scholarly journals A novel Bacillus ligniniphilus catechol 2,3-dioxygenase shows unique substrate preference and metal requirement

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Peter Adewale ◽  
Alice Lang ◽  
Fang Huang ◽  
Daochen Zhu ◽  
Jianzhong Sun ◽  
...  
Keyword(s):  
Phylogenetic Analyses ◽  
Gel Filtration ◽  
Fold Increase ◽  
Ligninolytic Enzyme ◽  
Substrate Preference ◽  
Activity Increase ◽  
Family Protein ◽  
Single Carbon Source ◽  
Native Condition ◽  
Monomeric Structure

AbstractIdentification of novel enzymes from lignin degrading microorganisms will help to develop biotechnologies for biomass valorization and aromatic hydrocarbons degradation. Bacillus ligniniphilus L1 grows with alkaline lignin as the single carbon source and is a great candidate for ligninolytic enzyme identification. The first dioxygenase from strain L1 was heterologously expressed, purified, and characterized with an optimal temperature and pH of 32.5 °C and 7.4, respectively. It showed the highest activity with 3-ethylcatechol and significant activities with other substrates in the decreasing order of 3-ethylcatechol > 3-methylcatechol > 3-isopropyl catechol > 2, 3-dihydroxybiphenyl > 4-methylcatechol > catechol. It did not show activities against other tested substrates with similar structures. Most reported catechol 2,3-dioxygenases (C23Os) are Fe2+-dependent whereas Bacillus ligniniphilus catechol 2,3-dioxygenase (BLC23O) is more Mn2+- dependent. At 1 mM, Mn2+ led to 230-fold activity increase and Fe2+ led to 22-fold increase. Sequence comparison and phylogenetic analyses suggested that BL23O is different from other Mn-dependent enzymes and uniquely grouped with an uncharacterized vicinal oxygen chelate (VOC) family protein from Paenibacillus apiaries. Gel filtration analysis showed that BLC23O is a monomer under native condition. This is the first report of a C23O from Bacillus ligniniphilus L1 with unique substrate preference, metal-dependency, and monomeric structure.

scholarly journals Purification and Characterization of 2,6-β-d-Fructan 6-Levanbiohydrolase fromStreptomyces exfoliatus F3-2

2000 ◽  
Vol 66 (1) ◽  
pp. 252-256 ◽  
Author(s):  
Katsuichi Saito ◽  
Kazuya Kondo ◽  
Ichiro Kojima ◽  
Atsushi Yokota ◽  
Fusao Tomita
Keyword(s):  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Gel Filtration ◽  
Extracellular Enzyme ◽  
Molecular Weights ◽  
Sds Page ◽  
Monomeric Structure ◽  
Ph Range ◽  
Hydrolysis Of

ABSTRACT Streptomyces exfoliatus F3-2 produced an extracellular enzyme that converted levan, a β-2,6-linked fructan, into levanbiose. The enzyme was purified 50-fold from culture supernatant to give a single band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The molecular weights of this enzyme were 54,000 by SDS-PAGE and 60,000 by gel filtration, suggesting the monomeric structure of the enzyme. The isoelectric point of the enzyme was determined to be 4.7. The optimal pH and temperature of the enzyme for levan degradation were pH 5.5 and 60°C, respectively. The enzyme was stable in the pH range 3.5 to 8.0 and also up to 50°C. The enzyme gave levanbiose as a major degradation product from levan in an exo-acting manner. It was also found that this enzyme catalyzed hydrolysis of such fructooligosaccharides as 1-kestose, nystose, and 1-fructosylnystose by liberating fructose. Thus, this enzyme appeared to hydrolyze not only β-2,6-linkage of levan, but also β-2,1-linkage of fructooligosaccharides. From these data, the enzyme from S. exfoliatus F3-2 was identified as a novel 2,6-β-d-fructan 6-levanbiohydrolase (EC 3.2.1.64 ).

Interleukin-2-triggered Raf-1 expression, phosphorylation, and associated kinase activity increase through G1 and S in CD3-stimulated primary human T cells

1991 ◽  
Vol 11 (5) ◽  
pp. 2794-2803
Author(s):  
A Zmuidzinas ◽  
H J Mamon ◽  
T M Roberts ◽  
K A Smith
Keyword(s):  
T Cells ◽  
Kinase Activity ◽  
Interleukin 2 ◽  
Constitutive Expression ◽  
Fold Increase ◽  
Activity Increase ◽  
Phosphorylation State ◽  
Fourfold Increase ◽  
Protein Levels ◽  
Human T Cells

To gain further insight into the role of Raf-1 in normal cell growth, c-raf-1 mRNA expression, Raf-1 protein production, and Raf-1-associated kinase activity in normal human T cells were analyzed. In contrast to the constitutive expression of Raf-1 in continuously proliferating cell lines, c-raf-1 mRNA and Raf-1 protein levels were barely detectable in freshly isolated G0 T lymphocytes. Previous work with fibroblasts has suggested that Raf-1 plays a signaling role in the G0-G1 phase transition. In T cells, triggering via the T-cell antigen receptor (TCR)-CD3 complex (TCR/CD3) resulted in an approximately fourfold increase in c-raf-1 mRNA. In addition, the promotion of G1 progression by interleukin 2 (IL-2) was associated with a 5- to 10-fold immediate/early induction of c-raf-1 mRNA, resulting in up to a 12-fold increase in Raf-1 protein expression. TCR/CD3 activation did not alter the phosphorylation state of Raf-1, whereas interleukin 2 receptor stimulation resulted in a rapid increase in the phosphorylation state of a subpopulation of Raf-1 molecules progressively increasing throughout G1. These findings were complemented by assays for Raf-1-associated kinase activity which revealed a gradual accumulation of serine and threonine autokinase activity in Raf-1 immunoprecipitates during G1, which remained elevated throughout DNA replication.

scholarly journals Macromolecular Chromogenic Substrates for Measuring Proteinase Activity

2001 ◽  
Vol 47 (2) ◽  
pp. 215-222 ◽  
Author(s):  
Glen L Hortin ◽  
Ilka Warshawsky ◽  
Maryline Laude-Sharp
Keyword(s):  
Gel Filtration ◽  
Fold Increase ◽  
Coagulation Factors ◽  
Filtration Efficiency ◽  
Hydrodynamic Size ◽  
Chromogenic Substrates ◽  
Functional Assays ◽  
Α2 Macroglobulin ◽  
The Impact ◽  
Physiological Substrates

Abstract Background: Proteinase activities are often measured using chromogenic substrates that are much smaller than physiological substrates. Methods: The hydrodynamic size of macromolecular substrates (macrosubstrates) prepared by linking small chromogenic substrates to polyethylene glycol was determined by gel filtration. Efficiency of macrosubstrate cleavage by proteinases and α2-macroglobulin-proteinase complexes was monitored spectrophotometrically. Results: Macrosubstrates had hydrodynamic radii of ∼20 Å, similar to proteins with a molecular weight of 18 000. Different macrosubstrates served as efficient substrates for chymotrypsin, trypsin, and thrombin. Linking small substrates to a polymer variably affected substrate efficiency, with the impact on activity ranging from a 60-fold decrease to a 30-fold increase. Proteinases complexed with α2-macroglobulin had ∼10-fold lower activity vs macrosubstrates than small substrates. Conclusions: Macrosubstrates are efficient substrates that allow decreased measurement of sterically hindered proteinase molecules such as α2-macroglobulin-proteinase complexes. Thus, macrosubstrates may provide more accurate functional assays of proteinases such as coagulation factors.

scholarly journals A New Procedure for Purification of Crotalase From Crotalus Adamanteus Venom

1977 ◽  
Author(s):  
F.S. Markland ◽  
J. Chou ◽  
Y. Shih ◽  
H. Pirkle
Keyword(s):  
Sodium Chloride ◽  
Large Scale ◽  
Specific Activity ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Fold Increase ◽  
Tris Buffer ◽  
High Yield ◽  
Crude Venom ◽  
Diamondback Rattlesnake

A new procedure has been developed for large scale, rapid purification of crotalase, the thrombin-1ike enzyme from the venom of the eastern diamondback rattlesnake (Crotalus adamanteus). The three step procedure involves: (1) molecular sieve chromatography on Sephadex G-100 in 0.04 M Tris buffer containing 0.10 M sodium chloride, pH 7.1; (2) gradient elution from DEAE-cellulose with sodium acetate buffer, pH 7.0; and (3) affinity chromatography on p-aminobenzamidine Sepharose using a spacer of 6-aminohexanoic acid. Crotalase was eluted from the affinity resin by 0.05 M Tris buffer containing 0.10 M sodium chloride and 0.15 M benzamidine-hydrochloride, pH 9.0, after first washing with the Tris buffer containing 0.40 M sodium chloride. From the crude venom, pure enzyme was obtained with an overall recovery of 40-60% of clotting activity and a 90-100 fold increase in specific activity. Crotalase was shown to be pure by Polyacrylamide disk gel electrophoresis which gave one band. The molecular weight was estimated to be approximately 31,000 by gel filtration on a calibrated Sephadex G-100 column. Amino acid analysis was performed and the composition was shown to be very similar to that reported earlier (F.S. Markland and P.S. Damus, J. Biol. Chem. 246: 6460, 1971). Clotting activity of the enzyme was not inhibited by heparin, either with or without plasma, whereas, thrombin was rapidly inactivated by heparin in the presence of plasma. In conclusion, we have developed a rapid and reproducible procedure for isolation in high yield of large quantities of the thrombin-like enzyme from the venom of the eastern diamondback rattlesnake. Studies are continuing on the primary structure and possible clinical applications of this enzyme.

scholarly journals Structure-Function Studies of Factor VIII/Von Willebrand Protein(s)

1977 ◽  
Author(s):  
Patrick A. McKee
Keyword(s):  
Factor Viii ◽  
Single Molecule ◽  
Gel Filtration ◽  
Protein S ◽  
Fold Increase ◽  
Procoagulant Activity ◽  
Void Volume ◽  
Pas Staining ◽  
Von Willebrand ◽  
Willebrand Factor

Factor VIII (FVIII) procoagulant activity was initially thought to be a glycoprotein with a molecular weight (MW) >1 million and composed of disulfide-1inked ~200,000 MW subunits. A protein with similar properties, except lacking procoagulant activity, is in hemophilic plasma; it was identical to normal FVIII by SDS-gel analyses, isoelectric focusing, and PAS staining. Subsequently it was shown that the FVIII glycoprotein also has von Willebrand factor (vWF) activity, suggesting that both FVIII and vWF activities might be properties of the same molecule. When the FVIII/vWF protein(s) is rechromatographed on 4% agarose and 0.25 M CaCl2, virtually all the protein and vWF activity elute in the void volume, but most of the FVIII procoagulant activity elutes much later. The extent of separation of the two activities depends on the amount of protein applied to the column. Also, exposure of the FVIII/vWF to thrombin before gel filtration strikingly accentuates separation of the two activities. The reduced SDS-gel pattern of the void volume protein peak showed the 200,000 MW subunit while that of the procoagulant peak contained several subunit bands which ranged from ~30,000–100,000 MW. Removal of sialic acid from FVIII/vWF is associated with reduced ristocetin induced platelet aggregation and causes a 50-fold increase in the rate of clearance of protein from the circulation by the hepatocyte. Currently, our data suggest that FVIII procoagulant and vWF activities are properties of a single molecule composed of disulfide-bound identical subunits. Cleavage by thrombin then results in FVIII procoagulant activity. Additional cleavages, to which the molecule appears very sensitive, results in FVIII inactivation. The vWF activity is very stable—even to proteolysis—and it appears to be a function of the carbohydrate side chains of the molecule.

scholarly journals Genomic Analysis Revealed a Convergent Evolution of LINE-1 in Coat Color: A Case Study in Water Buffaloes (Bubalus bubalis)

2020 ◽  
Author(s):  
Dong Liang ◽  
Pengju Zhao ◽  
Jingfang Si ◽  
Lingzhao Fang ◽  
Erola Pairo-Castineira ◽  
...  
Keyword(s):  
Genome Wide Association Study ◽  
Population Genomics ◽  
Coat Color ◽  
Phylogenetic Analyses ◽  
Regulation Of Gene Expression ◽  
Fold Increase ◽  
Bubalus Bubalis ◽  
Proximal Promoter ◽  
Causative Mutation ◽  
Swamp Buffalo

Abstract Visible pigmentation phenotypes can be used to explore the regulation of gene expression and the evolution of coat color patterns in animals. Here, we performed whole-genome and RNA sequencing and applied genome-wide association study, comparative population genomics and biological experiments to show that the 2,809-bp-long LINE-1 insertion in the ASIP (agouti signaling protein) gene is the causative mutation for the white coat phenotype in swamp buffalo (Bubalus bubalis). This LINE-1 insertion (3′ truncated and containing only 5′ UTR) functions as a strong proximal promoter that leads to a 10-fold increase in the transcription of ASIP in white buffalo skin. The 165 bp of 5′ UTR transcribed from the LINE-1 is spliced into the first coding exon of ASIP, resulting in a chimeric transcript. The increased expression of ASIP prevents melanocyte maturation, leading to the absence of pigment in white buffalo skin and hairs. Phylogenetic analyses indicate that the white buffalo-specific ASIP allele originated from a recent genetic transposition event in swamp buffalo. Interestingly, as a similar LINE-1 insertion has been identified in the cattle ASIP gene, we discuss the convergent mechanism of coat color evolution in the Bovini tribe.

scholarly journals Expression of a Shiga-Like Toxin during Plastic Colonization by Two Multidrug-Resistant Bacteria, Aeromonas hydrophila RIT668 and Citrobacter freundii RIT669, Isolated from Endangered Turtles (Clemmys guttata)

2020 ◽  
Vol 8 (8) ◽  
pp. 1172 ◽  
Author(s):  
Seema G. Thomas ◽  
Maryah A. Glover ◽  
Anutthaman Parthasarathy ◽  
Narayan H. Wong ◽  
Paul A. Shipman ◽  
...  
Keyword(s):  
Aeromonas Hydrophila ◽  
Biofilm Formation ◽  
Phylogenetic Analyses ◽  
Regulatory Protein ◽  
Antibiotic Resistance Genes ◽  
Fold Increase ◽  
Resistant Bacteria ◽  
Citrobacter Freundii ◽  
Protein Subunit ◽  
Clemmys Guttata

Aeromonas hydrophila RIT668 and Citrobacter freundii RIT669 were isolated from endangered spotted turtles (Clemmys guttata). Whole-genome sequencing, annotation and phylogenetic analyses of the genomes revealed that the closest relative of RIT668 is A. hydrophila ATCC 7966 and Citrobacter portucalensis A60 for RIT669. Resistome analysis showed that A. hydrophila and C. freundii harbor six and 19 different antibiotic resistance genes, respectively. Both bacteria colonize polyethylene and polypropylene, which are common plastics, found in the environment and are used to fabricate medical devices. The expression of six biofilm-related genes—biofilm peroxide resistance protein (bsmA), biofilm formation regulatory protein subunit R (bssR), biofilm formation regulatory protein subunit S (bssS), biofilm formation regulator (hmsP), toxin-antitoxin biofilm protein (tabA) and transcriptional activator of curli operon (csgD)—and two virulence factors—Vi antigen-related gene (viaB) and Shiga-like toxin (slt-II)—was investigated by RT-PCR. A. hydrophila displayed a >2-fold increase in slt-II expression in cells adhering to both polymers, C. freundii adhering on polyethylene displayed a >2-fold, and on polypropylene a >6-fold upregulation of slt-II. Thus, the two new isolates are potential pathogens owing to their drug resistance, surface colonization and upregulation of a slt-II-type diarrheal toxin on polymer surfaces.

scholarly journals Tyrosine phosphorylation of protein kinase CK2 by Src-related tyrosine kinases correlates with increased catalytic activity

2003 ◽  
Vol 372 (3) ◽  
pp. 841-849 ◽  
Author(s):  
Arianna DONELLA-DEANA ◽  
Luca CESARO ◽  
Stefania SARNO ◽  
Maria RUZZENE ◽  
Anna Maria BRUNATI ◽  
...  
Keyword(s):  
Catalytic Activity ◽  
Protein Kinase ◽  
Tyrosine Phosphorylation ◽  
Tyrosine Kinases ◽  
Fold Increase ◽  
Jurkat Cells ◽  
Src Inhibitor ◽  
Phosphatase Treatment ◽  
Family Protein ◽  
Kinase Ck2

Casein kinase-2 (CK2) is a pleiotropic and constitutively active serine/threonine protein kinase composed of two catalytic (α and/or α´) and two regulatory β-subunits, whose regulation is still not well understood. In the present study, we show that the catalytic subunits of human CK2, but not the regulatory β-subunits, are readily phosphorylated by the Src family protein tyrosine kinases Lyn and c-Fgr to a stoichiometry approaching 2 mol phosphotyrosine/mol CK2α with a concomitant 3-fold increase in catalytic activity. We also show that endogenous CK2α becomes tyrosine-phosphorylated in pervanadate-treated Jurkat cells. Both tyrosine phosphorylation and stimulation of activity are suppressed by the specific Src inhibitor 4-amino-5-(4-chlorophenyl)-7-(t-butyl)pyrazolo[3,4-d]pyrimidine. By comparison, mutations giving rise to inactive forms of CK2α do not abrogate and, in some cases, stimulate Lyn and c-Fgr-dependent tyrosine phosphorylation of CK2. Several radiolabelled phosphopeptides could be resolved by HPLC, following tryptic digestion of CK2α that had been phosphoradiolabelled by incubation with [32P]ATP and c-Fgr. The most prominent phosphopeptide co-migrates with a synthetic peptide encompassing the 248–268 sequence, phosphorylated previously by c-Fgr at Tyr255in vitro. The identification of Tyr255 as a phosphorylated residue was also supported by MS sequencing of both the phosphorylated and non-phosphorylated 248–268 tryptic fragments from CK2α and by on-target phosphatase treatment. A CK2α mutant in which Tyr255 was replaced by phenylalanine proved less susceptible to phosphorylation and refractory to stimulation by c-Fgr.

EVIDENCE FOR THYROCALCITONIN BINDING TO PROTEIN IN PLASMA

1969 ◽  
Vol 43 (1) ◽  
pp. 73-81 ◽  
Author(s):  
J. LEGGATE ◽  
A. D. CARE ◽  
S. C. FRAZER
Keyword(s):  
Biological Activity ◽  
Plasma Concentration ◽  
Protein Content ◽  
Silica Gel ◽  
Plasma Proteins ◽  
Gel Filtration ◽  
Fold Increase ◽  
Simple Method ◽  
Preparative Ultracentrifugation ◽  
Venous Plasma

SUMMARY A simple method is described for concentrating thyrocalcitonin from plasma by adsorption onto finely divided silica gel. An approximately 20-fold increase in biological activity with respect to protein content has been obtained with recoveries of added material of about 80%, allowing subsequent fractionation and bioassay of the fractions. Porcine thyrocalcitonin was added to either porcine or human plasma to give concentrations within the range observed in porcine thyroid venous plasma. Concentration on silica gel followed by gel filtration on Sephadex G 50 resulted in separation of the biological activity into two fractions, one of which was associated with the plasma proteins. A similar result was obtained with porcine thyroid venous plasma containing endogenous thyrocalcitonin. Preparative ultracentrifugation of plasma rich in thyrocalcitonin also provided evidence suggestive of some protein binding of the hormone. It is concluded that thyrocalcitonin is carried in plasma partly free and partly bound to plasma protein.

A dipeptidylpeptidase secreted by Fasciola hepatica

Parasitology ◽  
1994 ◽  
Vol 109 (1) ◽  
pp. 113-118 ◽  
Author(s):  
C. Carmona ◽  
S. McGonigle ◽  
A. J. Dowd ◽  
A. M. Smith ◽  
S. Coughlan ◽  
...  
Keyword(s):  
Molecular Weight ◽  
Ion Exchange ◽  
Liver Fluke ◽  
Fasciola Hepatica ◽  
Gel Filtration ◽  
Serine Proteinase ◽  
Ion Exchange Chromatography ◽  
Exchange Chromatography ◽  
Substrate Preference ◽  
Proteolytic Digestion

SUMMARYA dipeptidylpeptidase (DPP) was isolated from Fasciola hepatica by gel-filtration and ion-exchange chromatography. The exoproteinase is secreted by newly excysted juveniles, immature and mature flukes. The liver fluke DPP is a serine proteinase of molecular weight > 200 kDa and differs from previously characterized mammalian DPPs in its substrate preference and susceptibility to inactivation by inhibitors. The parasite DPP may function in the latter stages of the proteolytic digestion of host macromolecules. In this manner, the enzyme may be important in providing the parasite with dipeptides that could be absorbed through the intestine as nutrient.

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