Natural plant enzyme inhibitors V. A trypsin/chymotrypsin inhibitor from Alocasia macrorhiza tuber

doi:10.1016/0005-2744(77)90204-2

Biochimica et Biophysica Acta (BBA) - Enzymology

Volume 485, Issue 1, 23 November 1977, Pages 167-178

https://doi.org/10.1016/0005-2744(77)90204-2 Get rights and content

Abstract

A trypsin/chymotrypsin inhibitor was isolated from the tubers of Alocasia macrorhiza by extraction at pH 7.6, heat treatment at 80°C, ammonium sulphate precipitation and successive column chromatography on CM-cellulose, DEAE-Sephadex A-50 and Sephadex G-100. The inhibitor was pure by cellulose acetate electrophoresis. The molecular weight was approximately 32 000 as determined by gel filtration on Sephadex G-100. The inhibitor acted on bovine trypsin, human trypsin and bovine chymotrypsin. It had no action on human chymotrypsin, subtilisin BPN, pronase, Aspergillus oryzae protease, human and porcine pepsins. The bindings sites for bovine trypsin and chymotrypsin are not mutually exclusive. The inhibitor was stable over a pH range of 1–10. The purified inhibitor was far more thermostable than the crude inhibitor. The purified inhibitor lost only 33% of activity on heat treatment at 95°C for 2 h. Trinitrobenzene sulphonate treatment resulted in the loss of antichymotryptic activity faster than the antitrypsin activity of the inhibitor

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Citation Excerpt :
Three replicates were maintained for all the tests performed for both the oxidizers, respectively. Modification of lysine and arginine residues of CbTI–2 was done by using 2,4,6-trinitrobenzene sulfonic acid (TNBS) [43] and cyclohexane-1,2-dione (CHD) [44], respectively. CbTI–2 (520 μg) was treated with 3.3 mg of TNBS in the presence of 400 μmol of phosphate buffer, pH 7.6, in a volume of 4.0 ml at 40 °C.
Seeds of tropical legumes posses a repertoire of proteinase inhibitors (PI) and the current study highlights some structural/functional features of a strong serine PI from the seeds of Caesalpinia bonduc (CbTI–2). Following purification, N-terminal sequence of CbTI–2 revealed over 40% similarity with a few serine PIs of Caesalpinioideae subfamily. Upon exposure to metal ions and ionic/non ionic surfactants, CbTI–2 showed immense variation in the levels of antitryptic activity. Exposure of CbTI–2 to 1,4-Dithiothreitol, Guanidinium HCl, H₂O₂ and Dimethyl sulfoxide led to a steady loss of inhibitory activity. Chemical modification of amino acids suggested an arginine as the active site residue. Circular Dichroism spectrum of native CbTI–2 revealed an unordered state. Secondary structure composition of CbTI–2 following exposure to extreme conditions (heat, acidic/alkaline environment, Guanidine hydrochloride and DTT) showed considerable perturbations that caused severe loss of antiproteolytic activity. DLS studies yielded a hydrodynamic radius of ∼2.2 nm for CbTI–2 and also reconfirmed 1:1 stoichiometry for the trypsin-CbTI–2 complex. Initial studies indicated CbTI–2 to be a potent antiplasmodial agent by being highly toxic towards growth, schizont rupture process and erythrocytic invasion of Plasmodium falciparum.
Exploring the protease mediated conformational stability in a trypsin inhibitor from Archidendron ellipticum seeds
2006, Plant Physiology and Biochemistry
Citation Excerpt :
BSA (1 mg/ml) was used as the standard protein. Lysine and arginine residues of AeTI were modified by TNBS [15] and CHD [16], respectively. A sample (520 ug of protein) of AeTI was treated with 3.3 mg of TNBS in the presence of 400 μmol of phosphate buffer, pH 7.6, in a volume of 4.0 ml at 40 °C.
A Kunitz proteinase inhibitor from Archidendron ellipticum seeds (AeTI) was purified and complexed with bovine trypsin and chymotrypsin. The stoichiometric stability of AeTI with its interacting proteinases was then investigated using spectrophotometric, size exclusion chromatography (HPLC system), Western blotting and circular dichroism (CD) studies. All the methods were remarkably similar in revealing the preference of trypsin over chymotrypsin by AeTI for complex formation. Both Western blotting as well as spectrophotometry based assays for competition experiments indicated that trypsin displaces chymotrypsin from a previously formed AeTI–chymotrypsin complex. Chemical modification of lysine and arginine by TNBS and CHD treatments, respectively, suggested a lysine as the active site residue and also indicated the presence of a single protease-binding site for AeTI. CD of native AeTI showed a sharp minimum at 200 nm and deconvolution of the CD spectra revealed it to be an unordered protein possessing high β-sheet content. Complex formation of AeTI with trypsin induces a fractional switchover of its unordered structure towards the β-sheet fraction but lacked any such conversion in the presence of chymotrypsin. Prolonged exposure of excess trypsin generates conformational modifications both in the secondary and the tertiary structures.
Multiple trypsin inhibitors from Momordica cochinchinensis seeds, the Chinese drug mubiezhi
2004, Peptides
Five trypsin inhibitors, with N-terminal sequences demonstrating homology to each other and exhibiting a molecular weight of 5100, 4800, 4400, 4100, and 3900, respectively, were isolated from Momordica cochinchinensis seeds with a protocol involving acid extraction, ion exchange chromatography on SP-Sepharose chromatography, and RP-HPLC on a C18 column. Specific inhibitory activity against trypsin was demonstrated by the trypsin isoinhibitors with K_i values ranging from 5.3×10⁻⁸ to 1.8×10⁻⁶ M. None of the isoinhibitors could be cleaved by trypsin.
Trypsin inhibitors from Colocasia esculenta, Alocasia macrorrhiza and Cyrtosperma chamissonis
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Trypsin inhibitors from three edible aroids of the family Araceae, viz. taro (Colocasia esculenta) var. esculenta) giant taro (Alocasia macrorrhiza) and giant swamp taro (Cyrtosperma chamissonis) obtained from the Pacific region, were isolated by affinity chromatography and purified by gel filtration. The M_r of these inhibitors, as determined by gel filtration were 35 000–38 000, but were ca 20 000 by SDS gradient PAGE. A time course of heating in SDS showed a ready dissociation of the native protein into subunits of equal size. Further experiments showed that there were no disulfide bonds between these subunits. A single N-terminal sequence was found for each inhibitor showing that the two subunits had similar primary structure. Each of the N-terminal sequences showed homology with that of soybean trypsin inhibitor. To our knowledge, this finding follows only one other example of a Kunitz family inhibitor being located in a monocotyledonous, rather than dicotyledonous, plant species, and indicates that the ancestral gene from which Kunitz family inhibitors originate predates the evolutionary divergence of flowering plants into monocotyledons and dicotyledons.
Purification and characterization of an acidic trypsin/subtilisin inhibitor from tortoise egg white
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Egg whites of three species of tortoise and turtle have been compared by gel chromatography for inhibitory activity against proteases. The egg white of Geomda trijuga trijuga Schariggar contains trypsin/subtilisin inhibitor while the egg white of Caretta caretta Linn. contains both trypsin and chymotrypsin inhibitors. No protease inhibitory activity has been detected in the egg white of Trionyx gangeticus Cuvier. An acidic trypsin/subtilisin inhibitor has been purified to homogeneity from the egg white of tortoise (G. trijuga trijuga). It is a single polypeptide chain of 100 amino acid residues, having a molecular weight of 11 700. It contains six disulphide bonds and is devoid of methionine and carbohydrate moiety. Its isoelectric point is at pH 5.95 and is stable at 100°C for 4 h at neutral pH. The inhibitor inhibits both trypsin and subtilisin by forming enzyme-inhibitor complexes at a molar ratio close to unity. Their dissociation contants are 7.2·10⁻⁹ M for bovine trypsin adn 5.5·10⁻⁷ M for subtilisin. Chemical modification of amino groups with trinitrobenzene sulfonate has reduced its inhibitory activities against both trypsin and subtilisin, but the loss of its trypsin inhibitory activity is faster than of its subtilisin inhibitory activity. It has independent binding sites for inhibition of trypsin and subtilisin.
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Natural plant enzyme inhibitors V. A trypsin/chymotrypsin inhibitor from Alocasia macrorhiza tuber

Abstract

J. Biol. Chem.

Biochem. Biophys. Res. Commun.

J. Biol. Chem.

J. Biol. Chem.

Arch. Biochem. Biophys.

J. Biol. Chem.

Biochem. Biophys. Acta

Z. Physiol. Chem.

Biochemistry

Biochem. Biophys. Acta

Ind. J. Biochem. Biophys.