Physical inactivity and platelet function in humans and brown bears: A comparative study.

Physical inactivity increases the risk of thromboembolism. However, good standardized human models on inactivity are in short supply and experimental models are few. Our objective was to investigate how standardized bed rest affects platelet aggregation in humans and to investigate if aggregation is altered in a translational model system - the hibernating brown bear (Ursus arctos). We collected blood from (1) healthy male volunteers participating in a 21-day bed rest study in head-down tilt position (-6°) 24 h a day; (2) free-ranging brown bears captured during winter hibernation and again during active state in summer. We analyzed platelet function using multiple electrode platelet aggregometry. In total, 9 healthy male volunteers (age 31.0 ± 6.4 years) and 13 brown bears (7 females and 6 males, age 2.8 ± 0.6 years) were included. In hibernating bears adenosine diphosphate, arachidonic acid, thrombin receptor activating peptide, and collagen impedance aggregometry tests were all halved compared to summer active state. In human volunteers no statistically significant changes were found between baseline and the end of bed rest. In human male volunteers 3 weeks of bed rest did not affect platelet function. In hibernating brown bears platelet aggregation was halved compared to summer and we hypothesize that this is a protective measure to avoid formation of thrombi under periods of low blood flow.

Bacterial catalase-peroxidases are gene duplicated members of the plant peroxidase superfamily.

Bacterial catalase-peroxidases are enzymes containing 0.5-1.0 heme per subunit. The identical subunits are generally 80 kDa in size, and the sequenced subunits of E. coli, S. typhimurium and B. stearothermophilus contain 726-731 amino acid residues per subunit. The heme-containing peroxidases of plants, fungi and yeast are monomeric, homologous and 290-350 residues in size. Analyses of the amino acid sequences indicate that the double length of the bacterial peroxidases can be ascribed to gene duplication. Each half is homologous to eukaryotic, monomeric peroxidase and can be modelled into the high-resolution crystal structure of yeast cytochrome c peroxidase. The comparisons and modelling have predicted: (1) the C-terminal half does not bind heme, and bacterial peroxidases have one heme per subunit; (2) the ten dominating helices observed in the yeast enzyme are highly conserved and connected by surface loops which are often longer in the bacterial peroxidases; and (3) yeast cytochrome c peroxidase has evolved more slowly than other known peroxidases. The study has revealed ten invariant residues and a number of highly conserved residues present in peroxidases of the plant peroxidase superfamily and provides a basis for rationally engineered peroxidases.

Adapting protein solubility by glycosylation. N-glycosylation mutants of Coprinus cinereus peroxidase in salt and organic solutions.

Protein solubility is a fundamental parameter in biology and biotechnology. In the present study we have constructed and analyzed five mutants of Coprinus cinereus peroxidase (CIP) with 0, 1, 2, 4 and 6 N-glycosylation sites. All mutants contain Man(x)(GlcNAc)(2) glycans. The peroxidase activity was the same for wild-type CIP and all the glycosylation mutants when measured with the large substrate 2,2'-azino-bis(-3-ethylbenzthiazoline-6-sulfonic acid). The solubility of the five CIP mutants showed a linear dependence on the number of carbohydrate residues attached to the protein in buffered solution of both ammonium sulfate (AMS) and acetone, increasing in AMS and decreasing in acetone. Moreover, the change in free energy of solvation appears to be a constant, though with opposite signs in these solvents, giving DeltaDeltaG degrees (sol)=-0.32+/-0.05 kJ/mol per carbohydrate residue in 2.0 M AMS, a value previously obtained comparing ordinary and deglycosylated horseradish peroxidase, and 0. 37+/-0.10 kJ/mol in 60 v/v% acetone.

Reactions of soybean peroxidase and hydrogen peroxide pH 2.4-12.0, and veratryl alcohol at pH 2.4.

Peroxidase from soybean seed coat (SBP) has properties that makes it particularly suited for practical applications. Therefore, it is essential to know its fundamental enzymatic properties. Stopped-flow techniques were used to investigate the pH dependence of the reaction of SBP and hydrogen peroxide. The reaction is linearly dependent on hydrogen peroxide concentration at acidic and neutral pH with the second order rate constant k(1)=2.0x10(7) M(-1) s(-1), pH 4-8. From pH 9.3 to 10.2 the reaction is biphasic, a novel observation for a peroxidase at alkaline pH. A fast reaction has the characteristics of the reaction at neutral pH, and a slow reaction shows hyperbolic dependence on hydrogen peroxide concentration. At pH >10.5 only the slow reaction is seen. The shift in mechanism is coincident with the change in haem iron co-ordination to a six-coordinate low spin hydroxy ligated alkaline form. The pK(a) value for the alkaline transition was observed at 9.7+/-0.1, 9.6+/-0.1 and 9.9+/-0.2 by spectrophotometric titration, the fast phase amplitude, and decrease in the apparent second order rate constant, respectively. An acidic pK(a) at 3.2+/-0.3 was also determined from the apparent second order rate constant. The reactions of soybean peroxidase compounds I and II with veratryl alcohol at pH 2.44 give very similar second order rate constants, k(2)=(2.5+/-0.1)x10(4) M(-1) s(-1) and k(3)=(2.2+/-0.1)x10(4) M(-1) s(-1), respectively, which is unusual. The electronic absorption spectra of compounds I, II and III at pH 7.07 show characteristic bands at 400 and 651 nm (compound I), 416, 527 and 555 nm (compound II), and 414, 541 and 576 nm (compound III). No additional intermediates were observed.

Kinetic evidence for surface residues influencing the active site of Coprinus cinereus peroxidase: analysis of the pH dependence of G154E, P90H and P90H-G154E substrate entrance mutants.

Three mutants of Coprinus cinereus peroxidase (CIP) were made to mimic the substrate entrance histidine 82-glutamic acid 146 pair of the substrate channel in lignin peroxidase (LIP). Compound I formation of LIP has a low pH optimum around pH 3, while optimal formation of CIP compound I is obtained at pH 6-11. The mutants were glycine 154-->glutamic acid (G154E), proline 90-->histidine (P90H) and the double mutant P90H-G154E. All three showed kinetics of compound I formation similar to that of wt CIP between pH 3 and 9. However, the stability of compound I was strongly affected by these mutations. In wt CIP compound I is stable for approximately 30 min, while compound I of the mutants were stable for 5 s or less. The P90H and P90H-G154E mutants showed pK(a) values for the alkaline transition at least one pH unit lower than for wt CIP and the G154E mutant. We suggest that the changed electrostatic field results in destabilisation of the oxidised heme in compound I and II and that the P90H residue increases the electrostatic potential in the distal cavity thereby decreasing the pK(a) for the alkaline transition.

Analysis of two incompletely spliced Arabidopsis cDNAs encoding novel types of peroxidase.

Most known class III peroxidase genes contain three introns at conserved positions. Two Arabidopsis cDNAs (ESTs), encoding novel type peroxidases ATP9a and ATP15a were sequenced, and found to contain inserts for intron 2. PCR and sequence analysis of genomic DNA revealed that the atp9a gene contains all three introns, whereas atp15a contains only introns 2 and 3. The ATP15a cDNA intron contained a single base substitution reducing the splicing potential significantly as compared with the genomic sequence. The putative enzymes share essential catalytic and structural features with horseradish peroxidase, despite a pair-wise sequence identity of only 40-45% among the three.

Comparison of structure and activities of peroxidases from Coprinus cinereus, Coprinus macrorhizus and Arthromyces ramosus.

Initial structural and kinetic data suggested that peroxidases from Coprinus cinereus, Coprinus macrorhizus and Arthromyces ramosus were similar. Therefore they were characterized more fully. The three peroxidases were purified to RZ 2.5 and showed immunochemical identity as well as an identical M(r) of 38,000, pI about 3.5 and similar amino acid compositions. The N-termini were blocked for amino acid sequencing. The peroxidases had similar retention volumes by anion-exchange and gel-filtration chromatography. All peroxidases showed multiple peaks by Concanavalin A-Sepharose chromatography. The Concanavalin A-Sepharose profiles were different and depended furthermore on a fermentation batch. Tryptic peptide maps were very similar except for one peptide. This peptide contained an N-linked glycan composed of varying ratios of glucosamine and mannose for the three peroxidases. Rate constants and their pH dependence were the same for the three peroxidases using guaiacol or iodide as reducing substrates. We conclude that peroxidases from Coprinus cinereus, Coprinus macrorhizus and Arthromyces ramosus are most likely identical in their amino acid sequences, but deviate in glycosylation which, apparently, has no influence on the reaction rates of the enzyme. We suggest, that the Coprinus fungi express one peroxidase only in contrast to the lignin-degrading white-rot Basidiomycetes, which produce multiple peroxidase isozymes.

Crystallization and preliminary X-ray diffraction studies of a peroxidase from barley grain.

Crystals suitable for X-ray diffraction analysis of both glycosylated and non-glycosylated forms of a barley peroxidase have been grown. The crystals of the glycosylated peroxidase have been grown by the hanging drop vapour diffusion method using polyethylene glycol 4000 as the precipitant in the presence of n-propanol and potassium iodide at pH 8.5. The crystals are needles belonging to the orthorhombic spacegroup P2(1)2(1)2(1) with unit cell dimensions a = 62.95 A, b = 66.24 A and c = 70.78 A. There is one barley peroxidase molecule in the asymmetric unit. The crystals contain approximately 38% solvent and appear to be stable to lengthy X-ray exposure. They diffract to beyond 1.9 A.

Crystallization and X-ray diffraction analysis of recombinant Coprinus cinereus peroxidase.

Crystals suitable for an X-ray diffraction investigation have been obtained of recombinant Coprinus cinereus peroxidase expressed in Aspergillus oryzae. The crystals were grown by the hanging drop method with polyethylene glycol 6000 as the precipitant. A pH range from 6.2 to 8.0 and CaCl2 or MgCl2 present at a concentration of 0.35 M were essential for the crystal growth. A metastable monoclinic modification can be obtained under certain conditions, and with variations in temperature they are transformed into a stable orthorhombic modification. With CaCl2 as the additive, the unit cell dimensions were a = 74.9 A, b = 76.8 A and c = 128.2 A. With two peroxidase molecules per asymmetric unit, the solvent content is 49% (v/v). In the diffraction pattern, the reflections Okl are systematically very weak for k = 2n + 1. Combined with an analysis of the Patterson function, this showed that the two independent molecules are related by the pseudotranslational symmetry 0.29a + 0.5b. The possible space groups are P2(1)2(1)2(1) or P2(1)22(1) because of this pseudosymmetry. The crystals diffract to a resolution of 2.9 A.

Amino acid sequences and structures of chicken and turkey beta 2-microglobulin.

The complete amino acid sequences of chicken and turkey beta 2-microglobulins have been determined by analyses of tryptic, V8-proteolytic and cyanogen bromide fragments, and by N-terminal sequencing. Mass spectrometric analysis of chicken beta 2-microglobulin supports the sequence-derived Mr of 11,048. The higher apparent Mr obtained for the avian beta 2-microglobulins as compared to human beta 2-microglobulin by SDS-PAGE is not understood. Chicken and turkey beta 2-microglobulin consist of 98 residues and deviate at seven positions: 60, 66, 74-76, 78 and 82. The chicken and turkey sequences are identical to human beta 2-microglobulin at 46 and 47 positions, respectively, and to bovine beta 2-microglobulin at 47 positions, i.e. there is about 47% identity between avian and mammalian beta 2-microglobulins. The known X-ray crystallographic structures of bovine beta 2-microglobulin and human HLA-A2 complex suggest that the seven chicken to turkey differences are exposed to solvent in the avian MHC class I complex. The key residues of beta 2-microglobulin involved in alpha chain contacts within the MHC class I molecule are highly conserved between chicken and man. This explains that heterologous human beta 2-microglobulin can substitute the chicken beta 2-microglobulin in exchange studies with B-F (chicken MHC class I molecule), and suggests that the MHC class I structure is conserved over long evolutionary distances.

Human complement component C2: production and characterization of polyclonal and monoclonal antibodies against C2.

Rabbit antibodies to human complement component C2 were produced by immunization of rabbits with precipitates from line immunoelectrophoresis, and the antibodies were used to monitor a classical chromatographic purification of C2 and for affinity purification of C2. Twelve monoclonal antibodies with specificity for human complement component C2 were produced by fusion of myeloma cells with spleen cells from mice immunized with the affinity purified C2. The specificity of the monoclonal antibodies was confirmed by their reaction with antigen-antibody precipitates where C2 was the antigen, and by their specific reaction with C2 after separation in SDS-PAGE followed by immunoblotting. The affinity of the monoclonal antibodies varied as demonstrated by the titration curves in ELISA. The antibodies will be of importance for immunospecific purification of human C2 and C2 fragments, for specific depletion of C2 from human serum, and for quantification of C2 for clinical purposes.

Isolation and characterization of chicken and turkey beta 2-microglobulin.

Chicken and turkey beta 2-m were isolated from citrated plasma in sequential use of three chromatographic steps: affinity chromatography, gel filtration chromatography and anion-exchange chromatography. The purified protein was identified as beta 2-m by reaction with a beta 2-m specific monoclonal antibody and by the ability to recombine with the chicken MHC class I heavy chain. The purity was estimated by SDS-PAGE and IEF. The pI was between 5.1 and 5.3 for chicken beta 2-m and 4.7 and 4.8 for turkey beta 2-m, which fact is reflected in their different electrophoretic mobilities in agarose gel (turkey migrates in the alpha and chicken migrates in the beta region). The mol. wt of both chicken and turkey beta 2-m was 14,500 estimated by SDS-PAGE whereas calculations based on the amino acid compositions gave mol. wts of 11,000. EM280 was 15.9 for chicken beta 2-m and 16.4 for turkey beta 2-m. The amino acid compositions and sequences of the two avian beta 2-m molecules have been compared with earlier data from the literature. The sequence of the 23 N-terminal amino acids was found to be identical in our preparations from both chicken and turkey, namely DLTPKVQVYSRFPASAGTKNVLN, and is incompatible with a previously published sequence also thought to be from turkey beta 2-m. Reasons for our opinion that the molecules isolated and sequenced in this paper are the correct ones are given.

Structure of soybean seed coat peroxidase: a plant peroxidase with unusual stability and haem-apoprotein interactions.

Soybean seed coat peroxidase (SBP) is a peroxidase with extraordinary stability and catalytic properties. It belongs to the family of class III plant peroxidases that can oxidize a wide variety of organic and inorganic substrates using hydrogen peroxide. Because the plant enzyme is a heterogeneous glycoprotein, SBP was produced recombinant in Escherichia coli for the present crystallographic study. The three-dimensional structure of SBP shows a bound tris(hydroxymethyl)aminomethane molecule (TRIS). This TRIS molecule has hydrogen bonds to active site residues corresponding to the residues that interact with the small phenolic substrate ferulic acid in the horseradish peroxidase C (HRPC):ferulic acid complex. TRIS is positioned in what has been described as a secondary substrate-binding site in HRPC, and the structure of the SBP:TRIS complex indicates that this secondary substrate-binding site could be of functional importance. SBP has one of the most solvent accessible delta-meso haem edge (the site of electron transfer from reducing substrates to the enzymatic intermediates compound I and II) so far described for a plant peroxidase and structural alignment suggests that the volume of Ile74 is a factor that influences the solvent accessibility of this important site. A contact between haem C8 vinyl and the sulphur atom of Met37 is observed in the SBP structure. This interaction might affect the stability of the haem group by stabilisation/delocalisation of the porphyrin pi-cation of compound I.

The oxygen binding site of cytochrome oxidase. Structural predictions on subunit I from amino acid sequences.

Analyses of heme-attached amino acid sequences in known hemoprotein superfamilies provide a basis for prediction of such sequences in hemoproteins of unknown three-dimensional structure. Among 11 histidine residues conserved in subunit I of 3 mammalian and 2 fungal cytochrome oxidases the sequence around His-233 (human) is the most conserved and shows remarkable similarity to the sequence of the oxygen binding site in globins. Furthermore, the gene coding for subunit I in Saccharomyces cerevisiae and the gene for leghemoglobin in soybean are both split by introns right after these similar histidine sequences. The predicted distal histidine sequence of subunit I provides for heme a3 and Cua3 binding and has an extraordinarily high content of aromatic residues. These aromatic groups may serve as a molecular electron capacitor. Transmembrane sequences and electron transfer sequences are proposed.

Amino acid sequence analysis of the glycopeptides from human complement component C3.

Human complement component C3 has been cleaved completely by trypsin in the presence of 2-propanol. The hydrolysate was fully solubilized and fractionated by reversed-phase HPLC. Two peptides only contained glucosamine, Unambiguous sequence analyses identified Asn-63 of the beta-chain and Asn-268 of the alpha-chain as the sites of carbohydrate attachment. A third potential Asn-Xaa-Thr/Ser glycosylation site, Asn-946 of the alpha-chain, is not modified. The different states of glycosylation of the sites cannot be explained by differences in exposure or secondary structure. All three are predicted reverse turn.

Glycosylation and thermodynamic versus kinetic stability of horseradish peroxidase.

The influence of N-linked glycans on the stability of glycoproteins has been studied using horseradish peroxidase isoenzyme C (HRP), which contains eight asparagine-linked glycans. HRP was deglycosylated (d-HRP) with trifluoromethanesulfonic acid and purified to an enzymatically active homogeneous protein containing (GlcNAc)2 glycans. The thermal stability of HRP and d-HRP at pH 6.0, measured by residual activity, was indistinguishable and showed transition midpoints at 57 degrees C, whereas the unfolding in guanidinium chloride at pH 7.0, 23 degrees C was 2-3-fold faster for d-HRP than for HRP. The results are compatible with a glycan-induced decrease in the dynamic fluctuation of the polypeptide chain.

The potato tuber transcriptome: analysis of 6077 expressed sequence tags.

This is the first report of the biosynthetic potential of a tuber storage organ investigated by expressed sequence tag sequencing. A cDNA library was generated from the mature tuber of field grown potato (Solanum tuberosum var. Kuras). Partial sequences obtained from 6077 clones were assembled into 828 clusters and 1533 singletons. The average read length was 592 bp, and 2254 clones were full length. 5717 clones showed homology to genes from other organisms. Genes involved in protein synthesis, protein destination and cell defense predominated in tuber compared to stolon, shoot and leaf organs. 1063 clones were unique to tuber. Transcripts of starch metabolizing enzymes showed similar relative levels in tuber and stolon.

Structure and organ specificity of an anionic peroxidase from Arabidopsis thaliana cell suspension culture.

The predominant peroxidase (pI 3.5) (E.C. 1.11.1.7) of an Arabidopsis thaliana cell suspension culture was purified and partially sequenced. Oligonucleotides were designed and a specific probe was obtained. A cDNA clone was isolated from an Arabidopsis cell suspension cDNA library and completely sequenced. The cDNA clone comprised 1194 bp and encodes a 30 residue signal peptide and a 305 residue mature protein (Mr 31,966). The sequence of the mature protein is 95% identical to the well-characterized horseradish peroxidase HRP A2 and has therefore been designated ATP A2. Three introns at positions identical to those found in Arabidopsis and horseradish genes encoding cationic peroxidases were identified. RT-PCR analysis revealed root-specific expression.

Unique reaction of a barley peroxidase with hydrogen peroxide.

The reaction of barley peroxidase BP 1 with H2O2 is markedly different from that of other peroxidases. Saturation kinetics and a strong pH dependence over the accessible pH range from 3.09 to 5.08 are observed. At pH 3.8, native BP 1 has maxima at 401, 498 and 635 nm, cpd 1 at 403 nm, and cpd II at 407 and 521 nm with a shoulder at 553 nm. Both cpds I and II appear to be incompletely formed. Isosbestic points between native BP 1 and cpd I occur at 365 and 416 nm, while an isosbestic point in the Soret region between cpd I and cpd II has been observed at 410 nm. Between cpd II and a not yet identified intermediate isosbestic points have been observed at 408, 455 and 526 nm.

Computational analyses and annotations of the Arabidopsis peroxidase gene family.

Classical heme-containing plant peroxidases have been ascribed a wide variety of functional roles related to development, defense, lignification, and hormonal signaling. More than 40 peroxidase genes are now known in Arabidopsis thaliana for which functional association is complicated by a general lack of peroxidase substrate specificity. Computational analysis was performed on 30 near full-length Arabidopsis peroxidase cDNAs for annotation of start codons and signal peptide cleavage sites. A compositional analysis revealed that 23 of the 30 peroxidase cDNAs have 5' untranslated regions containing 40-71% adenine, a rare feature observed also in cDNAs which predominantly encode stress-induced proteins, and which may indicate translational regulation.