Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

A cDNA (C2C-Prx) corresponding to a 2Cys-peroxiredoxin (2Cys-Prx) was isolated from a leaf cDNA library of Chinese cabbage. The predicted amino acid sequence of C2C-Prx has 2 conserved cysteines and several peptide domains present in most of the 2Cys-Prx subfamily members. It shows the highest sequence homology to the 2Cys-Prx enzymes of spinach (88%) and Arabidopsis (86%). Southern analysis using the cDNA insert of C2C-Prx revealed that it consists of a small multigene family in Chinese cabbage genome. RNA blot analysis showed that the gene was predominantly expressed in the leaf tissue of Chinese cabbage seedlings, but the mRNA was generally expressed in most tissues of mature plant, except roots. The expression of C2C-Prx was slightly induced by treatment with H2O2 (100 microM) or Fe3+/O2/DTT oxidation system, but not by ABA (50 microM) or GA3 (10 microM). The C2C-Prx is encoded as a preprotein of 273 amino acids containing a putative chloroplast-targeting signal of 65 amino acids at its N-terminus. The N-terminally truncated recombinant protein (deltaC2C-Prx) migrates as a dimer in a non-reducing SDS-polyacrylamide gel and as a monomer in a reducing condition. The deltaC2C-Prx shows no immuno cross-reactivity to antiserum of the yeast thiol-specific antioxidant protein, and vice versa. The deltaC2C-Prx prevents the inactivation of glutamine synthetase and the DNA cleavage in the metal-catalyzed oxidation system. In the yeast thioredoxin system containing thioredoxin reductase, thioredoxin, and NADPH, the deltaC2C-Prx exhibits peroxidase activity on H2O2.
Plant Mol Biol 1999 Jul
PMID:Molecular cloning, expression, and functional characterization of a 2Cys-peroxiredoxin in Chinese cabbage. 1048 17

Myocardial stunning is characterized by the impairment of excitation-contraction coupling via a decrease in myofilament Ca2+ responsiveness, thought to be triggered by hydroxyl radicals (*OH) generated upon reperfusion. Since peroxynitrite is also expected to be produced during reperfusion, we examined whether it can induce a stunned myocardium-like impairment of cardiac myocytes. Its effect on cultured cardiac myocytes was compared with that of hydrogen peroxide (H2O2), *OH source. Infusion of peroxynitrite (0.2 mM) induced a decrease in cell motion and a complete arrest in diastole at 2.9 +/- 0.3 min, which coincided with an elevation in [Ca2+]i. Arrest induced by infusion of H2O2 (10 mM) was not associated with an increase in [Ca2+]i. The ATP content was unaffected by peroxynitrite (control, 34.3 +/- 3.4: + peroxynitrite, 32.9 +/- 3.5 nmol/mg protein) and the cells remained viable. Sulfhydryl (SH) content was decreased by peroxynitrite, but not by H2O2. The membrane fluidity (a measure of peroxidation of the membrane lipids) was not affected by peroxynitrite, but was decreased by H2O2. Onset time of arrest was unaffected by deferoxamine (0.2 mM), but was delayed by DTT (10 mM) (from 2.9 +/- 0.3 to 19.2 +/- 1.6 min). Nitrotyrosine content was unchanged by peroxynitrite, and its augmentation with Fe3+/EDTA (1 mM) was not associated with a shortened onset time of arrest. The function of the Na+/Ca2+ exchanger was impaired by peroxynitrite, but not by H2O2. Peroxynitrite and H2O2 each induce arrest, but only the former increases [Ca2+]i. One of the mechanisms of the increase in [Ca2+]i is Na/Ca2+ exchanger dysfunction. The impairments were induced through SH oxidation by peroxynitrite, but through lipid peroxidation by H2O2. Myocardial stunning may be induced by both species in concert.
Mol Cell Biochem 1999 Aug
PMID:Distinct roles of peroxynitrite and hydroxyl radical in triggering stunned myocardium-like impairment of cardiac myocytes in vitro. 1049 75

A Cortland saline-perfused rainbow trout (Oncorhynchus mykiss) liver model was used to study aspects of T4 efflux from the intact organ system. There was a consistent efflux of T4 in the absence of T4 in the perfusate, and the T4 efflux was increased in the presence of T4 in the perfusate, but the efflux was not T4-dose dependent. The addition of the thiol-containing compound dithiothreitol (DTT, 2 mM) to the perfusate had no significant effect on the flux of T4 from the liver, whereas the addition of cysteine (2 mM), a thiol-containing amino acid suppressed T4 efflux. The results are consistent with the known mechanisms of thyroid hormone trafficking across cell membranes, and suggest that organ systems, such as the liver, may act as a major reserve of hormone, thus participating in plasma thyroid hormone homeostasis.
Comp Biochem Physiol B Biochem Mol Biol 1999 Oct
PMID:Efflux of T4 from the in situ perfused liver of rainbow trout: effect of T4, dithiothreitol and cysteine in the perfusate. 1058

In this study the in vitro development of porcine nuclear transfer (NT) embryos was investigated. Transgenic fetal fibroblast cells that were frozen after 5 days of serum starvation were injected immediately after thawing into enucleated metaphase II (MII) oocytes. Reconstructed embryos were activated by incubation in 200 microM thimerosal followed by a 30-min treatment of 8 mM DTT. The embryos were subsequently cultured in NCSU23, supplemented with 4 mg/ml BSA for 7 days. The actual cleavage rate (embryos showing > or =2 nuclei) in 6 replicates was 33% (ranging from 15% to 50%). Three blastocysts with cell numbers of 14, 15, and 18 were obtained. The blastocyst rate was significantly lower for NT embryos as opposed to parthenogenetically activated embryos (1% vs. 5%; P<0.05). The neomycin-resistance gene was amplified by PCR in all three NT embryos, indicating their origin from the injected transgenic fibroblasts. Efforts are now being directed in improvements in the nuclear transfer technology, whereby viable fetuses or offspring can be produced from these NT-embryos.
Mol Reprod Dev 2000 Jun
PMID:Production of transgenic porcine blastocysts by nuclear transfer. 1081 45

The major oxidative folding pathways of bovine pancreatic ribonuclease A at pH 8.0 and 25 degrees C involve a pre-equilibrium steady state among ensembles of intermediates with zero, one, two, three and four disulfide bonds. The rate-determining steps are the reshuffling of the unstructured three-disulfide ensemble to two native-like three-disulfide species, des-[65-72] and des-[40-95], that convert to the native structure during oxidative formation of the fourth disulfide bond. Under the same regeneration conditions, with oxidized and reduced DTT, used previously for kinetic oxidative-folding studies of this protein, the addition of 4 microM protein disulfide isomerase (PDI) was found to lead to catalysis of each disulfide-formation step, including the rate-limiting rearrangement steps in which the native-like intermediates des-[65-72] and des-[40-95] are formed. The changes in the distribution of intermediates were also determined in the presence and absence of PDI at three different temperatures (with the DTT redox system) as well as at 25 degrees C (with the glutathione redox system). The results indicate that the acceleration of the formation of native protein by PDI, which we observed earlier, is due to PDI catalysis of each of the intermediate steps without changing the overall pathways or folding mechanism.
J Mol Biol 2000 Jul 21
PMID:Catalysis of the oxidative folding of bovine pancreatic ribonuclease A by protein disulfide isomerase. 1089 Dec 84

Creatine kinase (ATP:creatine N-phosphotransferase, EC 2.7.3.2) is a good model for studying dissociation and reassociation during unfolding and refolding. This study compares self-reassociated CK dimers and CK dimers that contain hybrid dimers under proper conditions. Creatine kinase forms a monomer when denatured in 6 M urea for 1 h which will very quickly form a dimer when the denaturant is diluted under suitable conditions. After modification by DTNB, CK was denatured in 6 M urea to form a modified CK monomer. Dimerization of this modified subunit of CK occurred upon dilution into a suitable buffer containing DTT. Therefore, three different types of reassociated CK dimers including a hybrid dimer can be made from two different CK monomers in the proper conditions. The CK monomers are from a urea-denatured monomer of DTNB-modified CK and from an unmodified urea dissociated monomer. Equal enzyme concentration ratios of these two monomers were mixed in the presence of urea, then diluted into the proper buffer to form the three types of reassociated CK dimers including the hybrid dimer. Reassociated CK dimers including all three different types recover about 75% activity following a two-phase course (k1 = 4.88 x 10(-3) s(-1), k2 = 0.68 x 10(-3) s(-1)). Intrinsic fluorescence spectra of the three different CK monomers which were dissociated in 6 M urea, dissociated in 6 M urea after DTNB modification, and a mixture of the first two dissociated enzymes were studied in the presence of the denaturant urea. The three monomers had different fluorescence intensities and emission maxima. The intrinsic fluorescence maximum intensity changes of the reassociated CK dimers were also studied. The refolding processes also follow biphasic kinetics (k1 = 3.28 x 10(-)3 s(-1), k2 = 0.11 x 10(-3) S(-1)) after dilution in the proper solutions. Tsou's method [Tsou (1988), Adv. Enzymol. Rel. Areas Mol. Biol. 61, 381-436] was also used to measure the kinetic reactivation rate constants for the different three types of reassociated CK dimers, with different kinetic reactivation rate constants observed for each type. CK dissociation and reassociation schemes are suggested based on the results.
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PMID:Reactivation and refolding of reassociated dimers of rabbit muscle creatine kinase. 1098 10

I. The serotonin1A (5-HT1A) receptors are members of a superfamily of seven-transmembrane-domain receptors that couple to G-proteins. They appear to be involved in various behavioral and cognitive functions. Mutagenesis and modeling studies point out that the ligand-binding sites in serotonin receptors are located in the transmembrane domain. However, these binding sites are not very well characterized. Since disulfide bonds and sulfhydryl groups have been shown to play vital roles in the assembly, organization, and function of various G-protein-coupled receptors, we report here the effect of disulfide and sulfhydryl group modifications on the agonist and antagonist binding activity of 5-HT1A receptors from bovine hippocampus. 2. DTT or NEM treatment caused a concentration-dependent reduction in specific binding of the agonist and antagonist in 5-HT1A receptors from bovine hippocampal native and solubilized membranes. This is supported by a concomitant reduction in binding affinity. 3. Pretreatment of the receptor with unlabeled ligands prior to chemical modifications indicate that the majority of disulfides or sulfhydryl groups that undergo modification giving rise to inhibition in binding activity could be at the vicinity of the ligand-binding sites. 4. In addition, ligand-binding studies in presence of GTP-gamma-S, a nonhydrolyzable analogue of GTP, indicate that sulfhydryl groups (and disulfide bonds to a lesser extent) are vital for efficient coupling between the 5-HT1A receptor and the G-protein. 5. Our results point out that disulfide bonds and sulfhydryl groups could play an important role in ligand binding in 5-HT1A receptors.
Cell Mol Neurobiol 2000 Dec
PMID:Role of disulfides and sulfhydryl groups in agonist and antagonist binding in serotonin1A receptors from bovine hippocampus. 1110 Sep 75

Superoxide dismutase (SOD; EC 1.15.1.1) is an enzyme that protects against oxidative stress from superoxide radicals in living cells. This enzyme has been isolated, purified and partially characterized from chicken liver. The following steps were carried out in order to purify chicken liver SOD. Initially, the liver was homogenized and hemoglobin was removed. Subsequently protein precipitation was effected with (NH(4))(2)SO(4), methanol, (NH(4))(2)SO(4)-methanol and polyethylene glycol methods. The product from polyethylene glycol-3350 precipitation was found to have the highest SOD activity. Polyethylene glycol was removed by chromatography using a PD-10 column. After passing through an ultrafilter, the superoxide dismutase was fractionated by DEAE-ion chromatography and then Sephadex G-75 gel filtration chromatography. During this purification procedure, a specific activity of 4818.2 IU/mg was reached, corresponding to 285.8-fold purification. The purified enzyme, which was characterized as cyanide-sensitive SOD, contained two subunits having Cu and Zn elements with a molecular weight of 16000+/-500 for each. The optimum pH of purified CuZnSOD was determined to be 8.9. The enzyme was found to have good pH stability in the pH range 6.0-7.5 at 25 degrees C over a 2-h incubation period and displayed good thermal stability up to 45 degrees C at pH 7.4 over a 1-h incubation period. The SOD enzyme was not inhibited by DTT and beta-mercaptoethanol, but inhibited by CN(-) and H(2)O(2). In the presence of 2 mM iodoacetamide, the enzyme showed an approximately 40% activity loss. Finally, the inhibitory effect of ionic strength on SOD was also investigated.
Comp Biochem Physiol B Biochem Mol Biol 2001 Feb
PMID:Purification and characterization of superoxide dismutase from chicken liver. 1120 34

Guanidine hydrochloride-denatured creatine kinase (CK) can very quickly form a dimer with reactivity when the denaturant is diluted into the reaction system in the presence of DTT or EDTA. Tsou's method and its applied equation [Tsou (1988), Adv. Enzymol. Rel. Areas Mol. Biol. 61, 381-436; Yang and Zhou (1998), Biochim. Biophys. Acta 1388, 190-198] were used to measure the kinetic reactivation rate constants and the reactivation degree for reassociated CK dimers. Partial reactivation (about 50% at best) occurred following a monophasic course during the substrate reaction when compared with previous time interval measurements. The reactivation degree increased with increasing DTT (0.1-5 mM) and EDTA (0.1-1 mM) concentrations. The apparent forward rate constants do not change with concentration, showing that the reactivation is a reversible first-order reaction, but not of complex formation type. However, the apparent forward rate constants do change with EDTA concentration, showing that the reactivation with EDTA is a reversible first-order reaction as well as of complex formation type. Excess DTT concentrations have an inhibitory effect, indicating that the excessive EDTA acts as a metal chealate not only for free Mg2+, but also for MgATP during the enzyme catalysis. This study shows that additional information about the reactivation of CK can be obtained from examining the substrate reaction. The possible refolding pathway of CK is discussed.
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PMID:Reactivation kinetics of guanidine hydrochloride-denatured creatine kinase measured using the substrate reaction. 1133 Mar 50

Intracellular pH has recently been shown to increase during parthenogenetic activation of the porcine oocyte. In the following set of experiments, intracellular pH was monitored during activation and pronuclear development was assessed following activation treatments with calcium, in the absence of calcium, and in oocytes loaded with the calcium chelator BAPTA-AM in calcium-free medium. Intracellular pH increase was not different among groups when treating with 7% ethanol or 50 microM calcium ionophore, or during treatment with thimerosal for 12 or 25 min. Activation with thimerosal (200 microM, 12 min) followed by 8 mM dithiothreitol (DTT, 30 min) resulted in a decreased pronuclear development in calcium-free medium with or without BAPTA-AM loaded oocytes as compared to controls. Activation with 50 microM calcium ionophore resulted in pronuclear development that was different between the calcium-free and BAPTA-AM loaded oocytes in calcium-free medium. Similar incidences of pronuclear formation were observed in all ethanol treatment groups. It was concluded that external calcium as well as large changes in intracellular free calcium are not necessary for the increase in intracellular pH, but normal intracellular calcium signaling is critical for normal levels of pronuclear development. Finally, oocytes were measured for intracellular pH changes for 30 min following subzonal sperm injection. Intracellular pH did not increase, although pronuclear formation was observed 6 hr post SUZI. This suggested that major differences were still present between sperm-induced and parthenogenetic activation of the porcine oocyte.
Mol Reprod Dev 2001 Jun
PMID:Porcine oocyte activation: differing roles of calcium and pH. 1138 59


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