Blocking factors and the isolation of glutathione transferases from Hymenolepis diminuta (Cestoda: Cyclophyllidea)

SummaryFour acidic glutathione (GSH) transferase forms were isolated from the cytosol of the adult cestode Hymenolepis diminuta by hydroxylapatite chromatography, glutathione-affinity chromatography and chromatofocusing, pH 7–5. The enzymes were dimers of subunit size approximately 24 kDa and accounted for at least 3% of the total soluble protein. The major GSH transferase had limited catalytic activity but may interact with a range of ligands and function as a binding/passive detoxification protein. An endogenous factor interfered with the binding of the crude cytosolic GSH transferase activity to glutathione-dependent affinity matrices but, following partial purification, the GSH transferase activity successfully interacted with the glutathione affinity matrix.

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Partial purification and characterization of a soluble acid phosphatase from the tapeworm, Hymenolepis diminuta

Journal of Helminthology ◽

10.1017/s0022149x00010543 ◽

1991 ◽

Vol 65 (2) ◽

pp. 103-110

Author(s):

Michael J. Bumbulis ◽

Peter W. Pappas

Keyword(s):

Acid Phosphatase ◽

Cellular Localization ◽

Specific Activity ◽

Sodium Dodecyl ◽

Hymenolepis Diminuta ◽

Partial Purification ◽

Transferase Activity ◽

Crude Enzyme Preparation ◽

Ammonium Sulphate Precipitation ◽

And Function

ABSTRACTAn acid phosphatase activity (APA; EC 3.1.3.2) was demonstrated in homogenates of adult Hymenolepis diminuta. The APA was soluble based on the observation that it did not sediment at 130 000 g. APA was partially purified using a combination of differential centrifugation, ammonium sulphate precipitation, chloroform extraction, and gel and fast-protein-liquid-chromatography. This combination of techniques resulted in a preparation with a specific activity approximately 500 times greater than the crude enzyme preparation. The temperature and pH optima of the partially purified APA were 44°C and pH 5·0. The enzyme appeared to be a monomer with a molecular weight of approximately 62 000. APA had a higher affinity for a greater activity towards aromatic than aliphatic phosphoesters and phosphoryl transferase activity was demonstrable using 1-butanol and ethylene glycol as acceptors. APA was inhibited significantly by sodium dodecyl sulphate, fluoride, molybdate and tartrate, but CuSO4 and Fast Garnet GBC were poor inhibitors. The precise cellular localization and function of this enzyme remains unknown since it possesses characteristics of both cytoplasmic and lysosomal APA's of other organisms.

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Altering the Receptor−Effector Ratio by Transgenic Overexpression of Type V Adenylyl Cyclase: Enhanced Basal Catalytic Activity and Function without Increased Cardiomyocyte β-Adrenergic Signalling†

Biochemistry ◽

10.1021/bi991619k ◽

1999 ◽

Vol 38 (50) ◽

pp. 16706-16713 ◽

Cited By ~ 47

Author(s):

Nicole M. Tepe ◽

John N. Lorenz ◽

Atsuko Yatani ◽

Rajesh Dash ◽

Evangelia G. Kranias ◽

...

Keyword(s):

Catalytic Activity ◽

Adenylyl Cyclase ◽

Type V ◽

And Function

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Functional characterization of the non-catalytic ectodomains of the nucleotide pyrophosphatase/phosphodiesterase NPP1

Biochemical Journal ◽

10.1042/bj20021943 ◽

2003 ◽

Vol 371 (2) ◽

pp. 321-330 ◽

Cited By ~ 38

Author(s):

Rik GIJSBERS ◽

Hugo CEULEMANS ◽

Mathieu BOLLEN

Keyword(s):

Catalytic Activity ◽

Catalytic Domain ◽

Transmembrane Domain ◽

Functional Characterization ◽

Structure Stability ◽

Subunit Structure ◽

Terminal Domain ◽

Nucleotide Pyrophosphatase ◽

Domain Truncation ◽

And Function

The ubiquitous nucleotide pyrophosphatases/phosphodiesterases NPP1–3 consist of a short intracellular N-terminal domain, a single transmembrane domain and a large extracellular part, comprising two somatomedin-B-like domains, a catalytic domain and a poorly defined C-terminal domain. We show here that the C-terminal domain of NPP1–3 is structurally related to a family of DNA/RNA non-specific endonucleases. However, none of the residues that are essential for catalysis by the endonucleases are conserved in NPP1–NPP3, suggesting that the nuclease-like domain of NPP1–3 does not represent a second catalytic domain. Truncation analysis revealed that the nuclease-like domain of NPP1 is required for protein stability, for the targeting of NPP1 to the plasma membrane and for the expression of catalytic activity. We also demonstrate that 16 conserved cysteines in the somatomedin-B-like domains of NPP1, in concert with two flanking cysteines, mediate the dimerization of NPP1. The K173Q polymorphism of NPP1, which maps to the second somatomedin-B-like domain and has been associated with the aetiology of insulin resistance, did not affect the dimerization or catalytic activity of NPP1, and did not endow NPP1 with an affinity for the insulin receptor. Our data suggest that the non-catalytic ectodomains contribute to the subunit structure, stability and function of NPP1–3.

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Tunable assembly of biomimetic peptoids as templates to control nanostructure catalytic activity

Nanoscale ◽

10.1039/c8nr03852j ◽

2018 ◽

Vol 10 (26) ◽

pp. 12445-12452 ◽

Cited By ~ 13

Author(s):

Nicholas A. Merrill ◽

Feng Yan ◽

Haibao Jin ◽

Peng Mu ◽

Chun-Long Chen ◽

...

Keyword(s):

Catalytic Activity ◽

Structure And Function ◽

And Function

Tunable peptoid assembly directs the control over structure and function of Pd nanomaterial catalysts.

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Alteration in the status of glutathione transferase of the water snail, Bulinus globosus, during aestivation and recovery

Animal Biology ◽

10.1163/157075610x491680 ◽

2010 ◽

Vol 60 (2) ◽

pp. 145-155 ◽

Cited By ~ 1

Author(s):

Yetunde Adedolapo Ojopagogo ◽

Isaac Olusanjo Adewale

Keyword(s):

Glutathione Transferase ◽

Specific Activity ◽

Glutathione Transferases ◽

Partial Purification ◽

Major Protein ◽

High Concentration ◽

Cibacron Blue ◽

The Status ◽

Bulinus Globosus ◽

Specific Activities

AbstractThe varying status of glutathione transferases (GSTs) in water snail, Bulinus globosus, an intermediate host of disease-causing Schistosoma haematobium (Bilharz 1852) has been investigated. The expression of GST isoenzymes in the water snail appears seasonal with about three isoenzymes appearing during raining season, when the organism is active, which may reduce to a single peak of one isoenzyme during aestivation, when the organism is inactive. GST isoenzyme is present in high concentration in all the tissues investigated namely: haemolymph, foot muscle and hepatopancreas with specific activities of 0.006 ± 0.002, 0.45 ± 0.021 and 1.33 ± 0.103 units/mg protein respectively for 1-chloro-2,4-dinitrobenzene (CDNB) as substrate. With this substrate, the specific activity of GST from the hepatopancreas appears higher than the specific activities that have been previously reported for GSTs from molluscs. Partial purification of the isoenzymes using Tris acrylic acid-based resins enabled us to observe that GST appears to be the major protein in the hepatopancreas of this organism. We also found indications for the presence of an endogenous GST inhibitor in the cytosol, whose function is yet unknown. All the traditional GST inhibitors such as cibacron blue, hematin, bromosulfophthalein and S-hexylglutathione were able to inhibit the isoenzymes effectively, with cibacron blue being the most potent. The isoenzymes however have narrow substrate specificity. We conclude that different isoenzymes of GST are expressed in the same class of molluscs, even when they belong to the same genus or species, and that the expression may depend on whether the snails are on aestivation or not.

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Implications of Stisa2 catalytic residue restoration through site directed mutagenesis

Turkish Journal of Biochemistry ◽

10.1515/tjb-2016-0169 ◽

2016 ◽

Vol 42 (2) ◽

Author(s):

Hasnain Hussain ◽

Nikson Fatt Ming Chong

Keyword(s):

Catalytic Activity ◽

Catalytic Triad ◽

Site Directed Mutagenesis ◽

Amino Acid Residues ◽

Qualitative And Quantitative Analysis ◽

Conserved Residues ◽

Overlap Extension Pcr ◽

Overlap Extension ◽

Catalytic Network ◽

And Function

AbstractObjective:Restoration of catalytic activity of Isa2 fromMethods:The six conserved amino acid residues absent in the Stisa2 gene were restored by mutation using the overlap extension PCR and the asymmetrical overlap extension PCR methods. Next, mutant Stisa2 with restored catalytic residues was expressed inResults:Both qualitative and quantitative analysis showed that the restoration of the conserved residues in the catalytic site did not restore starch debranching activity. Molecular modeling showed greater than expected distances between the catalytic triad in mutant Stisa2. These additional distances are likely to prevent hydrogen bonding which stabilizes the reaction intermediate, and are critical for catalytic activity.Conclusions:These results suggest that during evolution, mutations in other highly conserved regions have caused significant changes to the structure and function of the catalytic network. Catalytically inactive Isa2, which is conserved in starch-producing plants, has evolved important non-catalytic roles such as in substrate binding and in regulating isoamylase activity.

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Cloning and heterologous expression of cDNA encoding class Alpha rat glutathione transferase 8-8, an enzyme with high catalytic activity towards genotoxic α,β-unsaturated carbonyl compounds

Biochemical Journal ◽

10.1042/bj2840313 ◽

1992 ◽

Vol 284 (2) ◽

pp. 313-319 ◽

Cited By ~ 56

Author(s):

G Stenberg ◽

M Ridderström ◽

Å Engström ◽

S E Pemble ◽

B Mannervik

Keyword(s):

Catalytic Activity ◽

Cdna Clone ◽

Carbonyl Compounds ◽

Glutathione Transferase ◽

Ethacrynic Acid ◽

Glutathione Transferases ◽

High Catalytic Activity ◽

Reading Frame ◽

Dna Fragment ◽

Polymerase Chain

A cDNA clone, lambda GTRA8, encoding rat glutathione transferase subunit 8 has been isolated from a lambda gt10 rat hepatoma cDNA library. The previously known amino acid sequence of the enzyme was used to design primers for a polymerase chain reaction that yielded a 0.3 kb DNA fragment from the hepatoma library. The 0.3 kb fragment was used as a probe for screening and a 0.9 kb cDNA clone containing a complete open reading frame was obtained. After DNA sequencing and subcloning into an expression vector, the enzyme was expressed in Escherichia coli and purified. Specific activities and kcat./Km values were determined for a number of substrates, including alpha, beta-unsaturated carbonyl compounds. The highest activity was obtained with 4-hydroxyalkenals and with acrolein, genotoxic products of lipid peroxidation. In addition, the rat class Alpha glutathione transferase 8-8 displays high catalytic activity in the reaction between glutathione and the diuretic drug ethacrynic acid, a compound normally considered as a substrate characteristic for class Pi glutathione transferases.

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Human Glutathione Transferase T2-2 Discloses Some Evolutionary Strategies for Optimization of the Catalytic Activity of Glutathione Transferases

Journal of Biological Chemistry ◽

10.1074/jbc.m002818200 ◽

2000 ◽

Vol 276 (8) ◽

pp. 5432-5437 ◽

Cited By ~ 10

Author(s):

Anna Maria Caccuri ◽

Giovanni Antonini ◽

Philip G. Board ◽

Jack Flanagan ◽

Michael W. Parker ◽

...

Keyword(s):

Catalytic Activity ◽

Glutathione Transferase ◽

Glutathione Transferases ◽

Evolutionary Strategies

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Partial purification of two lithocholic acid-binding proteins from rat liver 100000g supernatants

Biochemical Journal ◽

10.1042/bj1650425 ◽

1977 ◽

Vol 165 (3) ◽

pp. 425-429 ◽

Cited By ~ 29

Author(s):

R C Strange ◽

R Cramb ◽

J D Hayes ◽

I W Percy-Robb

Keyword(s):

Ion Exchange ◽

Binding Proteins ◽

Lithocholic Acid ◽

Gel Filtration ◽

Ion Exchange Chromatography ◽

Exchange Chromatography ◽

Anion Binding ◽

Partial Purification ◽

Transferase Activity ◽

Glutathione S Transferase

1. The partial purification of two lithocholic acid-binding proteins from liver 100 000g supernatants is described. 2. Gel-filtration, (NH4)2SO4 fractionation, Ca3(PO4)2 fractionation and ion-exchange chromatography were used. 3. Both proteins exhibited glutathione S-transferase activity; one may be the non-specific anion-binding protein ligandin. 4. Glutathione S-transferase activity of one of the binding proteins was inhibited by lithocholic acid.

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Hymenolepis diminuta and H. microstoma: uptake of cyclosporin A and drug binding to parasite cyclophilins

Parasitology ◽

10.1017/s0031182000077076 ◽

1995 ◽

Vol 111 (5) ◽

pp. 591-597 ◽

Cited By ~ 4

Author(s):

H. C. Roberts ◽

J. M. Sternberg ◽

L. H. Chappell

Keyword(s):

Cyclosporin A ◽

Mode Of Action ◽

Drug Binding ◽

Hymenolepis Diminuta ◽

Isomerase Activity ◽

Intracellular Compartments ◽

And Function ◽

Hymenolepis Microstoma

SUMMARYCyclosporin A (CsA) acts as a powerful immunosuppressant through its binding to the cytosolic isomerase, cyclophilin (CyP), forming a complex which inhibits the phosphatase activity of calcineurin. The drug is also selectively anti-parasitic but its mode of action remains unknown. The mouse tapeworm, Hymenolepis microstoma is sensitive to CsA, but the rat tapeworm, H. diminuta is not susceptible either in rats, mice or in vitro. Using these two tapeworm models, the uptake and binding of CsA were examined in relation to parasite cyclophilins. Uptake and compartmentalization of the drug were markedly different in the two species: H. microstoma takes up more drug than does H. diminuta and sequesters more drug into intracellular compartments. Characterization of cyclophilins using both CsA binding and isomerase activity assays reveals that H. microstoma possesses two cyclophilin isoforms (Mr 17700 and 21400) with isomerase activity that is inhibited by CsA. Using identical assays, we have been unable to demonstrate CsA-binding proteins or CsA-sensitive isomerase activity in H. diminuta. These data suggest that the anthelmintic action of CsA relates in some way to the presence and function of parasite cyclophilins.

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