Catalytic properties of tryptophanless recombinant horseradish peroxidase

Heme-containing plant peroxidases (EC 1.11.1.7) contain a highly conserved single tryptophan residue. Its replacement with Phe in recombinant horseradish peroxidase (rHRP) increased the stability of the mutant enzyme in acid media. The kinetic properties of native, wild-type, and W117F mutant recombinant horseradish peroxidase in the reactions of ammonium 2, 2;-azino-bis(3-ethylbenzthiazoline-6-sulfonate) (ABTS), guaiacol, and o-phenylenediamine oxidation are very similar. However, significant changes in the reaction rate constant characteristic for the monomolecular rate-limiting step ascribed either to product dissociation from its complex with the enzyme or electron transfer from the substrate to the active site within the Michaelis complex were observed. The data indirectly indicate the participation of the single Trp residue in oxidation of ABTS and guaiacol and possible differences in kinetic mechanisms for oxidation of ABTS, guaiacol, and o-phenylenediamine.     

Effects of double ligation of Stensen's duct on the rabbit parotid gland

Salivary gland duct ligation is an alternative to gland excision for treating sialorrhea or reducing salivary gland size prior to tumor excision. Duct ligation also is used as an approach to study salivary gland aging, regeneration, radiotherapy, sialolithiasis and sialadenitis. Reports conflict about the contribution of each salivary cell population to gland size reduction after ductal ligation. Certain cell populations, especially acini, reportedly undergo atrophy, apoptosis and proliferation during reduction of gland size. Acini also have been reported to de-differentiate into ducts. These contradictory results have been attributed to different animal or salivary gland models, or to methods of ligation. We report here a bilateral double ligature technique for rabbit parotid glands with histologic observations at 1, 7, 14, 30, 60 days after ligation. A large battery of special stains and immunohistochemical procedures was employed to define the cell populations. Four stages with overlapping features were observed that led to progressive shutdown of gland activities: 1) marked atrophy of the acinar cells occurred by 14 days, 2) response to and removal of the secretory material trapped in the acinar and ductal lumens mainly between 30 and 60 days, 3) reduction in the number of parenchymal (mostly acinar) cells by apoptosis that occurred mainly between 14–30 days, and 4) maintenance of steady-state at 60 days with a low rate of fluid, protein, and glycoprotein secretion, which greatly decreased the number of leukocytes engaged in the removal of the luminal contents. The main post- ligation characteristics were dilation of ductal and acinar lumens, massive transient infiltration of mostly heterophils (rabbit polymorphonuclear leukocytes), acinar atrophy, and apoptosis of both acinar and ductal cells. Proliferation was uncommon except in the larger ducts. By 30 days, the distribution of myoepithelial cells had spread from exclusively investing the intercalated ducts pre-ligation to surrounding a majority of the residual duct-like structures, many of which clearly were atrophic acini. Thus, both atrophy and apoptosis made major contributions to the post-ligation reduction in gland size. Structures also occurred with both ductal and acinar markers that suggested acini differentiating into ducts. Overall, the reaction to duct ligation proceeded at a considerably slower pace in the rabbit parotid glands than has been reported for the salivary glands of the rat.     

AN INDIRECT ENZYME-LINKED IMMUNOSORBENT ASSAY FOR THE DETECTION OF LEPTOSPIRA INTERROGANS SEROVAR POMONA ANTIBODIES IN BOVINE SERA

An indirect ELISA was developed and initially evaluated for the detection of bovine antibodies to Leptospira interrogans serovar pomona. The antigen used in this ELISA was extracted from a serovar pomona culture supernatant by a combination of centrifugation, digestion with proteinase K and ultra-centrifugation. The antigen showed little cross-reaction with immune rabbit sera to L. interrogans serovars copenhageni, grippotyphosa, hardjo and sejroe and, Leptospira biflexa serovar patoc. Some cross-reaction was observed with immune rabbit serum to L. interrogans serovar canicola. The relative sensitivity of the ELISA was 94.76% confidence interval =± 3.32%) when estimated with bovine sera (n=172) with serovar pomona microscopic agglutination test (MAT) titers of 100. The relative specificity of the ELISA was 99.28% (95% confidence interval = 1.40%) when estimated with bovine sera (n=139) with MAT titers of <100 to L. interrogans serovars canicola, copenhageni, grippotyphosa, hardjo, pomona and sejroe. Thirty six of 258 field sera (13.95%) with serovar pomona MAT titers of <100, gave positive reactions in the ELISA.     

DEVELOPMENT AND EVALUATION OF A RAPID ENZYME-LINKED IMMUNOFILTRATION ASSAY (ELIFA) AND AN ENZYME-LINKED IMMUNOSORBENT ASSAY (ELISA) FOR THE DETECTION OF STAPHYLOCOCCAL ENTEROTOXIN B

An enzyme-linked immunofiltration assay (ELIFA) and a microtitre plate enzyme-linked immunosorbent assay (ELISA) were developed and compared for their ability to detect staphylococcal enterotoxin B (SEB). The double antibody capture format was used for both assays. Factors which improved the sensitivity of the ELIFA system were (1) addition of casein and thimerosal to the antigen dilution buffer; (2) addition of polyethylene glycol (MW 6000) to the detection and conjugate antibody dilution buffers; and (3) washing with diethanolamine buffer prior to addition of the substrate/chromogen. The ELIFA system had a turnaround time of approximately 1 h and a detection limit of 1 ng/mL of purified SEB. The ELISA had a total turnaround time of 21 h, or 3 h using plates pre-coated overnight with the capture antibody. The detection limit of the ELISA for purified SEB was 0.05 ng/mL. The detection limit of SEB in cheese samples spiked with purified enterotoxin and subjected to a simple extraction procedure was 1 ng/mL and 0.1 ng/mL of extract, with the ELIFA and the ELISA, respectively.     

Tryptophanless recombinant horseradish peroxidase: stability and catalytic properties.

The tryptophanless mutant of horseradish peroxidase, W117F, has been constructed and expressed in Escherichia coli. The mutation affects enzyme folding and stability. The optimum composition of the refolding medium requires the presence of ammonium sulfate. The yield of mutant is ca. 8000 U per liter of the optimized refolding medium with the specific activity of 1100-1500 U/mg (compared to 25, 000 U per liter and 2000 U/mg for the recombinant wild-type enzyme). The mutant is more stable in acid media, in the reaction course and toward irradiation. The effect of hydrogen peroxide pretreatment on radiation-induced inactivation of the wild-type and mutant enzyme indirectly indicates participation of Trp-117 in electron transfer pathways through the enzyme molecule. This is in agreement with the steady-state kinetic data interpreted in terms of Trp-117 participation in electron transfer within the Michaelis complex.     

Genetic diversity and population structure of <Emphasis Type="Italic">Taxus cuspidata</Emphasis> Sieb. et Zucc. ex Endl. (Taxaceae) in Russia according to data of the nucleotide polymorphism of intergenic spacers of the chloroplast genome

The genetic diversity and population structure of Taxus cuspidata Sieb. et Zucc. ex Endl. on the Russian part of its range was studied for the first time on the basis of the nucleotide polymorphism of intergenic spacers psbA–trnH, trnL–trnF, and trnS–trnfM of chloroplast DNA. A high level of gene (h = 0.807) and nucleotide (π = 0.0227) diversity was revealed. The data of AMOVA showed that the interpopulation component accounted for 12% of genetic variability (F _ST = 0.12044, P = 0.0000). We revealed 15 haplotypes, four of which were shown to be unique. The presence of common haplotypes in the majority of populations, the absence of phylogenetic structure, and low values or even the absence of nucleotide divergence show that the T. cuspidata populations studied are fragments of the once common ancestor population. Geographical isolation, which resulted from climatic changes in the Pleistocene–Holocene, as well as from human activities, did not produce a significant effect on the genetic structure of the species.     

Expression and Refolding of Tobacco Anionic Peroxidase from E. coli Inclusion Bodies

Coding DNA of the tobacco anionic peroxidase gene was cloned in pET40b vector. The problem of 11 arginine codons, rare in procaryotes, in the tobacco peroxidase gene was solved using E. coli BL21(DE3) Codon Plus strain. The expression level of the tobacco apo-peroxidase in the above strain was approximately 40% of the total E. coli protein. The tobacco peroxidase refolding was optimized based on the earlier developed protocol for horseradish peroxidase. The reactivation yield of recombinant tobacco enzyme was about 7% with the specific activity of 1100-1200 U/mg towards 2,2;-azino-bis(3-ethylbenzothiazoline-6-sulfonate) (ABTS). It was shown that the reaction of ABTS oxidation by hydrogen peroxide catalyzed by recombinant tobacco peroxidase proceeds via the ping-pong kinetic mechanism as for the native enzyme. In the presence of calcium ions, the recombinant peroxidase exhibits a 2.5-fold decrease in the second order rate constant for hydrogen peroxide and 1.5-fold decrease for ABTS. Thus, calcium ions have an inhibitory effect on the recombinant enzyme like that observed earlier for the native tobacco peroxidase. The data demonstrate that the oligosaccharide part of the enzyme has no effect on the kinetic properties and calcium inhibition of tobacco peroxidase.     

Glutamic acid-141: a heme 'bodyguard' in anionic tobacco peroxidase

The role of the conserved glutamic acid residue in anionic plant peroxidases with regard to substrate specificity and stability was examined. A Glu141Phe substitution was generated in tobacco anionic peroxidase (TOP) to mimic neutral plant peroxidases such as horseradish peroxidase C (HRP C). The newly constructed enzyme was compared to wild-type recombinant TOP and HRP C expressed in E. coli. The Glu141Phe substitution supports heme entrapment during the refolding procedure and increases the reactivation yield to 30% compared to 7% for wild-type TOP. The mutation reduces the activity towards ABTS, o-phenylenediamine, guaiacol and ferrocyanide to 50% of the wild-type activity. No changes are observed with respect to activity for the lignin precursor substrates, coumaric and ferulic acid. The Glu141Phe mutation destabilizes the enzyme upon storage and against radical inactivation, mimicking inactivation in the reaction course. Structural alignment shows that Glu141 in TOP is likely to be hydrogen-bonded to Gln149, similar to the Glu143-Lys151 bond in Arabidopsis A2 peroxidase. Supposedly, the Glu141-Gln149 bond provides TOP with two different modes of stabilization: (1) it prevents heme dissociation, i.e., it 'guards' heme inside the active center; and (2) it constitutes a shield to protect the active center from solvent-derived radicals.     

Engineering of substrate specificity of D-amino acid oxidase from the yeast Trigonopsis variabilis: Directed mutagenesis of Phe258 residue

Natural D-amino acid oxidases (DAAO) are not suitable for selective determination of D-amino acids due to their broad substrate specificity profiles. Analysis of the 3D-structure of the DAAO enzyme from the yeast Trigonopsis variabilis (TvDAAO) revealed the Phe258 residue located at the surface of the protein globule to be in the entrance to the active site. The Phe258 residue was mutated to Ala, Ser, and Tyr residues. The mutant TvDAAOs with amino acid substitutions Phe258Ala, Phe258Ser, and Phe258Tyr were purified to homogeneity and their thermal stability and substrate specificity were studied. These substitutions resulted in either slight stabilization (Phe258Tyr) or destabilization (Phe258Ser) of the enzyme. The change in half-inactivation periods was less than twofold. However, these substitutions caused dramatic changes in substrate specificity. Increasing the side chain size with the Phe258Tyr substitution decreased the kinetic parameters with all the D-amino acids studied. For the two other substitutions, the substrate specificity profiles narrowed. The catalytic efficiency increased only for D-Tyr, D-Phe, and D-Leu, and for all other D-amino acids this parameter dramatically decreased. The improvement of catalytic efficiency with D-Tyr, D-Phe, and D-Leu for TvDAAO Phe258Ala was 3.66-, 11.7-, and 1.5-fold, and for TvDAAO Phe258Ser it was 1.7-, 4.75-, and 6.61-fold, respectively.