Affinity chromatography of Ruta graveolens L. O-methyltransferases. Studies demonstrating the potential of the technique in the mechanistic investigation of O-methyltransferases

1979 ◽  
Vol 57 (7) ◽  
pp. 986-994 ◽  
Author(s):  
Satish K. Sharma ◽  
Stewart A. Brown
Keyword(s):  
Ferulic Acid ◽  
Affinity Chromatography ◽  
Caffeic Acid ◽  
Kinetic Mechanism ◽  
Ruta Graveolens ◽  
Methyl Transferase ◽  
Methyl Group ◽  
Acid Ligand ◽  
Mechanistic Investigation ◽  
Sepharose 4B

Two discrete furanocoumarin (5- and 8-) O-methyltransferases and a caffeic acid 3-O-methyl-transferase from cell cultures of Ruta graveolens L. have been copurified by affinity chromatography on 1,6-diaminohexane agarose (AH-Sepharose 4B) linked with 5-adenosyl-L-homocysteine (SAH). The furanocoumarin O-methyltransferases, which transfer a methyl group from S-adenosyl-L-methionine (SAM) to the 5- or 8-hydroxyls of linear furanocoumarins, were not retarded by 5-(3-carboxypropanamido)-xanthotoxin (CPAX) immobilized to AH-Sepharose 4B, but addition of SAM to the irrigant buffer led to complete retardation of both enzymes on this affinity system. An analogous phenomenon was observed for the caffeic acid O-methyltransferase, with a ferulic acid ligand coupled to the same insoluble support. SAH was as effective as SAM in promoting binding of the furanocoumarin O-methyltransferases to CPAX and caffeic acid 3-O-methyltransferase to immobilized ferulic acid, respectively. The strong and specific adsorption of these enzymes was abolished by exclusion of SAM or SAH from the irrigant buffer. It is concluded that the enzymes bind first to SAM or SAH, and that this binding process in turn induces the binding site for their specific phenolic substrates or their analogs. Based on these findings, a compulsory–ordered kinetic mechanism for the action of these O-methyltransferases is postulated.

Quantitative Assessment of Soluble Fibrin in Plasma by Affinity Chromatography – A Comparative Study with desAA-Fibrin, desAABB-Fibrin and Fibrinogen

1985 ◽  
Vol 54 (02) ◽  
pp. 533-538 ◽  
Author(s):  
Wilfried Thiel ◽  
Ulrich Delvos ◽  
Gert Müller-Berghaus
Keyword(s):  
Affinity Chromatography ◽  
Calcium Ions ◽  
Quantitative Assessment ◽  
Fluid Phase ◽  
Soluble Fibrin ◽  
Binding Characteristics ◽  
Different Temperatures ◽  
Immobilized Proteins ◽  
Sepharose 4B

SummaryA quantitative determination of soluble fibrin in plasma was carried out by affinity chromatography. For this purpose, desAA-fibrin and fibrinogen immobilized on Sepharose 4B were used at the stationary side whereas batroxobin-induced 125I-desAA-fibrin or thrombin-induced 125I-desAABB-fibrin mixed with plasma containing 131I-fibrinogen represented the fluid phase. The binding characteristics of these mixtures to the immobilized proteins were compared at 20° C and 37° C. Complete binding of both types of fibrin to the immobilized desAA-fibrin was always seen at 20° C as well as at 37° C. However, binding of soluble fibrin was accompanied by substantial binding of fibrinogen that was more pronounced at 20° C. Striking differences depending on the temperature at which the affinity chromatography was carried out, were documented for the fibrinogen-fibrin interaction. At 20° C more than 90% of the applied desAA-fibrin was bound to the immobilized fibrinogen whereas at 37° C only a mean of 17% were retained at the fibrinogen-Sepharose column. An opposite finding with regard to the tested temperature was made with the desAABB-fibrin. Nearly complete binding to insolubilized fibrinogen was found at 37° C (95%) but only 58% of the desAABB-fibrin were bound at 20° C. The binding patterns did not change when the experiments were performed in the presence of calcium ions. The opposite behaviour of the two types of soluble fibrin to immobilized fibrinogen at the different temperatures, together with the substantial binding of fibrinogen in the presence of soluble fibrin to insolubilized fibrin in every setting tested, devaluates affinity chromatography as a tool in the quantitative assessment of soluble fibrin in patients’ plasma.

Сrude Plant Extracts Mediated Polyphenol Oxidation Reactions in the Presence of 3-Methyl-2-Benzothiazolinone Hydrazone for the Determination of Total Polyphenol Content in Beverages

2018 ◽  
Vol 15 (1) ◽  
pp. 11-20 ◽  
Author(s):  
Maria A. Morosanova ◽  
Anton S. Fedorov ◽  
Elena I. Morosanova
Keyword(s):  
Hydrogen Peroxide ◽  
Ferulic Acid ◽  
Gallic Acid ◽  
Caffeic Acid ◽  
Crude Extract ◽  
Reaction Rates ◽  
Green Bean ◽  
Polyphenol Content ◽  
Total Polyphenol ◽  
Total Polyphenol Content

Background: The consumption of antioxidants, including phenolic compounds, is considered important for preventing the oxidative damage diseases and ageing. The total polyphenol content (TPC) is the parameter used to estimate the quality of plant-derived products. Methods: Phenol oxidase activity of green bean (Phaseolus vulgaris) crude extract (in the presence of hydrogen peroxide) and banana (Musa sp.) pulp crude extract has been studied spectrophotometrically using catechol, gallic acid, caffeic acid, ferulic acid, and quercetin as substrates. All studied compounds have been oxidized in the presence of green bean crude extract and hydrogen peroxide; all studied compounds except ferulic acid have been oxidized in the presence of banana pulp crude extract. Michaelis constants (Km) and maximum reaction rates (Vmax) have been determined for oxidation in the presence of green bean crude extract and hydrogen peroxide (Km are 3.8×10-4 M, 1.6×10-3 M, 2.2×10-4 M, 2.3×10-4 M, 1.4×10-4 M and Vmax are 0.046 min-1, 0.102 min-1, 0.185 min-1, 0.053 min-1, 0.041 min-1 for catechol, gallic acid, caffeic acid, ferulic acid, and quercetin, respectively) and for oxidation in the presence of banana pulp crude extract (Km are 1.6×10-3 M, 3.8×10-3 M, 2.2×10-3 M, 4.2×10-4 M and Vmax are 0.058 min-1, 0.025 min-1, 0.027 min-1, 0.015 min-1 for catechol, gallic acid, caffeic acid, and quercetin, respectively). The influence of 3-methyl-2-benzothiazolinone hydrazone (MBTH) on the oxidation reactions kinetics has been studied: Michaelis constants values decrease and maximum reaction rates increase, which contributes to the increase in sensitivity of the determination. Results: Kinetic procedures of Total Polyphenol Content (TPC) determination using crude plants extracts in the presence of MBTH have been proposed (time of analysis is 1 min). For gallic acid (used as a standard for TPC determination) detection limit is 5.3×10-5 M, quantitation limit is 1.8×10-4 M, and linear range is 1.8×10-4 - 1.3×10-3 M for green bean crude extract; detection limit is 2.9×10-5 M, quantitation limit is 9.5×10-5 M, and linear range is 9.5×10-5 - 2.4×10-3 M for banana pulp crude extract. Proposed procedures are characterized by higher interference thresholds for sulfites, ascorbic acid, and citric acid compared to pure enzymes (horseradish peroxidase and mushroom tyrosinase) in the same conditions. Compared with standard Folin-Ciocalteu (FC) method the procedures described in this work are also characterized by less interference and more rapid determination. Conclusion: The procedures have been applied to TPC determination in tea, coffee, and wine samples. The results agree with the FC method for tea and coffee samples and are lower for wine samples, probably, due to sulfites interference.

Caffeic acid: O-methyltransferases and the biosynthesis of ferulic acid in primary cell walls of wheat seedlings

1996 ◽  
Vol 41 (6) ◽  
pp. 1507-1510 ◽  
Author(s):  
Thi Bach Tuyet Lam ◽  
Kenji Iiyama ◽  
Bruce A. Stone
Keyword(s):  
Ferulic Acid ◽  
Caffeic Acid ◽  
Cell Walls ◽  
Primary Cell ◽  
Wheat Seedlings

scholarly journals Caffeic acid and ferulic acid improves diet induced metabolic syndrome in mice

2012 ◽  
Vol 26 (S1) ◽  
Author(s):  
Barbara Miranda Leite Costa Bocco ◽  
Fernanda Beraldo Lorena ◽  
Gustavo Werpel Fernandes ◽  
Roberta Monterazzo Cysneiros ◽  
Miriam Oliveira Ribeiro
Keyword(s):  
Metabolic Syndrome ◽  
Ferulic Acid ◽  
Caffeic Acid

scholarly journals Affinity chromatography and inhibition of chorismate mutase-prephenate dehydrogenase by derivatives of phenylalanine and tyrosine

1977 ◽  
Vol 165 (1) ◽  
pp. 121-126 ◽  
Author(s):  
G D Smith ◽  
D V Roberts ◽  
A Daday
Keyword(s):  
Affinity Chromatography ◽  
Competitive Inhibitor ◽  
Chorismate Mutase ◽  
Prephenate Dehydrogenase ◽  
Step Procedure ◽  
One Step ◽  
High Degree ◽  
Sepharose 4B ◽  
Derivatives Of

Several derivatives of phenylalanine and tyrosine were prepared and tested for inhibition of chorismate mutase-prephenate dehydrogenase (EC 1.3.1.12) from Escherichia coli K12 (strain JP 232). The best inhibitors were N-toluene-p-sulphonyl-L-phenylalanine, N-benzenesulphonyl-L-phenylalanine and N-benzloxycarbonyl-L-phenylalanine. Consequently two compounds, N-toluene-sulphonyl-L-p-aminophenylalanine and N-p-aminobenzenesulphonyl-L-phenylalanine, were synthesized for coupling to CNBr-activated Sepharose-4B. The N-toluene-p-sulphonyl-L-p-aminophenylalanine-Sepharose-4B conjugate was shown to bind the enzyme very strongly at pH 7.5. The enzyme was not eluted by various eluents, including 1 M-NaCl, but could be quantitatively recovered by washing with buffer of pH9. Elution was more effective in the presence of 10 mM-1-adamantaneacetic acid, a competitive inhibitor of the enzyme. This affinity-chromatography procedure results in a high degree of purification of the enzyme and can be used to prepare the enzyme in a one-step procedure from the bacterial crude extract. Such a procedure may therefore prove useful in studying this enzyme in a state that closely resembles that in vivo.

scholarly journals Rhizobial NodL O-Acetyl Transferase and NodS N-Methyl Transferase Functionally Interfere in Production of Modified Nod Factors

2001 ◽  
Vol 183 (11) ◽  
pp. 3408-3416 ◽  
Author(s):  
Isabel M. López-Lara ◽  
Dimitris Kafetzopoulos ◽  
Herman P. Spaink ◽  
Jane E. Thomas-Oates
Keyword(s):  
Nodule Formation ◽  
Signal Molecules ◽  
Nod Factors ◽  
Rhizobium Tropici ◽  
Methyl Transferase ◽  
Mesorhizobium Loti ◽  
Methyl Group ◽  
Rhizobial Species ◽  
Acetyl Group

ABSTRACT The products of the rhizobial nodulation genes are involved in the biosynthesis of lipochitin oligosaccharides (LCOs), which are host-specific signal molecules required for nodule formation. The presence of an O-acetyl group on C-6 of the nonreducingN-acetylglucosamine residue of LCOs is due to the enzymatic activity of NodL. Here we show that transfer of the nodLgene into four rhizobial species that all normally produce LCOs that are not modified on C-6 of the nonreducing terminal residue results in production of LCOs, the majority of which have an acetyl residue substituted on C-6. Surprisingly, in transconjugant strains ofMesorhizobium loti, Rhizobium etli, and Rhizobium tropici carrying nodL, such acetylation of LCOs prevents the endogenous nodS-dependent transfer of theN-methyl group that is found as a substituent of the acylated nitrogen atom. To study this interference betweennodL and nodS, we have cloned thenodS gene of M. loti and used its product in in vitro experiments in combination with purified NodL protein. It has previously been shown that a chitooligosaccharide N deacetylated on the nonreducing terminus (the so-called NodBC metabolite) is the preferred substrate for NodS as well as for NodL. Here we show that the NodBC metabolite, acetylated by NodL, is not used by the NodS protein as a substrate while the NodL protein can acetylate the NodBC metabolite that has been methylated by NodS.

scholarly journals Purification of cathepsin B by a new form of affinity chromatography

1986 ◽  
Vol 235 (3) ◽  
pp. 731-734 ◽  
Author(s):  
D H Rich ◽  
M A Brown ◽  
A J Barrett
Keyword(s):  
Affinity Chromatography ◽  
Cathepsin B ◽  
Single Step ◽  
Starting Material ◽  
High Yield ◽  
Cysteine Proteinases ◽  
New Form ◽  
Human Cathepsin ◽  
Sepharose 4B

Human cathepsin B was purified by affinity chromatography on the semicarbazone of Gly-Phe-glycinal linked to Sepharose 4B, with elution by 2,2′-dipyridyl disulphide at pH 4.0. The product obtained in high yield by the single step from crude starting material was 80-100% active cathepsin B. The possibility that this new form of affinity chromatography may be of general usefulness in the purification of cysteine proteinases is discussed.

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