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1.
The interactions of chymotrypsin, subtilisin and trypsin with a low MW proteinase inhibitor from potatoes were investigated. The Ki value calculated for the binding of inhibitor to chymotrypsin was 1.6 ± 0.9 × 10?10M, while the second-order rate constant for association was 6 × 105 M?1/sec. Although binding was not observed to chymotrypsin which had been treated with diisopropyl fluorophosphate or with l-tosylamide-2-phenylethyl chloromethyl ketone, the 3-methylhistidine-57 derivative bound inhibitor with a Ki value of 9.6 × 10?9 M. The inhibitor also exhibited a tight association with subtilisin (Ki < 4 × 10?9 M). In contrast, little inhibition of trypsin was observed, and this was believed to be due to low levels of a contaminant in our preparations. No evidence for reactive site cleavage was observed after incubation of the inhibitor with catalytic amounts of chymotrypsin or subtilisin at acid pH. 相似文献
2.
The Ser-170 residue of a trypsin-like enzyme from Streptomyces fradiae (SFT), which is considered to be the active-site serine, was replaced with alanine by site-directed mutagenesis to improve
the affinity chromatography step for a Kazal-type trypsin inhibitor, pancreatic secretory trypsin inhibitor (PSTI). The resulting
mutant SFT, designated as [S170A]SFT, was expressed in Streptomyces lividans and purified to homogeneity. [S170A]SFT was catalytically inactive, but still had the ability to bind tightly to PSTI and
to soybean trypsin inhibitor with dissociation constants of 3.1×10-7 M and 1.9×10-8 M respectively. We further demonstrated that recombinant human PSTI secreted into Saccharomyces cerevisiae culture broth could be purified to homogeneity with a one-step [S170A]SFT-affinity column. The purified PSTI contained no
molecules intramolecularly cleaved by active trypsin, which are found when trypsin-affinity chromatography is used for the
purification. This eliminated the need for further separation of intact PSTI from intramolecularly cleaved PSTI by high-performance
liquid chromatography, thus simplifying and improving its purification process.
Received: 29 November 1995/Received revision: 24 January 1996/Accepted: 17 March 1996 相似文献
3.
Amanda Fernandes de Medeiros Izael de Sousa Costa Fabiana Maria Coimbra de Carvalho Sumika Kiyota Beatriz Blenda Pinheiro de Souza Daniel Nogoceke Sifuentes 《Journal of enzyme inhibition and medicinal chemistry》2018,33(1):334-348
A trypsin inhibitor isolated from tamarind seed (TTI) has satietogenic effects in animals, increasing the cholecystokinin (CCK) in eutrophy and reducing leptin in obesity. We purified TTI (pTTI), characterised, and observed its effect upon CCK and leptin in obese Wistar rats. By HPLC, and after amplification of resolution, two protein fractions were observed: Fr1 and Fr2, with average mass of [M?+?14H]+?=?19,594,690?Da and [M?+?13H]+?=?19,578,266?Da, respectively. The protein fractions showed 54 and 53 amino acid residues with the same sequence. pTTI presented resistance to temperature and pH variations; IC50 was 2.7?×?10?10?mol.L?1 and Ki was 2.9?×?10?11?mol.L?1. The 2-DE revealed spots with isoelectric points between pH 5 and 6, and one near pH 8. pTTI action on leptin decrease was confirmed. We conclude that pTTI is a Kunitz trypsin inhibitor with possible biotechnological health-related application. 相似文献
4.
A trypsin inhibitor, termed ovostatin, has been purified approximately 265-fold with 82% yield, from unfertilized eggs of the sea urchin Strongylocentrotus intermedius, using trypsin coupled Sepharose 4B as an affinity column for chromatography. The isolated ovostatin is homogeneous in sodium dodecyl sulfate/polyacrylamide gel electrophoresis, the estimated molecular weight being 20K–21.5K. Ovostatin inhibits preferentially trypsin-like endogenous protease purified from the eggs of the same species and bovine pancreatic trypsin and also bovine pancreatic chymotrypsin. Values of IC50 (amount causing 50% inhibition of enzymes) for trypsin-like protease purified from eggs of the same species, bovine pancreatic trypsin, and bovine pancreatic chymotrypsin, are 0.91 ± 0.13 μg/ml (4.55 ± 0.65 × 10?8 M), 3.0 ± 0.28 μg/ml (1.5 ± 0.14 × 10?7 M), and 4.8 ± 0.2 μg/ml (2.4 ± 0.1 × 10?7 M), respectively, in the experimental condition used. Kinetic studies indicate that ovostatin is a noncompetitive inhibitor of trypsin. The inhibitor is relatively heat labile. NaCl (0.025–0.01 M) enhances the inhibitor activity, whereas KCl is inhibitory. Ovostatin requires a low concentration of Ca2+ for activity. The activity is higher in unfertilized eggs than in fertilized eggs; total activity and specific activity in unfertilized eggs is about 1.67-fold and 1.85-fold higher than those in fertilized eggs, respectively. We believe that ovostatin may regulate the function of the cortical granule protease and other trypsin-like proteases that are activated in sea urchin eggs during fertilization. 相似文献
5.
《Journal of enzyme inhibition and medicinal chemistry》2013,28(4):553-560
Context: The Bowman-Birk inhibitors (BBIs) are currently investigated with renewed interest due to their therapeutic properties in cancer and other inflammatory disease treatment. The molecular mass of the BBI is a limitation, as sufficient amounts of the inhibitor do not reach the organs outside the gastrointestinal tract when administered orally.Method: The anti-tryptic domain of HGI-III of horsegram (Dolichos biflorus) was cloned using the vector pET-20b (+) and expressed in E. coli BL21 (DE3) pLysS.Results: Kinetic analysis of this anti-tryptic peptide (recombinant trypsin inhibitory domain (rTID)) reveals that it is a potent inhibitor of trypsin and human tryptase. The Ki (3.2?±?0.17?×?10?8 M) establishes a very high affinity to bovine trypsin. rTID inhibited human lung tryptase (IC50 3.78?±?0.23?×?10?7 M). The rTID is resistant to the digestive enzymes found in humans and animals.Conclusion: These properties propagate further research on the use of rTID as a therapeutic for cancer and other related inflammatory diseases. 相似文献
6.
Chandan Shee Ashwani K. Sharma 《Journal of enzyme inhibition and medicinal chemistry》2013,28(1):115-120
A protein with trypsin inhibitory activity was purified to homogeneity from the seeds of Murraya koenigii (curry leaf tree) by ion exchange chromatography and gel filtration chromatography on HPLC. The molecular mass of the protein was determined to be 27 kDa by SDS-PAGE analysis under reducing conditions. The solubility studies at different pH conditions showed that it is completely soluble at and above pH 7.5 and slowly precipitates below this pH at a protein concentration of 1 mg/ml. The purified protein inhibited bovine pancreatic trypsin completely in a molar ratio of 1:1.1. Maximum inhibition was observed at pH 8.0. Kinetic studies showed that Murraya koenigii trypsin inhibitor is a competitive inhibitor with an equilibrium dissociation constant of 7 × 10? 9 M. The N-terminal sequence of the first 15 amino acids showed no similarity with any of the known trypsin inhibitors, however, a short sequence search showed significant homology to a Kunitz-type chymotrypsin inhibitor from Erythrina variegata. 相似文献
7.
Parul Gupta Kamal Dhawan Sarla P Malhotra Randhir Singh 《Acta Physiologiae Plantarum》2000,22(4):433-438
A trypsin inhibitor from seeds of faba bean (Vicia faba L.) was purified to near homogeneity as judged by native-PAGE with about 11 % recovery using ammonium sulphate fractionation,
ion-exchange chromatography on DEAE-cellulose and gel filtration through Sephadex G-100. The inhibitor had a molecular weight
of 18 kD as determined by SDS-PAGE and Sephadex G-100. The inhibitor inhibited trypsin and chymotrypsin to the extent of 48
and 12 %, respectively. The inhibtion was of non-competitive type with dissociation constant for the enzyme inhibitor complex
in the region of 0.07 mg·ml−1. The inhibtor was stable between pH 4 and 5. It completely lost its activity when heated at 125 °C for 1 h or at 100 °C for
2 h. The inhibitor also lost its activity on exposure to 2-mercaptoethanol. Based on these properties, it could be concluded
that Vicia faba trypsin inhibitor belongs to Bowman-Birk type of inhibitors, as it has molecular weight lower than generally observed for
Kunitz type inhibitors. 相似文献
8.
Osamu Johdo Akihiro Yoshimoto Tomoyuki Ishikura Hiroshi Naganawa Tomio Takeuchi Hamao Umezawa 《Bioscience, biotechnology, and biochemistry》2013,77(6):1657-1659
We studied the effects of peptidylarginine deiminase, a Ca2+-dependent protein-modulating enzyme that catalyzes the deimination of arginyl residues in protein on two major trypsin inhibitors in mouse plasma. One of these inhibitors was α-1-antitrypsin and the other was a recently characterized trypsin inhibitor termed contrapsin (H. Takahara and H. Sinohara, J. Biol. Chem., 257, 2438 (1982)). The enzyme abolished the trypsin-inhibiting activity of contrapsin in a process that was pseudo-first order with the rate dependent on enzyme concentration (second order rate constant = 3.4 × 103 m-1 · s-1), but no detectable changes in the activity was noted for α-1-antitrypsin. Millimolar Ca2+ and dithiothreitol were absolutely required for the inactivation of contrapsin by peptidylarginine deiminase. Although no significant alterations of the charge and the size in modified contrapsin were observed, the modified inhibitor indicated that 1 arginyl residue was converted to a citrullyl residue. Modified contrapsin whose anti-tryptic activity was lost inhibited chymotrypsin much more strongly than the native inhibitor. These data suggest that a vital amino acid residue for the anti-tryptic activity of mouse contrapsin is an arginyl residue and the conversion of this arginyl residue to a citrullyl residue causes the functional changes of the inhibitor. 相似文献
9.
Heisaburo Shindo 《Biopolymers》1980,19(3):509-522
31P-Nmr relaxation parameters (spin-lattice relaxation time, linewidth, and nuclear Overhauser effect) were obtained at three different frequencies for poly(U) and a well-defined (145 ± 3 base-pair) fragment of DNA in solution. Data sets for the two samples were analyzed by theories which included relaxation by the mechanisms of 31P chemical shift anisotropy as well as by 1H-31P dipole–dipole interaction. Neither data set could be satisfactorily described by a single correlation time. A model of a rigid rotor most nearly fits the data for the DNA molecule. Parameters obtained from the least-square fit indicate (1) that the DNA undergoes anisotropic reorientation with a correlation time τ0 = 6.5 × 10?7 sec for the end-to-end motion, (2) the ratio of diffusion constants D∥/D⊥ is 91, and (3) that the linewidth is due to chemical shift dispersion to the extent of 0.5 ppm. Some deviations of the calculated from the observed values suggested that significant torsional and bending motions may also take place for this DNA. Another model which contains isotropic motion but with a broad distribution of correlation times was required to fit the data for poly(U). A log ? χ2 distribution function of correlation times [Scheafer, J. (1973) Macromolecules 6 , 881–888] described well the motion of poly(U) with the average correlation time τ = 3.3 × 10?9 sec and a distribution parameter p = 14. 相似文献
10.
Following determination of trypsin inhibitory activity, a serine protease inhibitor was purified and characterized from frog
Duttaphrynus melanostictus serum. It was identified as serum albumin, with molecular weight of 67 kDa (DmA-serum). Different from bovine serum albumin, DmA-serum potently inhibited trypsin with similar K
i
values around 1.6 × 10−7 M. No inhibitory effect on thrombin, chymotrypsin, elastase and subtilisin was observed under the assay conditions. The N-terminal
amino acid is EAEPHSRI. Subsequently, a protein with same N-terminal amino acid was purified from skin, termed as DmA-skin. However, DmA-skin is distinct from DmA-serum by binding of a haem b (0.5 mol/mol protein), and with low trypsin inhibitory activity. Frog albumin is distributed
in frog skin and exhibited trypsin inhibitory activity, suggesting that it plays important roles in skin physiological functions,
like water economy, metabolite exchange and osmoregulation, etc. 相似文献
11.
Young-Kwon Seo Doo-Hoon Lee Youn-Ho Shin Bo-Young You Kyung-Mi Lee Key-Yong Song Seong-Jun Seo Sung-Joo Whang Young-Jin Kim Eun-Kyung Yang Chang-Seo Park Ih-Seop Chang Jung-Keug Park 《Biotechnology and Bioprocess Engineering》2003,8(2):151-157
Obtaining a sufficient amount of healthy keratinocytes from a small tissue is difficult. However, ORS cells can be a good
source of epithelium since they are easily obtainable and patients do not have to suffer from scar formation at donor sites.
Accordingly, the current study modified the conventional primary culture technique to overcome the low propagation and easy
aging of epithelial cells during culturing. In a conventional primary culture, the average yield of human ORS cells is 2.1×103 cells/follicle based on direct incubation in a trypsin (0.1%)/EDTA (0.02%) solution for 15 min at 37°C, however, our modified
method was able to obtain about 6.9×103 cells/follicle using a two-step enzyme digestion method involving dispase (1.2 U/mL) and a trypsin (0.1%)/EDTA (0.02%) solution.
Thus, the yield of primary cultured ORS cells could be increasd three times higher. Furthermore, a total of 2.0×107 cells was obtained in a serum-free medium, while a modified E-medium with mitomycin C-treated feeder cells produced a total
of 6.3×107 cells over 17 days when starting with 7.5×104 cells. Finally, we confirmed the effectiveness of our ORS cell isolation method by presenting their ability for reconstructing
the bioartificial skin epitheliumin vitro 相似文献
12.
A.K. Ray M.K. Guha N.K. Sinha 《Biochimica et Biophysica Acta (BBA)/General Subjects》1982,716(2):126-132
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. 相似文献
13.
Purification and characterization of a proteinase inhibitor from field bean, Dolichos lablab perpureus L 总被引:2,自引:0,他引:2
A proteinase inhibitor resembling Bowman-Birk family inhibitors has been purified from the seeds of cultivar HA-3 of Dolichos lablab perpureus L. The protein was apparently homogeneous as judged by SDS–PAGE, PAGE, IEF, and immunodiffusion. The inhibitor had 12 mole% 1/2-cystine and a few aromatic amino acids, and lacks tryptophan. Field bean proteinase inhibitor (FBPI) exhibited a pI of 4.3 and an M
r of 18,500 Da. CD spectral studies showed random coiled secondary structure. Conformational changes were detected in the FBPI–trypsin/chymotrypsin complexes by difference spectral studies. Apparent K
a values of complexes of inhibitor with trypsin and chymotrypsin were 2.1 × 107 M–1 and 3.1 × 107 M–1, respectively. The binary and ternary complexes of FBPI with trypsin and chymotrypsin have been isolated indicating 1:1 stoichiometry with independent sites for cognate enzymes. Amino acid modification studies showed lysine and tyrosine at the reactive sites of FBPI for trypsin and chymotrypsin, respectively. 相似文献
14.
《Journal of enzyme inhibition and medicinal chemistry》2013,28(3):207-213
AbstractThe effect of pH and temperature on the apparent association equilibrium constant (Ka) for the binding of the bovine and porcine pancreatic secretory trypsin inhibitor (Kazal-type inhibitor, PSTI) to human leukocyte elastase has been investigated. At pH8.0, values of the apparent thermodynamic parameters for human leukocyte elastase: Kazal-type inhibitor complex formation are: bovine PSTT – Ka = 6.3 × 104M?1, δ5G° = -26.9kJ/mol, δH° = +11.7kJ/mol, and δS° = +1.3 × 102 entropy units; porcine PSTI –Ka = 7.0 × 103M?1,δG° = -21.5kJ/mol, δH° = +13.0kJ/mol, and δS° = +1.2 × 102 entropy units (values of Ka δG° and δS° were obtained at 21.0°C; values of δH° were temperature independent over the range (between 5.0°C and 45.0°C) explored). On increasing the pH from 4.5 to 9.5, values of Ka for bovine and porcine PSTI binding to human leukocyte elastase increase thus reflecting the acidic pK-shift of the His57 catalytic residue from ?7.0, in the free enzyme, to ?5.1, in the serine proteinase: inhibitor complexes. Thermodynamics of bovine and porcine PSTI binding to human leukocyte elastase has been analyzed in parallel with that of related serine (pro)enzyme/Kazal-type inhibitor systems. Considering the known molecular models, the observed binding behaviour of bovine and porcine PSTI to human leukocyte elastase was related to the inferred stereochemistry of the serine proteinase/inhibitor contact region(s). 相似文献
15.
I. N. Sokotun O. V. Gnedenko A. V. Leychenko M. M. Monastyrnaya E. P. Kozlovskaya A. A. Molnar A. S. Ivanov 《Biochemistry (Moscow) Supplemental Series B: Biomedical Chemistry》2007,1(2):139-142
The interaction of the inhibitor VJ (InhVJ), isolated from sea anemone R. macrodactylus, with different proteases was investigated using the method of biosensor analysis. The following enzymes were tested: serine proteases (trypsin, α-chymotrypsin, plasmin, thrombin, kallikrein), cysteina protease (papain) and aspartic protease (pepsin). In the rage of the concentrations studied (10–400 nM) inhibitor VJ interacted only with trypsin and α-chymotrypsin. The intermolecular complexes formation between inhibitor VJ and each of these enzymes was characterized by the following kinetic and thermodynamics parameters: KD = 7.38 × 10?8 M and 9.93 × 10?7 M for pairs InhVJ/trypsin and InhVJ/α-chymotrypsin, respectively. 相似文献
16.
KINETIC ASSESSMENT AND EFFECT ON DEVELOPMENTAL PHYSIOLOGY OF A TRYPSIN INHIBITOR FROM Eugenia jambolana (JAMBUL) SEEDS ON Helicoverpa armigera (HÜBNER)
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Dushyant Singh Farrukh Jamal Prabhash K. Pandey 《Archives of insect biochemistry and physiology》2014,85(2):94-113
A trypsin inhibitor was purified from the seeds of Eugenia jambolana (Jambul) with a fold purification of 14.28 and a yield recovery of 2.8%. Electrophoretic analysis of E. jambolana trypsin inhibitor (EjTI) revealed a molecular weight of approximately 17.4 kDa on 12% denaturing polyacrylamide gel electrophoresis with or without reduction. EjTI exhibited high stability over a wide range of temperatures (4–80 °C for 30 min) and pH (3.0–10.0) and inhibited trypsin‐like activities of the midgut proteinases of fourth instar Helicoverpa armigera larvae by approximately 86%. Feeding assays containing 0.05, 0.15, and 0.45 (% w/w) EjTI on functionally important fourth‐instar larvae indicated a dose‐dependent downfall in the larval body weight as well as on extent of survival. The nutritional analysis suggests that EjTI exerts toxic effects on H. armigera. Dixon plot analysis revealed competitive inhibition of larval midgut proteinases by EjTI, with an inhibition constant (Ki) of approximately 3.1 × 10?9 M. However, inhibitor kinetics using double reciprocal plots for trypsin inhibition demonstrated a mixed inhibition pattern. These observations suggest the potential of E. jambolana trypsin inhibitor protein in insect pest management. 相似文献
17.
Protease inhibitors control major biological protease activities to maintain physiological homeostasis. Marine bacteria isolated from oligotrophic conditions could be taxonomically distinct, metabolically unique, and offers a wide variety of biochemicals. In the present investigation, marine sediments were screened for the potential bacteria that can produce trypsin inhibitors. A moderate halotolerant novel marine bacterial strain of Oceanimonas sp. BPMS22 was isolated, identified, and characterized. The effect of various process parameters like salt concentration, temperature, and pH was studied on the growth of the bacteria and production of trypsin inhibitor. Further, the trypsin inhibitor was purified to near homogeneity using anion exchange, size exclusion, and affinity chromatography. The purified trypsin inhibitor was found to competitively inhibit trypsin activity with an inhibition coefficient, Ki, of 3.44?±?0.13 μM and second-order association rate constant, kass, of 1.08?×?103 M?1 S?1. The proteinaceous trypsin inhibitor had a molecular weight of approximately 30 kDa. The purified trypsin inhibitor showed anticoagulant activity on the human blood samples. 相似文献
18.
A proteinase inhibitor resembling Bowman-Birk family inhibitors has been purified from the seeds of cultivar HA-3 of Dolichos lablab perpureus L. The protein was apparently homogeneous as judged by SDS–PAGE, PAGE, IEF, and immunodiffusion. The inhibitor had 12 mole% 1/2-cystine and a few aromatic amino acids, and lacks tryptophan. Field bean proteinase inhibitor (FBPI) exhibited a pI of 4.3 and an M r of 18,500 Da. CD spectral studies showed random coiled secondary structure. Conformational changes were detected in the FBPI–trypsin/chymotrypsin complexes by difference spectral studies. Apparent K a values of complexes of inhibitor with trypsin and chymotrypsin were 2.1 × 107 M?1 and 3.1 × 107 M?1, respectively. The binary and ternary complexes of FBPI with trypsin and chymotrypsin have been isolated indicating 1:1 stoichiometry with independent sites for cognate enzymes. Amino acid modification studies showed lysine and tyrosine at the reactive sites of FBPI for trypsin and chymotrypsin, respectively. 相似文献
19.
Macedo ML Diz Filho EB Freire MG Oliva ML Sumikawa JT Toyama MH Marangoni S 《The protein journal》2011,30(1):9-19
The present paper describes the purification, characterization and determination of the partial primary structure of the first
trypsin inhibitor isolated from the family Sapindaceae. A highly stable, potent trypsin inhibitor (SSTI) was purified to homogeneity.
SDS–PAGE analysis revealed that the protein consists of a two-polypeptide chain with molecular masses of approximately 15
and 3 kDa. The purified inhibitor inhibited bovine trypsin at a 1:1 M ratio. Kinetic analysis revealed that the protein is
a competitive inhibitor with an equilibrium dissociation constant of 10−9 M for trypsin. The partial NH2- terminal sequence of 36 amino acids in SSTI indicates homology with other members of the trypsin-inhibitor family from different
sources. This inhibitor is highly stable in the presence of denaturing agents. SSTI showed significant inhibitory activity
against trypsin-like proteases present in the larval midgut on Anagasta kuehniella, Corcyra cephalonica, Diatreae saccharalis and Anticarsia gemmatalis. 相似文献
20.
Zhuang Kang Jia-hong Jiang Dong Wang Ke Liu Lin-fang Du 《Biochemistry. Biokhimii?a》2009,74(1):102-109
The trypsin inhibitor SOTI was isolated from Spinacia oleracea L. seeds through ammonium sulfate precipitation, Sepharose 4B-trypsin affinity chromatography, and Sephadex G-75 chromatography.
This typical Kunitz inhibitor showed remarkable stability to heat, pH, and denaturant. It retained 80% of its activity against
trypsin after boiling for 20 min, and more than 90% activity when treated with 6 M guanidine hydrochloride. The formation
of stable SOTI-trypsin complex (K
i
= 2.3·10−6 M) is consistent with significant inhibitory activity of SOTI against trypsin-like proteinases present in the larval midgut
of Pieris rapae. Sequences of SOTI fragments showed homology with other inhibitors.
Published in Russian in Biokhimiya, 2009, Vol. 74, No. 1, pp. 131–140. 相似文献