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.

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 The synthesis and hydrolysis of long-chain fatty acyl-coenzyme A thioesters by soluble and microsomal fractions from the brain of the developing rat

1976 ◽  
Vol 160 (2) ◽  
pp. 247-251 ◽  
Author(s):  
P J Brophy ◽  
D E Vance
Keyword(s):  
Fatty Acid ◽  
Microsomal Fraction ◽  
Specific Activity ◽  
Arachidic Acid ◽  
Fatty Acyl ◽  
Chain Elongation ◽  
Fatty Acid Chain ◽  
Specific Activities ◽  
Coa Hydrolase ◽  
Long Chain

1. The specific activities of long-chain fatty acid-CoA ligase (EC6.2.1.3) and of long-chain fatty acyl-CoA hydrolase (EC3.1.2.2) were measured in soluble and microsomal fractions from rat brain. 2. In the presence of either palmitic acid or stearic acid, the specific activity of the ligase increased during development; the specific activity of this enzyme with arachidic acid or behenic acid was considerably lower. 3. The specific activities of palmitoyl-CoA hydrolase and of stearoyl-CoA hydrolase in the microsomal fraction decreased markedly (75%) between 6 and 20 days after birth; by contrast, the corresponding specific activities in the soluble fraction showed no decline. 4. Stearoyl-CoA hydrolase in the microsomal fraction is inhibited (99%) by bovine serum albumin; this is in contrast with the microsomal fatty acid-chain-elongation system, which is stimulated 3.9-fold by albumin. Inhibition of stearoyl-CoA hydrolase does not stimulate stearoyl-CoA chain elongation. Therefore it does not appear likely that the decline in the specific activity of hydrolase during myelogenesis is responsible for the increased rate of fatty acid chain elongation. 5. It is suggested that the decline in specific activity of the microsomal hydrolase and to a lesser extent the increase in the specific activity of the ligase is directly related to the increased demand for long-chain acyl-CoA esters during myelogenesis as substrates in the biosynthesis of myelin lipids.

TESTOSTERONE METABOLISM IN SUBCELLULAR FRACTIONS OF RAT PROSTATE

1973 ◽  
Vol 73 (3) ◽  
pp. 585-598 ◽  
Author(s):  
Kaoru Nozu ◽  
Bun-ichi Tamaoki
Keyword(s):  
Microsomal Fraction ◽  
Specific Activity ◽  
Hydroxysteroid Dehydrogenase ◽  
Inhibitory Effect ◽  
Subcellular Fractions ◽  
Hydrogenase Activity ◽  
Nuclear Fraction ◽  
Cytosol Fraction ◽  
Testosterone Metabolism ◽  
Major Activity

ABSTRACT Homogenates of rat ventral prostates were fractionated into the nuclear (purified from the precipitate at 800× g), mitochondrial (precipitate at 1000–6000 ×g), microsomal (precipitate at 10 000–105 000× g) and cytosol (supernatant fluid at 105 000 × g) fractions, which were morphologically identified under an electron-microscope. Among the subcellular fractions, the highest specific activity of Δ4-5α-hydrogenase was localized in the nuclear and microsomal fractions, while 3α-hydroxysteroid dehydrogenase (E. C. 1. 1. 1.50) activity was concentrated exclusively in the cytosol fraction. By addition of the cytosol fraction, the Δ4-5α-hydrogenase activity in the microsomal fraction was markedly reduced, whereas the activity in the nuclear fraction was scarcely inhibited. By heating the cytosol fraction at 100°C for 20 min, its inhibitory effect was significantly diminished. The inhibitory principle in the cytosol fraction against the Δ4-5α-hydrogenase was mostly concentrated in the precipitate at 0–60% saturation of ammonium sulphate, while the major activity of the 3α-hydroxysteroid dehydrogenase was localized in the precipitate at 40–100% saturation of the salt. According to the enzyme kinetics, the cytosol 0–40 % fraction showed the competitive type of inhibition with the Δ4-5α-hydrogenase activity in the microsomal fraction for testosterone.

scholarly journals Activation and inhibition of microsomal glutathione transferase from mouse liver

1988 ◽  
Vol 249 (3) ◽  
pp. 819-823 ◽  
Author(s):  
C Andersson ◽  
M Söderström ◽  
B Mannervik
Keyword(s):  
Molecular Mass ◽  
Mouse Liver ◽  
Glutathione Transferase ◽  
Cross Reactivity ◽  
Steep Slope ◽  
Glutathione Transferases ◽  
Inhibitor Concentration ◽  
Cytosolic Glutathione ◽  
Concentration Curves ◽  
Microsomal Glutathione

Mouse liver microsomal glutathione transferase was purified in an N-ethylmaleimide-activated as well as an unactivated form. The enzyme had a molecular mass of 17 kDa and a pI of 8.8. It showed cross-reactivity with antibodies raised against rat liver microsomal glutathione transferase, but not with any of the available antisera raised against cytosolic glutathione transferases. The fully N-ethylmaleimide-activated enzyme could be further activated 1.5-fold by inclusion of 1 microM-bromosulphophthalein in the assay system. The latter effect was reversible, which was not the case for the N-ethylmaleimide activation. At 20 microM-bromosulphophthalein the activated microsomal glutathione transferase was strongly inhibited, while the unactivated form was activated 2.5-fold. Inhibitors of the microsomal glutathione transferase from mouse liver showed either about the same I50 values for the activated and the unactivated form of the enzyme, or significantly lower I50 values for the activated form compared with the unactivated form. The low I50 values and the steep slope of the activity-versus-inhibitor-concentration curves for the latter group of inhibitors tested on the activated enzyme indicate a co-operative effect involving conversion of activated enzyme into the unactivated form, as well as conventional inhibition of the enzyme.

scholarly journals Cytosolic glutathione transferases from rat liver. Primary structure of class alpha glutathione transferase 8-8 and characterization of low-abundance class Mu glutathione transferases

1989 ◽  
Vol 261 (2) ◽  
pp. 531-539 ◽  
Author(s):  
P Alin ◽  
H Jensson ◽  
E Cederlund ◽  
H Jörnvall ◽  
B Mannervik
Keyword(s):  
Rat Liver ◽  
Glutathione Transferase ◽  
Glutathione Transferases ◽  
Rat Lung ◽  
Cytosol Fraction ◽  
Methionine Residue ◽  
Isoelectric Points ◽  
Cytosolic Glutathione ◽  
Lung And Kidney

Six GSH transferases with neutral/acidic isoelectric points were purified from the cytosol fraction of rat liver. Four transferases are class Mu enzymes related to the previously characterized GSH transferases 3-3, 4-4 and 6-6, as judged by structural and enzymic properties. Two additional GSH transferases are distinguished by high specific activities with 4-hydroxyalk-2-enals, toxic products of lipid peroxidation. The most abundant of these two enzymes, GSH transferase 8-8, a class Alpha enzyme, has earlier been identified in rat lung and kidney. The amino acid sequence of subunit 8 was determined and showed a typical class Alpha GSH transferase structure including an N-acetylated N-terminal methionine residue.

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.

Transformation of leukotriene A4 methyl ester to leukotriene C4 monomethyl ester by cytosolic rat glutathione transferases

FEBS Letters ◽  
1984 ◽  
Vol 175 (2) ◽  
pp. 289-293 ◽  
Author(s):  
Bengt Mannervik ◽  
Helgi Jensson ◽  
Per Ålin ◽  
Lars Örning ◽  
Sven Hammarström
Keyword(s):  
Methyl Ester ◽  
Glutathione Transferases ◽  
Leukotriene C4 ◽  
Leukotriene A4 ◽  
Monomethyl Ester

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.

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.

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