Purification and Characterization of Proteinase Inhibitors from Winged Bean (Psophocarpus tetragonolobus (L.) DC.) Seeds1

Shibata, Hiroshi; Hara, Saburo; Ikenaka, Tokuji; Abe, Jiro

doi:10.1093/oxfordjournals.jbchem.a135578

Cited by 70 publications

(58 citation statements)

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“…The stoichiometry of ECI with chymotrypsin was estimated to be 1 :2 by the titration patterns of its inhibitory activity, suggesting that one ECI molecule binds two chymotrypsin molecules. Although similar observations were reported on chymotrypsin inhibitors from Erythrina latissima seeds [ 191, Erythrina caffra seeds [20] and winged bean [15], no further study of their interaction with chymotrypsin was made. As for 'double heads' serine proteinase inhibitors, Bowman-Birk family proteinase inhibitors which form a stable ternary complex consisting of one inhibitor and two proteinase molecules are well known [22].…”

Section: Discussionmentioning

confidence: 73%

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Analysis of the Complex Formed by Erythrina variegata Chymotrypsin Inhibitor with Chymotrypsin and Properties of the Peptides Prepared from the Inhibitor by a Limited Proteolysis

Kimura

Harada

Iwanaga

et al. 1997

European Journal of Biochemistry

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The stoichiometry of Erythrina vuriegata chymotrypsin inhibitor (ECI) and chymotrypsin interaction was previously estimated to be 1 : 2 by a titration of inhibitory activity. In the present study, gel-permeation chromatography and reverse-phase HPLC (RP-HPLC) were employed to analyze the complex formed by the inhibitor and enzyme. The results showed that ECI and chymotrypsin molecules undergo aggregation in the complex-forming buffer simultaneously with a binary complex consisting of one ECI and one chymotrypsin molecules in a soluble form.A mild lysylendopeptidase digestion of ECI produced two peptides in high yield, which were separated by RP-HPLC and characterized in terms of their structures and inhibitory activities. The N-terminal peptide, ECI-( 1 -107)-peptide, containing the primary reactive site retained a slight inhibitory activity, while the C-terminal peptide, ECI-( 108 -179)-peptide, exhibited no inhibitory activity. The inhibitory potency of the ECI-(l-107)-peptide was enhanced by the presence of the ECI-(108-179)-peptide in reconstituted mixture. Recovery of the native-like structure of the reconstituted complex was further indicated by fluorescence spectra, which showed strong conformational interaction between the two peptides ; their dissociation constant Kd was calculated to be 209 nM. Taken together with the previous result obtained by chymotryptic digestion, it is suggested that the primary binding loop in ECI interacts with chymotrypsin not only by a standard mechanism but also by a non-substrate-like manner. Alternatively, ECI might have an additional binding segment in the N-terminal region which interacts with chymotrypsin by a non-substrate-like manner. Further, it is shown that the C-terminal region may support the native conformation of the binding loop(s) in the N-terminal region as an intramolecular chaperone.Keywords: chymotrypsin inhibitor; Erythrina variegutu ; Kunitz-family protein.Since Sweet et al. elucidated the tertiary structure of a complex consisting of soybean trypsin inhibitor (STI) and porcine trypsin [I], a vast amount of structural information has been accumulated for protein structures of plant serine proteinase inhibitors [2]. The crystal structures of these complexes with the cognate proteinases indicate that they all possess an exposed binding loop of a characteristic canonical conformation which associates with the catalytic residues in a similar manner to that of productively bound substrates. Thus, these studies have provided valuable insights into the nature of protein-protein interaction and also into the catalytic mechanism of serine proteinase.Apart from structural studies on plant proteinase inhibitors, the current interest in plant proteinase inhibitors is their application for transgenic plants with resistances to insects and pathogenes. Several positive results employing potato inhibitors I and I1 [3] and cowpea inhibitors [4] were reported. However, a recent study showed that Spodoptera exigua larvae adapt to the exogenous proteinase inhibitor by indu...

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Section: Discussionmentioning

confidence: 73%

“…Inhibiton of chymotrypsin by ECI and its derivatives was assayed, as described by Shibata et al [15], in 96-well microtiterplate. Briefly, chymotrypsin was dissolved in 1 mM HCI containing 20 mM CaCl, at the concentration of 0.1 mg/ml.…”

Section: Methodsmentioning

confidence: 99%

Analysis of the Complex Formed by Erythrina variegata Chymotrypsin Inhibitor with Chymotrypsin and Properties of the Peptides Prepared from the Inhibitor by a Limited Proteolysis

Kimura

Harada

Iwanaga

et al. 1997

European Journal of Biochemistry

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“…However, the technique suffered from the same serious drawback as that of Kortt that ended up with cleaved inhibitors by the cognate proteases. Again, purification method by Shibata et al [12] suffered being laborious and time consuming. However, this presently described purification method involves only a single column, is less time consuming and results in high yield of purified protein.…”

Section: Discussionmentioning

confidence: 99%

“…seeds are a rich source of Kunitz type protease inhibitors and are reported to contain at least nine trypsin or chymotrypsin inhibitors [5,12] . Its high protein and oil content has earned it a name "soybean of tropics [10] ".…”

Section: Introductionmentioning

confidence: 99%

“…To avoid such unwanted cleavage of the inhibitors, Yamamoto et al [14] purified two inhibitors from winged bean using DEAE cellulose column followed by SPSephadex C-25 column. Shibata et al [12] purified seven inhibitors including their isoinhibitors from winged bean using a large number of conventional ionexchange and gel filtration columns. All these inhibitors were Kunitz type having molecular weights of around 20 kDa, including four half cysteine residues.…”

Section: Introductionmentioning

confidence: 99%

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Purification of Chymotrypsin-Trypsin Inhibitor from Winged Bean Seeds using Single Step Immunoaffinity Column

Roy¹,

Dutta²

2009

American J. of Biochemistry and Biotechnology

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Problem statement: Winged bean is a rich source of protease inhibitors. The seeds of winged bean contain several serine protease inhibitors of which winged bean chymotrypsin-trypsin inhibitor (WbCTI) is of significant importance due to its dual inhibition of both chymotrypsin and trypsin. The purification of WbCTI has always been difficult and a time taking process that involved large number of steps and various purification columns. Approach: This study was focused on the single step method of purification of WbCTI to homogeneity from winged bean seeds using immunoaffinity column chromatography. Results: WbCTI was purified to homogeneity using antiWbCTI antibody column in a single step which gave a single band on SDS-PAGE analysis and with almost one and half times more yield of purified protein than obtained from previously mentioned purification methods. Conclusion: This method using immunoaffinity column is high yielding and time saving process of purification.

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Refined crystal structure (2.3 �) of a double-headed winged bean ?-chymotrypsin inhibitor and location of its second reactive site

et al. 1999

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The crystal structure of a doubleheaded ␣-chymotrypsin inhibitor, WCI, from winged bean seeds has now been refined at 2.3 Å resolution to an R-factor of 18.7% for 9,897 reflections. The crystals belong to the hexagonal space group P6 1 22 with cell parameters a ‫؍‬ b ‫؍‬ 61.8 Å and c ‫؍‬ 212.8 Å .The final model has a good stereochemistry and a root mean square deviation of 0.011 Å and 1.14°from ideality for bond length and bond angles, respectively. A total of 109 ordered solvent molecules were localized in the structure. This improved structure at 2.3 Å led to an understanding of the mechanism of inhibition of the protein against ␣-chymotrypsin. An analysis of this higher resolution structure also helped us to predict the location of the second reactive site of the protein, about which no previous biochemical information was available. The inhibitor structure is spherical and has twelve antiparallel ␤-strands with connecting loops arranged in a characteristic ␤-trefoil fold common to other homologous serine protease inhibitors in the Kunitz (STI) family as well as to some non homologous functionally unrelated proteins. A wide variation in the surface loop regions is seen in the latter ones. Proteins 1999;35:321-331.1999 Wiley-Liss, Inc.

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Purification and Characterization of Proteinase Inhibitors from Winged Bean (Psophocarpus tetragonolobus (L.) DC.) Seeds1

Cited by 70 publications

References 0 publications

Analysis of the Complex Formed by Erythrina variegata Chymotrypsin Inhibitor with Chymotrypsin and Properties of the Peptides Prepared from the Inhibitor by a Limited Proteolysis

Analysis of the Complex Formed by Erythrina variegata Chymotrypsin Inhibitor with Chymotrypsin and Properties of the Peptides Prepared from the Inhibitor by a Limited Proteolysis

Purification of Chymotrypsin-Trypsin Inhibitor from Winged Bean Seeds using Single Step Immunoaffinity Column

Refined crystal structure (2.3 �) of a double-headed winged bean ?-chymotrypsin inhibitor and location of its second reactive site

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