Effect of lead concentration on the level of glutathione, glutathione S-transferase, reductase and peroxidase in human blood.

Incubation of human whole blood for 24 h at 37 degrees C in the presence of 100-400 microg/dl lead chloride or lead acetate caused a concentration-dependent decrease in the level of reduced glutathione up to 40%. Similarly, the activities of glutathione reductase, glutathione peroxidase and glutathione S-transferase were decreased up to 25%, 50%, and 19%, respectively. Moreover, 100 microg/dl lead chloride or lead acetate slowed the process of glutathione regeneration, and delayed the time for complete regeneration from 20 to 40 min. When glutathione S-transferase was purified by affinity chromatography on Sepharose-linked glutathione, incubated with lead chloride or lead acetate, a concentration-dependent inhibition of the enzymatic activity was observed reaching 50% inhibition at a lead salt concentration of 6000 microg/dl.

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.

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. STUDY DESIGN: A total of 181 male and female babies born at Princess Basma Teaching Hospital, randomly selected, and cord blood samples were collected, and the erythrocyte G6PD activity was measured, and the hemoglobin electrophoresis for blood lysate was conducted and scanned for HbS scanning. RESULTS: The frequencies of two major red cell genetic defects, sickle hemoglobin (HbS) and deficiency G6PD was determined, of the studied subjects 10 (11%) females and 11 (12%) males were found to be deficient in the G6PD gene. The frequency of HbS carriers among the females was 4% while it was 6% among males. The coincidence of both G6PD deficiency and sickle cell hemoglobin in the samples was 1%. No coincidence was found between G6PD deficiency and hyperbilirubinemia. CONCLUSION: A better understanding of the distributions of these genetic disorders has the potential to aid in the more efficient utilization of health care resources and improved planning.

Effects of iron, zinc, calcium, and vitamins on the activity and contents of human placental copper/zinc and manganese superoxide dismutases.

One hundred seventy-nine pregnant women, ages 15-45 yr, were divided into three groups. Group A was orally given one spansule per day containing 150 mg dried ferrous sulfate, 61.8 mg zinc sulfate, and 500 micrograms folic acid, starting from the first 4 wk of pregnancy and ending at the day of delivery. Similarly, group B was given one tablet containing 625 mg calcium carbonate, 1000 mg vitamin C, 300 IU Vitamin D, 1350 mg citric acid, and 15 mg Vitamin B6. Group C was without any supplements and served as a control. Mothers who received iron/zinc supplements (group A) during pregnancy had significantly higher copper/zinc superoxide dismutase activity in their placentae than calcium/vitamin-supplemented mothers (group B) or unsupplemented mothers (group C). The enzyme activity increased with age of the mothers from 15 to 40 yr, then decreased after in both supplemented groups, whereas this increase and decrease occurred at early age in the unsupplemented group. Immunochemical quantitation of the enzyme contents showed no significant difference between the supplemented and unsupplemented groups, suggesting that the observed increase in the enzyme activity might arise from posttranslational processing of the enzyme. The placental manganese superoxide dismutase activity and contents, however, were similar in the supplemented groups, whereas they were slightly higher in the unsupplemented group; the overall superoxide dismutase-like activities in the placentae were the highest in iron-zinc supplemented group and the lowest in the unsupplemented group.

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.

Studies on human placental glutathione S-transferase. Multiplicity and mother age influence.

Human placental glutathione S-transferase was purified to apparent homogeneity by direct application of the crude homogenate into glutathione linked sepharose affinity chromatography. Chromatofocusing analysis in the presence of reduced glutathione resolved the enzyme into three acidic peaks eluted at pH 6.0, 5.7 and 5.5. About 36% of the initial activity was recovered in the isozyme fraction eluted at pH 6.0 whereas the isozymes eluted at pH 5.7 and 5.5 accounted for 20% and 25% of the activity respectively. Disc gel electrophoresis in the presence of sodium dodecyl sulfate revealed the presence of a single protein band in all the three separated isozymes. These isozymes were homodimers with an apparent relative molecular mass of 44.000 and subunit molecular mass of 21.000. The isozymes were immunologically related to each other and to the enzyme from goat and sheep placentae. Mother age had no influence in the placental glutathione S-transferase activity, albeit the activity was slightly higher in placenta obtained from younger women.

A comparison of cardiac glutathione S-transferases from wild and domestic animals.

1. Cardiac glutathione S-transferases from wild animals; hyena, red fox, porcupine, coypu and mountain gazelle were purified and compared with the enzymes from domestic animals; cow, camel, goat and sheep. 2. By using 1-chloro-2,4-dinitrobenzene as a substrate, domestic hearts expressed higher glutathione conjugating activity than wild animals hearts. 3. In all the studied hearts, the bulk of the activity was associated with near neutral and acidic glutathione S-transferase isozymes with pI values ranging from 4 to 7.4. 4. The enzymes from domestic animals displayed homodimeric structure of 25,000 mol. wt subunit while of the wild animals both hyena and coypu displayed homodimers of 26,500 mol. wt subunit and the rest exhibited heterodimers of 25,000 and 28,000 mol. wt subunits.

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.

A novel hemolysin from the lichen Parmelia pulla.

A hitherto unknown hemolysin from the lichen Parmelia pulla was discovered and a method was developed for its purification to apparent homogeneity. Saline phosphate buffer pH 7.2 extracted the bulk of the hemolysin from the lichen thalli. From this extract the hemolysin was purified by ammonium sulfate precipitation and gel filtration with Sepharose 6-B column. The overall recovery was about 75% and the purified hemolysin appeared to be electrophoretically homogenous and had a native molecular weight of 32,600. The purified hemolysin had a pH optimum around 5.5, stable at room temperature and gradually loses its activity upon freezing and thawing. Polyacrylamide gel electrophoresis of the purified hemolysin in the presence of sodium dodecyl sulfate revealed the presence of two types of subunits with apparent molecular weight of 18,000 and 14,000 respectively, indicating a dimeric (alpha beta) type of structure. Immunoblotting analysis demonstrated the presence of this hemolysin in crude extract prepared from P tinictina but not in crude extract from P tiliacea and P acetabulum. The purified hemolysin lyses rabbit erythrocytes and the rate of hemolysis was linear dependence on protein concentration. Erythrocytes obtained from various species including human were also lysed by the purified hemolysin in a concentration dependent manner.

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.

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.

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 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.

Source of methylmalonyl-coenzyme A for erythromycin synthesis: methylmalonyl-coenzyme A mutase from Streptomyces erythreus.

Streptomyces erythreus produces erythromycin, presumably from methylmalonyl-coenzyme A (CoA), which could be generated by the isomerization of succinyl-CoA. In S. erythreus cultures, [1,4-14C,2,3-3H]succinate was incorporated into erythromycin with a doubling of the 3H/14C ratio. This result is consistent with the hypothesis that succinyl-CoA is isomerized to methylmalonyl-CoA before incorporation into the macrocyclic lactone of erythromycin. The presence of methylmalonyl-CoA mutase, which catalyzes this isomerization, was demonstrated in cell-free extracts prepared from this organism. Consistent with the suggested role for this enzyme, methylmalonyl-CoA mutase activity increased over 12-fold at the time of the most rapid antibiotic production, and the activity level drastically declined when the antibiotic production ceased. The mutase was partially purified from this organism with DEAE-cellulose, ammonium sulfate precipitation, and affinity chromatography on a B12-coenzyme Sepharose column. The enzyme was stimulated 2.5-fold by the addition of B12-coenzyme. The enzyme showed a typical Michaelis-Menten type substrate saturation patterns, with KmS of 0.31 mM and 0.09 microM for methylmalonyl-CoA and B12-coenzyme, respectively, and a V of 0.5 mumol/min per mg. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified enzyme showed a major band with a molecular weight of 63,000. The properties of this enzyme appear to be fairly similar to those of the mutase previously obtained from other sources.