scholarly journals Polyphenol oxidase from wheat bran is a serpin.

2008 ◽  
Vol 55 (2) ◽  
pp. 325-328 ◽  
Author(s):  
Yoshiki Yamasaki ◽  
Haruyoshi Konno ◽  
Kazuhiko Noda
Keyword(s):  
Polyphenol Oxidase ◽  
Column Chromatography ◽  
Wheat Bran ◽  
Serine Proteases ◽  
Batch Adsorption ◽  
Amino Acid Residues ◽  
Protein Z ◽  
Spin Column ◽  
Sds Page ◽  
Relative Molecular Weight

Polyphenol oxidase (PPO; EC 1.10.3.2) was isolated from wheat bran by a procedure that included ammonium sulfate fractionation, batch adsorption by DEAE-cellulofine, CM-cellulofine column chromatography, DEAE-cellulofine column chromatography, preparative isoelectric focusing, adsorption on the membrane of a Vivapure Q Maxi H spin column, and heat treatment. These procedures led to 150-fold purification with 4.2% recovery. The PPO was homogeneous by SDS/PAGE. The relative molecular weight of the PPO was estimated to be 37,000 based on its mobility in SDS/PAGE. The isoelectric point of the PPO was 4.4. The K(m) values of the PPO for caffeic acid, chlorogenic acid, pyrocatechol, 4-methyl catechol and l-DOPA as substrates were 0.077, 0.198, 1.176, 1.667 and 4.545 mM. The PPO was strongly inhibited by tropolone. The K(i) value for tropolone is 2.2 x 10(-7) M. The sequence of the 15 N-terminal amino-acid residues was determined to be ATDVRLSIAHQTRFA, which was identical to those of serpin from Triticum aestivum and protein Z from Hordeum vulgare. The PPO strongly inhibited the activity of trypsin, which is an enzyme of serine proteases; 50% inhibition was observed with 1.5 x 10(-7) M PPO. The K(i) value for PPO is 2.3 x 10(-8) M. The wheat bran PPO should be a very important protein for protecting wheat against disease, virus, insect and herbivore damages by both the activities of PPO and protease inhibitor.

scholarly journals Subcellular localization and purification of a p-hydroxyphenylpyruvate dioxygenase from cultured carrot cells and characterization of the corresponding cDNA

1997 ◽  
Vol 325 (3) ◽  
pp. 761-769 ◽  
Author(s):  
Isabelle GARCIA ◽  
Matthew RODGERS ◽  
Catherine LENNE ◽  
Anne ROLLAND ◽  
Alain SAILLAND ◽  
...  
Keyword(s):  
Nucleotide Sequence ◽  
Gel Filtration ◽  
Peptide Sequence ◽  
Amino Acid Residues ◽  
Carrot Cells ◽  
Expression Studies ◽  
Sds Page ◽  
Molecular Masses ◽  
The Difference ◽  
Dioxygenase Activity

p-Hydroxyphenylpyruvate dioxygenase catalyses the transformation of p-hydroxyphenylpyruvate into homogentisate. In plants this enzyme has a crucial role because homogentisate is the aromatic precursor of all prenylquinones. Furthermore this enzyme was recently identified as the molecular target for new families of potent herbicides. In this study we examine precisely the localization of p-hydroxyphenylpyruvate dioxygenase activity within carrot cells. Our results provide evidence that, in cultured carrot cells, p-hydroxyphenylpyruvate dioxygenase is associated with the cytosol. Purification and SDS/PAGE analysis of this enzyme revealed that its activity is associated with a polypeptide of 45–46 kDa. This protein specifically cross-reacts with an antiserum raised against the p-hydroxyphenylpyruvate dioxygenase of Pseudomonas fluorescens. Gel-filtration chromatography indicates that the enzyme behaves as a homodimer. We also report the isolation and nucleotide sequence of a cDNA encoding a carrot p-hydroxyphenylpyruvate dioxygenase. The nucleotide sequence (1684 bp) encodes a protein of 442 amino acid residues with a molecular mass of 48094 Da and shows specific C-terminal regions of similarity with other p-hydroxyphenylpyruvate dioxygenases. This cDNA encodes a functional p-hydroxyphenylpyruvate dioxygenase, as evidenced by expression studies with transformed Escherichia coli cells. Comparison of the N-terminal sequence of the 45–46 kDa polypeptide purified from carrot cells with the deduced peptide sequence of the cDNA confirms that this polypeptide supports p-hydroxyphenylpyruvate dioxygenase activity. Immunodetection studies of the native enzyme in carrot cellular extracts reveal that N-terminal proteolysis occurs during the process of purification. This proteolysis explains the difference in molecular masses between the purified protein and the deduced polypeptide.

scholarly journals Localization of regions of a Bordetella pertussis autotransporter, Vag8, interacting with C1 inhibitor

2020 ◽  
Author(s):  
Naoki Onoda ◽  
Yukihiro Hiramatsu ◽  
Shihono Teruya ◽  
Koichiro Suzuki ◽  
Yasuhiko Horiguchi
Keyword(s):  
Amino Acid ◽  
Bordetella Pertussis ◽  
Serine Proteases ◽  
Whooping Cough ◽  
Amino Acid Residues ◽  
C1 Inhibitor ◽  
Passenger Domain ◽  
Complement Regulator ◽  
Function Relationship ◽  
Relationship Of

AbstractBordetella pertussis is the causative agent of pertussis (whooping cough), a contagious respiratory disease that has recently seen a resurgence despite high vaccination coverage, necessitating improvement of current pertussis vaccines. An autotransporter of B. pertussis, virulence-associated gene 8 (Vag8), has been proposed as an additional component to improve pertussis vaccines. Vag8 is known to play a role in evasion of the complement system and activation of the contact system by inactivating the complement regulating factor, C1 inhibitor (C1 Inh), which inhibits serine proteases, such as plasma kallikrein (PK). However, the nature of the molecular interaction between Vag8 and C1 Inh remains to be determined. In the present study, we attempted to determine the minimum region of Vag8 that interacts with C1 Inh by examining the differently–truncated Vag8 derivatives for the ability to bind and inactivate C1 Inh. The region of Vag8 from amino–acid residues 102 to 548 was found to bind C1 Inh and cancel its inhibitory action on the protease activity of PK at the same level as a Vag8 fragment from amino–acid residues 52 to 648 covering the passenger domain, which carries its extracellular function. In contrast, the truncated Vag8 containing amino–acid residues 102 – 479 or 202 – 648 barely interacted with C1 Inh. These results indicated that the two separate regions of amino–acid residues 102 – 202 and 479 – 548 are likely required for the interaction with C1 Inh.ImportancePertussis is currently reemerging worldwide, and is still one of the greatest disease burdens in infants. B. pertussis produces a number of virulence factors, including toxins, adhesins, and autotransporters. One of the autotransporters, Vag8, which binds and inactivates the complement regulator C1 Inh, is considered to contribute to the establishment of B. pertussis infection. However, the nature of the interaction between Vag8 and C1 Inh remains to be explored. In this study, we narrowed down the region of Vag8 that interacts with C1 Inh and demonstrated that at least two separate regions of Vag8 are necessary for the interaction with C1 Inh. Our results provide insight into the structure–function relationship of the Vag8 molecule and information to determine its potential role in the pathogenesis of B. pertussis.

scholarly journals Purification, Characterization, and Potential of Saline Waste Water Remediation of a Polyextremophilicα-Amylase from an Obligate HalophilicAspergillus gracilis

2014 ◽  
Vol 2014 ◽  
pp. 1-7 ◽  
Author(s):  
Imran Ali ◽  
Ali Akbar ◽  
Benjawan Yanwisetpakdee ◽  
Sehanat Prasongsuk ◽  
Pongtharin Lotrakul ◽  
...  
Keyword(s):  
Waste Water ◽  
Water Activity ◽  
Column Chromatography ◽  
Specific Activity ◽  
Single Band ◽  
Water Remediation ◽  
Saline Waste ◽  
Sds Page ◽  
Nacl Concentration ◽  
Burk Plot

An obligate halophilicAspergillus graciliswhich was isolated from a hypersaline man-made saltern from Thailand was screened for its potential of producing extracellularα-amylase in the previous studies. In this study theα-amylase was extracted and purified by the help of column chromatography using Sephadex G-100 column. Presence of amylase was verified by SDS-PAGE analysis, showing a single band of approximately 35 kDa. The specific activity of the enzyme was found to be 131.02 U/mg. The Lineweaver-Burk plot showed theVmaxandKmvalues of 8.36 U/mg and 6.33 mg/mL, respectively. The enzyme was found to have the best activity at 5 pH, 60°C, and 30% of NaCl concentration, showing its polyextremophilic nature. The use of various additives did not show much variation in the activity of enzyme, showing its resilience against inhibitors. The enzyme, when tested for its use for synthetic waste water remediation by comparing its activity with commercial amylase in different salt concentrations showed that theα-amylase fromA. graciliswas having better performance at increasing salt concentrations than the commercial one. This shows its potential to be applied in saline waste water and other low water activity effluents for bioremediation.

Properties of wheat bran polyphenol oxidase

Nahrung/Food ◽  
2004 ◽  
Vol 48 (1) ◽  
pp. 5-8 ◽  
Author(s):  
Çi??dem Soysal ◽  
Zerrin Söylemez
Keyword(s):  
Polyphenol Oxidase ◽  
Wheat Bran

Protease activities of Acanthamoeba polyphaga and Acanthamoeba castellanii

2006 ◽  
Vol 52 (1) ◽  
pp. 16-23 ◽  
Author(s):  
José de Jesús Serrano-Luna ◽  
Isaac Cervantes-Sandoval ◽  
Jesús Calderón ◽  
Fernando Navarro-García ◽  
Victor Tsutsumi ◽  
...  
Keyword(s):  
Protease Activity ◽  
Serine Proteases ◽  
Biochemical Characterization ◽  
Acanthamoeba Castellanii ◽  
Cysteine Proteases ◽  
Skin Lesions ◽  
Sds Page ◽  
Serine Protease Family ◽  
Proteolytic Activities ◽  
Amoebic Keratitis

Acanthamoeba spp. are free-living amoebae that cause amoebic granulomatous encephalitis, skin lesions, and ocular amoebic keratitis in humans. Several authors have suggested that proteases could play a role in the pathogenesis of these diseases. In the present work, we performed a partial biochemical characterization of proteases in crude extracts of Acanthamoeba spp. and in conditioned medium using 7.5% SDS–PAGE copolymerized with 0.1% m/v gelatin as substrate. We distinguished a total of 17 bands with proteolytic activity distributed in two species of Acanthamoeba. The bands ranged from 30 to 188 kDa in A. castellanii and from 34 to 144 kDa in A. polyphaga. Additionally, we showed that the pattern of protease activity differed in the two species of Acanthamoeba when pH was altered. By using protease inhibitors, we found that the proteolytic activities belonged mostly to the serine protease family and secondly to cysteine proteases and that the proteolytic activities from A. castellanii were higher than those in A. polyphaga. Furthermore, aprotinin was found to in hibit crude extract protease activity on Madin–Darby canine kidney (MDCK) monolayers. These data suggest that protease patterns could be more complex than previously reported.Key words: Acanthamoeba spp., amoebic keratitis, serine proteases, cysteine proteases, cytopathic effect.

Evidence for Biosynthetical Equivalence of the Epimeric Isoleucine Residues in Angolide

1972 ◽  
Vol 50 (4) ◽  
pp. 428-439 ◽  
Author(s):  
R. O. Okotore ◽  
D. W. Russell
Keyword(s):  
Amino Acid ◽  
Physical Properties ◽  
Acid Hydrolysis ◽  
Column Chromatography ◽  
Mass Spectra ◽  
Mathematical Treatment ◽  
Amino Acid Residues ◽  
Analytical Results ◽  
Cyclic Intermediate

Pithomyces sacchari was grown on media containing L-valine. Instead of the metabolite angolide (cyclo-L-α-hydroxyisovaleryl1-erythro-L-isoleucyl2-L-α-hydroxyisovaleryl3-threo-D-isoleucyl4), synthesized on un-supplemented media, it produced a mixture with very similar physical properties. Acid hydrolysis liberated erythro-L- and threo-D-isoleucines (1:1), and DL-valine in amounts proportional to the exogenous valine concentration; L-α-hydroxyisovaleric acid was the only other component. Radioactivity from L-valine-U-14C was incorporated without dilution into valine, and with extensive dilution into the hydroxy acid.Partial fractionation of the mixture was achieved by column chromatography. Mass spectra of two fractions, containing different proportions of valine, were compared with the spectrum of angolide. A simple mathematical treatment showed that the fractions contained angolide, its divaline homologue, and a homologue with only one isoleucine replaced by valine. This must be a 1: 1 mixture of the two possible isomers, since any deviation from this ratio is inconsistent with the analytical results. It follows that the erythro-L- and the threo-D-isoleucine residues of angolide are equivalent with respect to their replacement by valine. It is inferred that angolide biosynthesis involves a cyclic intermediate containing both amino acid residues in the L configuration.

scholarly journals Purification and Characterization ofα -l-Arabinopyranosidase andα -l-Arabinofuranosidase from Bifidobacterium breve K-110, a Human Intestinal Anaerobic Bacterium Metabolizing Ginsenoside Rb2 and Rc

2003 ◽  
Vol 69 (12) ◽  
pp. 7116-7123 ◽  
Author(s):  
Ho-Young Shin ◽  
Sun-Young Park ◽  
Jong Hwan Sung ◽  
Dong-Hyun Kim
Keyword(s):  
Ammonium Sulfate ◽  
Column Chromatography ◽  
Specific Activity ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Bifidobacterium Breve ◽  
Ammonium Sulfate Fractionation ◽  
Apparent Homogeneity ◽  
Sds Page ◽  
Ginsenoside Rb2

ABSTRACT Two arabinosidases, α-l-arabinopyranosidase (no EC number) and α-l-arabinofuranosidase (EC 3.2.1.55), were purified from ginsenoside-metabolizing Bifidobacterium breve K-110, which was isolated from human intestinal microflora. α-l-Arabinopyranosidase was purified to apparent homogeneity, using a combination of ammonium sulfate fractionation, DEAE-cellulose, butyl Toyopearl, hydroxyapatite Ultrogel, QAE-cellulose, and Sephacryl S-300 HR column chromatography, with a final specific activity of 8.81 μmol/min/mg.α -l-Arabinofuranosidase was purified to apparent homogeneity, using a combination of ammonium sulfate fractionation, DEAE-cellulose, butyl Toyopearl, hydroxyapatite Ultrogel, Q-Sepharose, and Sephacryl S-300 column chromatography, with a final specific activity of 6.46 μmol/min/mg. The molecular mass ofα -l-arabinopyranosidase was found to be 310 kDa by gel filtration, consisting of four identical subunits (77 kDa each, measured by sodium dodecyl sulfate-polyacrylamide gel electrophoresis [SDS-PAGE]), and that ofα -l-arabinofuranosidase was found to be 60 kDa by gel filtration and SDS-PAGE. α-l-Arabinopyranosidase and α-l-arabinofuranosidase showed optimal activity at pH 5.5 to 6.0 and 40°C and pH 4.5 and 45°C, respectively. Both purified enzymes were potently inhibited by Cu2+ and p-chlormercuryphenylsulfonic acid.α -l-Arabinopyranosidase acted to the greatest extent on p-nitrophenyl-α-l-arabinopyranoside, followed by ginsenoside Rb2. α-l-Arabinofuranosidase acted to the greatest extent on p-nitrophenyl-α-l-arabinofuranoside, followed by ginsenoside Rc. Neither enzyme acted on p-nitrophenyl-β-galactopyranoside or p-nitrophenyl-β-d-fucopyranoside. These findings suggest that the biochemical properties and substrate specificities of these purified enzymes are different from those of previously purified α-l-arabinosidases. This is the first reported purification ofα -l-arabinopyranosidase from an anaerobic Bifidobacterium sp.

Sepharose spin column chromatography

1994 ◽  
Vol 1 (1) ◽  
pp. 105-108 ◽  
Author(s):  
Jac A. Nickoloff
Keyword(s):  
Column Chromatography ◽  
Spin Column

scholarly journals Expression and characterization of a truncated murine Fc gamma receptor.

1988 ◽  
Vol 167 (3) ◽  
pp. 1195-1210 ◽  
Author(s):  
Z X Qu ◽  
J Odin ◽  
J D Glass ◽  
J C Unkeless
Keyword(s):  
B Cells ◽  
Cell Line ◽  
Immune Complexes ◽  
Disulfide Bonds ◽  
Peritoneal Macrophages ◽  
Eukaryotic Expression ◽  
Amino Acid Residues ◽  
Gamma Receptor ◽  
Sds Page ◽  
And Function

We have isolated a recombinant secreted Fc gamma R beta molecule by deletion of the transmembrane and cytoplasmic domains encoding sequence from a Fc gamma R beta 1 cDNA clone, and insertion of the truncated cDNA into a eukaryotic expression vector, pcEXV-3. To express and amplify the production of the truncated Fc gamma R beta molecule, we transfected the truncated cDNA plasmid into a dihydrofolate reductase-minus CHO cell line along with a dhfr minigene, and amplified the gene products with methotrexate. The resulting cell line secretes 2-3 micrograms/ml/24 h of truncated Fc gamma R beta, which can be readily purified by affinity chromatography on IgG-Sepharose. The truncated Fc gamma R beta has a Mr of 31-33,000 on SDS-PAGE and is glycosylated. N-glycosidase F cleavage reduces the Mr to 19,000, consistent with the size of the truncated product, 176 amino acid residues. There are two disulfide bonds in the protein. Binding of immune complexes formed between DNP20BSA and anti-DNP mAbs reveals better binding of IgG1 aggregates than that of IgG2b and IgG2a aggregates. The binding of the immune complexes was somewhat better at more acidic pH, in contrast to previous experiments with binding of purified Fc gamma R to immune complex-coated beads. We were surprised to observe that the truncated Fc gamma R beta did not react with the anti-Fc gamma R mAb 6B7C. Previous work had shown that 6B7C reacts with Fc gamma R on immunoblots, fails to bind to the surface of resting B cells and peritoneal macrophages, but does bind to macrophage cell lines and LPS-stimulated B cells. We show, by binding of mAb 6B7C to a peptide conjugate, that the 6B7C epitope lies within residues 169-183 of the intact Fc gamma R beta, which is just outside the plasma membrane. The availability of the truncated Fc gamma R beta in microgram quantities should facilitate further analysis of structure and function of these receptors.

scholarly journals Isolation, subunit composition and interaction of the NDH-1 complexes from Thermosynechococcus elongatus BP-1

2005 ◽  
Vol 390 (2) ◽  
pp. 513-520 ◽  
Author(s):  
Pengpeng Zhang ◽  
Natalia Battchikova ◽  
Virpi Paakkarinen ◽  
Hirokazu Katoh ◽  
Masako Iwai ◽  
...  
Keyword(s):  
Column Chromatography ◽  
Electron Flow ◽  
Co2 Uptake ◽  
Quinone Oxidoreductase ◽  
Complex Interactions ◽  
His Tag ◽  
Sds Page ◽  
Molecular Masses ◽  
One Step

NDH (NADH-quinone oxidoreductase)-1 complexes in cyanobacteria have specific functions in respiration and cyclic electron flow as well as in active CO2 uptake. In order to isolate NDH-1 complexes and to study complex–complex interactions, several strains of Thermosynechococcus elongatus were constructed by adding a His-tag (histidine tag) to different subunits of NDH-1. Two strains with His-tag on CupA and NdhL were successfully used to isolate NDH-1 complexes by one-step Ni2+ column chromatography. BN (blue-native)/SDS/PAGE analysis of the proteins eluted from the Ni2+ column revealed the presence of three complexes with molecular masses of about 450, 300 and 190 kDa, which were identified by MS to be NDH-1L, NDH-1M and NDH-1S respectively, previously found in Synechocystis sp. PCC 6803. A larger complex of about 490 kDa was also isolated from the NdhL-His strain. This complex, designated ‘NDH-1MS’, was composed of NDH-1M and NDH-1S. NDH-1L complex was recovered from WT (wild-type) cells of T. elongatus by Ni2+ column chromatography. NdhF1 subunit present only in NDH-1L has a sequence of -HHDHHSHH- internally, which appears to have an affinity for the Ni2+ column. NDH-1S or NDH-1M was not recovered from WT cells by chromatography of this kind. The BN/SDS/PAGE analysis of membranes solubilized by a low concentration of detergent indicated the presence of abundant NDH-1MS, but not NDH-1M or NDH-1S. These results clearly demonstrated that NDH-1S is associated with NDH-1M in vivo.

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