Nucleotide sequence of cDNA for Acacia confusa trypsin inhibitor and implication of post-translation processing.

Synthetic oligonucleotides corresponding to all possible sequences of N-terminal and C-terminal region of Acacia confusa trypsin inhibitor were used to generate ACTI-related sequences using the polymerase chain reaction on the cDNAs encoding ACTI of the seeds of legume, A. confusa. The deduced amino acid sequence agreed with that determined by the peptide analysis except an extra amino acid residue, serine, was found at the junction of A and B chain, which was removed by post-translation processing with specific protease(s). The substrate specificity of the protease(s) was found to cleave at the C-terminal sites of asparagine and serine, which was also shown to be the same case for another plant protein, abrin, isolated from legume, Abrus precatorius.     

Chimeric protein: abrin B chain-trypsin inhibitor conjugate as a new antitumor agent

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.     

Biochemical characterization of Acacia schweinfurthii serine proteinase inhibitor

Abstract

One of the many control mechanisms of serine proteinases is their specific inhibition by protein proteinase inhibitors. An extract of Acacia schweinfurthii was screened for potential serine proteinase inhibition. It was successfully purified to homogeneity by precipitating with 80% (v/v) acetone and sequential chromatographic steps, including ion-exchange, affinity purification and reversed-phase high performance liquid chromatography. Reducing sodium dodecyl sulphate polyacrylamide gel electrophoresis conditions revealed an inhibitor (ASTI) consisting of two polypeptide chains A and B of approximate molecular weights of 16 and 10?kDa, respectively, and under non-reducing conditions, 26?kDa was observed. The inhibitor was shown to inhibit bovine trypsin (K_i of 3.45?nM) at an approximate molar ratio of inhibitor:trypsin (1:1). The A- and B-chains revealed complete sequences of 140 and 40 amino acid residues, respectively. Sequence similarity (70%) was reported between ASTI A-chain and ACTI A-chain (Acacia confusa) using ClustalW. The B-chain produced a 76% sequence similarity between ASTI and Leucaena leucocephala trypsin inhibitor.     

Primary structure of potato kunitz-type serine proteinase inhibitor

The serine proteinase inhibitor (PSPI-21) isolated from potato tubers (Solanum tuberosum L.) comprises two protein species with pI 5.2 and 6.3, denoted as PSPI-21-5.2 and PSPI-21-6.3, respectively. They were separated by anion exchange chromatography on a Mono Q FPLC column. Both species tightly inhibit human leukocyte elastase, whereas their interaction with trypsin and chymotrypsin is substantially weaker. The sequences of both PSPI-21-5.2 and PSPI-21-6.3 were determined by analysis of overlapping peptides obtained from the oxidized or reduced and S-pyridylethylated proteins after digestion with trypsin or pepsin. Both species of PSPI-21 are composed of two chains, named chains A and B, which are linked by a disulfide bridge between Cys(146) and Cys(157). The other disulfide bridge is located within the A chains between Cys(48) and Cys(97). The amino acid sequences of the large A chains of the two forms, consisting of 150 amino acids residues each, differ in a single residue at position 52. The small chains B, containing 37 and 36 residues in PSPI-21-6.3 and PSPI-21-5.2, respectively, have nine different residues. The entire amino acid sequences of the two inhibitors show a high degree of homology to the other Kunitz-type proteinase inhibitors from plants.     

The complete amino acid sequence of the major Kunitz trypsin inhibitor from the seeds of Prosopsis juliflora.

The major inhibitor of trypsin in seeds of Prosopsis juliflora was purified by precipitation with ammonium sulphate, ion-exchange column chromatography on DEAE- and CM-Sepharose and preparative reverse phase HPLC on a Vydac C-18 column. The protein inhibited trypsin in the stoichiometric ratio of 1:1, but had only weak activity against chymotrypsin and did not inhibit human salivary or porcine pancreatic alpha-amylases. SDS-PAGE indicated that the inhibitor has a Mr of ca 20,000, and IEF-PAGE showed that the pI is 8.8. The complete amino acid sequence was determined by automatic degradation, and by DABITC/PITC microsequence analysis of peptides obtained from enzyme digestions of the reduced and S-carboxymethylated protein with trypsin, chymotrypsin, elastase, the Glu-specific protease from S. aureus and the Lys-specific protease from Lysobacter enzymogenes. The inhibitor consisted of two polypeptide chains, of 137 residues (alpha chain) and 38 residues (beta chain) linked together by a single disulphide bond. The amino acid sequence of the protein exhibited homology with a number of Kunitz proteinase inhibitors from other legume seeds, the bifunctional subtilisin/alpha-amylase inhibitors from cereals and the taste-modifying protein miraculin.     

The amino acid sequence of coagulogen isolated from southeast Asian horseshoe crab, Tachypleus gigas

The amino acid sequence of coagulogen isolated from Southeast Asian horseshoe crab (Tachypleus gigas) has been determined. The NH2-terminal sequence of the first 51 residues was obtained by automated Edman degradation. The intact protein was then treated with a Tachypleus clotting enzyme, to form a gel and to remove an internal peptide C (28 residues) located near the NH2-terminal portion. The gel protein, which consisted of A chain (18 residues) and B chain (129 residues), was S-alkylated and the resulting two chains were separated by acetone precipitation. Among these segments, A chain and peptide C were assigned to the NH2-terminal portion of whole coagulogen, as judged from their amino acid compositions. On the other hand, the covalent structure of B chain was determined by sequencing the peptides obtained from its tryptic digest. The alignments of the tryptic peptides were deduced from the sequence homology in comparison with the previously established B chain sequence of Japanese horseshoe crab (T. tridentatus) coagulogen. T. gigas coagulogen had a total of 175 amino acids and a calculated molecular weight of 19,770. When the sequence was compared with those of Japanese and American horseshoe crab (Limulus polyphemus) coagulogens, extensive structural homology was found: T. tridentatus/T. gigas, 87% and L. polyphemus/T. gigas, 67%. This comparison suggests that Japanese and Southeast Asian horseshoe crabs have a crab, based on amino acid sequence data.     

Isolation and primary structure of proteinase inhibitors from Erythrina variegata (Linn.) var. Orientalis seeds.

The Kunitz-type trypsin inhibitors, ETIa and ETIb, and chymotrypsin inhibitor ECI were isolated from the seeds of Erythrina variegata. The proteins were extracted from a defatted meal of seeds with 10 mM phosphate buffer, pH 7.2, containing 0.15 M NaCl, and purified by DEAE-cellulose and Q-Sepharose column chromatographies. The stoichiometry of trypsin inhibitors with trypsin was estimated to be 1:1, while that of chymotrypsin inhibitor with chymotrypsin was 1:2, judging from the titration patterns of their inhibitory activities. The complete amino acids of the two trypsin inhibitors were sequenced by protein chemical methods. The proteins ETIa and ETIb consist of 172 and 176 amino acid residues and have M(r) 19,242 and M(r) 19,783, respectively, and share 112 identical amino acid residues, which is 65% identity. They show structural features characteristic of the Kunitz-type trypsin inhibitor (i.e., identical residues at about 45% with soybean trypsin inhibitor STI). Furthermore, the trypsin inhibitors show a significant homology to the storage proteins, sporamin, in sweet potato and the taste-modifying protein, miraculin, in miracle fruit, having about 30% identical residues.     

Human granulocyte elastase is inhibited by the urinary trypsin inhibitor

Two forms of urinary trypsin inhibitor, A and B, were purified from the urine of pregnant women. Form A was the only inhibitor present in fresh urine and inhibitor B arose from degradation of A upon storage of urine. The molecular masses of A and B were about 44 and 20 kDa, respectively, as judged from dodecyl-sulfate polyacrylamide gel electrophoresis, but about 60 kDa and 30 kDa, respectively, as judged from gel filtration analysis. The discrepancy can perhaps be explained by the carbohydrate content amounting to about 10% of each inhibitor. After reduction with mercaptoethanol, inhibitor A and inhibitor B had identical apparent molecular masses of about 20 kDa on dodecyl-sulfate gel electrophoresis. These results and the results of amino acid analysis suggest that one molecule of inhibitor A yields two molecules of inhibitor B. On agarose gel electrophoresis inhibitor A migrated as a rather broad band in the prealbumin region and inhibitor B as 3 well defined bands in the beta-region. Specific antisera were raised against inhibitor A and B. The two inhibitors showed the immunologic reaction of identity with each other and with the plasma inter-alpha-trypsin inhibitor, when using either antiserum. The inhibitors both gave quantitative inhibition of bovine trypsin, the results indicating a 4/1 trypsin/inhibitor molar ratio for A and a 2/1 ratio for B. The two substances also effectively inhibited granulocyte elastase. No inhibition of porcine pancreatic elastase was demonstrable.     

Characterization of a trypsin inhibitor from equine urine.

A trypsin inhibitor was isolated from pregnant mares' urine by adsorption on bentonite and elution with aqueous pyridine followed by batch DEAE-cellulose treatment and column chromatography. Final purification to an electrophoretically homogenous glycoprotein was achieved by gel permeation chromatography. This equine urinary trypsin inhibitor (E-UTI) is acid- and heat-stable, has a molecular weight of 22 to 23 kDa, an isoelectric point of 4.55, forms a 1:1 molar complex with trypsin and has serine as its N-terminal amino acid. The N-terminal amino acid sequence of this protein is almost identical with that of EI-14, the inhibitor obtained from horse serum by tryptic treatment, except for two extra amino acid residues, Ser-Lys- on the N-terminal end of E-UTI. In its isoelectric point E-UTI differs from EI-14 and the inhibitor from human urine.     

Isolation and characterization of the trypsin inhibitors from winged bean seed (Psophocarpus tetragonolobus (L) Dc.).

The trypsin inhibitors from winged bean seed were isolated by affinity chromatography on trypsin-Sepharose 4B and the components fractionated by chromatography on SP-Sephadex C-25 and Sephadex G-100. The major components, inhibitors 2 and 3 were found to be homogeneous proteins with molecular weights of about 20,000. The inhibitors stoichiometrically inhibited bovine trypsin in the molar ratio of 1 : 1 whereas the inhibition of bovine alpha-chymotrypsin was weak and non-stoichiometric. Amino acid analysis indicated that both the inhibitors contain four cysteine residues and are rich in aspartic acid, glutamic acid, glycine, valine and leucine; however, inhibitor 3 lacks histidine and methionine while inhibitor 2 contains one histidine and three methionines. A minor trypsin inhibitor fraction was also isolated which contained at least three proteins with a molecular weight of about 10,000 and a high content of half-cystine.     

Amino acid sequences of two trypsin inhibitors from winged bean seeds (Psophocarpus tetragonolobus (L)DC.)

The trypsin inhibitor (WTI-1) purified from winged bean seeds is a Kunitz type protease inhibitor having a molecular weight of 19,200. WTI-1 inhibits bovine trypsin stoichiometrically, but not bovine alpha-chymotrypsin. The approximate Ki value for the trypsin-inhibitor complex is 2.5 X 10(-9) M. The complete amino acid sequence of WTI-1 was determined by conventional methods. Comparison of the sequence with that of soybean trypsin inhibitor (STI) indicated that the sequence of WTI-1 had 50% homology with that of STI. WTI-1 was separated into 2 homologous inhibitors, WTI-1A and WTI-1B, by isoelectric focusing. The isoelectric points of WTI-1A and WTI-1B were 8.5 and 9.4, respectively, and their sequences were presumed from their amino acid compositions.     

Purification and characteristics of an endogenous alpha-amylase inhibitor from barley kernels

An inhibitor of malted barley (Hordeum vulgare cv Conquest) α-amylase II was purified 125-fold from a crude extract of barley kernels by (NH₄)₂SO₄ fractionation, ion exchange chromatography on DEAE-Sephacel, and gel filtration on Bio-Gel P 60. The inhibitor was a protein with an approximate molecular weight of 20,000 daltons and an isoelectric point of 7.3. The protein was homogeneous, as assessed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Amino acid analysis indicated the presence of about 9 half-cystine residues per mole. The neutral isoelectric point of the inhibitor suggested that some of the apparently acidic residues (glutamic and aspartic) existed in the amide form. The first twenty N-terminal amino acids were sequenced. Some homology appeared to exist between the α-amylase II inhibitor and trypsin inhibitor from barley. Complex formation between α-amylase II and the inhibitor was detected by the appearance of a new molecular weight species after gel filtration on Bio-Gel P 100. Enzyme and inhibitor had to be preincubated for 5 min, prior to assaying for enzyme activity before maximum inhibition was attained. Inhibition increased at higher pH values. At pH 5.5, an approximately 1100 molar excess of inhibitor over α-amylase II produced 40% inhibition, whereas, at pH 8.0, a 1:1 molar ratio of inhibitor to enzyme produced the same degree of inhibition.     

Purification and some properties of a trypsin inhibitor from carp (Cyprinus carpio) muscle

A trypsin inhibitor was purified from carp muscle to apparent homogeneity by the successive chromatographies of DEAE-cellulose, DEAE-Sepharose CL-6B, Con A-Sepharose, Ultrogel AcA 44 and hydroxylapatite. The mol. wt of the inhibitor was estimated to be 58,000 by SDS-polyacrylamide gel electrophoresis or 50,000 by gel filtration. The inhibitor seemed to form a 1:1 stoichiometric complex with trypsin, alpha-chymotrypsin and elastase, respectively. Carp muscle trypsin inhibitor was likely to be identical with serum alpha 1-proteinase inhibitor judging from its glycoprotein nature, mol. wt and the inhibition stoichiometry.     

Incorporation of Streptidine-C6-phosphate into Phosphorylated Streptomycin by Resting Cell of Streptomyces griseus

The Kunitz-type trypsin inhibitors, ETIa and ETIb, and chymotrypsin inhibitor ECI were isolated from the seeds of Erythrina variegata. The proteins were extracted from a defatted meal of seeds with 10 mM phosphate buffer, pH 7.2, containing 0.15 M NaCl, and purified by DEAE-cellulose and Q-Sepharose column chromatographies. The stoichiometry of trypsin inhibitors with trypsin was estimated to be 1:1, while that of chymotrypsin inhibitor with chymotrypsin was 1:2, judging from the titration patterns of their inhibitory activities.

The complete amino acids of the two trypsin inhibitors were sequenced by protein chemical methods. The proteins ETIa and ETIb consist of 172 and 176 amino acid residues and have M_r 19,242 and M_r 19,783, respectively, and share 112 identical amino acid residues, which is 65% identity. They show structural features characteristic of the Kunitz-type trypsin inhibitor (i.e., identical residues at about 45%) with soybean trypsin inhibitor STI). Furthermore, the trypsin inhibitors show a significant homology to the storage proteins, sporamin, in sweet potato and the taste-modifying protein, miraculin, in miracle fruit, having about 30% identical residues.     

Purification and properties of the proteinase inhibitors from Erythrina caffra (coast erythrina) seed

• 1.1. Four proteinase inhibitors (DE-1 to DE-4) were purified from Erythrinu caffra seed by gel filtration on Sephadex G-50 followed by ion-exchange chromatography involving DEAE-cellulose and DEAE-sepharose.
• 2.2. They comprise 164–166 amino acid residues (mol. wt 18,100) including 4 half-cystine residues and resemble the Kunitz-type proteinase inhibitors.
• 3.3. The N-terminal primary structure of DE-3 revealed also homology with those of the Kunitz-type inhibitors. For DE-1, DE-2 and DE-4 no free N-terminal amino acid was found.
• 4.4. DE-1 contains a potent inhibitor for both porcine trypsin and bovine α-chymotrypsin. Whereas DE-2 inhibits a-chymotrypsin strongly and has practically no action on trypsin, DE-3 inhibits both trypsin and a-chymotrypsin strongly. DE-4 is a potent inhibitor for trypsin but it binds a-chymotrypsin only weakly.

     

Isolation and characterization of a new trypsin inhibitor from Crotalaria paulina seeds

A new trypsin inhibitor (CPTI) has been isolated from Crotalaria paulina seeds. Purification of the inhibitor was carried out by gel filtration, ion-exchange chromatography, and subsequent reversed-phase HPLC. The presence of a single polypeptide chain, with a molecular mass of 20 kDa and isoelectric point 4.0, was detected. The trypsin inhibitor had a Ki value of 4.5 x 10(-8) M and was capable of acting on human, bovine, and porcine trypsin and weakly on bovine chymotrypsin. Amino acid analysis showed that CPTI has a high content of aspartate, glutamate, leucine, serine, and glycine, having 177 amino acid residues in its composition. These data suggest that the protein belongs to the Kunitz-type trypsin inhibitors.     

The sequence of amino acids in insulin isolated from islet tissue of the cod (Gadus callarias)

1. S-Aminoethylcysteinyl derivatives of the A and B chains of cod insulin were prepared from the individual S-sulpho chains. 2. Studies on small peptides derived from the S-aminoethylated peptide chains by treatment with trypsin allowed the amino acid sequences in the region of the cysteinyl residues of the A and B peptide chains to be defined. 3. The six amide groups in cod insulin were located by complete digestion of small peptides from the A and B chains with aminopeptidase followed by amino acid analyses. 4. The results, together with previous studies on the oxidized A and B chains, define the sequences of the 51 amino acids that constitute cod insulin.     

Trypsin inhibitor from Dimorphandra mollis seeds: purification and properties

A trypsin inhibitor from Dimorphandra mollis seeds was isolated to apparent homogeneity by a combination of ammonium sulfate precipitation, gel filtration, ion-exchange and affinity chromatographic techniques. SDS-PAGE analysis gave an apparent molecular weight of 20 kDa, and isoelectric focusing analysis demonstrated the presence of three isoforms. The partial N-terminal amino acid sequence of the purified protein showed a high degree of homology with various members of the Kunitz family of inhibitors. This inhibitor, which inhibited trypsin activity with a Ki of 5.3 x 10(-10) M, is formed by a single polypeptide chain with an arginine residue in the reactive site.     

A novel serine protease inhibitor from Bungarus fasciatus venom

By Sephadex G-50 gel filtration, cation-exchange CM-Sephadex C-25 chromatography and reversed phase high-performance liquid chromatography (HPLC), a novel serine protease inhibitor named bungaruskunin was purified and characterized from venom of Bungarus fasciatus. Its cDNA was also cloned from the cDNA library of B. fasciatus venomous glands. The predicted precursor is composed of 83 amino acid (aa) residues including a 24-aa signal peptide and a 59-aa mature bungaruskunin. Bungaruskunin showed maximal similarity (64%) with the predicted serine protease inhibitor blackelin deduced from the cDNA sequence of the red-bellied black snake Pseudechis porphyriacus. Bungaruskunin is a Kunitz protease inhibitor with a conserved Kunitz domain and could exert inhibitory activity against trypsin, chymotrypsin, and elastase. By screening the cDNA library, two new B chains of beta-bungarotoxin are also identified. The overall structures of bungaruskunin and beta-bungarotoxin B chains are similar; especially they have highly conserved signal peptide sequences. These findings strongly suggest that snake Kunitz/BPTI protease inhibitors and neurotoxic homologs may have originated from a common ancestor.     

Cloning and characterization of a novel trypsin inhibitor (BTIomega1) gene from Fagopyrum esculentum.

Based on the amino acid information of trypsin inhibitor of buckwheat (Fagopyrum Esculentum Moench), degenerated primers were designed and a full-length cDNA sequence named BTIomega1 (Buckwheat Trypsin Inhibitor) was amplified from the leaves RNA by using RT-PCR and rapid amplification of cDNA ends (RACE) methods. Sequence analysis shows that the 392 bp cDNA contained an open reading frame (ORF) of 216 bp, encoding 72 amino acids residues. The deduced amino acid sequence exhibits 96 and 93% homology with BWI-1 and BTI-2, a natural trypsin inhibitor from buckwheat seeds. Southern blotting suggested that three copies of BTIomega1 gene existed in the buckwheat genome. Moreover, a predicted secondary structure and 3D-structural model was constructed by homology modeling. To our knowledge, this is the first all-round report of the gene BTIomega1. The novel BTIomega1 gene has been submitted to the GeneBank under Accession No. DQ289792.