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

2010 ◽  
Vol 60 (2) ◽  
pp. 145-155 ◽  
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.

scholarly journals Leukotriene C synthase in mouse mastocytoma cells. An enzyme distinct from cytosolic and microsomal glutathione transferases

1988 ◽  
Vol 250 (3) ◽  
pp. 713-718 ◽  
Author(s):  
M Söderström ◽  
S Hammarström ◽  
B Mannervik
Keyword(s):  
Microsomal Fraction ◽  
Glutathione Transferase ◽  
Specific Activity ◽  
Glutathione Transferases ◽  
Leukotriene C4 ◽  
Cytosol Fraction ◽  
Mastocytoma Cells ◽  
Specific Activities ◽  
Leukotriene A4 ◽  
Microsomal Glutathione

Leukotriene C4 synthesis was studied in preparations from mouse mastocytoma cells. Enzymic conjugation of leukotriene A4 with glutathione was catalysed by both the cytosol and the microsomal fraction. The specific activity of the microsomal fraction (7.8 nmol/min per mg of protein) was 17 times that of the cytosol fraction. The cytosol fraction of the mastocytoma cells contained two glutathione transferases, which were purified to homogeneity and characterized. A microsomal glutathione transferase was purified from mouse liver; this enzyme was shown by immunoblot analysis to be present in the mastocytoma microsomal fraction at a concentration one-tenth or less of that in the liver microsomal fraction. Both the cytosolic and the microsomal glutathione transferases in the mastocytoma cells were identified with enzymes previously characterized, by determining specific activities with various substrates, sensitivities to inhibitors, reactions with antibodies, and physical properties. The purified microsomal glutathione transferase from liver was inactive with leukotriene A4 or its methyl ester as substrate. The cytosolic enzymes displayed activity with leukotriene A4, but their specific activities and intracellular concentrations were too low to account for the leukotriene C4 formation in the mastocytoma cells. The microsomal fraction of the cells contained an enzyme distinguishable by various criteria from the previously studied glutathione transferases. This membrane-bound enzyme, leukotriene C synthase (leukotriene A4:glutathione S-leukotrienyltransferase), appears to carry the main responsibility for the biosynthesis of leukotriene C4.

scholarly journals Partial purification of presynaptic plasma membrane by immunoadsorption.

1982 ◽  
Vol 94 (1) ◽  
pp. 88-96 ◽  
Author(s):  
G P Miljanich ◽  
A R Brasier ◽  
R B Kelly
Keyword(s):  
Plasma Membrane ◽  
Synaptic Vesicle ◽  
Nerve Terminal ◽  
Specific Activity ◽  
Electric Organ ◽  
Partial Purification ◽  
Vesicle Membrane ◽  
Specific Activities ◽  
Active Zones ◽  
Presynaptic Plasma Membrane

During transmitter release, synaptic vesicle membrane is specifically inserted into the nerve terminal plasma membrane only at specialized sites or "active zones." In an attempt to obtain a membrane fraction enriched in active zones, we have utilized the electric organ of the marine ray. From this organ, a fraction enriched in nerve terminals (synaptosomes) was prepared by conventional means. These synaptosomes were bound to microscopic beads by an antiserum to purified electric organ synaptic vesicles (anti-SV). The success of this immunoadsorption procedure was demonstrated by increased specific activities of bead-bound nerve terminal cytoplasmic markers and decreased specific activities of markers for contaminating membranes. To obtain a presynaptic plasma membrane (PSPM) fraction, we lysed the bead-bound synaptosomes by hypoosmotic shock and sonication, resulting in complete release of cytoplasmic markers. When the synaptosomal fraction was surface-labeled with iodine before immunoadsorption, 10% of this label remained bead-bound after lysis, compared with 2% of the total protein, indicating an approximately fivefold enrichment of bead-bound plasma membrane. Concomitantly, the specific activity of bead-bound anti-SV increased approximately 30-fold, indicating an enrichment of plasma membrane which contained inserted synaptic vesicle components. This PSPM preparation is not simply synaptic vesicle membrane since two-dimensional electrophoresis revealed that the polypeptides of the surface-iodinated PSPM preparation include both vesicle and numerous nonvesicle components. Secondly, antiserum to the PSPM fraction is markedly different from anti-SV and binds to external, nonvesicle, nerve terminal components.

scholarly journals Distribution and partial purification of a liver membrane protein capable of inactivating cytosol enzymes

1980 ◽  
Vol 186 (2) ◽  
pp. 571-579 ◽  
Author(s):  
G L Francis ◽  
F J Ballard
Keyword(s):  
Plasma Membrane ◽  
Membrane Protein ◽  
Plasma Membranes ◽  
Specific Activity ◽  
Gel Filtration ◽  
Exchange Chromatography ◽  
Partial Purification ◽  
Major Protein ◽  
Major Protein Band ◽  
Glucose 6 Phosphate Dehydrogenase

1. The inactivation of cytosol enzymes in liver extracts was carried out by several subcellular fractions, with plasma membranes having the highest specific activity. Rough and smooth microsomal fractions were both active, whereas lysosmal inactivation capacity appeared to be derived entirely from contaminating plasma-membrane fragments. 2. Inactivation capacity in liver fractions was derived from parenchymal cells. Of the non-liver cells tested, plasma membranes from H35 hepatoma cells were able to inactivate glucose 6-phosphate dehydrogenase (EC 1.1.1.49), adipocyte “ghosts” showed slight activity and erythrocyte and reticulocyte “ghosts” were inactive. 3. Liposomes prepared from pure lipids with net negative, positive or neutral charge did not possess inactivation capacity. 4. Liver plasma-membrane inactivation capacity was destroyed by heating at 50 degrees C. 5. Inactivation factor solubilized from membranes by trypsin plus Triton X-100 treatment was partially purified by (NH4)2SO4 fractionation, gel filtration, ion-exchange chromatography and hydroxyapatite chromatography. 6. Partially purified inactivation factor analysed by gel electrophoresis gave a major protein band that co-migrated with capacity for inactivation of glucose 6-phosphate dehydrogenase. 7. It is concluded that inactivation factor is a membrane protein whose intracellular distribution and other properties are consistent with a possible role for this activity in the initial step of protein degradation.

scholarly journals Partial purification of (Ca2+ + Mg2+)-dependent ATPase from pig smooth muscle and reconstitution of an ATP-dependent Ca2+-transport system

1981 ◽  
Vol 198 (2) ◽  
pp. 265-271 ◽  
Author(s):  
F Wuytack ◽  
G De Schutter ◽  
R Casteels
Keyword(s):  
Enzyme Activity ◽  
Smooth Muscle ◽  
Transport System ◽  
Microsomal Fraction ◽  
Specific Activity ◽  
Partial Purification ◽  
Total Enzyme Activity ◽  
Dependent Atpase ◽  
Specific Activities ◽  
Total Enzyme

(CaMg)ATPase [(Ca2+ + Mg2+)-dependent ATPase] was partially purified from a microsomal fraction of the smooth muscle of the pig stomach (antrum). Membranes were solubilized with deoxycholate, followed by removal of the detergent by dialysis. The purified (CaMg)ATPase has a specific activity (at 37 degrees C) of 157 +/- 12.1 (7)nmol.min-1.mg-1 of protein, and it is stimulated by calmodulin to 255 +/- 20.9 (7)nmol.min.mg-1. This purification of the (CaMg)ATPase resulted in an increase of the specific activity by approx. 18-fold and in a recovery of the total enzyme activity of 55% compared with the microsomal fraction. The partially purified (CaMg)ATPase still contains some Mg2+-and (Na+ + K+)-dependent ATPase activities, but their specific activities are increased relatively less than that of the (CaMg)ATPase. The ratios of the (CaMg)ATPase to Mg2+- and (Na+ + K+)-dependent ATPase activities increase from respectively 0.14 and 0.81 in the crude microsomal fraction to 1.39 and 9.07 in the purified preparation. During removal of the deoxycholate by dialysis, vesicles were reconstituted which were capable of ATP-dependent Ca2+ transport.

scholarly journals Altered glutathione transferase levels in rat skin inflamed due to contact hypersensitivity: induction of the Alpha-class subunit 1

1998 ◽  
Vol 335 (3) ◽  
pp. 605-610 ◽  
Author(s):  
Junya KIMURA ◽  
Makoto HAYAKARI ◽  
Takayuki KUMANO ◽  
Hajime NAKANO ◽  
Kimihiko SATOH ◽  
...  
Keyword(s):  
Glutathione Transferase ◽  
Specific Activity ◽  
Contact Hypersensitivity ◽  
Glutathione Transferases ◽  
Rat Skin ◽  
Enzyme Modification ◽  
Control Value ◽  
Control Skin ◽  
Gst Activity

Since glutathione transferases (GSTs) are suggested to be involved in the prevention of tissue damage by oxidative stress, quantitative and qualitative alterations of GST forms were examined in rat skin after induction of inflammation by 0.6 and 1% 1-chloro-2,4-dinitrobenzene (CDNB) treatment. With 0.6% CDNB, the GST activity in supernatant preparations was 1.8-fold higher than that for control skin, with most GSTs in both cases being bound to S-hexyl-GSH–Sepharose. Major GST subunits of control skin were identified as subunits 7, 4 and 2 by HPLC and chromatofocusing at pH 11–7. These subunits were increased in inflamed skin by 0.6% CDNB and, in addition, the subunit 1 of the Alpha class and subunit 6, both hardly detectable in control skin, were expressed. The specific activity value for GST 7-7 from the inflamed skin by 0.6% CDNB was 2.4-fold lower than that from control skin. However, in the case of inflamed skin after application of 1% CDNB, GST activity was decreased to 69% of the control value and most activity was recovered in fractions binding to a GSH–Sepharose but not a S-hexyl-GSH–Sepharose column. GSTs eluted from the former column demonstrated a restored capacity to bind to the latter, suggesting the GSTs in inflamed skin to be partly inactivated and that they regained activity on exposure to GSH. The Km and Vmax values for GSH of GST 4-4 from inflamed skin after 1% CDNB treatment were 6-fold and 2-fold higher, respectively, than those for the enzyme from control skin, suggesting partial enzyme modification. These results suggest that not only quantitative but also qualitative alterations of GST subunits occur with CDNB-induced inflammation in vivo.

Partial purification and properties of a β-glucosidase from Erwinia herbicola Y46

1975 ◽  
Vol 21 (4) ◽  
pp. 513-520 ◽  
Author(s):  
A. Garibaldi ◽  
L. N. Gibbins
Keyword(s):  
Specific Activity ◽  
Polyacrylamide Gel Electrophoresis ◽  
Gel Filtration ◽  
Deae Cellulose ◽  
Basal Medium ◽  
Plant Diseases ◽  
Partial Purification ◽  
Major Protein ◽  
Erwinia Herbicola ◽  
Major Protein Band

A constitutive β-glucosidase of Erwinia herbicola Y46 was studied as a prerequisite to an assessment of its significance in the release of bacteriotoxic aglycones from plant β-glucosides, and the possible effects of the aglycones on the course of such plant diseases as "fire-blight." The enzyme was purified 86.5-fold from crude extracts of cells grown on yeast beef broth. Ammonium sulfate precipitation, DEAE-cellulose fractionation, and gel filtration through Sephadex G-100 resulted in a preparation having one peak of activity on isoelectrofocussing, on gel filtration through Sephadex G-200, and on polyacrylamide gel electrophoresis. The latter techniques demonstrated, in addition to the major protein band associated with activity, a single minor impurity. The enzyme was active against p-nitrophenyl-β-glucoside (p-NPG) and phloridzin, but showed only very slight activity against salicin and arbutin, and no detectable activity against β-methyl-D-glucoside, cellobiose, lactose, and esculin. The production of β-glucosidase was maximum at the late log phase of growth on yeast beef broth medium and declined somewhat thereafter. The incorporation of inducers (carbohydrates) in defined basal medium resulted in only small variations in specific activity in the resulting cells. The activity (p-NPG substrate) was not inhibited by D-glucose, phloretin, esculin, salicin, arbutin, lactose, or cellobiose, but was slightly inhibited by 1.0 mM phloridzin. Slight inhibition was observed in the presence of sulfhydryl reagents (iodoacetamide, p-chloromercuribenzoate), but sodium azide, ethylene-diaminetetraacetic acid, Cu2+, and Zn2+ ions produced no effect. The activity was stable, in both crude and purified preparations, over the pH ranges 6.0–7.5 (100% activity) and 4.5–>8.5 (50% activity). The enzyme retained 80% activity after 30 min at 50 °C, but only 25% after 30 min at 60 °C. The enzyme had a mean Km value (phloridzin) of 1.35 × 10−4 M, an isoelectric point of 4.75, a molecular weight, determined by Sephadex G-200 gel filtration, of about 122 000, and an optimum pH for activity of 6.5–7.0.

An International Collaborative Study to Investigate Standardisation of Hirudin Potency

1993 ◽  
Vol 69 (05) ◽  
pp. 430-435 ◽  
Author(s):  
Colin Longstaff ◽  
Man-Yu Wong ◽  
Patrick J Gaffney
Keyword(s):  
Specific Activity ◽  
Collaborative Study ◽  
Residual Activity ◽  
Quality Standard ◽  
Upper Limit ◽  
Assay Procedure ◽  
Specific Activities ◽  
International Collaborative ◽  
Range Of Values ◽  
International Collaborative Study

SummaryAn international collaborative study has been carried out to investigate the reproducibility of hirudin assays in 13 laboratories using four recombinant hirudins and one natural, sulphated product. A simple assay procedure was proposed involving the titration of α-thrombin with inhibitor and measurement of residual activity using a chromogenic substrate. A standard α-thrombin preparation was supplied to ensure that this reagent was of uniform quality throughout the study. The method appeared to present no difficulties and laboratories reported similar potencies for the 5 hirudin samples, in line with expected values. This gave 200–222 Thrombin Inhibitory Units/ampoule (TIU/ampoule) of lyophilised hirudin, with geometric coefficient of variation (gcv) values ranging from 10.15–15.97%. This corresponds to specific activities of approximately 14,300–15,900 TIU/mg protein. This is close to the upper limit of previously reported values of specific activity. We conclude that the precision of this determination compared with the wider range of values in the literature (8,000–16,000 thrombin inhibitory units [TIU]/mg) results from the use of good quality standard α-thrombin by all laboratories. This study has important implications for hirudin standardisation.

scholarly journals A novel PET tracer 18F-deoxy-thiamine: synthesis, metabolic kinetics, and evaluation on cerebral thiamine metabolism status

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Changpeng Wang ◽  
Siwei Zhang ◽  
Yuefei Zou ◽  
Hongzhao Ma ◽  
Donglang Jiang ◽  
...  
Keyword(s):  
High Performance ◽  
Specific Activity ◽  
High Accumulation ◽  
Metabolic Kinetics ◽  
Thiamine Metabolism ◽  
Pet Tracer ◽  
The Status ◽  
The Brain

Abstract Background Some neuropsychological diseases are associated with abnormal thiamine metabolism, including Korsakoff–Wernicke syndrome and Alzheimer’s disease. However, in vivo detection of the status of brain thiamine metabolism is still unavailable and needs to be developed. Methods A novel PET tracer of 18F-deoxy-thiamine was synthesized using an automated module via a two-step route. The main quality control parameters, such as specific activity and radiochemical purity, were evaluated by high-performance liquid chromatography (HPLC). Radiochemical concentration was determined by radioactivity calibrator. Metabolic kinetics and the level of 18F-deoxy-thiamine in brains of mice and marmosets were studied by micro-positron emission tomography/computed tomography (PET/CT). In vivo stability, renal excretion rate, and biodistribution of 18F-deoxy-thiamine in the mice were assayed using HPLC and γ-counter, respectively. Also, the correlation between the retention of cerebral 18F-deoxy-thiamine in 60 min after injection as represented by the area under the curve (AUC) and blood thiamine levels was investigated. Results The 18F-deoxy-thiamine was stable both in vitro and in vivo. The uptake and clearance of 18F-deoxy-thiamine were quick in the mice. It reached the max standard uptake value (SUVmax) of 4.61 ± 0.53 in the liver within 1 min, 18.67 ± 7.04 in the kidney within half a minute. The SUV dropped to 0.72 ± 0.05 and 0.77 ± 0.35 after 60 min of injection in the liver and kidney, respectively. After injection, kidney, liver, and pancreas exhibited high accumulation level of 18F-deoxy-thiamine, while brain, muscle, fat, and gonad showed low accumulation concentration, consistent with previous reports on thiamine distribution in mice. Within 90 min after injection, the level of 18F-deoxy-thiamine in the brain of C57BL/6 mice with thiamine deficiency (TD) was 1.9 times higher than that in control mice, and was 3.1 times higher in ICR mice with TD than that in control mice. The AUC of the tracer in the brain of marmosets within 60 min was 29.33 ± 5.15 and negatively correlated with blood thiamine diphosphate levels (r = − 0.985, p = 0.015). Conclusion The 18F-deoxy-thiamine meets the requirements for ideal PET tracer for in vivo detecting the status of cerebral thiamine metabolism.

scholarly journals Gut Digestive Function and Microbiome after Correction of Experimental Dysbiosis in Rats by Indigenous Bifidobacteria

2021 ◽  
Vol 9 (3) ◽  
pp. 522
Author(s):  
Lyudmila V. Gromova ◽  
Elena I. Ermolenko ◽  
Anastasiya L. Sepp ◽  
Yulia V. Dmitrieva ◽  
Anna S. Alekseeva ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Digestive Enzymes ◽  
Specific Activity ◽  
Control Group ◽  
Enteropathogenic Escherichia Coli ◽  
Beneficial Bacteria ◽  
Opportunistic Bacteria ◽  
Dyspeptic Symptoms ◽  
Specific Activities ◽  
Impaired Motor Function

In recent years, great interest has arisen in the use of autoprobiotics (indigenous bacteria isolated from the organism and introduced into the same organism after growing). This study aimed to evaluate the effects of indigenous bifidobacteria on intestinal microbiota and digestive enzymes in a rat model of antibiotic-associated dysbiosis. Our results showed that indigenous bifidobacteria (the Bf group) accelerate the disappearance of dyspeptic symptoms in rats and prevent an increase in chyme mass in the upper intestine compared to the group without autoprobiotics (the C1 group), but significantly increase the mass of chyme in the colon compared to the C1 group and the control group (healthy animals). In the Bf group in the gut microbiota, the content of opportunistic bacteria (Proteus spp., enteropathogenic Escherichia coli) decreased, and the content of some beneficial bacteria (Bifidobacterium spp., Dorea spp., Blautia spp., the genus Ruminococcus, Prevotella, Oscillospira) changed compared to the control group. Unlike the C1 group, in the Bf group there was no decrease in the specific activities of maltase and alkaline phosphatase in the mucosa of the upper intestine, but the specific activity of maltase was decreased in the colon chyme compared to the control and C1 groups. In the Bf group, the specific activity of aminopeptidase N was reduced in the duodenum mucosa and the colon chyme compared to the control group. We concluded that indigenous bifidobacteria can protect the microbiota and intestinal digestive enzymes in the intestine from disorders caused by dysbiosis; however, there may be impaired motor function of the colon.

scholarly journals THE Ah PHENOTYPE. SURVEY OF FORTY-EIGHT RAT STRAINS AND TWENTY INBRED MOUSE STRAINS

Genetics ◽  
1982 ◽  
Vol 100 (1) ◽  
pp. 79-87
Author(s):  
Daniel W Nebert ◽  
Nancy M Jensen ◽  
Hisashi Shinozuka ◽  
Heinz W Kunz ◽  
Thomas J Gill
Keyword(s):  
Specific Activity ◽  
Inbred Mouse ◽  
Inbred Mouse Strain ◽  
Mouse Strains ◽  
Ah Receptor ◽  
Inbred Mouse Strains ◽  
Sprague Dawley ◽  
Rat Strains ◽  
Specific Activities ◽  
Inbred Rat

ABSTRACT Forty-four inbred and four randombred rat strains and 20 inbred mouse strains were examined for their Ah phenotype by determining the induction of liver microsomal aryl hydrocarbon (benzo[a]pyrene) hydroxylase activity (EC 1.14.14.1) by intraperitoneal treatment with either β-naphthoflavone or 3-methylcholanthrene. All 48 rat strains were found to be Ah-responsive. The maximally induced hydroxylase specific activities of the ALB/Pit, MNR/Pit, MR/Pit, SHR/Pit, and Sprague-Dawley strains were of the same order of magnitude as the basal hydroxylase specific activities of the ACI/Pit, F344/Pit, OKA/Pit, and MNR/N strains. Six of the 20 mouse strains were Ah-nonresponsive (i.e. lacking the normal induction response and presumably lacking detectable amounts of the Ah receptor). The basal hydroxylase specific activities of the BDL/N, NFS/N, STAR/N, and ST/JN mouse strains were more than twice as high as the maximally induced hydroxylase specific activity of the CBA/HT strain.——To date, 24 Ah-nonresponsive mouse strains have been identified, out of a total of 68 known to have been characterized. The reasons for not finding a single Ah-nonresponsive inbred rat strain—as compared with about one Ah-nonresponsive inbred mouse strain found for every three examined—remain unknown.

Sign in / Sign up

Export Citation Format

Share Document