scholarly journals Probing enzymatic activity – a radical approach

2020 ◽  
Vol 11 (11) ◽  
pp. 2967-2972 ◽  
Author(s):  
Neil C. Taylor ◽  
Gary Hessman ◽  
Holger B. Kramer ◽  
Joanna F. McGouran
Keyword(s):  
Enzymatic Activity ◽  
Specific Binding ◽  
Deubiquitinating Enzymes ◽  
Binding Interaction ◽  
Latent Activity ◽  
Radical Approach

Latent activity-based probes have been developed for deubiquitinating enzymes using a thiol–ene strategy, labelling following a specific binding interaction.

Differential Digestion of Different LMWHs by Heparinase-I and Heparinase-II: Drug Developmental Implications.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 4084-4084
Author(s):  
Jyothi Maddineni ◽  
Walter P. Jeske ◽  
Omer Iqbal ◽  
Debra A. Hoppensteadt ◽  
Jawed Fareed
Keyword(s):  
Molecular Weight ◽  
Enzymatic Activity ◽  
High Performance ◽  
Uronic Acid ◽  
Specific Binding ◽  
Average Molecular Weight ◽  
Uv Detection ◽  
Molecular Profile ◽  
Heparinase I ◽  
Differential Digestion

Abstract The purpose of this study was to determine the differential digestion of two different batches of branded enoxaparin (Aventis, USA), three generic versions of enoxaparin (GlandPharma, India; Lazar, Argentina; and Biochemie, Brazil), dalteparin (Pfizer, USA) and tinzaparin (Leo, USA) by heparinase-I and heparinase-II. Heparinase-I (Ibex Tech., Montreal, Canada) and heparinase-II (Ram Sasisekharan, MIT, Cambridge, MA) were isolated from Flavobacterium heparinum. The substrate specificity of these enzymes has been inferred from the reducing and non-reducing terminal structures of the di and oligosaccharides formed by digesting heparin. Heparinase-I cleaves the glucosaminidic linkage in GlcN (N-sulfate) a 1–4 IdceA (2-sulfate) and endures C-6 sulfation of hexosamine unit. More susceptibility of polymers such as heparin than oligomers to this enzymatic depolymerization indicates the size dependency of this enzymatic activity. Heparinase-II cleaves all the glucosaminidic linkages in heparin independent of O-and/or N-sulfation as well as the type of uronic acid residue. The non-sulfated substrates are somewhat resistant to this enzyme. The glucosaminidic linkage adjacent to a 3-O-sulfated GlcN residue and the innermost glucosaminidic linkage right next to the glycosaminoglycan-protein linkage region of proteoglycan are resistant to this enzymatic activity (Sugahara et al., 1994, Glycobiology 4, 535–544). In this study, several low molecular weight heparins (LMWHs) produced from different depolymerization methods of unfractionated heparin were digested with heparinase-I and heparinase-II to determine the differential digestion of these two enzymes. Eight different LMWHs with average molecular weight (MW) ranging from 3425 to 6281 Da (in UV detection at 205nm) were prepared at a concentration of 10mg/ml in 0.3M Na2SO4. Each LMWH was incubated with these enzymes (1.0 U/mL) separately for 30 minutes at 37° C in the presence of calcium Following the incubation, the samples were heated at 100° C to inactivate enzymatic activity. The molecular weight profiling of these samples was determined by using a gel permeation chromatography-high performance liquid chromatography (GPC-HPLC) system with UV detection at 205nm. A narrow range calibration method comprised of oligosaccharides and polysaccharides was used to determine the relative molecular profile of the native and digested products. The LMWHs were digested to LMW oligosaccharides with average MW of 1510± 275 Da with heparinase-I and 3071± 1044 Da with heparinase-II. The extent of digestion observed with all the LMWHs was more with heparinase-I than heparinase-II. This may be due to the different specific binding sites available for these enzymes and the requirement of sulfation at different positions in GlcN/uronic acid residues. All the LMWHs were equally digested into oligosaccharides (di, tetra and hexa) with heparinase-I. However heparinase-II resulted in the formation of only hexa, octa and decasaccharides. These results show that the LMWHs were more susceptible to heparinase-I than heparinase-II. The possible reason for the less susceptibility of these compounds to heparinase-II is likely due to the oligosaccharide composition and degree and pattern of sulfation in GlcN/uronic acid residues of these compounds. The heparinase-I and heparinase-II digestion can therefore be used in the profiling of these agents.

scholarly journals Binding of NAD+-Glycohydrolase to Streptolysin O Stabilizes Both Toxins and Promotes Virulence of Group AStreptococcus

mBio ◽  
2017 ◽  
Vol 8 (5) ◽  
Author(s):  
Jorge J. Velarde ◽  
Maghnus O’Seaghdha ◽  
Buket Baddal ◽  
Benedicte Bastiat-Sempe ◽  
Michael R. Wessels
Keyword(s):  
Enzymatic Activity ◽  
Mammalian Cells ◽  
Catalytic Domain ◽  
Critical Role ◽  
Point Mutations ◽  
Full Length ◽  
Missense Mutations ◽  
Binding Interaction ◽  
Nad Glycohydrolase ◽  
Streptolysin O

ABSTRACTThe globally dominant, invasive M1T1 strain of group AStreptococcus(GAS) harbors polymorphisms in the promoter region of an operon that contains the genes encoding streptolysin O (SLO) and NAD+-glycohydrolase (NADase), resulting in high-level expression of these toxins. While both toxins have been shown experimentally to contribute to pathogenesis, many GAS isolates lack detectable NADase activity. DNA sequencing of such strains has revealed that reduced or absent enzymatic activity can be associated with a variety of point mutations innga, the gene encoding NADase; a commonly observed polymorphism associated with near-complete abrogation of activity is a substitution of aspartic acid for glycine at position 330 (G330D). However,ngahas not been observed to contain early termination codons or mutations that would result in a truncated protein, even when the gene product contains missense mutations that abrogate enzymatic activity. It has been suggested that NADase that lacks NAD-glycohydrolase activity retains an as-yet-unidentified inherent cytotoxicity to mammalian cells and thus is still a potent virulence factor. We now show that expression of NADase, either enzymatically active or inactive, augments SLO-mediated toxicity for keratinocytes. In culture supernatants, SLO and NADase are mutually interdependent for protein stability. We demonstrate that the two proteins interact in solution and that both the translocation domain and catalytic domain of NADase are required for maximal binding between the two toxins. We conclude that binding of NADase to SLO stabilizes both toxins, thereby enhancing GAS virulence.IMPORTANCEThe global increase in invasive GAS infections in the 1980s was associated with the emergence of an M1T1 clone that harbors a 36-kb pathogenicity island, which codes for increased expression of toxins SLO and NADase. Polymorphisms in NADase that render it catalytically inactive can be detected in clinical isolates, including invasive strains. However, such isolates continue to produce full-length NADase. The rationale for this observation is not completely understood. This study characterizes the binding interaction between NADase and SLO and reports that the expression of each toxin is crucial for maximal expression and stability of the other. By this mechanism, the presence of both toxins increases toxicity to keratinocytes and is predicted to enhance GAS survival in the human host. These observations provide an explanation for conservation of full-length NADase expression even when it lacks enzymatic activity and suggest a critical role for binding of NADase to SLO in GAS pathogenesis.

Binding Analysis of a Novel Peptide to Plasmodium falciparum Knob-Associated Histidine Rich Protein (KAHRP)

2007 ◽  
Vol 1 (1) ◽  
Author(s):  
J. Preston ◽  
L. Zeng ◽  
C. G. Takoudis ◽  
X. Li ◽  
A. Chishti
Keyword(s):  
Plasmodium Falciparum ◽  
Amino Acid ◽  
Red Blood Cells ◽  
Blood Vessels ◽  
Real Time ◽  
Optical Sensor ◽  
Blood Cells ◽  
Specific Binding ◽  
Binding Interaction ◽  
The Real

Knob-associated histidine rich protein (KAHRP) is secreted by Plasmodium falciparum in infected red blood cells. This protein is required for the production of surface protrusions called knobs, which have been shown to be crucial for the adherence of P. falciparum-infected erythrocytes (Pf-IRBC) to the endothelia of small blood vessels. KP-AP, a 10-amino acid (AA) peptide (FITRANDTSK), binds specifically with KAHRP in preliminary studies. KP-AP is expected to disrupt knob formation and prohibit adherence of Pf-IRBC to blood vessels and greatly reduce the pathogenicity of the parasite. This paper describes an investigation into the binding interaction between biotinylated KP-AP (Biotin-AP) and a segment of KAHRP. ELISA and the real-time bio-interaction optical sensor, BIAcore are the methods of detection. The specific binding was confirmed with ELISA and the KD value was estimated to be 1.2 μM. Binding was not detected with BIAcore, most likely due to the reduced flexibility of Biotin-AP while immobilized on the sensor chip.

Study on Specific Binding Interaction Between Protein and DNA Aptamer via Dynamic Force Spectroscopy

2013 ◽  
Author(s):  
Xiao Ma ◽  
Pranav Shrotriya
Keyword(s):  
Loading Rate ◽  
Specific Binding ◽  
Dna Aptamer ◽  
Dynamic Force ◽  
Binding Interaction ◽  
Force Microscopy ◽  
Bond Breakage ◽  
Coagulation Protein ◽  
Molecular Bond ◽  
Thiolated Aptamer

Recently the need to design nanoscale, sensitive and flexible bio-sensors or biotic-abiotic interface keeps increasing. One of the essential challenges on this objective is to grasp a thorough understanding of the mechanism governing binding interaction between bio-molecules. In this study we aim to demonstrate the binding specificity and reveal force interaction between the anti-coagulation protein thrombin and the single-stranded DNA thrombin aptamer by application of Atomic Force Microscopy (AFM). The thiolated aptamer was deposited onto gold substrate, and then repeatedly brought into contact with a thrombin-coated AFM tip, and force drop-offs during the pull-off were measured to determine the unbinding force between the thrombin-aptamer pair. The results from experiment show that the thrombin-aptamer pair has specific binding and the force between the pair exhibits loading rate dependence. It was shown that the binding forces of the thrombin-aptamer interaction increases with growth of loading rates. The average binding force for a single thrombin/aptamer pair increased from 20 pN to 40 pN, with loading rate changes from 500pN/s to 13500pN/s. Distribution of the unbinding forces measured for each loading rate can be explained on the basis of single energy barrier model for molecular bond breakage.

scholarly journals SPECIFIC BINDING OF NERVE GROWTH FACTOR (NGF) BY MURINE C 1300 NEUROBLASTOMA CELLS

1974 ◽  
Vol 140 (2) ◽  
pp. 437-451 ◽  
Author(s):  
R. Revoltella ◽  
L. Bertouni ◽  
M. Pediconi ◽  
E. Vigneti
Keyword(s):  
Nerve Growth Factor ◽  
Growth Factor ◽  
Binding Capacity ◽  
Specific Binding ◽  
Membrane Surface ◽  
Neuroblastoma Cells ◽  
Molecular Heterogeneity ◽  
High Avidity ◽  
Binding Interaction ◽  
Nerve Growth

Murine C 1300 neuroblastoma cells bind with high avidity on their membrane surface the nerve growth factor (NGF), a protein capable of inducing differentiation of sympathetic nerve cells. The total binding capacity of NGF by the cells was quantitatively measured by a radioimmunoassay technique, using 125I-labeled NGF. An average number of about 106 molecules of NGF could be bound, at saturation, by each cell with an average relative association constant of about 107 liters/mol. Using synchronized cells, it was found, however, that either the number of molecules of ligand bound or the avidity of the binding interaction between NGF and cells varied depending upon their growth cycle, the maximal-binding occurring during the G1 and early S phase. Binding of [125I]NGF was suppressed by trypsin treatment of the cells, however new receptor sites were rapidly replaced onto the membrane surface within 1–2 h. Cells exposed to 3 M KCl released into the supernate a protein product exhibiting similar high avidity for NGF. Acrylamide gel electrophoresis suggested a restricted molecular heterogeneity of this product, with a major component in the 52,000 mol wt region. Antibodies made specific to this protein were capable, in the absence of the complement, of inhibiting the binding of [125I]NGF by the cells and in the presence of the complement they killed them.

Antihypertensive activity of a quinoline appended chalcone derivative and its site specific binding interaction with a relevant target carrier protein

RSC Advances ◽  
2015 ◽  
Vol 5 (80) ◽  
pp. 65496-65513 ◽  
Author(s):  
Himank Kumar ◽  
Vinod Devaraji ◽  
Ritika Joshi ◽  
Manojkumar Jadhao ◽  
Piyush Ahirkar ◽  
...  
Keyword(s):  
Therapeutic Target ◽  
Specific Binding ◽  
Transport Protein ◽  
Clear Picture ◽  
Antihypertensive Activity ◽  
Carrier Protein ◽  
Binding Interaction ◽  
Site Specific ◽  
Chalcone Derivative ◽  
Delivery Mechanism

The usefulness of heterocyclic chalcone derivative as a therapeutic target in controlling hypertension and its site specific binding interaction with model transport protein to get a clear picture about its delivery mechanism.

scholarly journals Functional Bacillus thuringiensis Cyt1Aa Is Necessary To Synergize Lysinibacillus sphaericus Binary Toxin (Bin) against Bin-Resistant and -Refractory Mosquito Species

2020 ◽  
Vol 86 (7) ◽  
Author(s):  
Nathaly Alexandre Nascimento ◽  
Mary Carmen Torres-Quintero ◽  
Samira López Molina ◽  
Sabino Pacheco ◽  
Tatiany Patrícia Romão ◽  
...  
Keyword(s):  
Bacillus Thuringiensis ◽  
Insect Resistance ◽  
Mosquito Control ◽  
Specific Binding ◽  
Mosquito Species ◽  
Binary Toxin ◽  
Binding Interaction ◽  
Data Set ◽  
Lysinibacillus Sphaericus ◽  
Content Type

ABSTRACT The binary (Bin) toxin from Lysinibacillus sphaericus is effective to mosquito larvae, but its utilization is threatened by the development of insect resistance. Bin toxin is composed of the BinB subunit required for binding to midgut receptors and the BinA subunit that causes toxicity after cell internalization, mediated by BinB. Culex quinquefasciatus resistance to this toxin is caused by mutations that prevent expression of Bin toxin receptors in the midgut. Previously, it was shown that the Cyt1Aa toxin from Bacillus thuringiensis subsp. israelensis restores Bin toxicity to Bin-resistant C. quinquefasciatus and to Aedes aegypti larvae, which are naturally devoid of functional Bin receptors. Our goal was to elucidate the mechanism involved in Cyt1Aa synergism with Bin in such larvae. In vivo assays showed that the mixture of Bin toxin, or its BinA subunit, with Cyt1Aa was effective to kill resistant larvae. However, no specific binding interaction between Cyt1Aa and the Bin toxin, or its subunits, was observed. The synergy between Cyt1Aa and Bin toxins is dependent on functional Cyt1Aa, as demonstrated by using the nontoxic Cyt1AaV122E mutant toxin affected in oligomerization and membrane insertion, which was unable to synergize Bin toxicity in resistant larvae. The synergism correlated with the internalization of Bin or BinA into anterior and medium midgut epithelial cells, which occurred only in larvae treated with wild-type Cyt1Aa toxin. This toxin is able to overcome failures in the binding step involving BinB receptor by allowing the internalization of Bin toxin, or its BinA subunit, into the midgut cells. IMPORTANCE One promising management strategy for mosquito control is the utilization of a mixture of L. sphaericus and B. thuringiensis subsp. israelensis insecticidal toxins. From this set, Bin and Cyt1Aa toxins synergize and display toxicity to resistant C. quinquefasciatus and to A. aegypti larvae, whose midgut cells lack Bin toxin receptors. Our data set provides evidence that functional Cyt1Aa is essential for internalization of Bin or its BinA subunit into such cells, but binding interaction between Bin and Cyt1Aa is not observed. Thus, this mechanism contrasts with that for the synergy between Cyt1Aa and the B. thuringiensis subsp. israelensis Cry toxins, where active Cyt1Aa is not necessary but a specific binding between Cry and Cyt1Aa is required. Our study established the initial molecular basis of the synergy between Bin and Cyt1Aa, and these findings enlarge our knowledge of their mode of action, which could help to develop improved strategies to cope with insect resistance.

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