressed the activities of trypsin and alpha-amylase in the upper, middle and lower parts of the intestine of the rats, but the lipase activity was increased in the middle part and remained unaffected in the upper and lower parts. It is presumed that the tannins have little affinity for lipase. 4. Digestion trials, when the microflora level of the alimentary canal of rats was reduced by dietary antibiotic treatment, revealed that the inclusion of tannins in the diet (20 g/kg) depressed the digestibilities of proximate constituents, except crude fat, and increased faecal bile acid excretion.
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PMID:Effects of condensed tannins prepared from leaves of fodder plants on digestive enzymes in vitro and in the intestine of rats. 246 30

Abrin B chain and trypsin inhibitor isolated from Acacia confusa (ACTI) were covalently linked to form a chimeric protein (ANB-ACTI) with N-succinimidyl-3-(-2-pyridyldithio)propionate. The chimeric protein had 31% of trypsin inhibitory activity of ACTI and 7% of hemagglutinating activity of abrin B chain, but no inhibition on protein biosynthesis. ANB-ACTI had strong inhibitory effects on the growth of sarcoma 180 cells and Hela cell culture while the mixture of an equivalent amount of free abrin B chain and ACTI did not. The results suggests that abrin B chain of chimeric protein may act as a vector to carry ACTI into the tumor cells. ACTI into the tumor cells. ACTI in the chimeric protein potentiates its antitumor activity as well as its resistance to tryptic digestion.
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PMID:Chimeric protein: abrin B chain-trypsin inhibitor conjugate as a new antitumor agent. 281 99

Concanavalin A (Con A) and trypsin inhibitor isolated from Acacia confusa were covalently linked with N-succinimidyl-3-(2-pyridyldithio)propionate. Con A-A. confusa trypsin inhibitor (ACTI) conjugate covalently bound (Con A-ACTI) retained about 42% of the trypsin inhibitory activity present in the native ACTI and had a higher hemagglutinating activity than did the native Con A. Con A-ACTI had a greater resistance to tryptic digestion than did the mixture of Con A and ACTI. The conjugate entered sarcoma 180 tumor cells, whereas the free ACTI did not. A single dose of the conjugate injected ip into noninbred N:NIH(S) white mice bearing sarcoma 180 had a remarkable effect of increasing the survival of tumor-bearing mice, while the mixture of an equivalent dose of free Con A and ACTI was not effective.
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PMID:Antitumor lectin-trypsin inhibitor conjugate. 388 55

Two trypsin inhibitors from the seed of Acacia elata, a legume of the subfamily Mimosoideae, were isolated by affinity chromatography on trypsin-Sepharose 4B and separated by chromatography on SP-Sephadex C-25 and Sephadex G-100. The two inhibitors, with molecular weights of about 20 000, were composed of two polypeptide chains linked by a disulfide bond. The two inhibitors had essentially the same amino acid compositions and both contained four half-cystine residues, no methionine and were rich in aspartic acid, glutamic acid, glycine and leucine. The inhibitors had isoelectric points of 6.4 and 5.9. The inhibitors stoichiometrically inhibited trypsin in the molar ratio of 1:1, alpha-chymotrypsin was inhibited also in a 1:1 molar ratio but the binding of the enzyme by the inhibitor was weaker and the inhibitor-chymotrypsin complex dissociated during the assay. Both enzymes are probably inhibited at an identical site. Amino-terminal sequence analysis of the two polypeptide chains of the inhibitors revealed extensive homology with the trypsin inhibitor from the silk tree (another Mimosoideae legume); both these inhibitors are homologous with the soybean trypsin inhibitor (Kunitz).
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PMID:Acacia proteinase inhibitors. Purification and properties of the trypsin inhibitors from Acacia elata seed. 723 19

Native Acacia confusa trypsin inhibitor (ACTI) contains two disulphide bonds; one is an intrachain disulphide bond (Cys40-Cys86), located in the A-chain, while the other is an interchain disulphide bond (Cys133-Cys141) connecting the A- and B-chain; the inhibitor consists of 175 amino acid residues, 136 residues in the A-chain and 39 residues in the B-chain. The putative reactive site of ACTI is located at Lys64, while for all other Kunitz family trypsin inhibitors it is at Arg64. When the Lys64 residue of ACTI was converted into Ile or Arg by site-specific mutagenesis, the K64I mutant completely lost its inhibitory activity but the K64R mutant retained most of its inhibitory activity. The C133G mutant lost its inhibitory activity while the C40G mutant did not. This suggests that the interchain disulphide bond (Cys133-Cys141) linking two beta-strands of the six-strand beta-barrel is essential for ACTI inhibitory activity, while the intrachain disulphide bond (Cys40-Cys86) connecting the two loops is non-essential.
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PMID:Inactivation of Acacia confusa trypsin inhibitor by site-specific mutagenesis. 795 82

The complete amino acid sequence of a Kunitz-type two-chain trypsin inhibitor was determined for the first time. The sequence of the inhibitor from Acacia confusa (ACTI) was determined by analysis of peptides obtained from the reduced and S-carboxymethylated protein by digestion with endopeptidase Lys-C, endopeptidase Arg-C, and V8 endopeptidase. ACTI is comprised of two chains, namely A and B chains linked by the disulfide bridge between Cys(133) and Cys(141), and the inhibitor consists of 175 amino acid residues, 136 residues in the A-chain and 39 residues in the B-chain. The N-terminal amino acid sequence of ACTI shows extensive homology to the trypsin inhibitors from Acacia elata and Albizzia julibrissin, while the whole amino acid sequence of ACTI has a high degree of homology to the other Kunitz-type trypsin inhibitors from soybeans, winged bean seeds [Psophocarpus tetragonolobus (L) DC.], and seeds of Erythrina species.
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PMID:The complete amino acid sequence of a Kunitz family trypsin inhibitor from seeds of Acacia confusa. 846 33

A recombinant plasmid containing the coding regions for Acacia confusa trypsin inhibitor (ACTI) has been constructed and expressed in Escherichia coli cells, as a fusion protein between ACTI and glutathione S-transferase (GST). The GST-fusion was produced as a soluble protein which did not require denaturing agents such as urea to solubilize it. The recombinant ACTI (reACTI) was obtained by treating the GST-fusion protein with thrombin. Both the reACTI and fusion protein have a strong inhibitory effect on trypsin activity without post-translational proteolysis.
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PMID:Cloning and expression of the gene encoding Acacia confusa trypsin inhibitor that is active without post-translational proteolysis. 850 Jul 64

We report for the first time the isolation and characterization of a protease inhibitor from the seeds of Pithecellobium dulce, which is a Leguminosae tree native to Mexico. The purification of the P. dulce trypsin inhibitor (PDTI) was a direct process. After its extraction (pH 8.0) and precipitation (80% (NH(4))(2)SO(4)), the pH was adjusted to 4.0, the supernatant was loaded onto a CM-Sepharose column, and a single peak of trypsin inhibitory activity was eluted (CM-TIA). The main component of CM-TIA was PDTI, a protein composed of two polypeptide chains joined by disulfide bridge(s), with a pI of 4.95 and a molecular weight determined by electrospray mass spectrometry of 19 614 Da. The N-terminal sequence of PDTI has the highest similarity with the seed inhibitor of Acacia confusa. PDTI lacks chymotrypsin inhibitory activity. A low rate of cytotoxicity of CM-TIA toward RINm5F cells contrasted with a high rate of the active fraction G75-TIA (gel filtration chromatography; LC(50) of 0.04 mg/mL).
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PMID:Isolation and properties of a Kunitz-type protein inhibitor obtained from Pithecellobium dulce seeds. 1545 75

This study was aimed at investigating the purification, biological activity, and some structural properties of three serine protease inhibitors isoforms, denoted ApTIA, ApTIB, and ApTIC from Acacia plumosa Lowe seeds. They were purified from the saline extract of the seeds, using Superdex-75 gel filtration and Mono-S ion exchange chromatography. They were further investigated by mass spectrometry, spectroscopic measurements, surface plasmon resonance, and inhibition assays with proteases and phytopathogenic fungi. The molecular mass of each isoform was estimated at ca. 20 kDa. Each contained two polypeptide chains linked by a disulfide bridge, with different isoelectric points that are acidic in nature. The N-terminal sequences of both chains indicated that they were Kunitz-type inhibitors. Circular dichroism (CD) analyses suggested the predominance of both disordered and beta-strands on ApTI isoforms secondary structure, as expected for beta-II proteins. In addition, it was observed that the proteins were very stable, even at either extreme pH values or at high temperature, with denaturation midpoints close to 75 degrees C. The isoinhibitors could delay, up to 10 times, the blood coagulation time in vitro and inhibited action of trypsin (Ki 1.8 nM), alpha-chymotrypsin (Ki 10.3 nM) and kallikrein (Ki 0.58 microM). The binding of ApTIA, ApTIB, and ApTIC to trypsin and alpha-chymotrypsin, was investigated by surface plasmon resonance (SPR), this giving dissociation constants of 0.39, 0.56 and 0.56 nM with trypsin and 7.5, 6.9 and 3.5 nM with alpha-chymotrypsin, respectively. The growth profiles of Aspergillus niger, Thielaviopsis paradoxa and Colletotrichum sp. P10 were also inhibited by each isoforms. These three potent inhibitors from A. plumosa may therefore be of great interest as specific inhibitors to regulate proteolytic processes.
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PMID:Physico-chemical and antifungal properties of protease inhibitors from Acacia plumosa. 1944 1


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