scholarly journals Physico-Chemical and Antifungal Properties of a Trypsin Inhibitor from the Roots of Pseudostellaria heterophylla

Molecules ◽  
2018 ◽  
Vol 23 (9) ◽  
pp. 2388 ◽  
Author(s):  
Xixi Cai ◽  
Xiaoli Xie ◽  
Nanyan Fu ◽  
Shaoyun Wang
Keyword(s):  
Trypsin Inhibitor ◽  
Membrane Integrity ◽  
Gel Filtration ◽  
Kinetic Studies ◽  
Competitive Inhibitor ◽  
Exchange Chromatography ◽  
Antifungal Protein ◽  
Cell Membrane Integrity ◽  
Pseudostellaria Heterophylla ◽  
Α Helix

Plant peptidase inhibitors play essential roles in the defense systems of plants. A trypsin inhibitor (PHTI) with a molecular mass of 20.5 kDa was isolated from the fresh roots of the medicinal herb, Pseudostellaria heterophylla. The purification process involved ammonium sulfate precipitation, gel filtration chromatography on Sephadex G50, and ion-exchange chromatography on DEAE 650M. The PHTI contained 3.7% α-helix, 42.1% β-sheets, 21.2% β-turns, and 33% disordered structures, which showed similarity with several Kunitz-type trypsin inhibitors. Inhibition kinetic studies indicated that PHTI was a competitive inhibitor, with a Ki value of 3.01 × 10−9 M, indicating a high affinity to trypsin. The PHTI exhibited considerable stability over a broad range of pH (2–10) and temperatures (20–70 °C); however, metal ions, including Fe3+, Ba2+, Mn2+, and Al3+, could inactivate PHTI to different degrees. Results of fluorescence spectroscopy and circular dichroism showed that Fe3+ could bind to TI with an association constant of 2.75 × 105 M−1 to form a 1:1 complex, inducing conformation changes and inactivation of PHTI. In addition, PHTI could inhibit the growth of the phytopathogens, Colletotrichum gloeosporioides and Fusarium oxysporum, through disruption of the cell membrane integrity. The present study extended research on Pseudostellaria heterophylla proteins and makes PHTI an exploitable candidate as an antifungal protein for further investigation.

LUTI: a double-function inhibitor isolated from naked flax seeds

2019 ◽  
Vol 51 (10) ◽  
pp. 989-996
Author(s):  
Lei Wang ◽  
Yinglu Chen ◽  
Feng Wu ◽  
Shasha Wu ◽  
Xiaojun Hu ◽  
...  
Keyword(s):  
Trypsin Inhibitor ◽  
Lactic Acid Production ◽  
Gel Filtration ◽  
Glucose Consumption ◽  
Competitive Inhibitor ◽  
Size Exclusion ◽  
Kinetic Experiment ◽  
Glucosidase Inhibitors ◽  
Exclusion Chromatography ◽  
Inhibitor Type

Abstract Acute glucose fluctuation during the postprandial period causes a risk for type 2 diabetes mellitus (T2DM). α-Glucosidase inhibitors have been approved as therapeutic agents for diabetes. In the present study, a protein with α-glucosidase inhibitory activity from Flax (Linum usitatissimum) seeds was isolated using a one-step purification with Q-Sepharose4B column, followed by Sephacryl S-200 size-exclusion chromatography. It was identified as a trypsin inhibitor, named L. usitatissimum trypsin inhibitor (LUTI). The half maximal inhibitory concentration (IC50) of LUTI was 113.92 μM for α-glucosidase and 6.17 μM for trypsin. Lineweaver–Burk kinetic experiment showed that the protein exhibited two distinct inhibitory modes, a competitive inhibitor type for α-glucosidase and a non-competitive type for trypsin. The interaction between LUTI and α-glucosidase was detected through gel filtration chromatography and dynamic light scattering. Increased glucose consumption and lactic acid production were also observed following LUTI treatment in Caco-2 and HepG2 cells. LUTI inhibits not only the activity of trypsin but also the activity of α-glucosidase. It is expected that LUTI will become an oral hypoglycemic polypeptide drug for T2DM.

Purification of glysojanin, an antifungal protein, from the black soybean Glycine soja

2003 ◽  
Vol 81 (6) ◽  
pp. 387-394 ◽  
Author(s):  
Patrick H.K Ngai ◽  
T B Ng
Keyword(s):  
Liquid Chromatography ◽  
Ion Exchange ◽  
Glycine Soja ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Fast Protein Liquid Chromatography ◽  
Antifungal Protein ◽  
Black Soybean

A monomeric protein, with a molecular mass of 25 kDa and an N-terminal sequence resembling a segment of chitin synthase, was isolated from the seeds of the black soybean Glycine soja. The protein, designated glysojanin, demonstrated potent antifungal activity against the fungi Fusarium oxysporum and Mycosphaerella arachidicola. It inhibited HIV-1 reverse transcriptase with an IC50 of 47 µmol/L, [methyl-3H]thymidine incorporation by mouse spleen cells with an IC50 of 175 µmol/L, and translation in the rabbit reticulocyte lysate with an IC50 of 20 µmol/L. Glysojanin was purified using a procedure that involved ion-exchange chromatography on DEAE-cellulose, affinity chromatography on Affi-gel blue gel, ion-exchange chromatography by fast protein liquid chromatography on Mono S, and gel filtration by fast protein liquid chromatography on Superdex 75.Key words: antifungal protein, seeds, soybean, purification.

Characterization of a High Affinity Folate Binding Protein in Porcine Serum: Ionic Charge, Concentration—Dependent Polymerization and Ligand Binding Mechanism

2003 ◽  
Vol 23 (5-6) ◽  
pp. 339-351 ◽  
Author(s):  
Jan Holm ◽  
Steen Ingemann Hansen
Keyword(s):  
Affinity Chromatography ◽  
Binding Protein ◽  
Gel Filtration ◽  
Competitive Inhibitor ◽  
Exchange Chromatography ◽  
Ionic Charge ◽  
Folate Binding Protein ◽  
Porcine Serum ◽  
Hill Coefficients ◽  
Triton X

The folate binding protein in porcine serum, present at concentrations of 50-100 nM, is cationic at near neutral pH as evidenced by ion exchange chromatography. The gel filtration profile of the protein isolated from porcine serum by methotrexate affinity chromatography exhibited one peak at 48 kDa and an additional peak of 91 kDa at higher protein concentrations. This could suggest the involvement of concentration-dependent polymerization phenomena. Binding of [3H] folate was of a high-af.nity type with upward convex Scatchard plots and Hill coefficients >1.0 indicative of apparent positive cooperativity. However, binding to protein isolated from porcine serum after affinity chromatography was biphasic (high/low-affinity) in the absence of Triton X-100, 1 g/1. These findings which are similar to those reported for purified milk folate binding proteins are consistent with a model predicting association between unliganded and liganded monomers to weak-ligand affinity heterodimers. Amphiphatic substances, e.g. Triton X-100, form micelles which could separate hydrophobic unliganded monomers from hydrophilic liganded monomers (monomers are hydrophilic in the liganded state) thereby preventing heterodimerization. The folate analogue N10 methyl folate was a potent and competitive inhibitor of [3H] folate binding to the folate binding protein, and moreover changed the binding type to apparent negative cooperativity.

scholarly journals Purification and Characterization of a Novel Antifungal Flagellin Protein from Endophyte Bacillus methylotrophicus NJ13 against Ilyonectria robusta

2019 ◽  
Vol 7 (12) ◽  
pp. 605 ◽  
Author(s):  
Yun Jiang ◽  
Chao Ran ◽  
Lin Chen ◽  
Wang Yin ◽  
Yang Liu ◽  
...  
Keyword(s):  
Amino Acid ◽  
Amino Acid Sequence ◽  
Gene Sequence ◽  
Gel Filtration ◽  
Anion Exchange Chromatography ◽  
Inhibitory Effect ◽  
Exchange Chromatography ◽  
Antifungal Protein ◽  
Bacillus Methylotrophicus ◽  
Cloned Gene

Endophyte Bacillus methylotrophicus NJ13 was isolated from Panax ginseng. Its sterile fermentation liquid showed a significant inhibitory effect against Ilyonectria robusta, causing the rusty root rot of P. ginseng and P. quinquefolius. The antifungal protein was obtained after precipitation by 20% saturated ammonium sulfate, desalted by Sephadex G-25, weak anion exchange chromatography, and gel filtration chromatography. SDS-PAGE showed that the purified protein was approximately 29 KDa. The antifungal protein after desalting was not resistant to temperatures higher than 100 °C, resistant to acid conditions, and did not tolerate organic solvents and protease K. The amino acid sequence of purified antifungal protein had an identity of 76% to flagellin from Bacillus velezensis. The isoelectric point of the protein was 4.97 and its molecular mass was 27 KDa. Therefore, a specific primer G1 was designed based on the flagellin gene sequence, and a 770 bp gene sequence was cloned in NJ13 genomic DNA, which shared the same size of flagellin. There were ten base differences between the gene sequences of flagellin and the cloned gene, however, the amino acid sequence encoded by the cloned gene was identical to the flagellin. In conclusion, the antifungal protein produced by biocontrol agent NJ13 contained a flagellin protein.

scholarly journals Kinetic Studies of Na+/K+-ATPase in Tissue Aerobic Thyroid Patients

2019 ◽  
Vol 16 (3(Suppl.)) ◽  
pp. 0740
Author(s):  
Al Samurai Et al.
Keyword(s):  
Kinetic Characteristic ◽  
Thyroid Tissue ◽  
Gel Filtration ◽  
Kinetic Studies ◽  
Exchange Chromatography ◽  
Partial Purification ◽  
Old Patients ◽  
Physiological Regulation ◽  
Sds Page ◽  
Intracellular Regulation

Na+/K+-ATPase is a prevalent enzyme that maintains the Na+ and K+ gradients across the cell membrane by transporting three Na+ out and two K+ into the cell, the aim of this study  is to provide detailed mechanistic insights, potentially with important effects on physiological regulation of active Na and K transport in tissues of Aerobic Thyroid Patient. Thyroid tissues were obtained from a 35 year old patients, the operation was carried out at the Al-Hadi Specialist Hospital in Samarra city, the sample was stored at -20ºC until used. The purification protocol included Salt Precipitation, Ion Exchange Chromatography, Gel Filtration and Electrophoresis, a spectrophotometric method was   used to determine the enzyme activity. kinetic parameters  was also  obtained  for the enzyme. Partial purification of Na+/K+-ATPase revealed two isoenzymes (I ,II). The purity of separated isoenzymes were proved by SDS-PAGE electrophoresis. The kinetic characteristics of Na+/K+-ATPase showed that optimum substrate concentration about 1.5mM,  Km 1.052mM,  and Vmax 6.062, optimum temperature was 37 ºC, optimum pH  7.4 and optimum  time  in 25 min. Na+/K+-ATPase purified from Thyroid tissue  has distinct kinetic characteristic that reflects the importance of      intracellular regulation of specific Na+/K+-ATPase pump which gives cells the ability to precisely coordinate to their physiological requirements .                                                                                             

scholarly journals Characterization of a membrane protease from rat submaxillary-gland mitochondria that possess thrombin-like activity

1996 ◽  
Vol 313 (1) ◽  
pp. 193-199 ◽  
Author(s):  
Mausumi BHARADWAJ ◽  
Dwaipayan BHARADWAJ ◽  
Ratha N. HATI
Keyword(s):  
Trypsin Inhibitor ◽  
Gel Filtration ◽  
Submaxillary Gland ◽  
Kinetic Studies ◽  
Lima Bean ◽  
Maximum Activity ◽  
Reducing Conditions ◽  
Sds Page ◽  
Bean Trypsin Inhibitor

A membrane protease possessing thrombin-like activity was purified to homogeneity from mitochondria of rat submaxillary gland. The molecular mass of the enzyme was determined to be 45 kDa by SDS/PAGE under reducing conditions and by gel filtration on a Sephadex G-100 column. The enzyme is a glycoprotein and has an isoelectric point of 3.25. Maximum activity was observed at pH 10.5. Inhibition by di-isopropyl fluorophosphate, benzamidine, aprotinin and antipain suggested the enzyme to be a serine protease. Other inhibitors such as EDTA, soya-bean trypsin inhibitor, lima-bean trypsin inhibitor, TosLysCH2Cl and chymostatin did not alter the activity. The enzyme showed affinity towards different synthetic substrates (p-nitroanilide derivatives) containing arginine at the P1 position. Kinetic studies revealed that kcat./Km was highest with the substrate N-Bz-Phe-Val-Arg-p-nitroanilide. The enzyme exhibits significant plasma-coagulating activity. The coagulation initiated by the enzyme was not altered by concanavalin A, indicating that the carbohydrate moiety of the enzyme is not essential for this reaction. Further, this enzyme can catalyse the formation of fibrin clots from purified fibrinogen, which describes its thrombin-like activity. However, an antibody raised against the purified enzyme inhibited the plasma-clotting as well as fibrinogen-clotting activity of the enzyme. Fibrinogen coagulation by the enzyme was blocked in the presence of aprotinin, a protease inhibitor. Release of fibrinopeptides A and B from bovine fibrinogen by the enzyme has been shown by HPLC analysis. Our studies reveal that the enzyme reported here differs from trypsin, chymotrypsin and other mitochondrial proteases reported so far.

Purification and characterization of tonin

1976 ◽  
Vol 54 (9) ◽  
pp. 788-795 ◽  
Author(s):  
S. Demassieux ◽  
R. Boucher ◽  
C. Grisé ◽  
J. Genest
Keyword(s):  
Analytical Ultracentrifugation ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Potassium Phosphate ◽  
Kinetic Studies ◽  
Exchange Chromatography ◽  
Sedimentation Equilibrium ◽  
Angiotensin I ◽  
Ph Optimum ◽  
Ammonium Sulphate Precipitation

Tonin was purified from rat submaxillary glands by differential centrifugation, ammonium sulphate precipitation, gel filtration on Sephadex G150, and by ion-exchange chromatography on DEAE-cellulose, phospho-cellulose, SP-Sephadex C25, and SP-Sephadex C50. Purified tonin was shown to be homogeneous by analytical electrophoresis and by analytical ultracentrifugation analysis. Purified tonin was very stable when stored in buffers of low pH values or when incubated at high temperatures in neutral solutions. The molecular weight estimated by sedimentation equilibrium was 28 700. The pH optimum was near 6.8 in a 0.1 M potassium phosphate buffer. The Michaelis–Menten constant for tonin using angiotensin I as substrate was about 4 × 10−5 M. Tonin activity was strongly inhibited by plasma. Kinetic studies using angiotensin I as substrate showed that the inhibition of tonin by plasma was of the non-competitive type.

scholarly journals Purification and properties of an alginate lyase from a marine bacterium

1976 ◽  
Vol 159 (3) ◽  
pp. 707-713 ◽  
Author(s):  
I W Davidson ◽  
I W Sutherland ◽  
C J Lawson
Keyword(s):  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Alginate Lyase ◽  
Kinetic Studies ◽  
Defined Medium ◽  
Exchange Chromatography ◽  
Bacterial Cells ◽  
Purification And Properties ◽  
Primary Composition

An unidentified pseudomonad isolated by enrichment procedures from decomposing seaweed was grown in defined medium containing sodium alginate as the sole carbon source. The alginate lyase recovered from disrupted bacterial cells was purified by a procedure of (NH4)2SO4 precipitation, gel filtration and ion-exchange chromatography. From sodium dodecyl sulphate/polyacrylamide-gel-electrophoresis experiments a mol.wt. of about 50 000 was determined. The enzyme was active against both algal and bacterial alginate preparations. Kinetic studies together with analysis of the unsaturated oligouronide products of alginate lyase action indicated the enzyme was specific for guluronic acid-containing regions of the macromolecular substrate. The specificity of the enzyme can be used to give information about the primary composition of alginate samples.

Partial purification of dog angiotensinogen.

1979 ◽  
Vol 236 (6) ◽  
pp. E655
Author(s):  
B J Morris ◽  
B Moffat ◽  
I A Reid
Keyword(s):  
Gel Filtration ◽  
Kinetic Studies ◽  
Apparent Molecular Weight ◽  
Exchange Chromatography ◽  
Order Reaction ◽  
Partial Purification ◽  
First Order ◽  
Step Procedure ◽  
Dog Plasma

Dog angiotensinogen was purified 450-fold from the plasma of nephrectomized dogs by a simple four-step procedure involving precipitation between 1.5 and 2.3 M ammonium sulfate, gel filtration on Sephadex G-150, ion-exchange chromatography on DE-52 cellulose, and affinity chromatography on Concanavalin A-Sepharose. The purity of the final preparation was over 50%. The preparation of dog angiotensinogen had an apparent molecular weight of 80,000 determined by gel filtration on Sephadex G-100. Kinetic studies indicated that the Km of the reaction of dog renin with partially purified dog angiotensinogen (1,840 pmol/ml) was similar to that for the reaction with angiotensinogen in diluted dog plasma (1,820 pmol/ml). Thus the purification procedures employed did not alter the affinity of dog renin for the Leu10-Leu11 bond of dog angiotensinogen. Because the concentration of angiotensinogen in dog plasma is about 700 pmol/ml, a first order reaction with respect to substrate is indicated in vivo.

A Comparison of the Esterase (TAMe) and Clotting Activities of Human and Bovine Thrombin Preparations*

1965 ◽  
Vol 14 (01/02) ◽  
pp. 159-175
Author(s):  
G. F Lanchantin ◽  
C. A Presant ◽  
D. W Hart ◽  
J. A Friedmann
Keyword(s):  
Sodium Citrate ◽  
Specific Activity ◽  
Gel Filtration ◽  
Kinetic Studies ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Bovine Thrombin ◽  
Prolonged Contact ◽  
Human Thrombin ◽  
Low Ionic Strength

SummaryA comparison has been made between the clotting activity (C) and TAMe esterase activity (E) during prothrombin activation and subsequent purification of human thrombin. A limited number of studies were made using bovine thrombin for comparison purposes. Under certain circumstances, the ratio of C to E for human thrombin is not constant, particularly upon gel-filtration at low ionic strength and during prolonged contact with 25% sodium citrate. Under other circumstances, however, these enzymic parameters are stable and human thrombin appears to have a ratio of C to E of approximately 6 while bovine thrombin has a ratio of 3. This finding may confirm the observations of others concerning the higher specific activity of human thrombin than the bovine enzyme against bovine fibrinogen. Only a single component having C and E activity has been isolated from human prothrombin preparations activated to thrombin by sodium citrate or biologically and purified by either gel filtration or ion exchange chromatography. Kinetic studies on human thrombin preparations indicate that in all other respects they are similar in enzymic properties to those reported for the purified bovine enzyme.

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