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1.
Hog thyroid peroxidase: physical, chemical, and catalytic properties of the highly purified enzyme. 总被引:4,自引:0,他引:4
A B Rawitch A Taurog S B Chernoff M L Dorris 《Archives of biochemistry and biophysics》1979,194(1):244-257
Studies are reported on the purity and on the physical, chemical, and catalytic properties of a highly purified, stable, thyroid peroxidase (TPO). The enzyme was solubilized by treatment with deoxycholate and trypsin, and it was purified by a series of column treatments, including ion-exchange chromatography on DEAE-cellulose, gel filtration through Bio-Gel P-100, and hydroxylapatite chromatography. The final product, designated TPO VII, had a value for A410/A280 of 0.54, and its specific activity based on the guaiacol assay (794 μmol of guaiacol oxidized/min/mg) was considerably greater than that of any previously described TPO. Specific activity values based on other peroxidase-catalyzed reactions were also higher for TPO VII than for previous TPO preparations. Purity estimates for TPO VII, based on polyacrylamide disc gel electrophoresis and on isoelectric focusing in polyacrylamide gels, ranged from 80 to 95%. The molecular weight, determined by sedimentation equilibrium, was 93,000. Results of sodium dodecyl sulfate-gel electrophoresis also indicated a molecular weight of approximately 90,000. Sodium dodecyl sulfate-gel electrophoresis under reducing conditions indicated that TPO VII is composed of two peptide chains of unequal size, with the larger about 2.5-fold the size of the smaller. Carbohydrate analysis revealed that TPO is a glycoprotein containing about 10% by weight of carbohydrate. The predominant sugars were mannose and N-acetyl glucosamine. A significant amount of glucose was also found, along with small amounts of galactose, fucose, and xylose. The amino acid composition of TPO VII showed a high proline content, a predominance of arginine over lysine, and a ratio of [Asp] plus [Glu] to [Lys] plus [Arg] of over 2. Isoelectric focusing in polyacrylamide gels indicated an isoelectric pH of 5.75. In agreement with observations made on earlier preparations of TPO, heme spectral data showed significant differences between the pyridine hemochromogens of TPO VII and horseradish peroxidase, suggesting that the heme in TPO is not ferriprotoporphyrin IX. Circular dichroism measurements indicated that approximately 40% of TPO VII involves α helix or β structure. 相似文献
2.
Spectral scans in both the visible (650-450 nm) and the Soret (450-380 nm) regions were recorded for the native enzyme, Compound II, and Compound III of lactoperoxidase and thyroid peroxidase. Compound II for each enzyme (1.7 microM) was prepared by adding a slight excess of H2O2 (6 microM), whereas Compound III was prepared by adding a large excess of H2O2 (200 microM). After these compounds had been formed it was observed that they were slowly reconverted to the native enzyme in the absence of exogenous donors. The pathway of Compound III back to the native enzyme involved Compound II as an intermediate. Reconversion of Compound III to native enzyme was accompanied by the disappearance of H2O2 and generation of O2, with approximately 1 mol of O2 formed for each 2 mol of H2O2 that disappeared. A scheme is proposed to explain these observations, involving intermediate formation of the ferrous enzyme. According to the scheme, Compound III participates in a reaction cycle that effectively converts H2O2 to O2. Iodide markedly affected the interconversions between native enzyme, Compound II, and Compound III for lactoperoxidase and thyroid peroxidase. A low concentration of iodide (4 microM) completely blocked the formation of Compound II when lactoperoxidase or thyroid peroxidase was treated with 6 microM H2O2. When the enzymes were treated with 200 microM H2O2, the same low concentration of iodide completely blocked the formation of Compound III and largely prevented the enzyme degradation that otherwise occurred in the absence of iodide. These effects of iodide are readily explained by (i) the two-electron oxidation of iodide to hypoiodite by Compound I, which bypasses Compound II as an intermediate, and (ii) the rapid oxidation of H2O2 to O2 by the hypoiodite formed in the reaction between Compound I and iodide. 相似文献
3.
Purification and characterization of a large, tryptic fragment of human thyroid peroxidase with high catalytic activity 总被引:2,自引:0,他引:2
A Taurog M L Dorris N Yokoyama C Slaughter 《Archives of biochemistry and biophysics》1990,278(2):333-341
Thyroid peroxidase (TPO) was purified from human thyroid tissue, obtained at surgery from patients with Graves' disease, by a procedure similar to one that we had previously used for the purification of porcine TPO. The membrane-bound enzyme was solubilized by treatment of the thyroid particulate fraction with trypsin plus detergent. After precipitation with ammonium sulfate, the enzyme was purified by a series of column treatments, including ion-exchange chromatography on DEAE-cellulose, gel filtration through Bio-Gel P-100, and hydroxylapatite chromatography. Although a high degree of purification was achieved, the finally isolated product was considerably more heterogeneous than the TPO obtained from porcine thyroids. Several pools of active enzyme differing in values for A412/A280 and in specific activity were collected. Gel electrophoresis was performed under native, denaturing [sodium dodecyl sulfate (SDS)] and denaturing plus reducing conditions. Native gel electrophoresis indicated that the active enzyme (93 kDa) was heavily contaminated with an inactive 60-kDa fragment, which we were unable to remove by HPLC. The inactive fragment was highly antigenic when tested on immunoblots with an antibody to TPO. The presence of the inactive fragment greatly reduced values for A412/A280 in the finally purified human TPO. Two of the pools, with A412/A280 values of 0.159 and 0.273, were used for further testing. Catalytic activity was very similar in these two pools when measured on the basis of heme content by several different assays. Moreover, the specific activities of both, based on heme content, were very similar to those observed with a porcine TPO preparation with A412/A280 = 0.48. These findings indicate that the inactive 60-kDa fragment most likely did not contain heme. On SDS-polyacrylamide gel electrophoresis under reducing conditions, the 60-kDa fragment completely disappeared and was replaced by a 36- and a 24-kDa component. Amino terminal sequence information obtained on these components indicated that the 24-kDa component represents the amino terminal portion of the active 93-kDa fragment, whereas the 36-kDa fragment represents the carboxyl terminal portion. A model is proposed suggesting that the 60-kDa fragment was generated by trypsin cleavage of native TPO at two internal sites within a disulfide loop (res approximately 300 and res 564) and at one further internal site (res 280). In addition, trypsin cleavage is proposed at sites near the amino and carboxyl ends common to both the active 93-kDa and the inactive 60-kDa fragments.(ABSTRACT TRUNCATED AT 400 WORDS) 相似文献
4.
R Sinclair I Yamazaki J Bumpus B Brock C S Chang A Albo L Powers 《Biochemistry》1992,31(20):4892-4900
The wood-degrading fungus Phanerochaete chrysosporium secretes a number of extracellular enzymes called lignin peroxidases which are involved in the degradation of both lignin and a number of persistent environmental pollutants. Lignin peroxidase isozyme H2, a glycosylated protein of approximately 40 kDa, contains a single heme. X-ray absorption spectroscopy (XAS) has been used to probe the local environment of the iron in the active site of resting enzyme, reduced enzyme, and compound III. For the native and reduced forms, respectively, the average Fe-pyrrole nitrogen distances are 2.055 and 2.02 A (+/- 0.015 A); the Fe-proximal nitrogen distance is 1.93 and 1.91 A (+/- 0.02 A) while the Fe-distal ligand distance is 2.17 and 2.10 A (+/- 0.03 A). Although the results are not as well-defined, the active-site structure of compound III is largely 2.02 +/- 0.015 A for the average Fe-pyrrole nitrogen distance, 1.90 +/- 0.02 for the Fe-proximal nitrogen, and 1.74 +/- 0.03 A for the Fe-distal ligand distance. The heme iron-pyrrole nitrogen distance is more expanded in ligninase H2 than in other peroxidases. The possible significance of this is discussed in relation to other heme proteins. 相似文献
5.
Gardas A Watson PF Hobby P Smith A Weetman AP Sutton BJ Banga JP 《Redox report : communications in free radical research》2000,5(4):237-241
The enzyme, thyroid peroxidase (TPO), is a dominant antigen in thyroid autoimmune diseases. Autoantibodies recognised two major dominant conformational epitopes termed A and B. The epitopes have been defined by mAbs, but the amino acid residues which constitute these determinants remain unknown. Using a model of TPO, built from the structure of myeloperoxidase (MPO), we have synthesised peptides corresponding to exposed loops and generated rabbit antibodies to the peptides. Antisera to peptide sequence 599-617 (peptide 14) representing a highly protrusive loop on the TPO, showed the highest inhibition in 65 sera from patients positive with anti-TPO antibodies. The inhibition was by 15-80% (mean 41%), and no other antibody showed any inhibition. Binding of hFabs to the B determinant on TPO was inhibited by anti-peptide 14 antibodies more then 85%, but not Fabs to the A determinant. In conclusion, the peptide 14 defines a sequence taking part in building up the B major conformational epitope. None of generated anti-peptide antibodies alone inhibited the binding of human Fabs to the A epitope, however a combination of four anti-peptide antibodies (P1, P12, P14 and P18) inhibits Fabs binding to the A determinant by more then 60% and autoantibodies binding from 65% to 94%. Combination of antibodies reacting with peptides outside the surface defined by those four antipeptide antibodies did not give any inhibition of Fabs to TPO. The inhibition of Fabs and auto Abs to TPO by this combination of anti-peptide Abs is the result of steric hindrance as none of these Abs individually inhibited auto Abs' or Fabs' binding to TPO. The four peptides define an area on the enzyme surface where the A and B major conformational epitopes are localised. 相似文献
6.
《Redox report : communications in free radical research》2013,18(4):237-241
AbstractThe enzyme, thyroid peroxidase (TPO), is a dominant antigen in thyroid autoimmune diseases. Autoantibodies recognised two major dominant conformational epitopes termed A and B. The epitopes have been defined by mAbs, but the amino acid residues which constitute these determinants remain unknown. Using a model of TPO, built from the structure of myeloperoxidase (MPO), we have synthesised peptides corresponding to exposed loops and generated rabbit antibodies to the peptides.Antisera to peptide sequence 599–617 (peptide 14) representing a highly protrusive loop on the TPO, showed the highest inhibition in 65 sera from patients positive with anti-TPO antibodies. The inhibition was by 15–80% (mean 41%), and no other antibody showed any inhibition. Binding of hFabs to the B determinant on TPO was inhibited by anti-peptide 14 antibodies more then 85%, but not Fabs to the A determinant. In conclusion, the peptide 14 defines a sequence taking part in building up the B major conformational epitope.None of generated anti-peptide antibodies alone inhibited the binding of human Fabs to the A epitope, however a combination of four anti-peptide antibodies (P1, P12, P14 and P18) inhibits Fabs binding to the A determinant by more then 60% and autoantibodies binding from 65% to 94%. Combination of antibodies reacting with peptides outside the surface defined by those four anti-peptide antibodies did not give any inhibition of Fabs to TPO.The inhibition of Fabs and auto Abs to TPO by this combination of anti-peptide Abs is the result of steric hindrance as none of these Abs individually inhibited auto Abs' or Fabs' binding to TPO.The four peptides define an area on the enzyme surface where the A and B major conformational epitopes are localised. 相似文献
7.
Six tryptic peptides ranging in size from 3 to 126 residues were isolated from maleylated Fragment A of diphtheria toxin after tryptic hydrolysis. These peptides accounted for all 193 residues found by amino acid analysis. After demaleylation, the six peptides were purified by chromatography on Sephadex G-50, coupled with paper chromatography and electrophoresis, and were analyzed by various methods. The compositions and properties of the peptides are reported. Almost 70% of the residues were positioned within these peptides. 相似文献
8.
J H Martinez F Solano R Pe?afiel J D Galindo J L Iborra J A Lozano 《Comparative biochemistry and physiology. B, Comparative biochemistry》1986,83(3):633-636
The inhibition of tyrosinases from frog epidermis (Rana esculenta ridibunda), mushroom (Agaricus bisporus) and Harding-Passey mouse melanoma by halides is compared. In all cases, the inhibition is pH dependent, increasing when the pH decreases. The order of inhibition is I- greater than Br- greater than Cl- much greater than F- for frog epidermis tyrosinase, F- greater than I- greater than Cl- greater than Br- for mushroom tyrosinase and F- greater than Cl- much greater than Br- greater than I- for the mouse melanoma enzyme. These results are discussed in terms of the active site accessibility to exogenous ligands. The activation energies of the enzyme-catalysed L-dopa oxidation were also calculated, being the values 6.86, 17.01 and 20.25 kcal/mol for frog epidermis, mushroom and Harding-Passey mouse melanoma, respectively. A relationship between these values and the evolutionary adaptation of these enzymes is proposed. 相似文献
9.
Purified human erythrocyte membrane acetylcholinesterase was subjected to limited proteolysis with papain. This treatment generated a hydrophilic form of the enzyme as determined by charge-shift crossed immunoelectrophoresis and by binding to phenyl-Sepharose. The hydrophilic enzyme was stable and its activity was independent of the presence of amphiphiles. Electroimmunochemical analysis showed no antigenic difference between the two enzyme forms. Although the proteolytic treatment only brought about a small change in molecular weight, marked differences in the hydrodynamic properties were encountered. The Stokes radius decreased from 8.2 to 5.9 nm and the sedimentation coefficient increased from 6.3 to 7.0 S. The results are consistent with the view that a short hydrophobic peptide responsible for the amphipatic character of acetylcholinesterase is removed by the treatment with papain. 相似文献
10.
Porcine D-amino acid oxidase: production of the biologically active enzyme in Escherichia coli 总被引:4,自引:0,他引:4
E Ciccarelli M Massaer J P Guillaume A Herzog R Loriau A Cravador P Jacobs A Bollen 《Biochemical and biophysical research communications》1989,161(2):865-872
DNA molecules coding either for mature porcine D-amino acid oxidase or for truncated forms of the enzyme have been obtained by stepwise addition of synthetic oligonucleotides to a partial cDNA. Under the control of the lambda PL thermoregulatable promoter, these DNAs were respectively expressed in Escherichia coli as 36, 28 and 25 kilodalton polypeptides, specifically recognised by antibodies raised against the natural enzyme. None of the truncated proteins were biologically active whereas the mature recombinant species was able to hydrolyze D-alanine in vitro as efficiently as the natural product. 相似文献
11.
12.
Le Du MH Lamoure C Muller BH Bulgakov OV Lajeunesse E Ménez A Boulain JC 《Journal of molecular biology》2002,316(4):941-953
The crystal structure of three mutants of Escherichia coli alkaline phosphatase with catalytic activity (k(cat)) enhancement as compare to the wild-type enzyme is described in different states. The biological aspects of this study have been reported elsewhere. The structure of the first mutant, D330N, which is threefold more active than the wild-type enzyme, was determined with phosphate in the active site, or with aluminium fluoride, which mimics the transition state. These structures reveal, in particular, that this first mutation does not alter the active site. The second mutant, D153H-D330N, is 17-fold more active than the wild-type enzyme and activated by magnesium, but its activity drops after few days. The structure of this mutant was solved under four different conditions. The phosphate-free enzyme was studied in an inactivated form with zinc at site M3, or after activation by magnesium. The comparison of these two forms free of phosphate illustrates the mechanism of the magnesium activation of the catalytic serine residue. In the presence of magnesium, the structure was determined with phosphate, or aluminium fluoride. The drop in activity of the mutant D153H-D330N could be explained by the instability of the metal ion at M3. The analysis of this mutant helped in the design of the third mutant, D153G-D330N. This mutant is up to 40-fold more active than the wild-type enzyme, with a restored robustness of the enzyme stability. The structure is presented here with covalently bound phosphate in the active site, representing the first phosphoseryl intermediate of a highly active alkaline phosphatase. This study shows how structural analysis may help to progress in the improvement of an enzyme catalytic activity (k(cat)), and explains the structural events associated with this artificial evolution. 相似文献
13.
Carbamoyl-phosphate synthetase: an example of effects on enzyme properties of shifting an equilibrium between active monomer and active oligomer 总被引:1,自引:0,他引:1
P M Anderson 《Biochemistry》1986,25(19):5576-5582
Carbamoyl-phosphate synthetase from Escherichia coli is subject to allosteric activation by ornithine, allosteric inhibition by uridine 5'-phosphate (UMP), and reversible concentration-dependent self-association. Positive allosteric effectors, magnesium adenosine 5'-triphosphate (MgATP), K+, and inorganic phosphate facilitate association. The purpose of this study was to determine the state of association of carbamoyl-phosphate synthetase in the presence and absence of different substrates and effectors and to consider the basis for the observed effects of enzyme concentration on specific activity. Studies employing gel filtration chromatography have shown that when the concentration of carbamoyl-phosphate synthetase is low (less than 0.01 mg/mL), the enzyme exists as monomer under all conditions, including the presence of UMP in phosphate buffer and the presence of all substrates plus ornithine (conditions that support maximal catalytic activity). At higher enzyme concentrations (e.g., greater than 0.01 mg/mL) the specific activity increases with increasing enzyme concentration when MgATP is nonsaturating but is independent of enzyme concentration when MgATP is saturating or when ornithine is present with MgATP being either saturating or nonsaturating. These results indicate that the catalytic activity of this enzyme is not directly linked to oligomer formation. The theoretical properties and possible significance of a generalized model of enzyme association-dissociation in which the active monomeric form, in equilibrium with another monomeric form, is specifically subject to self-association but the different states of association have the same specific activity, are discussed.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
14.
Biochemical activities of highly purified, catalytically active human APOBEC3G: correlation with antiviral effect
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APOBEC3G (APO3G), a cytidine deaminase with two zinc finger domains, inhibits human immunodeficiency virus type 1 replication in the absence of Vif. Here, we provide a comprehensive molecular analysis of the deaminase and nucleic acid binding activities of human APO3G using a pure system containing only one protein component, i.e., highly purified, catalytically active enzyme expressed in a baculovirus system. We demonstrate that APO3G deaminates cytosines in single-stranded DNA (ssDNA) only, whereas it binds efficiently to ssDNA and ssRNA, about half as well to a DNA/RNA hybrid, and poorly to double-stranded DNA and RNA. In addition, the base specificities for deamination and binding of ssDNA are not correlated. The minimum length required for detection of APO3G binding to an ssDNA oligonucleotide in an electrophoretic mobility shift assay is 16 nucleotides. Interestingly, if nucleocapsid protein and APO3G are present in the same reaction, we find that they do not interfere with each other's binding to RNA and a complex containing the RNA and both proteins is formed. Finally, we also identify the functional activities of each zinc finger domain. Thus, although both zinc finger domains have the ability to bind nucleic acids, the first zinc finger contributes more to binding and APO3G encapsidation into virions than finger two. In contrast, deamination is associated exclusively with the second zinc finger. Moreover, zinc finger two is more important than finger one for the antiviral effect, demonstrating a correlation between deaminase and antiviral activities. 相似文献
15.
《The International journal of biochemistry》1977,8(1):11-16
- 1.1. Creatinine amidohydrolase from Pseudomonas sp. has a pH optimum of 8.0 and is activated by divalent metals manganese, magnesium, zinc and cobalt.
- 2.2. It is acid labile but shows good stability at 55°C in alkaline solutions.
- 3.3. It has a mol. wt in the region of 248,000 and Michaelis constants of 31.7mM and 80 mM for creatinine and creatine respectively.
- 4.4. Results indicate that the enzyme molecule contains 8 subunits of similar mol. wt.
16.
J N Siedow S Power F F de la Rosa G Palmer 《The Journal of biological chemistry》1978,253(7):2392-2399
A soluble enzymically active cytochrome b.c1 complex has been purified from baker's yeast mitochondria by a procedure involving solubilization in cholate, differential fractionation with ammonium sulfate, and ultracentrifugation. The resulting particle is free of both cytochrome c oxidase and succinate dehydrogenase activities. The complex contains cytochromes b and c1 in a ratio of 2:1 and quinone and iron-sulfur protein in amounts roughly stoichiometric with cytochrome c1. EPR spectroscopy has shown the iron-sulfur protein to be present mainly as the Rieske protein. EPR spectroscopy also shows a heterogeneity in the cytochrome b population with resonances appearing at g = 3.60 (cytochrome bK) and g = 3.76 (cytochrome bT). A third EPR resonance appearing in the region associated with low spin ferric hemes (g = 3.49) is assigned to cytochrome c1. Anaerobic titration of the complex with dithionite confirmed the heterogeneity in the cytochrome b population and demonstrated that the oxidation-reduction potential of the iron-sulfur protein is approximately 30 mV more positive than cytochrome c1. An intense EPR signal assigned to the coenzyme Q free radical appeared midway in the reductive titration; this signal disappeared toward the end of the titration. A conformational change in the iron-sulfur protein attendant on reduction of a low potential species was noted. 相似文献
17.
J Arnhold P G Furtmüller G Regelsberger C Obinger 《European journal of biochemistry》2001,268(19):5142-5148
The standard reduction potential of the redox couple compound I/native enzyme has been determined for human myeloperoxidase (MPO) and eosinophil peroxidase (EPO) at pH 7.0 and 25 degrees C. This was achieved by rapid mixing of peroxidases with either hydrogen peroxide or hypochlorous acid and measuring spectrophotometrically concentrations of the reacting species and products at equilibrium. By using hydrogen peroxide, the standard reduction potential at pH 7.0 and 25 degrees C was 1.16 +/- 0.01 V for MPO and 1.10 +/- 0.01 V for EPO, independently of the concentration of hydrogen peroxide and peroxidases. In the case of hypochlorous acid, standard reduction potentials were dependent on the hypochlorous acid concentration used. They ranged from 1.16 V at low hypochlorous acid to 1.09 V at higher hypochlorous acid for MPO and from 1.10 V to 1.03 V for EPO. Thus, consistent results for the standard reduction potentials of redox couple compound I/native enzyme of both peroxidases were obtained with all hydrogen peroxide and at low hypochlorous acid concentrations: possible reasons for the deviation at higher concentrations of hypochlorous acid are discussed. They include instability of hypochlorous acid, reactions of hypochlorous acid with different amino-acid side chains in peroxidases as well as the appearance of a compound I-chloride complex. 相似文献
18.
4-methyleneglutamine amidohydrolase from peanut leaves : preparation, catalytic properties, and immunological responses of a highly purified form of the enzyme 总被引:1,自引:0,他引:1
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4-Methyleneglutamine amidohydrolase has been extracted and purified over 1000-fold from 14-day-old peanut (Arachis hypogaea) leaves by modification of methods described previously. The purified enzyme shows two bands of activity and three to four bands of protein after electrophoresis on nondenaturing gels. Each of the active bands is readily eluted from gel slices and migrates to its original position on subsequent electrophoresis. Although they are electrophoretically distinct, the two forms of the enzyme are immunologically identical by Ouchterlony double-diffusion techniques and have similar catalytic properties. Activity toward glutamine that has a threefold lower Vmax and a four-fold higher Km value copurifies with MeGln aminohydrolase activity. 4-Methyleneglutamine and 4-methyleneglutamic acid inhibit the hydrolysis of glutamine while glutamine inhibits 4-methyleneglutamine hydrolysis, further indicating the identity of the activity toward both substrates. Amidohydrolase activity is stimulated up to threefold by preincubation with either ionic or non-ionic detergents (0.1%) and also by added proteins (0.5% bovine serum albumin or whole rabbit serum); it is inhibited 50% by 1 millimolar borate or the glutamine analog, albizziin (10 millimolar). Rabbit antiserum to the purified peanut enzyme cross-reacts with one or more proteins in extracts of some plants but not others; in no instance, however, was 4-methyleneglutamine amidohydrolase activity detected in other species. Overall, the results support the hypothesis that 4-methyleneglutamine supplies N, via its hydrolysis by the amidohydrolase, to the growing shoots of peanut plants, whereas glutamine hydrolysis is prevented by the prepon-derance of the preferred substrate. Some results also suggest that this amidohydrolase activity may be regulated by metabolites and/or by association with other cellular components. 相似文献
19.
B Czarnocka J Ruf M Ferrand P Carayon 《Comptes rendus de l'Académie des sciences. Série III, Sciences de la vie》1985,300(15):577-580
Pools of sera from patients with Graves' disease or Hashimoto's thyroiditis highly inhibit the binding to human thyroid membranes of one of 19 monoclonal antibodies raised against preparations of human thyroid membranes. This monoclonal antibody reacts with human and bovine thyroid peroxidase and bovine lactoperoxidase but not with human hemoglobin, cytochrome c and other related molecules. These results indicate that the thyroid peroxidase and the microsomal antigen are antigenically related. These data taken together with those from other groups, highly suggest that thyroid peroxidase is the microsomal antigen involved in autoimmune thyroid diseases. 相似文献
20.
Yasuyo Suzuki Keiko Soga Katsuhiko Yoshimatsu Yuzo Shioi 《Photochemical & photobiological sciences》2008,7(10):1260-1266
Formation of pyropheophorbide (PyroPheid) during chlorophyll metabolism in some higher plants has been shown to involve the enzyme pheophorbidase (PPD). This enzyme catalyzes the conversion of pheophorbide (Pheid) a to a precursor of PyroPheid, C-13(2)-carboxylPyroPheid a, by demethylation, and then the precursor is decarboxylated non-enzymatically to yield PyroPheid a. In this study, expression, purification, and biochemical characterization of recombinant PPD from radish (Raphanus sativus L.) were performed, and its properties were compared with those of highly purified native PPD. Recombinant PPD was produced using a glutathione S-transferase (GST) fusion system. The PPD and GST genes were fused to a pGEX-2T vector and expressed in Escherichia coli under the control of a T7 promoter as a fusion protein. The recombinant PPD-GST was expressed as a 55 kDa protein as measured by SDS-PAGE and purified by single-step affinity chromatography through a GSTrap FF column. PPD-GST was purified to homogeneity with a yield of 0.42 mg L(-1) of culture. The protein purified by this method was confirmed to be PPD by measuring its activity. The purified PPD-GST fusion protein revealed potent catalytic activity for demethylation of the methoxycarbonyl group of Pheid a and showed a pH optimum, substrate specificity, and thermal stability quite similar to the native enzyme purified from radish, except for the Km values toward Pheid a: 95.5 microM for PPD-GST and about 15 microM for native PPDs. 相似文献