Purification and characterization of glucose-6-phosphate dehydrogenase from Eisenia fetida and effects of some pesticides and metal ions

2020 ◽  
Vol 45 (4) ◽  
pp. 373-380
Author(s):  
Naciye Kayhan ◽  
Veysel Çomaklı ◽  
Sevki Adem ◽  
Caglar Güler
Keyword(s):  
Metal Ions ◽  
Eisenia Fetida ◽  
Biochemical Characterization ◽  
Inhibitory Effect ◽  
Biochemical Properties ◽  
Affinity Column ◽  
Glucose 6 Phosphate Dehydrogenase ◽  
Sepharose 4B

AbstractObjectivesEarthworms have a large impact on the soil ecosystem. They are quite sensitive to pollutants. Purification and biochemical characterization of glucose-6-phosphate dehydrogenases (G6PD) from the earthworm species Eisenia fetida were aimed. The determination of the toxicity potentials of some soil pollutants on G6PD activity was intended.MethodsG6PD was isolated using 2′,5′-ADP-Sepharose 4B affinity column. Enzyme purity and molecular mass were determined by SDS-PAGE. Its biochemical properties investigated. The effects of some soil pollutants on the enzyme were studied in vitro.ResultsEnzyme was purified with 28% yields and 232 fold. Optimum pH and buffer concentration, optimal and stable temperature was determined as pH: 8.5, 60 mM, 25 °C and 20 °C. Its molecular weight estimated as 36 kDa. The Ni2+, Hg2+, Pb2+, Cr2+, and Fe2+ ions with IC50 values in the range of 56 ± 06−120 ± 20 μM and the diniconazole, metalaxyl, methomyl, carboxyl, and oxamyl with IC50 values in the range of 7.6 ± 1.2−77 ± 12 μM exhibited an inhibitory effect on G6PD.ConclusionsG6PD was isolated and characterized from E. fetida. Its catalytic activity decreased with very low concentration by pesticides and metal ions. The results indicated that the inhibition of G6PD may be important in the toxicity mechanism of pollutants on this earthworm.

Functional and Biochemical Characterization of Immunologically Derived Equine Platelet-Activating Factor

1985 ◽  
Vol 22 (4) ◽  
pp. 375-386 ◽  
Author(s):  
H. C. Wimberly ◽  
D. O. Slauson ◽  
N. R. Neilsen
Keyword(s):  
Functional Activity ◽  
Biochemical Characterization ◽  
Platelet Activating Factor ◽  
Biochemical Properties ◽  
Naturally Occurring ◽  
Rabbit Platelets ◽  
Rapid Reaction ◽  
Specific Challenge

Antigen-specific challenge of equine leukocytes induced the non-lytic release of a platelet-activating factor in vitro. The equine platelet-activating factor stimulated the release of serotonin from equine platelets in a dose-responsive manner, independent of the presence of cyclo-oxygenase pathway inhibitors such as indomethacin. Rabbit platelets were also responsive to equine platelet-activating factor. The release of equine platelet-activating factor was a rapid reaction with near maximal secretion taking place in 30 seconds. Addition of equine platelet-activating factor to washed equine platelets stimulated platelet aggregation which could not be inhibited by the presence of aspirin or indomethacin. Platelets preincubated with equine platelet-activating factor became specifically desensitized to equine platelet-activating factor while remaining responsive to other platelet stimuli such as collagen and epinephrine. The following biochemical properties of equine platelet-activating factor are identical to those properties of 1-0-alkyl-2-acetyl-sn-glyceryl-3-phosphorylcholine (AGEPC): stability upon exposure to air and acid; loss of functional activity after basecatalyzed methanolysis with subsequent acylation that returned all functional activity; and identical relative mobilities on silica gel G plates developed with chloroform:methanol:water (65:35:6, volume/volume). The combined functional and biochemical characteristics of equine platelet-activating factor strongly suggest identity between this naturally occurring, immunologically derived equine factor and AGEPC.

Biochemical characterization of human DNA polymerase i provides clues to its biological function

2001 ◽  
Vol 29 (2) ◽  
pp. 183-187 ◽  
Author(s):  
A. Tissier ◽  
E. G. Frank ◽  
J. P. McDonald ◽  
A. Vaisman ◽  
A. R. Fernàndez deHenestrosa Henestrosa ◽  
...  
Keyword(s):  
Dna Polymerase ◽  
Biochemical Characterization ◽  
Short Review ◽  
Biochemical Properties ◽  
Dna Polymerase I ◽  
Polymerase I

The human RAD30B gene has recently been shown to encode a novel DNA polymerase, DNA polymerase i (poli). The role of poli within the cell is presently unknown, and the only clues to its cellular function come from its biochemical characterization in vitro. The aim of this short review is, therefore, to summarize the known enzymic activities of poli and to speculate as to how these biochemical properties might relate to its in vivo function.

scholarly journals Expression and biochemical characterization of human glucose-6- phosphate dehydrogenase in Escherichia coli: a system to analyze normal and mutant enzymes

Blood ◽  
1994 ◽  
Vol 83 (5) ◽  
pp. 1436-1441 ◽  
Author(s):  
TK Tang ◽  
CH Yeh ◽  
CS Huang ◽  
MJ Huang
Keyword(s):  
Escherichia Coli ◽  
Biochemical Characterization ◽  
Fold Increase ◽  
Binding Domain ◽  
Occurrence Rate ◽  
Substrate Analogues ◽  
Normal Enzyme ◽  
Glucose 6 Phosphate Dehydrogenase

We have developed a system to characterize normal and mutated glucose-6- phosphate dehydrogenase (G6PD) enzymes in vitro. Normal or mutant G6PD cDNA was subcloned into a pGEX-3X vector, which allowed production of a functional fusion protein in Escherichia coli. When we compared the recombinant normal enzyme with authentic human G6PD, indistinguishable Km values for glucose-6-phosphate (G6P) and NADP were obtained, and the utilization rates for two substrate analogues (2-deoxy G6P and deamino NADP) also showed no difference between the enzymes. This system was used to assay a biochemically uncharacterized variant, G6PD Taipei (493 A-->wG; 165 Asn-->Asp), plus two other known mutations (487 G-->A; 163 Gly-->Ser and 592 C-->T; 198 Arg-->Cys) that are located close to or within the putative G6P binding domain. Our results show that the G6PD activities of these three mutants were greatly reduced. No significant alteration in G6PD kinetics was observed for both 487 and 493 mutations. However, a drastic reduction in the Km for G6P (4-fold decrease) and tremendous increases in utilization rates of 2-deoxy G6P (32-fold increase) and deamino NADP (6-fold increase) were associated with the 592 mutation. This results suggests that arginine 198 in human G6PD, possibly located within the putative G6P binding domain, may play an important role in binding the substrate G6P. In addition, we and others have recently identified that at least nine different types of mutations are responsible for G6PD deficiency in Chinese. In this report, we also present the occurrence rate of each mutation present in the population of Taiwan.

The origin, evolution and diversification of multiple isoforms of light-dependent protochlorophyllide oxidoreductase (LPOR): focus on angiosperms

2020 ◽  
Vol 477 (12) ◽  
pp. 2221-2236
Author(s):  
Michal Gabruk ◽  
Beata Mysliwa-Kurdziel
Keyword(s):  
Biochemical Characterization ◽  
Duplication Event ◽  
Biochemical Properties ◽  
Protochlorophyllide Oxidoreductase ◽  
Independent Manner ◽  
Duplication Events ◽  
Multiple Species

Light-dependent protochlorophyllide oxidoreductase (LPOR) catalyzes the reduction of protochlorophyllide to chlorophyllide, which is a key reaction for angiosperm development. Dark operative light-independent protochlorophyllide oxidoreductase (DPOR) is the other enzyme able to catalyze this reaction, however, it is not present in angiosperms. LPOR, which evolved later than DPOR, requires light to trigger the reaction. The ancestors of angiosperms lost DPOR genes and duplicated the LPORs, however, the LPOR evolution in angiosperms has not been yet investigated. In the present study, we built a phylogenetic tree using 557 nucleotide sequences of LPORs from both bacteria and plants to uncover the evolution of LPOR. The tree revealed that all modern sequences of LPOR diverged from a single sequence ∼1.36 billion years ago. The LPOR gene was then duplicated at least 10 times in angiosperms, leading to the formation of two or even more LPOR isoforms in multiple species. In the case of Arabidopsis thaliana, AtPORA and AtPORB originated in one duplication event, in contrary to the isoform AtPORC, which diverged first. We performed biochemical characterization of these isoforms in vitro, revealing differences in the lipid-driven properties. The results prone us to hypothesize that duplication events of LPOR gave rise to the isoforms having different lipid-driven activity, which may predispose them for functioning in different locations in plastids. Moreover, we showed that LPOR from Synechocystis operated in the lipid-independent manner, revealing differences between bacterial and plant LPORs. Based on the presented results, we propose a novel classification of LPOR enzymes based on their biochemical properties and phylogenetic relationships.

scholarly journals Expression and biochemical characterization of human glucose-6- phosphate dehydrogenase in Escherichia coli: a system to analyze normal and mutant enzymes

Blood ◽  
1994 ◽  
Vol 83 (5) ◽  
pp. 1436-1441 ◽  
Author(s):  
TK Tang ◽  
CH Yeh ◽  
CS Huang ◽  
MJ Huang
Keyword(s):  
Escherichia Coli ◽  
Biochemical Characterization ◽  
Fold Increase ◽  
Binding Domain ◽  
Occurrence Rate ◽  
Substrate Analogues ◽  
Normal Enzyme ◽  
Glucose 6 Phosphate Dehydrogenase

Abstract We have developed a system to characterize normal and mutated glucose-6- phosphate dehydrogenase (G6PD) enzymes in vitro. Normal or mutant G6PD cDNA was subcloned into a pGEX-3X vector, which allowed production of a functional fusion protein in Escherichia coli. When we compared the recombinant normal enzyme with authentic human G6PD, indistinguishable Km values for glucose-6-phosphate (G6P) and NADP were obtained, and the utilization rates for two substrate analogues (2-deoxy G6P and deamino NADP) also showed no difference between the enzymes. This system was used to assay a biochemically uncharacterized variant, G6PD Taipei (493 A-->wG; 165 Asn-->Asp), plus two other known mutations (487 G-->A; 163 Gly-->Ser and 592 C-->T; 198 Arg-->Cys) that are located close to or within the putative G6P binding domain. Our results show that the G6PD activities of these three mutants were greatly reduced. No significant alteration in G6PD kinetics was observed for both 487 and 493 mutations. However, a drastic reduction in the Km for G6P (4-fold decrease) and tremendous increases in utilization rates of 2-deoxy G6P (32-fold increase) and deamino NADP (6-fold increase) were associated with the 592 mutation. This results suggests that arginine 198 in human G6PD, possibly located within the putative G6P binding domain, may play an important role in binding the substrate G6P. In addition, we and others have recently identified that at least nine different types of mutations are responsible for G6PD deficiency in Chinese. In this report, we also present the occurrence rate of each mutation present in the population of Taiwan.

scholarly journals The Glutathione/Glutaredoxin System Is Essential for Arsenate Reduction in Synechocystis sp. Strain PCC 6803

2009 ◽  
Vol 191 (11) ◽  
pp. 3534-3543 ◽  
Author(s):  
Luis López-Maury ◽  
Ana María Sánchez-Riego ◽  
José Carlos Reyes ◽  
Francisco J. Florencio
Keyword(s):  
Biochemical Characterization ◽  
Biochemical Properties ◽  
Arsenate Reductase ◽  
Arsenic Resistance ◽  
Arsenate Resistance ◽  
Mutant Strains ◽  
Pcc 6803

ABSTRACT Arsenic resistance in Synechocystis sp. strain PCC 6803 is mediated by an operon of three genes in which arsC codes for an arsenate reductase with unique characteristics. Here we describe the identification of two additional and nearly identical genes coding for arsenate reductases in Synechocystis sp. strain PCC 6803, which we have designed arsI1 and arsI2, and the biochemical characterization of both ArsC (arsenate reductase) and ArsI. Functional analysis of single, double, and triple mutants shows that both ArsI enzymes are active arsenate reductases but that their roles in arsenate resistance are essential only in the absence of ArsC. Based on its biochemical properties, ArsC belongs to a family that, though related to thioredoxin-dependent arsenate reductases, uses the glutathione/glutaredoxin system for reduction, whereas ArsI belongs to the previously known glutaredoxin-dependent family. We have also analyzed the role in arsenate resistance of the three glutaredoxins present in Synechocystis sp. strain PCC 6803 both in vitro and in vivo. Only the dithiolic glutaredoxins, GrxA (glutaredoxin A) and GrxB (glutaredoxin B), are able to donate electrons to both types of reductases in vitro, while GrxC (glutaredoxin C), a monothiolic glutaredoxin, is unable to donate electrons to either type. Analysis of glutaredoxin mutant strains revealed that only those lacking the grxA gene have impaired arsenic resistance.

Biochemical and functional characterization of the human tissue kallikrein 9

2017 ◽  
Vol 474 (14) ◽  
pp. 2417-2433 ◽  
Author(s):  
Panagiota S. Filippou ◽  
Sofia Farkona ◽  
Davor Brinc ◽  
Yijing Yu ◽  
Ioannis Prassas ◽  
...  
Keyword(s):  
Human Tissue ◽  
Biochemical Characterization ◽  
Functional Characterization ◽  
Squamous Carcinoma ◽  
Biochemical Properties ◽  
Tissue Kallikrein ◽  
Related Family ◽  
Squamous Carcinoma Cells

Human tissue kallikrein 9 (KLK9) is a member of the kallikrein-related family of proteases. Despite its known expression profile, much less is known about the functional roles of this protease and its implications in normal physiology and disease. We present here the first data on the biochemical characterization of KLK9, investigate parameters that affect its enzymatic activity (such as inhibitors) and provide preliminary insights into its putative substrates. We show that mature KLK9 is a glycosylated chymotrypsin-like enzyme with strong preference for tyrosine over phenylalanine at the P1 cleavage position. The enzyme activity is enhanced by Mg2+ and Ca2+, but is reversibly attenuated by Zn2+. KLK9 is inhibited in vitro by many naturally occurring or synthetic protease inhibitors. Using a combination of degradomic and substrate specificity assays, we identified candidate KLK9 substrates in two different epithelial cell lines [the non-tumorigenic human keratinocyte cells (HaCaT) and the tumorigenic tongue squamous carcinoma cells (SCC9)]. Two potential KLK9 substrates [KLK10 and midkine (MDK)] were subjected to further validation. Taken together, our data delineate some functional and biochemical properties of KLK9 for future elucidation of the role of this enzyme in health and disease.

scholarly journals Antibodies Raised against Bcvir15, an Extrachromosomal Double-Stranded RNA-Encoded Protein from Babesia canis, Inhibit the In Vitro Growth of the Parasite

2003 ◽  
Vol 71 (3) ◽  
pp. 1056-1067 ◽  
Author(s):  
P. Drakulovski ◽  
B. Carcy ◽  
K. Moubri ◽  
C. Carret ◽  
D. Depoix ◽  
...  
Keyword(s):  
Biochemical Characterization ◽  
Protozoan Parasite ◽  
Inhibitory Effect ◽  
Open Reading Frames ◽  
Babesia Canis ◽  
In Vitro Growth ◽  
Viral Origin ◽  
Double Stranded Rna

ABSTRACT As part of a search for homologous members of the Plasmodium falciparum Pf60 multigene family in the intraerythrocytic protozoan parasite Babesia canis, we report here the characterization of a cDNA of 1,115 bp, which was designated Bcvir for its potential viral origin. The Bcvir cDNA contained two overlapping open reading frames (ORFs) (ORF1 from nucleotide [nt] 61 to 486 and ORF2 from nt 417 to 919), where Bcvir15, the deduced ORF1 peptide (M1 to I141), is the main expressed product. The Bcvir cDNA was derived from an extrachromosomal dsRNA element of 1.2 kbp that was always found associated with a double-stranded RNA (dsRNA) of 2.8 kbp by hybridization, and no copy of this cDNA sequence was found in B. canis genomic DNA. Biochemical characterization of Bcvir15, by using polyclonal rabbit sera directed against recombinant proteins, indicated that it is a soluble protein which remained associated with the cytoplasm of the B. canis merozoite. Interestingly, purified immunoglobulins from the anti-glutathione S-transferase-Bcvir15 (at a concentration of 160 μg/ml) induced 50% inhibition of the in vitro growth of B. canis, and the inhibitory effect was associated with morphological damage of the parasite. Our data suggest that the extrachromosomal dsRNA-encoded Bcvir15 protein might interfere with the intracellular growth of the parasite rather than with the process of invasion of the host cell by the merozoite. Epitope mapping of Bcvir15 identified three epitopes that might be essential for the function of the protein.

scholarly journals Purification of Drosophila DNA polymerase ζ by REV1 protein-affinity chromatography

2004 ◽  
Vol 382 (2) ◽  
pp. 535-543 ◽  
Author(s):  
Ryo TAKEUCHI ◽  
Masahiko OSHIGE ◽  
Makiyo UCHIDA ◽  
Gen ISHIKAWA ◽  
Kei-ichi TAKATA ◽  
...  
Keyword(s):  
Dna Synthesis ◽  
Dna Polymerase ◽  
Biochemical Properties ◽  
Affinity Column ◽  
Protein Affinity ◽  
Pyrimidine Dimers ◽  
Multicellular Organisms ◽  
Mutation Assay

Studies on the biochemical properties of very-large-size eukaryotic DNA polymerases have been limited by the difficulty in obtaining sufficient purified forms of each enzyme. Our aim was to determine and elucidate the biochemical properties of one such polymerase, pol ζ (DNA polymerase ζ) from Drosophila melanogaster (Dmpol ζ). Using an REV1 (UV-revertible gene 1) protein-affinity column, we have isolated the enzyme directly from Drosophila embryos. Completely purified Dmpol ζ was found to have a molecular mass of approx. 240 kDa, and to be sensitive to aphidicolin and resistant to ddTTP (2′,3′-dideoxythymidine-5-triphosphate) and N-ethylmaleimide. The enzyme has a preference for poly(dA)/oligo(dT)10:1 as a template primer and has high processivity for DNA synthesis. Moreover, Dmpol ζ showed significantly higher fidelity compared with Rattus norvegicus DNA polymerase, an error-prone DNA polymerase, in an M13 forward mutation assay. The activities of bypassing pyrimidine dimers and (6-4) photoproducts and extending from mismatched primer-template termini in (6-4) photoproduct by Dmpol ζ were not detected. Drosophila REV7 interacted with Dmpol ζ in vitro, but did not influence the DNA synthesis activity of Dmpol ζ. The present study is the first report about characterization of purified pol ζ from multicellular organisms, and the second concerning the characterization of yeast pol ζ.

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