Reported channel formation by prion protein fragment 106-126 in planar lipid bilayers cannot be reproduced.
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Amino Acid Sequence↗
Biomedical subjects
P Christen
Publications and source records attributed to P Christen.
Rates of evolution of pyridoxal-5'-phosphate-dependent enzymes.
The pyridoxal-5'-phosphate-dependent enzymes (B(6) enzymes), that operate in the metabolism of amino acids, are of multiple evolutionary origin. To estimate their rates of evolution, a total of 180 sequences of 21 B(6) enzymes from distantly related eukaryotic species were compared. The enzymes belong to all four evolutionarily independent families of B(6) enzymes with different folds, i.e., the large alpha family, the beta family, the d-alanine aminotransferase family, and the alanine racemase family. Their unit evolutionary periods, i.e., the time for a 1% sequence difference to accumulate between branches, ranged from 4.6 to 45.1 million years. Both, fastest changing serine pyruvate aminotransferase and most slowly changing glutamate decarboxylase are members of the alpha family. The evolutionary rates of the few enzymes belonging to the other three families were interspersed among those of the alpha family members. Enzymes that catalyze the same reaction, e.g., transamination or amino acid decarboxylation, with different substrates show widely varying rates. The absence of correlations of the rate of evolution with either protein fold or type of catalyzed reaction suggests that individual functional constraints have determined the differential rates of evolution of B(6) enzymes.
Animals↗
Enantioseparation of atropine by capillary electrophoresis using sulfated beta-cyclodextrin: application to a plant extract.
A capillary zone electrophoresis (CZE) method, with sulfated beta-CD as chiral selector, was optimized by means of an experimental design for the enantioseparation of atropine. In this study, a central composite design was used and the following factors were varied simultaneously: buffer concentration, buffer pH and sulfated beta-CD concentration. The resolutions between littorine and its positional isomer ((-)-hyoscyamine) and between atropine enantiomers, as well as the separation time and generated current were established as responses. A model was obtained for each response by linear multiple regression of a second-degree mathematical expression. The most favorable conditions were determined by maximizing the resolution between atropine enantiomers and by setting the other responses at threshold values. Successful results were obtained with a 55 mM phosphate buffer at pH 7 in the presence of 2.9 mM sulfated-beta-CD at 20 degrees C and 20 kV. Under these optimized conditions, a baseline separation of littorine and atropine enantiomers was achieved in less than 5 min. Finally, the method allowed the enantiomeric separation of atropine in a pharmaceutical formulation and was also found to be suitable for the enantiomeric purity evaluation of (-)-hyoscyamine in plant extracts, in relation with the extraction procedure. It was demonstrated that supercritical fluid extraction induced less racemization than classical liquid-solid extraction procedures.
Atropine↗
Reply by the authors(1)
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Journal Article↗
The molecular evolution of pyridoxal-5'-phosphate-dependent enzymes.
The pyridoxal-5-phosphate-dependent enzymes (B6 enzymes) that act on amino acid substrates are of multiple evolutionary origin. The numerous common mechanistic features of B6 enzymes thus are not historical traits passed on from a common ancestor enzyme but rather reflect evolutionary or chemical necessities. Family profile analysis of amino acid sequences supported by comparison of the available three-dimensional (3-D) crystal structures indicates that the B6 enzymes known to date belong to four independent evolutionary lineages of homologous (or more precisely paralogous) proteins, of which the alpha family is by far the largest. The alpha family (with aspartate aminotransferase as the prototype enzyme) includes enzymes that catalyze, with several exceptions, transformations of amino acids in which the covalency changes are limited to the same carbon atom that carries the amino group forming the imine linkage with the coenzyme (i.e., Calpha in most cases). Enzymes of the beta family (tryptophan synthase beta as the prototype enzyme) mainly catalyze replacement and elimination reactions at Cbeta. The D-alanine aminotransferase family and the alanine racemase family are the two other independent lineages, both with relatively few member enzymes. The primordial pyridoxal-5-phosphate-dependent enzymes apparently were regio-specific catalysts that first diverged into reaction-specific enzymes and then specialized for substrate specificity. Aminotransferases as well as amino acid decarboxylases are found in two different evolutionary lineages. Comparison of sequences from eukaryotic, archebacterial, and eubacterial species indicates that the functional specialization of most B6 enzymes has occurred already in the universal ancestor cell. The cofactor pyridoxal-5-phosphate must have emerged very early in biological evolution; conceivably, organic cofactors and metal ions were the first biological catalysts. In attempts to stimulate particular steps of molecular evolution, oligonucleotide-directed mutagenesis of active-site residues and directed molecular evolution have been applied to change both the substrate and reaction specificity of existent B6 enzymes. Pyridoxal-5-phosphate-dependent catalytic antibodies were elicited with a screening protocol that applied functional selection criteria as they might have been operative in the evolution of protein-assisted pyridoxal catalysis.
Alanine Racemase↗
Use of borate complexation for the separation of non-UV-absorbing calystegines by capillary electrophoresis.
A capillary zone electrophoresis method was developed for the simultaneous analysis of seven closely related polyhydroxyalkaloids called calystegines. Successful results were obtained with a fused-silica capillary, 80 mM sodium tetraborate at pH 9.2 and temperature of 50 degrees C. Detection of non-UV-absorbing calystegines was achieved through in-situ complexation with borate ions. To further improve method sensitivity, a capillary with a bubble cell was used and detection performed at low wavelength (191 nm). Effects of buffer concentration, pH and temperature on migration times and efficiency are discussed. Migration behavior of selected compounds was significantly affected by their chemical structure (i.e., number and position of hydroxy groups). Under optimized conditions, baseline separation of the selected compounds was achieved in less than 12 min. Precision was evaluated by measuring repeatability and intermediate precision of migration times and corrected peak areas. Finally, the method was applied to the qualitative analysis of calystegines in plant extracts and results were confirmed by GC-MS.
Alkaloids↗
Quaternary indole alkaloids from the stem bark of Strychnos guianensis.
Two new quaternary alkaloids, 9-methoxy-Nb-methylgeissoschizol and guiachrysine together with the known compounds C-alkaloid O, fluorocurine, mavacurine, macusine B and C-profluorocurine, were isolated from Strychnos guianensis stembark. The structures of the compounds were elucidated on the basis of spectroscopic studies.
Alkaloids↗
Simultaneous determination of scopolamine, hyoscyamine and littorine in plants and different hairy root clones of Hyoscyamus muticus by micellar electrokinetic chromatography.
Hyoscyamus muticus hairy root clones were established following infection with Agrobacterium rhizogenes strains A4, LBA-9402 and 15834 and with A. tumefaciens strain C58C1pRTGus104. The accumulation of tropane alkaloids hyoscyamine, littorine and scopolamine was evaluated by micellar electrokinetic capillary electrophoresis. Littorine was reported for the first time in these clones as well as in the roots of the intact plant and confirmed by collision induced dissociation-mass spectrometry. Tropane alkaloid content in hairy roots was compared with leaves and roots of normal plants at two vegetative stages. Significant differences appeared between the alkaloid contents of the different clones. In particular, all the hairy root clones and the roots of the intact plant produced 1.5-3 and 4.5-9 times more littorine than scopolamine, respectively. The only exception was clone KB7, carrying the h6h gene, which overproduced scopolamine. The aerial parts of H. muticus plants did not contain any littorine, thus indicating different transportation or translocation mechanisms of the various tropane alkaloids.
Atropine↗
Amino acid sequences and hapten binding of catalytic and noncatalytic antibodies against N(alpha)-(5'-phosphopyridoxyl)-L-lysine.
Upon immunization with a transition-state analog, only a minority of the hapten-binding antibodies will possess catalytic activity, which will vary in efficacy and substrate specificity. Here, the amino acid sequences of the variable domains of two pyridoxal-5'-phosphate-dependent catalytic and five noncatalytic hapten-binding antibodies raised by immunization with protein-conjugated N(alpha)-(5'-phosphopyridoxyl)-L-lysine (Gramatikova, S., Christen, P., 1997. J. Biol. Chem. 272, 9779-9784) were determined by sequencing their cDNAs. The analysis revealed that the light chains of this set of antibodies were closely related (pairwise identity 65-80%), whereas the heavy chains could be traced back to two different but related groups (intergroup identity 50-54%). The majority of the antibodies proved not to be clonally related, a finding which correlates with their differences in enantiomeric selectivity in ligand binding and reaction specificity. Only one noncatalytic antibody was found to be clonally related with a catalytic antibody, the sequence identity being >95% in both the V(H) and V(L) domains. The complementarity-determining regions were invariably abundant in tyrosine residues. Nitration of three to four tyrosine residues with tetranitromethane abolished hapten binding and catalytic activity. Partial protection by pyridoxal-5'-phosphate against inactivation suggested the presence of functionally important tyrosine residues in the binding sites of the antibodies.
Amino Acid Sequence↗
Increased withdrawal volume per deposit for pre-operative autologous blood donation in adolescents.
OBJECTIVES: This study was undertaken to assess the feasibility, tolerance, haemodynamic and haematologic effects of an aggressive phlebotomy schedule for autologous blood donation (ABD) in adolescents undergoing major orthopaedic surgery. METHODS: Twenty adolescents were studied prospectively; 10 patients in group A donated 20% of the circulating blood volume on 2 occasions, whereas 10 patients in group B donated 10% on 4 occasions. RESULTS: The amount of blood donated, subjective tolerance, cardiovascular changes during the procedure and pre-operative haemoglobin level did not differ between the study groups (group A 111+/-16 vs. group B 106+/-10 g/l). The increase in erythropoietin was greater and occurred sooner in group A than in group B. CONCLUSION: In adolescents ABD is feasible with a reduced number of appointments as they demonstrate tolerance to phlebotomies with a volume which is double the standard per deposit.
Adolescent↗
Amino acid analysis by high-performance liquid chromatography after derivatization with 1-fluoro-2,4-dinitrophenyl-5-L-alanine amide (Marfey's reagent).
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Alanine↗
Conversion of aspartate aminotransferase into an L-aspartate beta-decarboxylase by a triple active-site mutation.
The conjoint substitution of three active-site residues in aspartate aminotransferase (AspAT) of Escherichia coli (Y225R/R292K/R386A) increases the ratio of L-aspartate beta-decarboxylase activity to transaminase activity >25 million-fold. This result was achieved by combining an arginine shift mutation (Y225R/R386A) with a conservative substitution of a substrate-binding residue (R292K). In the wild-type enzyme, Arg(386) interacts with the alpha-carboxylate group of the substrate and is one of the four residues that are invariant in all aminotransferases; Tyr(225) is in its vicinity, forming a hydrogen bond with O-3' of the cofactor; and Arg(292) interacts with the distal carboxylate group of the substrate. In the triple-mutant enzyme, k(cat)' for beta-decarboxylation of L-aspartate was 0.08 s(-1), whereas k(cat)' for transamination was decreased to 0.01 s(-1). AspAT was thus converted into an L-aspartate beta-decarboxylase that catalyzes transamination as a side reaction. The major pathway of beta-decarboxylation directly produces L-alanine without intermediary formation of pyruvate. The various single- or double-mutant AspATs corresponding to the triple-mutant enzyme showed, with the exception of AspAT Y225R/R386A, no measurable or only very low beta-decarboxylase activity. The arginine shift mutation Y225R/R386A elicits beta-decarboxylase activity, whereas the R292K substitution suppresses transaminase activity. The reaction specificity of the triple-mutant enzyme is thus achieved in the same way as that of wild-type pyridoxal 5'-phosphate-dependent enzymes in general and possibly of many other enzymes, i.e. by accelerating the specific reaction and suppressing potential side reactions.
Aspartate Aminotransferases↗
The aspartate aminotransferase-catalysed exchange of the alpha-protons of aspartate and glutamate: the effects of the R386A and R292V mutations on this exchange reaction.
1H-NMR was used to follow the aspartate aminotransferase-catalysed exchange of the alpha-protons of aspartate and glutamate. The effect of the concentrations of both the amino acids and the cognate keto acids on exchange rates was determined for wild-type and the R386A and R292V mutant forms of aspartate aminotransferase. The wild-type enzyme is found to be highly stereospecific for the exchange of the alpha-protons of L-aspartate and L-glutamate. The R386A mutation which removes the interaction of Arg-386 with the alpha-carboxylate group of aspartate causes an approximately 10,000-fold decrease in the first order exchange rate of the alpha-proton of L-aspartate. The R292V mutation which removes the interaction of Arg-292 with the beta-carboxylate group of L-aspartate and the gamma-carboxylate group of L-glutamate causes even larger decreases of 25,000- and 100,000-fold in the first order exchange rate of the alpha-proton of L-aspartate and L-glutamate respectively. Apparently both Arg-386 and Arg-292 must be present for optimal catalysis of the exchange of the alpha-protons of L-aspartate and L-glutamate, perhaps because the interaction of both these residues with the substrate is essential for inducing the closed conformation of the active site.
Aspartate Aminotransferases↗
Neurotoxicity of prion peptide 106-126 not confirmed.
Prion-related diseases are accompanied by neurodegeneration, astroglial proliferation and formation of proteinase K-resistant aggregates of the scrapie isoform of the prion protein (PrPSc). The synthetic PrP fragment 106-126 was reported to be neurotoxic towards cultured rat hippocampal neurons (Forloni, G., Angeretti, N., Chiesa, R., Monzani, E., Salmona, M., Bugiani, O. and Tagliavini, F. (1993) Nature 362, 543-546) and mouse cortical cells (Brown, D.R., Herms, J. and Kretzschmar, H.A. (1994) Neuroreport 5, 2057-2060). However, we found the viability of these and other neuronal cell types not to be impaired in the presence of PrP106-126 under widely varied sets of conditions. Aged preparations of the peptide as well as synthetic deamidated and isomerized derivatives that correspond to the aging products of the peptide proved also to lack neurotoxicity. Apparently, PrP106-126 cannot serve as a model for the interaction of PrP with neuronal cells.
Animals↗
Spontaneous deamidation and isomerization of Asn108 in prion peptide 106-126 and in full-length prion protein.
In prion-related encephalopathies, the cellular prion protein (PrP(C)) undergoes a change in conformation to become the scrapie prion protein (PrP(Sc)) which forms infectious deposits in the brain. Conceivably, the conformational transition of PrP(C) to PrP(Sc) might be linked with posttranslational alterations in the covalent structure of a fraction of the PrP molecules. We tested a synthetic peptide corresponding to residues 106-126 of human PrP for the occurrence of spontaneous chemical modifications. The only asparagine residue, Asn108, was deamidated to aspartic acid and isoaspartic acid with a half-life of about 12 days. The same posttranslational modifications were found in recombinant murine full-length protein. On aging, 0.8 mol of isoaspartyl residue per mole of protein was detected by the protein-l-isoaspartyl methyltransferase assay (t(1/2) approximately 30 days). Mass spectrometry and Edman degradation of Lys-C fragments identified Asn108 in the amino-terminal flexible part of the protein to be partially converted to aspartic acid and isoaspartic acid. A second modification was the partial isomerization of Asp226' which is only present in rodents.
Amino Acid Sequence↗
Recognizing very distant sequence relationships among proteins by family profile analysis.
Family profile analysis (FPA), described in this paper, compares all available homologous amino acid sequences of a target family with the profile of a probe family while conventional sequence profile analysis (Gribskov M, Lüthy R, Eisenberg D. Meth Enzymol 1990;183:146-159) considers only a single target sequence in comparison with the probe family. The increased input of sequence information in FPA expands the range for sequence-based recognition of structural relationships. In the FPA algorithm, Zscores of each of the target sequences, obtained from a probe profile search over all known amino acid sequences, are averaged and then compared with the scores for sequences of 100 reference families in the same probe family search. The resulting F-Zscore of the target family, expressed in "effective standard deviations" of the mean Zscores of the reference families, with value above a threshold of 3.5 indicates a statistically significant evolutionary relationship between the target and probe families. The sensitivity of FPA to sequence information was tested with several protein families where distant relationships have been verified from known tertiary protein architectures, which included vitamin B6-dependent enzymes, (beta/alpha)8-barrel proteins, beta-trefoil proteins, and globins. In comparison to other methods, FPA proved to be significantly more sensitive, finding numerous new homologies. The FPA technique is not only useful to test a suspected relationship between probe and target families but also identifies possible target families in profile searches over all known primary structures.
Algorithms↗
Conversion of tyrosine phenol-lyase to dicarboxylic amino acid beta-lyase, an enzyme not found in nature.
Tyrosine phenol-lyase (TPL), which catalyzes the beta-elimination reaction of L-tyrosine, and aspartate aminotransferase (AspAT), which catalyzes the reversible transfer of an amino group from dicarboxylic amino acids to oxo acids, both belong to the alpha-family of vitamin B6-dependent enzymes. To switch the substrate specificity of TPL from L-tyrosine to dicarboxylic amino acids, two amino acid residues of AspAT, thought to be important for the recognition of dicarboxylic substrates, were grafted into the active site of TPL. Homology modeling and molecular dynamics identified Val-283 in TPL to match Arg-292 in AspAT, which binds the distal carboxylate group of substrates and is conserved among all known AspATs. Arg-100 in TPL was found to correspond to Thr-109 in AspAT, which interacts with the phosphate group of the coenzyme. The double mutation R100T/V283R of TPL increased the beta-elimination activity toward dicarboxylic amino acids at least 10(4)-fold. Dicarboxylic amino acids (L-aspartate, L-glutamate, and L-2-aminoadipate) were degraded to pyruvate, ammonia, and the respective monocarboxylic acids, e.g. formate in the case of L-aspartate. The activity toward L-aspartate (kcat = 0.21 s-1) was two times higher than that toward L-tyrosine. beta-Elimination and transamination as a minor side reaction (kcat = 0.001 s-1) were the only reactions observed. Thus, TPL R100T/V283R accepts dicarboxylic amino acids as substrates without significant change in its reaction specificity. Dicarboxylic amino acid beta-lyase is an enzyme not found in nature.
Amino Acid Sequence↗