Single Band For Enzyme Research Articles

In vitro effects of some drugs on human erythrocyte glutathione reductase

The effects of dacarbazine, thiocolchicoside, methotrexate, olanzapine, pantoprazole sodium, and 5-fluorouracil on the enzyme activity of glutathione reductase (GR) were studied using human erythrocyte GR enzymes in in vitro studies, respectively. Materials and methods: The enzyme was purified 3333-fold from human erythrocytes in a yield of 44.44% with 4.0 U/mg. The purification procedure involved the preparation of hemolysate, ammonium sulfate precipitation, 2', 5'-ADP Sepharose 4B affinity chromatography, and Sephadex G-200 gel filtration chromatography. SDS-PAGE showed a single band for enzyme. Purified enzyme was used in the in vitro studies. Results: In the in vitro studies, IC50 values and Ki constants were 0.014 mM and 0.0121 ± 0.0026 mM for dacarbazine, and 0.054 mM and 0.0135 ± 0.0024 mM for thiocolchicoside, 0.31 mM and 0.1213 ± 0.040 mM for methotrexate, 0.35 mM and 0.3173 ± 0.0981 mM for olanzapine, 0.94 mM and 0.5413 ± 0.0728 mM for pantoprazole sodium, and 98.57 mM and 69.611 ± 9.690 mM for 5-fluorouracil, showing the inhibition effects on the purified enzyme. Inhibition types were non-competitive for dacarbazine, methotrexate, olanzapine, and 5-fluorouracil and competitive for thiocolchicoside and pantoprazole sodium. Conclusion: All the drugs indicated the inhibitory effects on the enzyme.

Effects of Some Metal Ions on Human Erythrocyte Glutathione Reductase:An In Vitro Study

In this study, we investigated inhibitory effects of some metal ions on human erythrocyte glutathione reductase. For this purpose, initially human erythrocyte glutathione reductase was purified 1051-fold in a yield of 41% by using 2', 5'-ADP Sepharose 4B affinity gel and Sephadex G-200 gel filtration chromatography. SDS polyacrylamide gel electrophoresis was done in order to control the purification of enzyme. SDS polyacrylamide gel electrophoresis showed a single band for enzyme. A constant temperature (4 degrees C) was maintained during the purification process. Enzyme activity was determined with the Beutler method by using a spectrophotometer at 340 nm. Hg(2+), Cd(2+), Pb(2+), Cu(2+), Fe(3+) and Al3+ exhibited inhibitory effects on the enzyme in vitro. K(i) constants and IC(50) values for metal ions were determined by Lineweaver-Burk graphs and plotting activity % vs. [I]. IC(50) values of Pb(2+), Hg(2+), Cu(2+), Cd(2+), Fe(3+) and Al(3+) were 0.011, 0.020, 0.0252, 0.0373, 0.209 and 0.229 mM, and the Ki constants 0.0254+/-0.0027, 0.0378+/-0.0043, 0.0409+/-0.0048, 0.0558+/-0.0083, 0.403+/-0.043 and 1.137+/-0.2 mM, respectively. While Pb(2+), Hg(2+), Cd(2+) and Fe(3+) showed competitive inhibition, others displayed noncompetitive inhibition.

Dehalogenation of 4 — Chlorobenzoic Acid by Pseudomonas isolates

Twenty three bacterial isolates either pure or consortium were initially screened on the basis of their ability to degrade as well as dechlorinate 4 - chlorobenzoic acid (4-CBA). Based on comparative growth response, three pure isolates Pseudomonas putida GVS-4, Pseudomonas aeruginosa GVS-18 and Pseudomonas aeruginosa GWS-19 and a consortium SW-2 was finally selected for further studies. The enzyme studies performed with cell free extracts revealed that dehalogenase activity was substrate specific with maximum activity at 300 μgml(-1) substrate concentration. Catechol 1,2 dioxygenase activity was found to be present in cell free extracts suggesting that 4 - chlorobenzoic acid (4-CBA) is catabolized by ortho-ring cleavage pathway. The dehalogenase enzyme profile showed single enzyme band in case of GVS-4 (Rm 0.76), GVS-18 (Rm 0.84), GWS -19 (Rm 0.85) and two bands in SW-2 (Rm 0.71 & 0.10).

Effects of some drugs on hepatic glucose 6-phosphate dehydrogenase activity in Lake Van Fish ( Chalcalburnus Tarischii Pallas, 1811)

Inhibitory effects of some drugs on hepatic glucose 6-phosphate dehydrogenase from Lake Van fish ( chalcalburnus tarischii pallas, 1811) were investigated. For this purpose, initially liver glucose 6-phosphate dehydrogenase was purified 899-fold in a yield of 46.24% by using 2′,5′-ADP Sepharose 4B affinity gel. In order to control the purification of enzyme was done SDS polyacrylamide gel electrophoresis. SDS polyacrylamide gel electrophoresis showed a single band for enzyme. A constant temperature (+4 °C) was maintained during the purification process. Enzyme activity was determined with the Beutler method by using a spectrophotometer at 340 nm. Vankomycine, sulfanylamide, sulfanylacetamide, nidazole, ciprofloxacin, amoxicillin and KMnO 4 were used as drugs. These drugs exhibited inhibitory effects on the enzyme. IC 50 values of vankomycine, sulfanylamide, sulfanylacetamide, nidazole, ciprofloxacin, amoxicillin and KMnO 4 were 1.88, 0.037, 0.032, 1.178, 2.26, 643.5 and 0.0002 mM, and the K i constants 1.18 ± 0.148, 0.119 ± 0.021, 0.075 ± 0.015, 1.15 ± 0.21, 7.69 ± 0.67, 1007 ± 69, and 0.001 ± 0.00022 mM, respectively. While vankomycine and nidazole showed competitive inhibition, others displayed noncompetitive inhibition. K i constants and IC 50 values for drugs were determined by Lineweaver–Burk graphs and plotting activity percentage versus [ I], respectively.

Purification and characterization of glucose 6-phosphate dehydrogenase from Lake Van fish (Chalcalburnus tarichii pallas, 1811) liver

Glucose 6-phosphate dehydrogenase (D-glucose 6-phosphate: NADP+ oxidoreductase, EC 1.1.1.49; G6PD) was purified from Lake Van fish (Chalcalburnus tarichii pallas, 1811) liver, using a simple and rapid method, and some characteristics of the enzyme were investigated. The purification procedure was composed of two steps: homogenate preparation and 2', 5'-ADP Sepharose 4B affinity gel chromatography, which took 7-8 hours. Thanks to the two consecutive procedures, the enzyme, having specific activity of 38 EU/mg protein, was purified with a yield of 44.39% and 1310 fold. In order to control the enzyme purification SDS polyacrylamide gel electrophoresis (SDS-PAGE) was done. SDS polyacrylamide gel electrophoresis showed a single band for enzyme. Optimal pH, stable pH, optimal temperature, Km and, Vmax values for NADP+ and glucose 6-phosphate (G6P) were also determined for the enzyme. In addition, molecular weight and subunit molecular weights were found by sodium dodecyl sulfate polyacrilamide gel electrophoresis (SDS-PAGE) and gel filtration chromatography respectively.

Purification and Characterization of Acetylcholinesterase from the Lake Van Fish (Chalcalburnus tarichii Pallas, 1811)

In this study, acetylcholinesterase (AChE; EC 3.1.1.7) was purified from plasma and erythrocytes in the Lake Van fish (Chalcalburnus tarichii P.1811) by affinity chromatography. Enzymatic activity was spectrophotometrically measured according to Ellman’s method, at 412 nm. Then, the optimal pH and temperature of the enzyme was determined. According to the results, the optimal pH and the optimum temperature were 8.0 and 25°C, respectively. In order to control the purification of the enzyme, sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE) was done. SDS-PAGE showed a single band for enzyme. The purification rates for plasma AChE and erythrocyte AChE are 3251.6 and 8500, respectively.

Purification of 6-Phosphogluconate Dehydrogenase from Parsley ( Petroselinum hortense ) Leaves and Investigation of Some Kinetic Properties

In this study, 6-phosphogluconate dehydrogenase (E.C.1.1.1.44; 6PGD) was purified from parsley (Petroselinum hortense) leaves, and analysis of the kinetic behavior and some properties of the enzyme were investigated. The purification consisted of three steps that are preparation of homogenate ammonium sulfate fractionation and on DEAE-Sephadex A50 ion exchange. The enzyme was obtained with a yield of 49% and had a specific activity of 18.3 U (mg proteins)−1 (Lehninger, A.L.; Nelson, D.L.; Cox, M.M. Principles of Biochemistry, 2nd Ed.; Worth Publishers Inc.: N.Y., 2000, 558–560). The overall purification was about 339-fold. A temperature of +4°C was maintained during the purification process. Enzyme activity was spectrophotometrically measured according to the Beutler method at 340 nm. In order to control the purification of the enzyme, SDS-polyacrylamide gel electrophoresis was carried out in 4% and 10% acrylamide for stacking and running gel, respectively. SDS-polyacrylamide gel electrophoresis showed a single band for enzyme. The molecular weight was found to be 97.5 kDa by Sephadex G-150 gel filtration chromatography. A protein band corresponding to a subunit molecular weight of 24.1 kDa was obtained on SDS-polyacrylamide gel electrophoresis. For the enzymes, the stable pH, optimum pH, and optimum temperature were found as 8.0, 8.0, and 50°C, respectively. In addition, KM and Vmax values for NADP+ and G6-P at optimum pH and 25°C were determined by means of Lineweaver-Burk plots.

PURIFICATION AND INVESTIGATION OF SOME KINETIC PROPERTIES OF GLUCOSE-6-PHOSPHATE DEHYDROGENASE FROM PARSLEY (PETROSELINUM HORTENSE LEAVES

ABSTRACT In this study, glucose-6-phosphate dehydrogenase (D-glucose-6-phosphate: NADP+ oxidoreductase, EC 1.1.1.49; G6PD) was purified from parsley (Petroselinum hortense) leaves, and analysis of the kinetic behavior and some properties of the enzyme were investigated. The purification consisted of three steps: preparation of homogenate, ammonium sulfate fractionation, and DEAE-Sephadex A50 ion exchange chromatography. The enzyme was obtained with a yield of 8.79% and had a specific activity of 2.146 U (mg protein)−1. The overall purification was about 58-fold. Temperature of +4°C was maintained during the purification process. Enzyme activity was spectrophoto metrically measured according to the Beutler method, at 340 nm. In order to control the purification of enzyme, SDS-polyacrylamide gel electrophoresis was carried out in 4% and 10% acrylamide for stacking and running gel, respectively. SDS-polyacrylamide gel electrophoresis showed a single band for enzyme. The molecular weight was found to be 77.6 kDa by Sephadex G-150 gel filtration chromatography. A protein band corresponding to a molecular weight of 79.3 kDa was obtained on SDS-polyacrylamide gel electrophoresis. For the enzymes, the stable pH, optimum pH, and optimum temperature were found to be 6.0, 8.0, and 60°C, respectively. Moreover, KM and Vmax values for NADP+ and G6-P at optimum pH and 25°C were determined by means of Lineweaver-Burk graphs. Additionally, effects of streptomycin sulfate and tetracycline antibiotics were investigated for the enzyme activity of glucose-6-phosphate dehydrogenase in vitro.

Purification and Characterization of Glucose 6-phosphate Dehydrogenase from Sheep Erythrocytes and Inhibitory Effects of some Antibiotics on Enzyme Activity

Glucose 6-phosphate dehydrogenase (d -glucose 6-phosphate: NADP + oxidoreductase, EC 1.1.1.49; G6PD) was purified from sheep erythrocytes, using a simple and rapid method. The purification consisted of three steps; preparation of haemolysate, ammonium sulphate fractionation and 2′, 5′-ADP Sepharose 4B affinity chromatography. The enzyme was obtained with a yield of 37.1% and had a specific activity of 4.64 U/mg proteins. Optimal pH, stable pH, molecular weight, and K M and V max values for NADP + and glucose 6-phosphate (G6-P) substrates were also determined for the enzyme. The overall purification was about 1,189-fold. A temperature of +4°C was maintained during the purification process. In order to control the purification of the enzyme SDS polyacrylamide gel electrophoresis (SDS-PAGE) was done in 4% and 10% acrylamide concentration for stacking and running gel, respectively. SDS-PAGE showed a single band for enzyme. Enzymatic activity was spectrophotometrically measured according to Beutler's method at 340 nm. In addition, in vitro effects of gentamicin sulphate, penicillin G potassium, amicasin on sheep red blood cell G6PD enzyme activity were investigated. These antibiotics showed inhibitory effects on enzyme activity. I 50 values were determined from Activity %-[Drug] graphs and K i values and the type of inhibition (noncompetitive) were determined by means of Lineweaver-Burk graphs.

Purification and some of the properties of alkaline phosphatase in guinea fowls (Numida meleagris galeata).

1. Alkaline phosphatase activity in the plasma of different strains of guinea fowls showed considerable variation both within and between sexes as well as within and between strains. 2. The enzymes from different strains of wild guinea fowls had different mobilities on disc polyacrylamide electrophoresis but each was characterised by a single band. 3. When the enzyme was purified 163-fold from the plasma of a domesticated grey breasted strain, both ion-exchange chromatography and gel-filtration purification steps yielded a single band of enzyme. 4. The purified enzyme had a molecular weight of 79,400 +/- 3,000 and was stable up to 60 degrees C at the optimum pH of 9.6. 5. Evidence is provided that guinea fowl alkaline phosphatase is a metalloenzyme.

Trichomonas species: Homocysteine desulphurase and serine sulphydrase activities

Homocysteine desulphurase (EC 4.4.1.2) and serine sulphydrase (EC 4.2.1.22) activities in various lines of Trichomonas vaginalis, both metronidazole resistant and sensitive, and other trichomonad species were assessed. T. vaginalis contained the highest homocysteine desulphurase and serine sulphydrase activities of all the species. Although the levels of the enzyme activity in T. vaginalis isolates differed, no correlation between the activities and sensitivity to metronidazole was apparent. T. vaginalis homocysteine desulphurase catalysed both the hydrolysis of homocysteine to hydrogen sulphide, ammonia, and 2-oxoacid, and an exchange reaction between homocysteine and 2-mercaptoethanol. Homocysteine desulphurase was detected as a single enzyme band on isoelectric focusing, whereas several isoenzymes of serine sulphydrase were found. There were large differences in serine sulphydrase isoenzyme patterns between T. vaginalis lines and between species. Several isoenzymes were amplified in cells grown with 10 −5 M dl-propargylglycine for 24 hr. T. vaginalis homocysteine desulphurase and serine sulphydrase activities were inhibited by bithionol, hexachlorophene, and dichlorophene. These compounds also inhibited growth in vitro of T. vaginalis at concentrations similar to those that inhibited the enzymes.

Isoenzymes of human prostate acid phosphatase.

The isoenzymes of human prostatic acid phosphatase have been studied by an isoelectric focusing technique. Purified acid phosphatase from malignant prostates contained eight isoenzymes with pI 4.4--5.3. The sera from patients with prostate cancer were shown to have similar acid phosphatase isoenzyme patterns at pI 4.0--5.5; as the serum enzyme activities increased, the pI of isoenzymes shifted to more acidic pH. These isoenzyme patterns of sera from patients with prostate cancer were different from those of patients with Gaucher's disease or from acid phosphatase of human erythrocytes, both of which exhibited only one enzyme band around pI 5.0 and 6.0, respectively. Treatment of serum sample of prostate cancer with neuraminidase did not result in a single enzyme band but alter the pI of isoenzymes, which shifted to a higher pH region. The significance of acid phosphatase activities and its isoenzyme patterns in prostate cancer merits further investigation.

A microchemical analysis of alkaline phosphatase in human malignant and benign breast tumours.

AbstractA microchemical technique has been adapted to the evaluation of the membrane‐bound enzyme alkaline phosphatase within human malignant and benign breast lesions. Frozen tissue sections were either stained or lyophilized in alternating sequence in order that samples for enzyme analysis could be chosen from well‐defined regions of epithelial proliferation. Microgram quantities of human tumors could then be examined with multiple replications to elucidate patterns of overall enzyme activity, the activity of tissue fractions, and the isoenzyme form present within individual tumors. A significant difference (p<0.001) was found to exist between the specific activity of alkaline phosphatase from human infiltrating ductal carcinomas and human fibroadenomas. The activity value determined for the malignant group of tumors was 2.12±1.80, while that for benign tumors was 7.52±4.31 mmoles/min/kg dry weight at 37° C. The percentage of alkaline phosphatase activity found within 29,000 g pellets of malignant tumors was variable and constituted approximately 60% of the total activity found in all fractions, while that for benign tumors was more constant and amounted to approximately 90% of the total found. The malignant and benign tumor alkaline phosphatase of both supernatant and pellet fractions was shown to be 90% labile to incubation at 65° C for 5 min and to be 50% inhibited by 20 mM 1‐phenylalanine. Polyacrylamide gel electrophoresis revealed a single enzyme band of identical Rf for each of these fractions from the two tumor types. When varying proportions of malignant and benign tissue samples were co‐incubated, no influence of tumor type on the total activity or the percentage membrane‐bound activity was found. However, a change in the proportion of enzyme activity pelleted at 29,000 g could be shown when the cellularity of the tissue sample was varied. While homogenization of the total sample or of the insoluble material could release some of this activity, greater enzyme activities were always found within insoluble, 480 g pellets and 29,000 g pellets taken from benign tumors. An entrapment of membrane proteins could account for these results and should be considered when tissues are analyzed in the manner described.

Polymorphism of sorbitol dehydrogenase and 6‐phosphogluconate dehydrogenase in swine (Sus scrofa)

SummaryMore than 300 kidneys from the local bacon factory were investigated and it was shown that the enzyme sorbitol dehydrogenase (EC 1.1.1.14) exhibits a genetically determined polymorphism. While homozygous individuals yield only a single enzyme band after starch gel electrophoresis, heterozygotes show multiple bands.On the same material also 6‐phosphogluconate dehydrogenase (EC 1.1.1.44) and lactate dehydrogenase were investigated, the former showed also clear genetic variants while the latter appeared only in one form.Gene frequencies for different breeds and types of pigs are given and the results discussed.

Esterase polymorphisms in Microtus ochrogaster: interaction and linkage.

Three polymorphic loci have been identified in the prairie vole, Microtus ochrogaster. Together they control a group of plasma esterases which can be separated using starch gel electrophoresis. A structural locus, Es-1, produces an enzyme which from genetic evidence appears to be a dimer. The allele Es-1a produces a wholly active subunit, and homozygotes give a single enzyme band. The product of the second allele, Es-1o, cannot form active enzyme on its own but will dimerize with the Es-1a subunit, giving a hybrid enzyme with a slower electrophoretic mobility than the pure Es-1a enzyme. The third allele, Es-1−, has no detectable product. A second structural locus, Es-2, is linked to Es-1. The allele Es-2a produces a single enzyme band, but the second allele Es-2− has no detectable product. A modifier locus, Me, changes the mobility of the Es-1 enzymes. Mef is dominant over mes, and in homozygotes for mes the mobility is reduced.