Partial purification and characterization of a peroxidase activity from human placenta.
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ABSTRACT:
SUBMITTER: Nelson JL
PROVIDER: S-EPMC1131502 | biostudies-other | 1990 Jun
REPOSITORIES: biostudies-other
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Project description:Glutathione peroxidase (glutathione--H2O2 oxidoreductase; EC 1.11.1.9) was purified to homogeneity from human placenta by using (NH4)2SO4 precipitation, ion-exchange chromatography, Sephadex gel filtration and preparative polyacrylamide-disc-gel electrophoresis. Glutathione peroxidase from human placenta is a tetramer, having 4g-atoms of selenium/mol of protein. The molecular weight of the enzyme is about 85000 with a subunit size of about 22,000. Kinetic properties of the enzyme are described. On incubation with cyanide, glutathione peroxidase is completely and irreversibly inactivated and selenium is released as a low-molecular-weight fragment. Reduced glutathione, beta-mercaptoethanol and dithiothreitol protect the enzyme from inactivation by cyanide and the release of selenium. Properties of human placental glutathione peroxidase are similar to those of isoenzyme A reported earlier by us from human erythrocytes. The presence of isoenzyme, B, reported earlier by us in human erythrocytes, was not detected in placenta. Also selenium-independent glutathione peroxidase (isoenzyme II), which is specific for cumene hydroperoxide, was not present in human placenta.
| S-EPMC1186396 | biostudies-other
Project description:Lipoxygenase possessing dual catalytic activities, i.e. dioxygenase and hydroperoxidase, was purified from the cytosols of term placentas from non-smoking women. Concanavalin A affinity chromatography followed by phenyl-Sepharose CL-4B chromatography resulted in the separation of one hydrophobic and one non-hydrophobic isoenzyme. The concanavalin A-purified enzyme was used in all subsequent experiments. The dioxygenase activity of the enzyme exhibited a Vmax. of 204.37 +/- 17.66 nmol/min per mg of protein and a Km of 0.79 mM for linoleic acid. The involvement of dioxygen in enzymic linoleic acid oxidation was confirmed by O2 uptake studies. Arachidonic acid and linolenic acid also served as substrates for the dioxygenase activity. The placental lipoxygenase co-oxidized benzidine in the presence of linoleic acid (hydroperoxidase activity). Both the dioxygenase and hydroperoxidase activities were significantly stimulated by Ca2+ (1-100 microM), ATP (10-400 nM) and H2O2 (1-10 nM). Similarly, these two activities were inhibited by nordihydroguaiaretic acid, 5,8,11-eicosatriynoic acid, gossypol, esculetin, butylated hydroxyanisole and butylated hydroxytoluene. Boiled enzyme was without significant dioxygenase and hydroperoxidase activities. Pyrogallol, 3,3'-dimethoxybenzidine, 3,3',5,5'-tetramethylbenzidine, tetramethylphenylenediamine and 2,2'-azino-bis-(3-ethylbenzothiazoline-6-sulphonic acid) were also co-oxidized by the placental lipoxygenase. These results suggest that: (i) lipoxygenase from human term placenta exhibits both dioxygenase and hydroperoxidase activities, and (ii) this enzyme represents an important pathway for chemical oxidation in the placentas of non-smoking women.
| S-EPMC1134323 | biostudies-other
Project description:1. Subcellular fractions of human placenta were prepared by nitrogen-bomb homogenization and differential centrifugation. 2. beta-Glucuronidase from placental lysosomes was purified 2100-fold on a protein basis. 3. The lysosomal enzyme, at different stages of purification, was characterized by using 4-methylumbelliferyl beta-d-glucuronide and phenolphthalein beta-d-glucuronide as substrates. 4. Only one isoenzyme of beta-glucuronidase was found in placenta; the enzyme in the endoplasmic reticulum appeared to be the same as the lysosomal enzyme. 5. The isoenzyme contained in normal plasma was different from that of the placenta. 6. The elevated beta-glucuronidase activity found in plasma obtained during pregnancy was due to increased activity of the normal plasma isoenzyme; no contribution was made by placental isoenzyme. 7. Plasma contained a heat-stable, non-diffusible activator of placental beta-glucuronidase. 8. A heat-stable competitive inhibitor of placental and plasma beta-glucuronidase was also present in plasma.
| S-EPMC1173564 | biostudies-other
Project description:Phosphatidylinositol synthase (CDP-1,2-diacyl-sn-glycerol:myoinositol 3-phosphatidyltransferase, EC 2.7.8.11) was purified from the microsomal fraction of human placenta. The Triton X-100-extracted enzyme was purified 8300-fold over the microsomal fraction by affinity chromatography on CDP-diacylglycerol-Sepharose followed by ion-exchange chromatography on Mono Q. The purified enzyme had a molecular mass of 24,000 Da on SDS/PAGE. The enzyme had a pH optimum at 9.0, required Mn2+ or Mg2+, and was inhibited by Ca2+ and Zn2+. The Km for myo-inositol was determined to be 0.28 mM. Optimal activity was obtained at 0.2-0.4 mM CDP-diacylglycerol; higher concentrations of the lipid substrate inhibited the enzyme reaction. The enzyme was inhibited by nucleoside di- and tri-phosphates, Pi and PPi. CDP competitively inhibited the enzyme reaction with a Kis of 4 mM. The optimal temperature for the PtdIns synthase reaction was 50 degrees C.
| S-EPMC1137864 | biostudies-other
Project description:S-Adenosylhomocysteine hydrolase (EC 3.3.1.1) was purified to homogeneity from human placenta by using S-adenosylhomocysteine-agarose affinity chromatography. The enzyme is a tetramer with a native Mr of 189 000 and subunit Mr of 47 000-48 000; there were nine cysteine residues per subunit and no disulphide bonds. The pI was 5.7. H.p.l.c. analysis revealed that the enzyme contained four molecules of tightly bound cofactor (NAD) per tetramer, of which 10-50% was in the reduced form. The enzyme had four binding sites per tetramer for adenosine, of which 10-35% were found to be occupied. Two types of adenosine-binding sites could be distinguished on the basis of differences in rates of dissociation of the enzyme-adenosine complex, and by examining binding of adenosine at 0 degree C and 37 degrees C. The enzyme catalysed the interconversion of adenosine and 4',5'-dehydroadenosine; the equilibrium constant for this reaction was 2.1 and favoured 4',5'-dehydroadenosine formation. Variability in the specific activity of preparations of S-adenosylhomocysteine hydrolase was related to the NAD+/NADH ratio of the preparation. The capacity to bind radioactively labelled adenosine depended on the adenosine content of the purified enzyme. The rate of adenosine binding and the sensitivity of S-adenosylhomocysteine hydrolase to inactivation by adenosine were both diminished in the absence of dithiothreitol.
| S-EPMC1152584 | biostudies-other
Project description:Spermidine synthase was purified to apparent homogeneity from human spleens (8700-fold) by affinity chromatography. The native enzyme was composed of two subunits of identical Mr (35,000) and showed an apparent Mr of 62,000 in pore-gradient gel electrophoresis. Its pI was 5.1, Spermine synthase was purified to apparent homogeneity from placenta (5300-fold) and from kidney (4600-fold). The native enzyme was composed of two subunits of identical Mr (45,000) and showed an apparent Mr of 78,000 in pore-gradient gel electrophoresis. In isoelectric focusing it revealed two bands, with pI values of 4.9 and 5.0. Both synthases were present in all human tissues studied, but revealed a clear tissue-specific pattern. Mouse antisera against spermidine synthase revealed only one band, of Mr 35,000, in all purified enzyme preparations and in crude human tissue extracts in immunoblotting. Antisera against spermine synthase showed an immunoreactive band corresponding to the Mr of the subunit of spermine synthase. These antisera did not indicate any cross-reactivity in immunoblotting. Thus spermine synthase and spermidine synthase do not share homologous antigenic sites and are totally different proteins.
| S-EPMC1138598 | biostudies-other
Project description:?-Glucosidase (EC 3.2.1.21) is a prominent member of the GH1 family of glycoside hydrolases. The properties of this ?-glucosidase appear to include resistance to temperature, urea, and iodoacetamide, and it is activated by 2-ME, similar to other members. ?-Glucosidase from chayote (Sechium edule) was purified by ionic-interchange chromatography and molecular exclusion chromatography. Peptides detected by LC-ESI-MS/MS were compared with other ?-glucosidases using the BLAST program. This enzyme is a 116 kDa protein composed of two sub-units of 58 kDa and shows homology with Cucumis sativus ?-glucosidase (NCBI reference sequence XP_004154617.1), in which seven peptides were found with relative masses ranging from 874.3643 to 1587.8297. The stability of ?-glucosidase depends on an initial concentration of 0.2 mg/mL of protein at pH 5.0 which decreases by 33% in a period of 30 h, and then stabilizes and is active for the next 5 days (pH 4.0 gives similar results). One hundred ?g/mL ?-D-glucose inhibited ?-glucosidase activity by more than 50%. The enzyme had a Km of 4.88 mM with p-NPG and a Kcat of 10,000 min(-1). The optimal conditions for the enzyme require a pH of 4.0 and a temperature of 50 °C.
| S-EPMC6332095 | biostudies-literature
Project description:Human erythrocytes contain a phosphatase that is highly specific for phosphoglycollate. It shows optimum pH of 6.7 and has Km 1 mM for phosphoglycollate. The molecular weight appears to be about 72000. The enzyme is a dimeric molecule having subunits of mol. wt. about 35000. It could be purified approx. 4000-fold up to a specific activity of 5.98 units/mg of protein. The activity of the enzyme is Mg2+-dependent. Co2+, and to a smaller extent Mn2+, may substitute for Mg2+. Half-maximum inhibition of the phosphatase by 5,5'-dithiobis-(2-nitrobenzoate), EDTA and NaF is obtained at 0.5 microM, 1 mM and 4 mM respectively. Moreover, it needs a univalent cation for optimum activity. Phosphoglycollate phosphatase is a cytoplasmic enzyme. Approx. 5% of its total activity is membrane-associated. This part of activity can be approx. 70% solubilized by freezing, thawing and treatment with 0.25% Triton X-100.
| S-EPMC1162188 | biostudies-other
Project description:In this study, peroxidase from Ziziphus jujuba was purified using ion exchange, and gel filtration chromatography resulting in an 18.9-fold enhancement of activity with a recovery of 20%. The molecular weight of Z. jujuba peroxidase was 56?kDa, as estimated by Sephacryl S-200. The purity was evaluated by SDS, which showed a single prominent band. The optimal activity of the peroxidase was achieved at pH 7.5 and 50?°C. Z. jujuba peroxidase showed catalytic efficiency (Kcat/Km) values of 25 and 43 for guaiacol and H2O2, respectively. It was completely inactivated when incubated with ?-mercaptoethanol for 15?min. Hg2+, Zn2+, Cd2+, and NaN3 (5?mM) were effective peroxidase inhibitors, whereas Cu2+ and Ca2+ enhanced the peroxidase activity. The activation energy (Ea) for substrate hydrolysis was 43.89?kJ?mol-1, while the Z value and temperature quotient (Q10) were found to be 17.3?°C and 2, respectively. The half-life of the peroxidase was between 117.46 and 14.15?min. For denaturation of the peroxidase, the activation energy for irreversible inactivation Ea*(d) was 120.9 kJmol-1. Thermodynamic experiments suggested a non-spontaneous (?G*d > 0) and endothermic reaction phase. Other thermodynamic parameters of the irreversible inactivation of the purified enzyme, such as ?H* and ?S*, were also studied. Based on these results, the purified peroxidase has a potential role in some industrial applications.
| S-EPMC7224213 | biostudies-literature
Project description:Phytase is a phosphatase enzyme widely used as feed additive to release inorganic phosphorus from plant phytate and enhance its uptake in monogastric animals. Although engineered fungal phytases are used most, a natural enzyme gives opportunity to understand novel properties, if any. In the current study, a novel fungal strain, Aspergillus foetidus MTCC 11682 was immobilized on poly urethane cubes and used for phytase production, purification and molecular characterization. Phytase produced by this method was partially purified by ammonium sulphate precipitation and Sephacryl S-200HR gel filtration to 23.4-fold (compared to crude extract) with recovery of 13% protein. Electrophoresis analysis revealed that phytase has molecular weight of 90.5 kDa on non-reducing and 129.6 kDa on reducing SDS-PAGE. The purified phytase exhibited a wider pH and temperature stability. Analysis of the cloned sequence showed that the gene has 1176 bp that encodes for a peptide of 391 amino acids of the core catalytic region. It was also found that phytase from A. foetidus has a sequence identity of 99% with the phytase gene of other Aspergillus species at nucleotide level and 100% at protein level in A. niger, A. awamori, A. oryzae. In silico analysis of sequence identified the presence of two consecutive and one non-consecutive intra chain disulfide bonds in the phytase. This probably contributed to the differential migration of phytase on reducing and non-reducing SDS-PAGE. There are predicted 11 O-glycosylation sites and 8 N-glycosylation sites, possibly contributed to an enhanced stability of enzyme produced by this organism. This study opened up a new horizon for exploring the novel properties of phytase for other applications.
| S-EPMC6320329 | biostudies-literature