Abdelrahim A. Hunaiti

Glutathione S-transferase from oxadiazon treated chickpea

Abstract Glutathione S -transferase was purified more than 150-fold with ca 70% recovery from chickpea shoots after treatment with 10 ppm of the herbicide oxadiazon. The purification steps involved ammonium sulphate precipitation, gel filtration and affinity chromatography. The M r weight of the native enzyme was 47 000 as determined by gel filtration and the enzyme was separated by ion exchange chromatography into five distinct isozymes named according to their elution order from a DEAE-Sephacel column as GST-I to GST-V. Sodium dodecyl sulfate-polyacrylamide electrophoresis analysis revealed the presence of one type of subunit for GST-I and GST-II with an apparent M r of 27 000 while the other three isozymes (GST-III, GST-IV and GST-V) displayed two types of subunits with M r s 29 000 and 27 000. Antibodies raised against the purified chickpea shoot glutathione S -transferase gave a single precipitin line with both wheat and corn extracts but only partial cross-reactivity was observed with purified human placenta enzyme.

Cytotoxicity, Mutagenicity and Antimicrobial Activity of Forty Jordanian Medicinal Plants

AbstractEthanolic extracts of forty Jordanian medicinal plants were examined for cytotoxicity, mutagenicity and antimicrobial activity. Among the 40 tested plants, 12 extracts were active in brine shrimp cytotoxicity bioassay with LC50 in the range of 74-748 μg/rnl. These positive extracts were further tested for their antitumor activity on human cell cultures. Nerium oleander and Calotropis procera showed an activity in the antitumor human cell line test with ED50 varied in the range of 0.008 to 2.13 μg/ml. depending upon the cell line. Regarding the mutagenic effect, ten extracts were active on Salmonella typhimurium strains TA98 and TA100. The most active extract was Peganum harmala which was mutagenic against both TA98 and TA100, while Ceratonia siliqua showed the lowest activity against both tested strains. Four extracts showed antibacterial activity on both Escherichia coli and Bacillus subtilis and two showed antifungal activity agains Candida albicans.

Developmental Studies on Drosophila melanogaster Glutathione S-transferase and its Induction by Oxadiazolone

Glutathione S-transferase activity toward 1-chloro-2,4-dinitrobenzene was detected in various developmental stages of Drosophila melanogaster. The specific activity of the enzyme was 110, 35, 25 and 15 nmol/min/mg protein in crude extracts prepared from eggs, larvae, pupae and adult stages respectively. The enzymes from larval, pupal and adult stages were purified and compared. Incorporation of the widely used herbicide oxadiazolone at concentrations of 375 and 563 part/million into the culture media caused 4- and 2.5-fold increase in the enzyme activity in pupal and adult stages respectively.

Purification and characterization of camel liver glutathione S-transferase

Glutathione S-transferases have been purified (18-fold) in 65-70% yield from the liver of one humped camel using affinity chromatography on glutathione-linked agarose. Chromatofocusing technique resolves the glutathione S-transferases into seven distinct isoenzymes with apparent pI of 8.7, 8.4, 8.0, 7.8, 7.3 and 6.5. The major isoenzyme (pI 8.7) which accounted for over 95% of the total activity was composed of two identical subunits of molecular mass 24,000 and was immunologically similar to the other six isoenzymes. The substrate specificities and the effect of various inhibitors on the activity of the abundant camel liver isoenzyme were also examined.

The Prevalence of Hemoglobin S and Glucose-6-Phosphate Dehydrogenase Deficiency in Jordanian Newborn

Objective: The aim of this study was to determine the incidence of HbS and glucose‐6‐phosphate dehydrogenase (G6PD) deficiency in Jordanian newborn.

The distribution and comparison of glutathione, glutathione reductase and glutathione S-transferase in various camel tissues

Extracts prepared from liver, kidney, lung and brain of camel contain glutathione, glutathione S-transferase and glutathione reductase. Liver had the highest level of glutathione (218.7 mumol/g wet weight) whereas brain had the lowest level (66.4 mumol/g wet weight). The highest activity for glutathione reductase was found in the kidney (2.6 mumol/min/mg protein) while the lowest activity was found in the lung (0.9 mumol/min/mg protein). Glutathione S-transferase activity was the highest in liver (4.2 mumol/min/mg protein) and the lowest in brain (1 mumol/min/mg protein). Purified glutathione S-transferases from lung, kidney, brain and liver were similar in their molecular size, subunit composition as well as immuno-reactivity and showed some differences in their response to heat and inhibitors.

A Rapid Purification Procedure for Camel Kidney Glutathione S-Transferase

Glutathione S-transferase was isolated from supernatant of camel kidney homogenate centrifugation at 37,000 xg by glutathione agarose affinity chromatography. The enzyme preparation has a specific activity of 44 mumol/min/mg protein and recovery was more than 85% of the enzyme activity in the crude extract. Glutathione agarose affinity chromatography resulted in a purification factor of about 49 and chromatofocusing resolved the purified enzyme into two major isoenzymes (pI 8.7 and 7.9) and two minor isoenzymes (pI 8.3 and 6.9). The homogeneity of the purified enzyme was analyzed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and gel filtration on Sephadex G-100. The different isoenzymes were composed of a binary combination of two subunits with molecular weight of 29,000 D and 26,000 D to give a native molecular weight of 55,000 D. The substrate specificities of the major camel kidney glutathione S-transferase isoenzymes were determined towards a range of substrates. 1-chloro-2,4-dinitrobenzene was the preferred substrate for all the isoenzymes. Isoenzyme III (pI 7.9) had higher specific activity for ethacrynic acid and isoenzyme II (pI 8.3) was the only isoenzyme that exhibited peroxidase activity. Ouchterlony double-diffusion analysis with rabbit antiserum prepared against the camel kidney enzyme showed fusion of precipitation lines with the enzymes from camel brain, liver and lung and no cross reactivity was observed with enzymes from kidneys of sheep, cow, rat, rabbit and mouse. Different storage conditions have been found to affect the enzyme activity and the loss in activity was marked at room temperature and upon repeated freezing and thawing.

Interplay between Glutathione-S-Transferase and Glucose-6-Phosphate Dehydrogenase in Neonatal Cord Blood

Three hundred neonatal cord blood samples were collected from two major cities in Jordan and assayed for glutathione-S-transferase (GST), glutathione, and glucose-6-phosphate dehydrogenase (G6PD). A significant positive correlation between the levels of G6PD and GST activities was detected. When the G6PD activity decreased, the GST activity declined. A similar concurrent decrease in the concentration of reduced glutathione was also observed. When the samples were divided into males and female samples, some significant variations in the levels of the two enzymes were observed. Female samples exhibited higher G6PD and GST activities as compared with male samples. Moreover, the incidence of severe and moderate G6PD deficiencies was higher in male as compared with female samples. Analyses of the samples for total bilirubin, red blood cells, hematocrit, and hemoglobin were also performed, and slight nonsignificant variations were noted.

interaction of Organic Azides with Purified Camel Glutathione S-Transferase'

A series of organic azides was synthesized and was tested as inhibitors of purified camel glutathione S-transferases. Enzymes purified from camel liver, lung, and kidney were inhibited reversibly by these compounds in a concentration-dependent pattern. The liver glutathione S-transferase was more sensitive to inhibition by most of these compounds and the lung enzyme was the least affected. The most effective reversible inhibitors of the tested organic azides for the purified camel liver enzyme were alkyl and allyl azides. The inhibition occurred immediately upon adding the inhibitors and remained constant during a further 30-min incubation period. The tested organic azides were found to inhibit the glutathione S-transferase catalyzed conjugation of glutathione with both 1-chloro-2,4-dinitrobenzene and 4-nitrobenzyl chloride and the kinetics of these inhibitions was qualitatively different, being competitive with some inhibitors and noncompetitive with others.

Camel brain glutathione S-transferase purification, properties, regional and subcellular distribution

1. Camel brain glutathione S-transferase was purified by glutathione-linked agarose affinity column and the different isozymes were separated by chromatofocusing. 2. The basic isozymes which comprise 45% of the total activity were immunologically indistinguishable from the near-neutral isozymes which constitute 55% of the activity. 3. Some differences were detectable among the basic and near-neutral isozymes in relation to substrate specificities and subunit composition. 4. Biochemical and immunological quantification of glutathione S-transferase revealed the presence of the enzyme in all camel brain regions tested and subcellular fractions. 5. The pons had the highest concentration of the enzyme and the cortex had the lowest, while more than 88% of the enzyme was present in the cytosol.