Purification and biochemical characterization of Hel a 6, a cross-reactive pectate lyase allergen from Sunflower (Helianthus annuus L.) pollen

AbstractSunflower pollen was reported to contain respiratory allergens responsible for occupational allergy and pollinosis. The present study describes the comprehensive characterization of a major sunflower allergen Hel a 6. Natural Hel a 6 was purified from sunflower pollen by anion exchange and gel filtration chromatography. Hel a 6 reacted with IgE-antibodies from 57% of 39 sunflower-sensitized patient sera suggesting it to be a major allergen. The patients were of Indian origin and suffering from pollinosis and allergic rhinitis. Hel a 6 exhibited allergenic activity by stimulating mediator release from basophils. Monomeric Hel a 6 displayed pectate lyase activity. The effect of various physicochemical parameters such as temperature, pH, and calcium ion on the functional activity of Hel a 6 revealed a stable nature of the protein. Hel a 6 was folded, and its melting curve showed reversible denaturation in which it refolded back to its native conformation from a denatured state. Hel a 6 displayed a high degree of sequence conservation with the pectate lyase allergens from related taxonomic families such as Amb a 1 (67%) and Art v 6 (57%). The IgE-cross reactivity was observed between Hel a 6 and its ragweed and mugwort homologs. The cross-reactivity was further substantiated by the mediator release when Hel a 6-sensitized effector cells were cross-stimulated with Art v 6 and Amb a 1. Several putative B cell epitopes were predicted and mapped on these 3 allergens. Two antigenic regions were found to be commonly shared by these 3 allergens, which could be crucial for cross-reactivity. In conclusion, Hel a 6 serves as a candidate molecule for diagnosis and immunotherapy for weed allergy.

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Purification and characterization of fat body lipase from the greater wax moth, Galleria mellonella (Lepidoptera: Pyralidae)

The Journal of Basic and Applied Zoology ◽

10.1186/s41936-019-0134-y ◽

2019 ◽

Vol 81 (1) ◽

Cited By ~ 1

Author(s):

Rahma R. Z. Mahdy ◽

Shaimaa A. Mo’men ◽

Marah M. Abd El-Bar ◽

Emad M. S. Barakat

Keyword(s):

Metal Ions ◽

Kinetic Parameters ◽

Biochemical Characterization ◽

Galleria Mellonella ◽

Fat Body ◽

Specific Activity ◽

Larval Instar ◽

Gel Filtration ◽

Molecular Weights

Abstract Background Insect lipid mobilization and transport are currently under research, especially lipases and lipophorin because of their roles in the production of energy and lipid transport at a flying activity. The present study has been conducted to purify intracellular fat body lipase for the first time, from the last larval instar of Galleria mellonella. Results Purification methods by combination of ammonium sulfate [(NH4)2SO4] precipitation and gel filtration using Sephadex G-100 demonstrated that the amount of protein and the specific activity of fat body lipase were 0.008633 ± 0.000551 mg/ml and 1.5754 ± 0.1042 μmol/min/mg protein, respectively, with a 98.9 fold purity and recovery of 50.81%. Hence, the sephadex G-100 step was more effective in the purification process. SDS-PAGE and zymogram revealed that fat body lipase showed two monomers with molecular weights of 178.8 and 62.6 kDa. Furthermore, biochemical characterization of fat body lipase was carried out through testing its activities against several factors, such as different temperatures, pH ranges, metal ions, and inhibitors ending by determination of their kinetic parameters with the use of p-nitrophenyl butyrate (PNPB) as a substrate. The highest activities of enzyme were determined at the temperature ranges of 35–37 °C and 37–40 °C and pH ranges of 7–9 and 7–10. The partially purified enzyme showed significant stimulation by Ca2+, K+, and Na+ metal ions indicating that fat body lipase is metalloproteinase. Lipase activity was strongly inhibited by some inhibitors; phenylmethylsulfonyl fluoride (PMSF), ethylene-diaminetetractic acid (EDTA), and ethylene glycoltetraacetic acid (EGTA) providing evidence of the presence of serine residue and activation of enzymes by metal ions. Kinetic parameters were 0.316 Umg− 1 Vmax and 301.95 mM Km. Conclusion Considering the purification of fat body lipase from larvae and the usage of some inhibitors especially ion chelating agents, it is suggested to develop a successful control of Galleria mellonella in near future by using lipase inhibitors.

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Ultrastructural and Partial Biochemical Characterization of Platelets from Chediak-Higashi Cattle

10.1055/s-0039-1680402 ◽

1977 ◽

Author(s):

K. M. Meyers ◽

C. I. Seacord ◽

G. Hopkins ◽

H. Holmsen

Keyword(s):

Electron Micrographs ◽

Biochemical Characterization ◽

Platelet Rich Plasma ◽

Gel Filtration ◽

Human Platelets ◽

Additional Information ◽

Storage Pool ◽

Calcium And Magnesium ◽

Chediak Higashi Syndrome

To provide additional information on the platelet defect which is associated with the Chediak-Higashi syndrome (CHS), platelet rich plasma from normal and CHS cattle was incubated with 14C-adenine. Platelets were then isolated by gel filtration and treated with thrombin. Both the resting amount and extent of secretion of ATP, ADP, several acid hydrolasis, serotonin, calcium and magnesium was determined. Nucleotide profiles and electron micrographs of resting and thrombin treated platelets were also obtained. The markedly reduced secretion of nucleotides, serotonin, and metals demonstrate that CHS cattle have a storage pool defect. Furthermore, there appears to be significant differences in both the resting amount and extent of secretion of several of these measured substances between normal cattle and human platelets.

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Molecular and biochemical characterization of the thermoactive family 1 pectate lyase from the hyperthermophilic bacterium Thermotoga maritima

Biochemical Journal ◽

10.1042/bj20021595 ◽

2003 ◽

Vol 370 (2) ◽

pp. 651-659 ◽

Cited By ~ 42

Author(s):

Leon D. KLUSKENS ◽

Gert-Jan W.M. van ALEBEEK ◽

Alphons G.J. VORAGEN ◽

Willem M. de VOS ◽

John van der OOST

Keyword(s):

Thermal Inactivation ◽

Biochemical Characterization ◽

Thermotoga Maritima ◽

Pectate Lyase ◽

Major Product ◽

Degree Of Polymerization ◽

Extracellular Medium ◽

Native Form ◽

Hyperthermophilic Bacterium

The ability of the hyperthermophilic bacterium Thermotoga maritima to grow on pectin as a sole carbon source coincides with the secretion of a pectate lyase A (PelA) in the extracellular medium. The pelA gene of T. maritima was functionally expressed in Escherichia coli as the first heterologously produced thermophilic pectinase, and purified to homogeneity. Gel filtration indicated that the native form of PelA is tetrameric. Highest activity (422units/mg, with a Km of 0.06mM) was demonstrated on polygalacturonic acid (PGA), whereas pectins with an increasing degree of methylation were degraded at a decreasing rate. In the tradition of pectate lyases, PelA demonstrated full dependency on Ca2+ for stability and activity. The enzyme is highly thermoactive and thermostable, operating optimally at 90°C and pH9.0, with a half-life for thermal inactivation of almost 2h at 95°C, and an apparent melting temperature of 102.5°C. Detailed characterization of the product formation with PGA indicated that PelA has a unique eliminative exo-cleavage pattern liberating unsaturated trigalacturonate as the major product, in contrast with unsaturated digalacturonate for other exopectate lyases known. The unique exo-acting mode of action was supported by progression profiles of PelA on oligogalacturonides (degree of polymerization, 3—8) and the examination of the bond cleavage frequencies.

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Biochemical characterization of guanidinium chloride-soluble dentine collagen from lathyritic-rat incisors

Biochemical Journal ◽

10.1042/bj1810667 ◽

1979 ◽

Vol 181 (3) ◽

pp. 667-676 ◽

Cited By ~ 9

Author(s):

M Wohllebe ◽

D J Carmichael

Keyword(s):

Biochemical Characterization ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Gel Filtration ◽

Neutral Salt ◽

Guanidinium Chloride ◽

Lysine Residues ◽

Cross Links ◽

Lysine Hydroxylation

alpha- and beta-Chains were isolated by sequential ion-exchange and gel-filtration chromatography of guanidinium chloride-soluble dentine collagen obtained from Tris/NaCl-extracted EDTA-demineralized lathyritic-rat incisors. The alpha-chains were identified as alpha 1 I and alpha 2 by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and amino acid analysis of the intact chains and their CNBr peptides. The dentine alpha-chains exhibited higher lysine hydroxylation and phosphate content, but lower hydroxylysine glycosylation, than alpha-chains from skin. Increased lysine hydroxylation was observed in the helical sequences. The alpha 1 I/alpha 2 ratio was approx. 3:1, and was presumably due to the presence of (alpha 1 I)3 molecules along with (alpha 1 I)2 alpha 2 molecules as shown recently for neutral-salt-soluble dentine collagen [Wohllebe & Carmichael (1978) Eur. J. Biochem. 92, 183–188]. In the borohydride-reduced beta 11- and beta 12-chains from guanidinium chloride-soluble dentine collagen, the reduced cross-links hydroxylysinohydroxynorleucine and hydroxylysinonorleucine were present. A higher proportion of hydroxylysinonorleucine in the reduced beta 12-chain probably reflects differences in extent of hydroxylation of specific lysine residues of the alpha 1 I- and alpha 2-chains.

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Molecular Characterization of Saccharomyces cerevisiae TFIID

Molecular and Cellular Biology ◽

10.1128/mcb.22.16.6000-6013.2002 ◽

2002 ◽

Vol 22 (16) ◽

pp. 6000-6013 ◽

Cited By ~ 78

Author(s):

Steven L. Sanders ◽

Krassimira A. Garbett ◽

P. Anthony Weil

Keyword(s):

Saccharomyces Cerevisiae ◽

Transcription Factor ◽

Molecular Mass ◽

Molecular Characterization ◽

Biochemical Characterization ◽

Gel Filtration ◽

Calculated Molecular Mass ◽

General Transcription Factor

ABSTRACT We previously defined Saccharomyces cerevisiae TFIID as a 15-subunit complex comprised of the TATA binding protein (TBP) and 14 distinct TBP-associated factors (TAFs). In this report we give a detailed biochemical characterization of this general transcription factor. We have shown that yeast TFIID efficiently mediates both basal and activator-dependent transcription in vitro and displays TATA box binding activity that is functionally distinct from that of TBP. Analyses of the stoichiometry of TFIID subunits indicated that several TAFs are present at more than 1 copy per TFIID complex. This conclusion was further supported by coimmunoprecipitation experiments with a systematic family of (pseudo)diploid yeast strains that expressed epitope-tagged and untagged alleles of the genes encoding TFIID subunits. Based on these data, we calculated a native molecular mass for monomeric TFIID. Purified TFIID behaved in a fashion consistent with this calculated molecular mass in both gel filtration and rate-zonal sedimentation experiments. Quite surprisingly, although the TAF subunits of TFIID cofractionated as a single complex, TBP did not comigrate with the TAFs during either gel filtration chromatography or rate-zonal sedimentation, suggesting that TBP has the ability to dynamically associate with the TFIID TAFs. The results of direct biochemical exchange experiments confirmed this hypothesis. Together, our results represent a concise molecular characterization of the general transcription factor TFIID from S. cerevisiae.

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Purification and characterization of fat body lipase from the Greater Wax Moth Galleria mellonella (Lepidoptera: Pyralidae)

10.1101/625129 ◽

2019 ◽

Author(s):

Rahma R.Z. Mahdy ◽

Shaimaa A. Mo’men ◽

Marah M. Abd El-Bar ◽

Emad M.S. Barakat

Keyword(s):

Metal Ions ◽

Kinetic Parameters ◽

Biochemical Characterization ◽

Galleria Mellonella ◽

Fat Body ◽

Specific Activity ◽

Larval Instar ◽

Gel Filtration ◽

Molecular Weights

AbstractLipid mobilization and transport in insects is under investigation, especially lipases and lipophorin because of their roles in energy production and transport of lipids at flying activity. The present study has been conducted to purify intracellular fat body lipase for the first time, from last larval instar of Galleria mellonella. Purification methods by combination of ammonium sulfate precipitation and gel filtration using Sephadex G-100 demonstrated that the amount of protein and the specific activity of fat body lipase were 0.008633±0.000551 mg/ml and 1.5754±0.1042 μmol/min/mg protein, respectively, with a 98.9 fold purity and recovery of 50.81%. Hence, the sephadex G-100 step was more effective in purification process. SDS-PAGE and zymogram revealed that fat body lipase showed two monomers with molecular weights of 178.8 and 62.6 kDa. Furthermore biochemical characterization of fat body lipase was carried out through testing its activities against several factors such as; different temperatures, pH ranges, metal ions and inhibitors ending by determination of their kinetic parameters with the use of p-Nitrophenyl butyrate (PNPB) as a substrate. The highest activities of enzyme were determined at the temperature ranges of 35-37°C and 37-40°C and pH ranges of 7-9 and 7–10. The partially purified enzyme showed significant stimulation by Ca2+, K+ and Na+ metal ions indicating that fat body lipase is metalloproteinase. Additionally, lipase activity was strongly inhibited by some inhibitors; phenylmethylsulfony fluoride (PMSF), ethylene-diaminetetractic acid (EDTA) and ethylene glycoltetraacetic acid (EGTA) providing an evidence of presence of serine residue and activation of enzymes by metal ions. Kinetic parameters were 301.95mM Km and 0.316 Umg−1 Vmax. By considering the purification of fat body lipase from larvae and using some inhibitors especially ion chelating agents, it is suggested to develop this study by using lipase inhibitors to reach a successful control of Galleria mellonella in the near future.

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Purification and characterization of casein kinase I from broccoli

Biochemical Journal ◽

10.1042/bj2930283 ◽

1993 ◽

Vol 293 (1) ◽

pp. 283-288 ◽

Cited By ~ 17

Author(s):

L J Klimczak ◽

A R Cashmore

Keyword(s):

Molecular Mass ◽

Specific Activity ◽

Gel Filtration ◽

Cross Reactivity ◽

Casein Kinase ◽

Casein Kinase I ◽

Native Molecular Mass ◽

Specificity And Sensitivity ◽

Sds Page

Casein kinase I from broccoli was purified approximately 65,000-fold by chromatography on phosphocellulose, phenyl-Sepharose, CM-Sephacel, and affinity chromatography on N-(2-aminoethyl)-5-chloroisoquinolone-8-sulphonamide (CKI-7)-Sepharose. The catalytic subunit of casein kinase I was identified as a 36-38 kDa polypeptide doublet by using the technique of activity gel assay after SDS/PAGE with casein as a gel-incorporated substrate. A silver-stained polypeptide doublet of the same molecular mass constituted at least 95% of the protein in the final preparation, corresponding to a specific activity of approximately 1800 nmol/min per mg of protein. The enzyme was found to be a monomer by gel filtration and glycerol gradient sedimentation; the native molecular mass was calculated to be 34.2 kDa. These characteristics, as well as other essential features of plant casein kinase I activity, such as substrate specificity and sensitivity to inhibitors, were found to be similar to those established for animal casein kinase I. Broccoli casein kinase I showed weak immunological cross-reactivity with antibodies raised against bovine casein kinase I.

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Biochemical characterization of aspartate aminotransferase allozymes from common wheat

Open Life Sciences ◽

10.2478/s11535-013-0240-7 ◽

2013 ◽

Vol 8 (12) ◽

pp. 1183-1193 ◽

Cited By ~ 1

Author(s):

Marcin Maciąga ◽

Michał Szkop ◽

Andrzej Paszkowski

Keyword(s):

Common Wheat ◽

Aspartate Aminotransferase ◽

Biochemical Characterization ◽

Gel Filtration ◽

Catalytic Efficiency ◽

Amino Acid Sequences ◽

Molecular Weights ◽

Sds Page ◽

Technique Comparison

AbstractSix allozymes of aspartate aminotransferase (AAT, EC 2.6.1.1): three plastidial (AAT-2 zone) and three cytosolic (AAT-3 zone) were isolated from common wheat (Triticum aestivum) seedlings and highly purified by a five-step purification procedure. The identity of the studied proteins was confirmed by mass spectrometry. The molecular weight of AAT allozymes determined by gel filtration was 72.4±3.6 kDa. The molecular weights of plastidial and cytosolic allozymes estimated by SDS-PAGE were 45.3 and 43.7 kDa, respectively. The apparent Michaelis constant (K m) values determined for four substrates appeared to be very similar for each allozyme. The values of the turnover number (k cat) and the k cat/K m ratio calculated for allozymes with L-aspartate as a leading substrate were in the range of 88.5–103.8 s−1/10,412–10,795 s−1 M−1 for AAT-2 zone and 4.6–7.0 s−1/527–700 s−1 M−1 for AAT-3 zone. These results clearly demonstrated much higher catalytic efficiency of AAT-2 allozymes. Therefore, partial sequences of cDNA encoding AATs from different zones were obtained using the RT-PCR technique. Comparison of the AAT-2 and AAT-3 amino acid sequences from active site regions revealed five non-conservative substitutions, which impact on the observed differences in the isozymes catalytic efficiency is discussed.

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Structural and biochemical characterization of a new type of lectin isolated from carp eggs

Biochemical Journal ◽

10.1042/bj20030413 ◽

2003 ◽

Vol 376 (2) ◽

pp. 433-440 ◽

Cited By ~ 30

Author(s):

Monica GALLIANO ◽

Lorenzo MINCHIOTTI ◽

Monica CAMPAGNOLI ◽

Alberto SALA ◽

Livia VISAI ◽

...

Keyword(s):

Horseshoe Crab ◽

Biochemical Characterization ◽

Carbohydrate Chain ◽

Gel Filtration ◽

Homologous Genes ◽

New Type ◽

Lectin Family ◽

Innate Defence ◽

Sepharose 4B

A previously unidentified glycoprotein present in the eggs of the carp (Cyprinus carpio) was isolated and structurally characterized. The protein binds to a Sepharose 4B matrix and can be eluted with 0.4 M N-acetylglucosamine. The protein has an apparent molecular mass of 26686.3 Da. On the basis of gel-filtration chromatography, the protein appears to be present in solution as a monomer. The sequence of its 238 amino acids, the position of its four disulphide bridges and the composition of its single N-linked carbohydrate chain were determined. The lectin shows a very low agglutinating activity for human A-type erythrocytes and interacts with both Gram-positive and -negative bacteria. These latter interactions are inhibited by N-acetylglucosamine. A database search shows that its amino acid sequence is similar to that of the members of an invertebrate lectin family that includes tachylectin-1. Tachylectin-1 is present in the amoebocytes of the horseshoe crab, Tachypleus tridentatus, and plays a role in the innate defence system of this species. Homologous genes are also present in other fish, having 85% identity with a gene expressed in the oocytes of the crucian carp (Carassius auratus gibelio) and 78% identity with a gene in the cDNA library of the zebrafish (Danio rerio).

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Rhizobium etli HrpW is a pectin-degrading enzyme and differs from phytopathogenic homologues in enzymically crucial tryptophan and glycine residues

Microbiology ◽

10.1099/mic.0.027599-0 ◽

2009 ◽

Vol 155 (9) ◽

pp. 3045-3054 ◽

Cited By ~ 18

Author(s):

Maarten Fauvart ◽

Natalie Verstraeten ◽

Bruno Dombrecht ◽

Ruth Venmans ◽

Serge Beullens ◽

...

Keyword(s):

Pathogenic Bacteria ◽

Plant Cell Wall ◽

Biochemical Characterization ◽

Sequence Data ◽

Functional Characterization ◽

Pectate Lyase ◽

Site Directed Mutagenesis ◽

Rhizobium Etli ◽

Lyase Activity

While establishing a nitrogen-fixing symbiosis with leguminous plants, rhizobia are faced with the problem of penetrating the plant cell wall at several stages of the infection process. One of the major components of this barrier is pectin, a heteropolysaccharide composed mainly of galacturonic acid subunits. So far, no enzymes capable of degrading pectin have been isolated from rhizobia. Here, we make an inventory of rhizobial candidate pectinolytic enzymes based on available genome sequence data and present an initial biochemical and functional characterization of a protein selected from this list. Rhizobium etli hrpW is associated with genes encoding a type III secretion system, a macromolecular structure that allows bacteria to directly inject so-called effector proteins into a eukaryotic host's cell cytosol and an essential virulence determinant of many Gram-negative pathogenic bacteria. In contrast to harpin HrpW from phytopathogens, R. etli HrpW possesses pectate lyase activity and is most active on highly methylated substrates. Through comparative sequence analysis, three amino acid residues crucial for the observed enzymic activity were identified: Trp192, Gly212 and Gly213. Their importance was confirmed by site-directed mutagenesis and biochemical characterization of the resulting proteins, with the tryptophan mutant showing no detectable pectate lyase activity and the double-glycine mutant's activity reduced by about 80 %. Surprisingly, despite hrpW expression being induced specifically on the plant root surface, a knockout mutation of the gene does not appear to affect symbiosis with the common bean Phaseolus vulgaris.

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