A 20-kDa protein with the GTP-binding and trypsin inhibitory activities from Glycine max

A 20-kDa protein (p20) with a GTP binding activity was purified from the cultured cells of Glycine max (soybean). The amino acid sequence of p20 showed 65% identity in a 23 amino acid overlap against the Kunitz-type trypsin inhibitor of soybean reported. Furthermore, it was found that a Kunitz-type soybean trypsin inhibitor of commercial origin also binds GTP.     

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.     

Amino acid sequence of a crystalline seed albumin (winged bean albumin-1) from Psophocarpus tetragonolobus (L.) DC. Sequence similarity with Kunitz-type seed inhibitors and 7S storage globulins

The complete amino acid sequence of winged bean albumin-1 (WBA-1) of Psophocarpus tetragonolobus (L.) DC has been determined. The protein consists of a single polypeptide chain of 175 amino acid residues, with one disulfide bond, corresponding to a molecular mass of 19333 Da. WBA-1 was found to be homologous with the Kunitz-type seed trypsin inhibitors. The similarity between WBA-1 and the trypsin inhibitors from soybean and winged bean was 38% and 28%, respectively; similarity was most marked in the C-terminal third of the sequence with identities of 47% and 37%, respectively. Significant similarity was found also between the 2S Kunitz-type proteins and the carboxy-terminal region of the 7S storage globulins, suggesting that these two groups of proteins are related and may have evolved from a common ancestral precursor. Circular dichroism measurements suggest a high content of beta sheet (52%) while secondary structure predictions based on amino acid sequence indicate a similar content and distribution of beta sheet to that found for soybean trypsin inhibitor by X-ray diffraction studies.     

Amino acid sequence of the acidic Kunitz-type trypsin inhibitor from winged-bean seed [Psophocarpus tetragonolobus (L.) DC]

The primary sequence of trypsin inhibitor-2 (WBTI-2) fromPsophocarpus tetragonolobus (L.) DC seeds was determined. This inhibitor consists of a single polypeptide chain of 182 amino acids, including four half-cystine residues, and an N-terminal residue of pyroglutamic acid. The sequence of WBTI-2 showed 57% identity to the basic trypsin inhibitor (WBTI-3) and 50% identity to the chymotrypsin inhibitor (WBCI) of winged bean, and 54% identity to the trypsin inhibitor DE-3 fromErythrina latissima seed. The similarity to the soybean Kunitz trypsin inhibitor (40%) and the other Kunitz-type inhibitors fromAdenanthera pavonina (30%) and wheat (26%) was much lower. Sequence comparisons indicate that thePsophocarpus andErythrina inhibitors are more closely related to each other than to other members of the Kunitz inhibitor family.     

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.     

Structural insights into the unique inhibitory mechanism of Kunitz type trypsin inhibitor from Cicer arietinum L.

Kunitz-type trypsin inhibitors bind to the active pocket of trypsin causing its inhibition. Plant Kunitz-type inhibitors are thought to be important in defense, especially against insect pests. From sequence analysis of various Kunitz-type inhibitors from plants, we identified CaTI2 from chickpea as a unique variant lacking the functionally important arginine residue corresponding to the soybean trypsin inhibitor (STI) and having a distinct and unique inhibitory loop organization. To further explore the implications of these sequence variations, we obtained the crystal structure of recombinant CaTI2 at 2.8Å resolution. It is evident from the structure that the variations in the inhibitory loop facilitates non-substrate like binding of CaTI2 to trypsin, while the canonical inhibitor STI binds to trypsin in substrate like manner. Our results establish the unique mechanism of trypsin inhibition by CaTI2, which warrant further research into its substrate spectrum. Abbreviations BApNA Nα-Benzoyl-L-arginine 4-nitroanilide

BPT bovine pancreatic trypsin

CaTI2 Cicer arietinum L trypsin inhibitor 2

DrTI Delonix regia Trypsin inhibitor

EcTI Enterolobium contortisiliquum trypsin inhibitor

ETI Erythrina caffra trypsin inhibitor

KTI Kunitz type inhibitor

STI soybean trypsin inhibitor

TKI Tamarindus indica Kunitz inhibitor

Communicated By Ramaswamy H. Sarma     

Purification and some properties of two proteinase inhibitors (DE-1 and DE-3) from Erythrina latissima (broad-leaved erythrina) seed

Two proteinase inhibitors, DE-1 and DE-3, were purified from Erythrina latissima seeds. Whereas DE-1 inhibits bovine chymotrypsin and not bovine trypsin, DE-3 inhibits trypsin but not chymotrypsin. The molecular weights and the amino acid compositions of the two inhibitors resemble the corresponding properties of the Kunitz-type proteinase inhibitors. The N-terminal primary structure of DE-3 showed homology with soybean trypsin inhibitor (Kunitz) and also with the proteinase inhibitors (A-II and B-II) from Albizzia julibrissin seed.     

Concurrent isolation of a Kunitz-type trypsin inhibitor with antifungal activity and a novel lectin from Pseudostellaria heterophylla roots

A simple purification protocol, involving ion exchange chromatography on DEAE-cellulose and CM-cellulose and fast protein liquid chromatography-gel filtration on Superdex 75, was employed to isolate a Kunitz-type trypsin inhibitor with antifungal activity and a novel lectin from Pseudostellaria heterophylla roots. Both the trypsin inhibitor and the lectin were unadsorbed on DEAE-cellulose and adsorbed on CM-cellulose. They could be separated from one another by gel filtration on Superdex 75 in which the 36-kDa lectin appeared as the first peak and the 20.5-kDa trypsin inhibitor as the second peak. P. heterophylla trypsin inhibitor exhibited a trypsin inhibitory potency similar to that of soybean trypsin inhibitor. It also demonstrated antifungal activity toward Fusarium oxysporum like aprotinin and Kunitz-type trypsin inhibitors from soybeans and lima beans. P. heterophylla lectin was devoid of antifungal activity and exhibited low thermostability and also lability in the presence of acid and alkali. The novel aspects of the present report include demonstration of antifungal activity in Kunitz-type trypsin inhibitors and isolation of a novel lectin as well as a trypsin inhibitor from roots.     

The papaya Kunitz-type trypsin inhibitor is a highly stable beta-sheet glycoprotein

The papaya Kunitz-type trypsin inhibitor, a 24-kDa glycoprotein, was purified to homogeneity. The purified inhibitor stoichiometrically inhibits bovine trypsin in a 1:1 molar ratio. Circular dichroism and infrared spectroscopy analyses demonstrated that the inhibitor contains extensive beta-sheet structures. The inhibitor was found to retain its full inhibitory activity over a broad pH range (1.5-11.0) and temperature (up to 80 degrees C), besides being stable at very high concentrations of strong chemical denaturants (e.g., 5.5 M guanidine hydrochloride). The inhibitor retained its compact structure over the pH range analyzed as shown by 8-anilino-1-naphtalenesulfonic acid binding characteristics, excluding the formation of some relaxed or molten state. Exposure to 2.5 mM dithiothreitol for 120 min caused a 33% loss of the inhibitory activity, while a loss of 75% was obtained in the presence of 20 mM of dithiothreitol during the same time period. A complete loss of the inhibitory activity was observed after incubation with 50 mM dithiothreitol for 5 min. Incubation of the inhibitor with general proteases belonging to different families revealed its extraordinary resistance to proteolysis in comparison with the soybean trypsin inhibitor, the archetypal member of the Kunitz-type inhibitors family. The inhibitor also exhibited a remarkable resistance to proteolytic degradation against pepsin for at least a 24-h incubation period. Instead, the soybean inhibitor was completely degraded after 2 h incubation with this aspartic protease. All these data demonstrated the high stability of the papaya trypsin inhibitor.     

Primary structure of kunitz-type trypsin inhibitor-2a (pI 5.9) fromPsophocarpus tetragonolobus (L.) DC seed

The primary structure of acidic trypsin inhibitor-2a (WBTI-2a,pI 5.9) fromPsophocarpus tetragonolobus (L.) DC seed was determined. This inhibitor consists of a single polypeptide chain of 180 amino acids including four half-cystine residues and has an N-terminal residue of pyroglutamic acid. The sequence of WBTI-2a,pI 5.9, showed 84% identity to acidic trypsin inhibitor-2 (WBTI-2,pI 5.1) but only 57% identity to the basic trypsin inhibitor (WBTI-1,pI 8.9) and 50% identity to the chymotrypsin inhibitor of winged bean. The data indicate that winged bean seed contains a family of three Kunitz-type inhibitors which have about 50% identity.     

Fragment of the gene encoding chymotrypsin and trypsin inhibitor protein of potato tubers

Product of polymerase chain reaction designated as PKPIJ-B was isolated after amplification from genomic DNA of potato (Solarium tuberosum L., Zhukov Jubilee cultivar) using the designed primers. Nucleotide sequence of PKPIJ-B was determined and amino acid sequence of protein was restored. Analysis of this sequence has allowed us to suggest that isolated gene fragment encodes chymotrypsin and trypsin inhibitor protein (PKCI-23 potato Kunitz-type chymotrypsin inhibitor) of potato tubers.     

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

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 V8 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.     

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.     

Purification and amino acid sequence of Kunitz-type protease inhibitor found in the hemocytes of horseshoe crab (Tachypleus tridentatus)

A low molecular weight protein protease inhibitor was purified from Japanese horseshoe crab (Tachypleus tridentatus) hemocytes. It consisted of a single polypeptide with a total of 61 amino acid residues. This protease inhibitor inhibited stoichiometrically the amidase activity of trypsin (Ki = 4.60 X 10(-10) M), and also had inhibitory effects on alpha-chymotrypsin (Ki = 5.54 X 10(-9) M), elastase (Ki = 7.20 X 10(-8) M), plasmin, and plasma kallikrein. However, it had no effect on T. tridentatus clotting enzyme and factor C, mammalian blood coagulation factors (activated protein C, factor Xa and alpha-thrombin), papain, and thermolysin. The complete amino acid sequence of this inhibitor was determined and its sequence was compared with those of bovine pancreatic trypsin inhibitor (BPTI) and other Kunitz-type inhibitors. It was found that the amino acid sequence of this inhibitor has a high homology of 47 and 43% with those of sea anemone inhibitor 5-II and BPTI, respectively. Thus, this protease inhibitor appeared to be one of the typical Kunitz-type protease inhibitors.     

Determination of the disulfide bond pairings in human tissue factor pathway inhibitor purified fromEscherichia coli

The disulfide bond assignments of human alanyl tissue factor pathway inhibitor purified fromEscherichia coli have been determined. This inhibitor of the extrinsic blood coagulation pathway possesses three Kunitz-type inhibitor domains, each containing three disulfide bonds. The disulfide bond pairings in domains 1 and 3 were determined by amino acid sequencing and mass spectrometry of peptides derived from a thermolysin digest. However, thermolysin digestion did not cleave any peptide bonds within domain 2. The disulfide bond pairings in domain 2 were determined by isolating it from the thermolysin treatment and subsequently cleaving it with pepsin and trypsin into peptides which yielded the three disulfide bond pairings in this domain. These results demonstrate that the disulfide pairings in each of the three domains of human tissue factor pathway inhibitor purified fromEscherichia coli are homologous to each other and also to those in bovine pancreatic trypsin inhibitor.     

Proteinase inhibitors from Erythrina corallodendron and Erythrina cristagalli seeds

Eight and five proteinase inhibitors were purified from Erythrina corallodendron and E. cristagalli seeds, respectively, by gel filtration followed by ion exchange chromatography on DEAE-cellulose and DEAE-sepharose. Each inhibitor consists of 161–163 amino acids (M_r 18 000) including four half-cystine residues and resembles the Kunitz-type proteinase inhibitors. The N-terminal amino acid sequence of trypsin inhibitor DE-7 from E. corallodendron seed resembles those of other Erythrina species. For the other inhibitors no free N-terminal amino acid was found. DE-1,-2,-3,-4 and -5 from the seed of E. corallodendron contain potent inhibitors for α-chymotrypsin and they have practically no action on trypsin. From the same seed, inhibitors DE-6, -7 and -8 strongly inhibit trypsin and also inhibit α-chymotrypsin to varying degrees. From the seeds of E. cristagalli, inhibitors DE-1 and -8 inhibit trypsin strongly and DE-2, -3 and -4 are strongly inhibitory for α-chymotrypsin. On summarizing the inhibitor characteristics of the Kunitz-type proteinase inhibitors from the seeds of eight different species of Erythrina, it was obvious that there is a relationship between the alanine content of the inhibitors and their activities. A high alanine content is associated with potent α-chymotrypsin activities and low alanine content with strong trypsin activities.     

Kunitz-type protease inhibitor found in rat mast cells. Purification, properties, and amino acid sequence

A low molecular weight serine protease inhibitor, named trypstatin, was purified from rat peritoneal mast cells. It is a single polypeptide with 61 amino acid residues and an Mr of 6610. Trypstatin markedly inhibits blood coagulation factor Xa (Ki = 1.2 x 10(-10) M) and tryptase (Ki = 3.6 x 10(-10) M) from rat mast cells, which have activities that convert prothrombin to thrombin. It also inhibits porcine pancreatic trypsin (Ki = 1.4 x 10(-8) M) and chymase (Ki = 2.4 x 10(-8) M) from rat mast cells, but not papain, alpha-thrombin, or porcine pancreatic elastase. Trypstatin forms a complex in a molar ratio of 1:1 with trypsin and one subunit of tryptase. The complete amino acid sequence of this inhibitor was determined and compared with those of Kunitz-type inhibitors. Trypstatin has a high degree of sequence homology with human and bovine inter-alpha-trypsin inhibitors, A4(751) Alzheimer's disease amyloid protein precursor, and basic pancreatic trypsin inhibitor. However, unlike other known Kunitz-type protease inhibitors, it inhibits factor Xa most strongly.     

Chymotrypsin and trypsin inhibitor isolated from potato tubers

Potato Kunitz-type chymotrypsin inhibitor (PKCI-23) was isolated from potato tubers (Solanum tuberosum L., cv. Zhukov’s Jubilee) and purified to a homogenous state. The protein was purified by gel-filtration chromatography and ion-exchange chromatography using Sephadex G-75 and CM-sepharose CL-6B, respectively. PKCI-23 protein has been shown to inhibit both chymotrypsin and trypsin with equal efficacy, forming equimolar complexes with these enzymes. However, much weaker inhibitory effect of PKCI-23 has been observed for subtilisin Carlsberg. The N-terminal 20 amino acid sequence of PKCI-23 has been sequenced. PKCI-23 has been shown to suppress, with different efficacy, the growth and development of pathogenic microorganisms Fusarium culmorum (Wm. G. Sm.) Sacc. and Phytophtora infestans (Mont.) de Bary that infect potato.     

Protein trypsin inhibitor from potato tubers

A protein of 22 kDa designated as PKTI-22 was isolated from potato tubers (Solanum tuberosum L., cv. Istrinskii) and purified to homogeneity using CM-Sepharose CL-6B ion-exchange chromatography. The protein efficiently suppressed the activity of trypsin, affected chymotrypsin less, and did not affect subtilisin Carlsberg. The N-terminal sequence of PKTI-22 (20 amino acid residues) was found to be highly homologous with the amino acid sequences of the potato Kunitz-type proteinase inhibitors of group B (PKPI-B) that were aligned from the corresponding gene sequences and was identical to the sequence (from the 2nd to the 20th residue) of the recombinant protein PKPI-B10. These data together with the observed similarity of the properties of two proteins indicate that the PKTI-22 protein is encoded by the PKPI-B10 gene.