bluuebunny/crossref_metadata_2025 huggingface dataset:
Selenomethionine Labelling of Phosphomannose Isomerase Changes its Kinetic Properties
Authors: Bernard Alain R. , Wells Timothy N. C. , Cleasby Anne , Borlat Frédéric , Payton Mark A. , Proudfoot Amanda E. I.
Published: March 2005

Phosphomannose isomerase (PMI) is an essential enzyme in the early steps of the protein glycosylation pathway in both prokaryotes and eukaryotes. Lack of the enzyme is lethal for fungal organisms and it is thus a potential fungicidal target. To facilitate the solution of the three‐dimensional structure of the enzyme from the pathogen Candida albicans, we have produced the recombinant selenomethionine‐labelled enzyme (SeMet‐PMI). DL41, a methionine auxotroph Escherichia coli strain, was transformed with a PMI expression plasmid and grown on an enriched selenomethionine‐containing medium to high‐cell densities. The SeMet‐PMI protein has been purified and found by amino acid analysis to have its methionine residues replaced by selenomethionine residues. Electrospray mass spectroscopy showed a major species of 49063 ± 10 Da for SeMet‐PMI compared to 48735 ± 6 Da for the normal recombinant enzyme, accounting for the incorporation of seven selenomethionine residues. SeMet‐PMI crystallised isomorphously with the normal PMI protein and the crystals diffract to 0.23 nm. Kinetic characterisation of SeMet‐PMI showed that its Km for the substrate mannose‐6‐phosphate was fourfold higher than that of its methionine‐containing counterpart. The inhibition constant for zinc ions was also increased by a similar factor. However, the Vmax was unaltered. These results suggested that one or more methionine residues must be in close proximity to the substrate‐binding pocket in the active site, rendering substrate access more difficult compared to the normal enzyme. This hypothesis was confirmed by the finding of four methionine residues lying along one wall of the active site.
Arrangement of Catalytic Sites in the Multifunctional Enzyme Enniatin Synthetase
Authors: Pieper Rembert , Haese Angela , Schröder Werner , Zocher Rainer
Published: March 2005

Enniatin synthetase is an N ‐methyl peptide synthetase comprising 3131 amino acids. Catalytic sites of the 347‐kDa multifunctional enzyme were mapped by N‐terminal sequencing of substrate affinity‐labelled enzyme fragments formed by proteolysis, and functional studies of purified enniatin synthetase fragments. An N‐terminal 200‐kDa fragment containing the cofactor 4′‐phosphopantetheine was able to activate d‐hydroxyisovaleric acid (d‐HOiVl) as a thioester. The N‐termini of two [14C]HOiVl‐labelled enzyme fragments could be assigned to amino acid position 429 within the N‐terminal conserved enniatin synthetase portion named EA. This portion of about 600 amino acids shares high similarity to microbial peptide synthetase regions. A 68‐kDa l‐[14C]Val‐labelled enniatin synthetase fragment was localized at amino acid position 2294 within the second C‐terminal conserved protein portion EB. Additionally enniatin synthetase was labelled with isovaleryl‐l‐[14C]Val, an analogue of the D‐hydroxyisovaleryl‐l‐Val intermediate in enniatin biosynthesis. The N‐terminus of a 30‐kDa isovaleryl‐l‐[14C]Val‐labelled enniatin synthetase fragment was mapped in a C‐terminal segment of the protein portion EA. The same N‐terminal sequence was obtained from a 60‐kDa enniatin synthetase fragment modified with [3H]βAla, a constituent of the cofactor 4′‐phosphopantetheine. This indicates the presence of the cofactor in this protein fragment. Localization of the methyltransferase function of enniatin synthetase in an amino acid portion integrated into region EB was achieved by N‐terminal sequencing of a photolabelled S‐[methyl ‐14C]adenosyl methio‐nine 45–kDa fragment and the identification of a photolabelled peptide Asn‐Leu‐Asn‐Pro‐Gly‐Leu‐Asn‐Ser‐Tyr.
Electron Transfer and Spectral α‐Band Properties of the Di‐Heme Protein Cytochrome c4 from Pseudomonas Stutzeri
Authors: Conrad Lars S. , Karlsson Jens J. , Ulstrup Jens
Published: March 2005

Cytochrome c4 is a 190‐residue protein active in the aerobic and anaerobic respiration of several bacteria. We have isolated Pseudomonas stutzeri (ATCC no. 11607) cytochrome c4 by an optimized growth procedure following factorial design. The ultraviolet/visible spectra of reduced cytochrome c4 have a composite α/β band which can be resolved into six components. One of these seems to be specific for the high‐potential heme group. The kinetics for full oxidation and reduction with the two inorganic redox couples, [Co(terpy)2]2+/3+ and [Co(bipy)3]2+/3+, is formally compatible with either bi‐ or tri‐exponential kinetics. The former would be in line with weak interaction between the heme groups, the latter with notable interaction effects. Arguments in favour of the latter and a cooperative two‐electron transfer pattern are given. All phases are approximately proportional to the Co‐complex concentration, implying that intramolecular electron transfer in this time range is unlikely. The rate constants are in the range (0.7–80) × 104 M−1 s−1 at pH = 7.6 (Tris) and 0.1 M NaCl and very little dependent on the ionic strength in the range 0.1–0.3 M. The reduction potentials could be calculated from the forward and reverse rate constant ratios. The values are 241 ± 5 and 328 ± 2 mV (Nernst hydrogen electrode) if bi‐exponential kinetics is used and interaction between the heme groups disregarded. The intrinsic microscopic reduction potential values are closer when the tri‐exponential, cooperative model is used as this model transfers 30–40 mV to electrostatically dominated interaction potentials. The overall electron transfer pattern can be related to the recently determined crystal structure of the P. stutzeri cytochrome c4.
The γ Subunit of Phosphorylase Kinase Contains a Pseudosubstrate Sequence
Authors: Lanciotti Robert A. , Bender Patrick K.
Published: March 2005

The catalytic subunit, γ, of phosphorylase kinase is regulated by a complex set of interactions involving the calcium‐binding protein calmodulin and two other subunits designated α and β. These interactions regulate γ activity that, at least for the calmodulin interactions, involves the regulatory domain in γ spanning residues 302–366. Within this regulatory domain, we report the identification of a sequence (residues 326–334) that resembles the phosphorylation site in γ substrates with the exception that a V residue (V332) occurs at the analogous position of the phosphorylated S/T residue. The inhibitory properties of the sequence were assayed with a 10‐amino‐acid peptide of the sequence. This peptide inhibits a truncated version of γ, residues 1–300, which is missing the regulatory domain, more potently than it inhibits full‐length γ, and it is a better inhibitor of the full‐length γ at pH 8.2 than at pH 6.8. A similar peptide of the same sequence, except for a S substitution of the V residue, is a good substrate with a comparable Km and better Vmax than peptides of similar length that represent the phosphorylation site in the substrate of the enzyme, glycogen phosphorylase. A mutant γ protein, with a S for V332 substitution ([V332S]γ), was prepared using the baculovirus expression system. [V332S]γ autophosphorylates by an intramolecular mechanism. This demonstrates that this sequence can occupy the catalytic site in the protein. Development of [V332S]γ affords an experimental model in which the effects of the regulatory factors on autophosphorylation can be determined.
Primary Structure of Acidic Oligosaccharide‐alditols Derived from the Jelly Coat of Ambystoma tigrinum
Authors: Maes Emmanuel , Plancke Yves , Delplace Florence , Strecker Gérard
Published: March 2005

Eleven O‐glycosidic carbohydrate units derived from the jelly coat of Ambystoma tigrinum were analyzed by 1H‐NMR spectroscopy. As previously shown for four other amphibian species, the glycans display a remarkable species specificity. As a characteristic feature, 3‐deoxy‐d‐glycero ‐d‐galacto ‐nonulo‐sonic acid (Kdn) was found difucosylated at C4 and C5, and α‐Gal attached to C3 of GalNAc‐ol. The most representative carbohydrate units are:
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Control of Glucose Metabolism by the Enzymes of the Glucose Phosphotransferase System in Salmonella typhimurium
Authors: Van Der Vlag Johan , Van't Hof Rechien , Van Dam Karel , Postma Pieter W.
Published: March 2005

The quantitative role of the phosphoenolpyruvate:glucose phosphotransferase system (glucose phosphotransferase system) in glucose uptake and metabolism, and phosphotransferase‐system‐mediated regulation of glycerol uptake, was studied in vivo in Salmonella typhimurium. Expression plasmids were constructed which contained the genes encoding enzyme I (ptsI), HPr (ptsH), IIAGlc (crr), and IICBGlc (ptsG) of the glucose phosphotransferase system behind inducible promoters. These plasmids allowed the controlled expression of each of the glucose phosphotransferase system proteins from about 30 % to about 300% of its wild‐type level. When enzyme I, HPr or IIAGlc were modulated between 30% and 300% of their wild‐type value, hardly any effects on the growth rate on glucose, the glucose oxidation rate, the rate of methyl α‐d‐glucopyranoside (a glucose analog) uptake or the phosphotransferase‐system‐mediated inhibition of glycerol uptake by methyl α‐d‐glucopyranoside were observed. Employing the method of metabolic control analysis, it was shown that the enzyme flux control coefficients of these phosphotransferase system components on the different measured processes were close to zero. The enzyme flux control coefficient of IICBGlc on growth on glucose or glucose oxidation was also close to zero. In contrast, the enzyme flux control coefficient of IICBGlc on the flux through the glucose phosphotransferase system (transport and phosphorylation) was 0.72. The experimentally determined enzyme flux control coefficients allowed us to calculate the flux control coefficients of the phosphoenolpyruvate/pyruvate and methyl α‐d‐glucopyranoside/methyl α‐d‐glucopyranoside 6‐phosphate couples and the process control coefficients of the phosphotransfer reactions of the glucose phosphotransferase system. We discuss the implications of these values and the possible control points in the glucose phosphotransferase system.
Characterization of Phospholipase A2 Activity in Reticulocyte Endocytic Vesicles
Authors: Bette‐Bobillo Pascale , Vidal Michel
Published: March 2005

Electron spin resonance spectroscopy was used to investigate the presence of phospholipase A2 activity in endocytic vesicles prepared from reticulocytes and to define some of its characteristics. Using spin‐labeled phospholipid analogues, we measured the hydrolysis rate of the ester bond at position 2 during incubation with reticulocyte endocytic vesicles. We have shown that this phospholipase A2 activity was membrane‐associated, enriched in endocytic vesicles as compared to cytosol and plasma membrane. En‐zymic activity was also observed in exosomes, vesicles coming from the endocytic compartment and released by reticulocytes during their maturation in erythrocytes. Neither the hydrolytic activity nor the membrane association was found to be Ca2+‐dependent. Spin‐labeled phospholipids with choline and serine polar heads were better substrates than glycerophosphoethanolamine analogues. Optimal pH was found to be close to neutral. 5,5′‐Dithiobis(2‐nitrobenzoic acid) and diisopropyl fluorophosphate very efficiently inhibited spin‐labeled phospholipid hydrolysis. This phospholipase A2 activity was confirmed using a radioactive assay, although with much lower sensitivity. (E)‐6‐(Bromometriylene)‐tetrahydro‐3‐(1‐naphthalenyl)‐2H‐pyran‐2‐one, a specific mechanism‐based inhibitor of calcium‐independent phospho‐lipases A2, was found to abolish the enzymic activity present in endocytic vesicles.
The Effect of Free DNA on the Interactions of the Estrogen Receptor Bound to Hormone, Partial Antagonist or Pure Antagonist with Target DNA
Authors: Aliau Sigrid , Groblewski Thierry , Borgna Jean‐Louis
Published: March 2005

Interactions between the lamb uterine estrogen receptor occupied by estradiol, 4‐hydroxytamoxifen (a non‐steroidal partial estrogen antagonist) or ICI 164,384 (a steroidal pure estrogen antagonist), and the vitellogenin A2 estrogen‐response element (vit ERE) were compared using a biotinylated 25‐base all‐palindromic double‐stranded oligonucleotide, containing vit ERE (b‐ERE), which allowed isolation of the b‐ERE · receptor · [3H]ligand assembly on streptavidin‐Sepharose. The results of saturation analyses of the three receptor · [3H]ligand complexes by increasing amounts of b‐ERE were quite similar for the proportion of complexes able to interact with b‐ERE (which varied from 30% to 65% according to experiments) and for the equilibrium dissociation constant [kd(0°C) ≈ 1.2 nM, assuming that the receptor interacted as a dirner with b‐ERE]. With each ligand, receptor binding to ERE did not change the rate of ligand dissociation from the receptor at 20°C. The rate of estrogen receptor dissociation from b‐ERE, measured at 20°C in the presence of a given concentration of ERE, did not vary according to the ligand bound to the receptor; however, this dissociation rate increased linearly over the ERE concentration range (0.5 – 10 μM). The experimental rate constant (k−) of estrogen receptor dissociation from b‐ERE appeared to be the sum of the basal dissociation‐rate constant (k−°≈ 0.011 min−1), corresponding to spontaneous dissociation which would occur in the absence of ERE, and of the ERE‐induced dissociation‐rate constant, proportional to the used concentration of ERE (ki−≈4500 CEREM−1 min−1, where CERE is the molar concentration of ERE). Non‐target DNA also induced receptor dissociation from b‐ERE, but its efficiency was 6–10‐fold lower than that of ERE. We conclude that, the two antiestrogens are as efficient as estradiol in promoting estrogen receptor binding to a single vit ERE; the low or nil ability of antiestrogens to induce estrogenic responses is probably not linked with the receptor DNA‐binding step; DNA binding does not seem to affect the conformation of the filled hormone‐binding site of the receptor at 20°C; interactions of receptor dimers with DNA seems to proceed by direct transfer of receptor dimers between DNA strands.
Role of Protein Phosphatase 2A in the Control of Glycogen Metabolism in Yeast
Authors: Clotet Josep , Posas Francesc , Hu Guo‐Zhen , Ronne Hans , Ariño Joaqum
Published: March 2005

The yeast homologues of mammalian protein phosphatase 2A (PP2A) are encoded by two genes, PPH21 and PPH22. To evaluate the role of these phosphatases in the control of glycogen metabolism, wild‐type cells and mutants carrying deletions of PPH21 or PPH22 were studied. Our results indicate that the lack of a single gene product does not result in significant changes in glycogen content, glycogen synthase, and glycogen phosphorylase activities. Since the double disruption is very detrimental to the cell, the effect of lack of PP2A was evaluated by using strain H336, which carries a deletion of the PPH21 gene and has the PPH22 gene placed under the control of the GAL1 promoter, under conditions that allowed either progressive depletion or overexpression of PPH22. When grown on galactose, H336 cells contain 2–3‐fold more PP2A activity than control cells. After 14 h in glucose, however, PP2A activity in strain H336 is markedly reduced. The decrease in PP2A activity correlates with a reduced accumulation of glycogen and a more pronounced inactivation of glycogen synthase while glycogen phosphorylase becomes more resistant to inactivation. These observations suggest a role for PP2A in controlling the activation states of both enzymes. The total amount of phosphorylase was also higher in the PP2A‐depleted cells, as determined by both enzymic and immunochemical techniques. However, Northern‐blot analysis revealed that this is not due to an increase in the phosphorylase mRNA, which is in fact reduced in these cells. In contrast, overexpression of PP2A causes an increased expression of glycogen phosphorylase and a resulting failure to accumulate glycogen. We conclude that PP2A is involved in regulating both the amounts and the activation states of glycogen synthase and glycogen phosphorylase.
Cytochrome‐c Oxidase in Developing Rat Heart Enzymic Properties and Amino‐terminal Sequences Suggest Identity of the Fetal Heart and the Adult Liver Isoform
Authors: Schägger Hermann , Noack Heiko , Halangk Walter , Brandt Ulrich , Von Jagow Gebhard
Published: March 2005

Perinatal development of cytochrome‐c oxidase (complex IV) and ubiquinol–cytochrome‐c reductase (complex III) was investigated in rat heart and liver by analysing catalytic properties, protein amounts, and subunit isoforms during the transition from the fetal to the adult state. The total amounts of complexes from milligram quantities of tissue, and the portions of isoforms of complex IV, were quantified densito‐metrically after isolation of the native complexes by blue native polyacrylamide gel electrophoresis and separation of the protein subunits by Tricine/SDS/PAGE [Schägger, H. & von Jagow, G. (1991) Anal. Biochem. 199, 223–231].

A parallel increase of protein amounts and catalytic activities during perinatal development was observed in heart and liver for complex III, but only in liver for complex IV. In heart, both a doubling of the turnover number of complex IV and a lowered Km for cytochrome c were observed. The altered enzymic properties correlated with the increase of heart type subunits VIa and VIII. The fetal enzymes from heart and liver seem to be identical to the adult liver isoform, as deduced from their enzymic properties and identical aminoterminal sequences of subunits VIa and VIII.
MATH‐2, a Mammalian Helix‐Loop‐Helix Factor Structurally Related to the Product ofDrosophilaProneural Geneatonal, is Specifically Expressed in the Nervous System
Authors: Shevlizu Chikara , Akazawa Chihiro , Nakanishi Shigetada , Kageyama Ryoichiro
Published: March 2005

InDrosophila, multiple helix‐loop‐helix (HLH) factors play an essential role in neural development. Mammalian homologues of suchDrosophilaHLH factors have been recently characterized and provide useful information for the analysis of the mechanisms of mammalian neurogenesis. We report here the molecular characterization of a novel mouse HLH factor, designated MATH‐2, that has a structural homol‐ogy to the product ofDrosophilaproneural geneatonal.MATH‐2 consists of 337 amino acid residues and contains an atonal‐related basic HLH domain. However, outside of this domain, there is no significant sequence similarity between MATH‐2 and Atonal. MATH‐2 expression occurs by embryonic day 11.5 (El 1.5), and is first detected in the wall of brain vesicles as well as in the spinal cord. It is expressed in the cortical plate and the mantle layer throughout the developing central nervous system but not in the ventricular zone. By E13.5, the expression becomes more prominent in the cortical plate of the cerebrum but decreases in the other regions. In the adult, the cerebrum produces a high level of MATH‐2 RNA but other neural tissues produce only low levels. MATH‐2 RNA is not detected in non‐neural tissues, indicating that MATH‐2 expression is specific to the nervous system. The gel mobility‐shift analysis shows that MATH‐2 can interact with several E‐box sequences in collaboration with E47, a ubiquitously expressed HLH factor. These results raise the possibility that MATH‐2 may be a Trans‐acting factor involved in the development and maintenance of the mammalian nervous system.
Role of Arginine Residues for the Activity of Fasciculin
Authors: Červeanský Carlos , Engström Åke , Karlsson Evert
Published: March 2005

The West African green mamba, Dendroaspis angusticeps, has two toxins, fasciculins, that are non‐competitive inhibitors of acetylcholinesterase. Arginine residues of fasciculin 2 were modified with 1,2‐cyclohexanedione. Two of these residues, Arg24 and Arg37, reacted very slowly or not at all. Modification of Arg28 reduced the activity only by 13%. Arg11 and Arg27 are unique for fasciculins; a comparison of the sequences of 175 snake toxins homologous to fasciculins showed that no other toxin has arginine in the corresponding positions. Modification of the two unique arginines had a large effect and decreased the activity by 73% (Arg11) and 85% (Arg27). This was apparently not due to structural perturbations, since the modification did not change the circular dichroic spectra. The two arginine residues probably participate in the binding to acetylcholinesterase. They are located on the same side of the toxin molecule and the distance between their α‐carbons is 2.7 nm. This may indicate binding to sites that are far apart and suggests that fasciculin covers a large area of the enzyme.
Purification and Characterization of an ATP‐dependent Amidohydrolase, N‐methylhydantoin Amidohydrolase, from Pseudomonas putida 77
Authors: Ogawa Jun , Kim Jong Min , Nirdnoy Warawadee , Amano Yasushi , Yamada Hideaki , Shimizu Sakayu
Published: March 2005

N‐Methylhydantoin amidohydrolase, an ATP‐dependent amidohydrolase involved in microbial degradation of creatinine, was purified 70‐fold to homogeneity, with a 62% overall recovery, and was crystallized from Pseudomonas putida 77. The enzyme has a relative molecular mass of 300000. It is a tetramer of two identical small subunits(Mr 70000) and two identical large subunits (Mr, 80000). The enzyme requires ATP for the amidohydrolysis of N‐methylhydantoin and vice versa. Mg2+, Mn2+ or Co2+, and K+, NH4+, Rb+ or Cs+, were absolutely required concomitantly for the enzyme activity as divalent and monovalent cations, respectively. The Km and Vmax values for N‐methylhydantoin were 32 μM and 9.0 μmol · min−1· mg protein−1. The hydrolysis of amide compounds and coupled hydrolysis of ATP were observed with hydantoin, dl‐5‐methylhydantoin, glutarimide and succimide in addition to N‐methyl‐hydantoin. 2‐Pyrrolidone, 2‐oxazolidone, δ‐valerolactam, 2,4‐thiazolidinedione, 2‐imidazolidone, d‐5‐oxoproline methyl ester, dl‐5‐oxoproline methyl ester, and naturally occurring pyrimidine compounds, i.e. dihydrouracil, dihydrothymine, uracil, and thymine, effectively stimulated ATP hydrolysis by the enzyme without undergoing detectable self‐hydrolysis.
The Ontogeny of Apolipoprotein Expression in Rat Liver
Authors: Hall Graham P. , Redgrave Trevor G. , Yeoh George C. T.
Published: March 2005

The pattern of apolipoprotein (apo) A‐I, A‐IV and E expression in developing rat liver was established by determining steady‐state levels of the respective mRNAs. Apo A‐I and A‐IV altered in a coordinate fashion; the transcripts were detected from day 13 of gestation, whereas apo E was first detected on day 19 of gestation. Apo A‐I and A‐IV mRNA levels increased with developmental age until day 19, then declined until birth, after which they increased. In contrast, apo E mRNA levels progressively increased from day‐13 gestation until 3 days postnatal at which time it reached adult levels.

In cultured hepatocytes established from immature (15‐day gestation) and near‐term (19‐day gestation) fetuses the difference in regulation between apo A‐I and A‐IV and apo E was also observed. In 3‐day‐old fetal hepatocyte cultures established from 19‐day gestation rats, dexamethasone, insulin, thyroxine and glucagon each substantially increased levels of apo A‐I and A‐IV mRNA but markedly decreased apo E mRNA. Thus fetal and adult hepatocytes respond similarly to the hormones tested with respect to apolipoprotein expression. Unexpectedly, 15‐day gestation hepatocytes expressed apo E in culture, even without hormone supplementation. The discrepancy between in vivo and in vitro data suggests that, in the fetus, apo E expression may be suppressed by high levels of circulating steroid, insulin and thyroxine and that establishment of the hepatocytes in culture removes the inhibition, thereby inducing apo E expression in these immature cells. The data are also consistent with the view that the same group of hormones may be responsible for regulating levels of apo A‐I and A‐IV in the perinatal period. Both apolipoproteins progressively increase as the fetus reaches term at a time when these hormones which induce their expression are also increasing.
Molecular Cloning of Human cDNA with a Sequence Highly Similar to that of the Dihydrofolate Reductase Gene in Brain Libraries Derived from Alzheimer's Disease Patients
Authors: Matsumoto Akira , Matsumoto Reiko , Fujiwara Yoshisada
Published: March 2005

A polyclonal antibody was raised against a serine protease purified from the extracellular fluid of familial Alzheimer's disease lymphoblastoid cells. Using this antibody, a cDNA library from familial Alzheimer's disease cells and two cDNA libraries from the brains of two Alzheimer's disease patients were screened independently. The familial Alzheimer's disease protein 1 (FADP1) cDNA clones isolated from these three libraries were subjected to DNA sequence analysis. The nucleotide sequence of FADP1 cDNA is highly similar to the 5′ portion of the human dihydrofolate reductase (DHFR) gene, however, the sequence corresponding to exon 1 of the DHFR gene is completely disrupted and contains a 247‐bp DNA insert with a sequence unique to FADP1. Moreover, FADP1 cDNA harbours a large open reading frame, including the unique insert, which has the potential to code an approximately 50‐kDa protein. The deduced amino acid sequence of this protein contains 12 cysteine residues potentially involved in six disulfide bonds, a proline‐rich segment and a hydrophobic segment. Northern‐blot analysis with the unique insert DNA probe verified that FADP1 protein is expressed in both lymphoblastoid and brain cells derived from Alzheimer's disease patients.
Calystegins of Physalts alkekengi var. Francheti (Solanaceae)
Authors: Asano Naoki , Kato Atsushi , Oseki Kengo , Kizu Haruhisa , Matsui Katsuhiko
Published: March 2005

Five calystegins were extracted from the roots of Physalis alkekengi var. francheti (Solanaceae) with hot water and purified to homogeneity by the combination of a variety of ion‐exchange column chromato‐graphies. Their structures have been determined from the 1H‐ and 13C‐NMR spectral data, and two of the compounds were identified as calystegins A3 and B2, which have been isolated from the roots of Calys‐tegia sepium (Convolvulaceae). Two of the remaining three were found to be lα,3α,4β‐trihydroxy‐nor‐tropane and 1α,2α,3α,4β‐tetrahydroxy‐nor‐tropane and given the trivial name calystegins A5 and B3, respectively. The last calystegin was assigned as 1α,2β,3α,6α‐tetrahydroxy‐nor‐tropane, which was the same as the relative configuration proposed in the literature for calystegin B1 isolated from C. sepium. However, the 13C‐NMR spectral data for the compound from C. sepium differed substantially from our results. From a personal communication with the authors of the original paper on calystegins, it was clarified that the 13C‐NMR chemical shifts of calystegin B1 in the original paper had been erroneous. Since their corrected 13C‐NMR data of calystegin b1 and its 1H‐NMR chemical shifts in the original paper are very close to our present data, we concluded that both compounds from C. sepium and P. alkekengi are identical. Calystegin B2 has been known to be a potent competitive inhibitor of almond β‐glucosidase (Ki= 1.2 μM) and coffee bean α‐galactosidase (Ki= 0.86 μM). In this study calystegin B1 (1α,2β,3α,6α‐tetrahydroxynor‐tropane) proved to be a potent competitive inhibitor of almond β‐glucosidase (Ki= 1.9 μM) and bovine liver β‐galactosidase Ki= 1.6 μM), but not an inhibitor of α‐galactosidases. Calystegin A3 was found to be a weaker inhibitor compared to calystegin B2 but with the same inhibitory spectrum. Calystegin A5, a 2‐deoxy derivative of calystegin B2, showed no activity against any glycosidases tested. Since calystegin B3, a 2‐epimer of calystegin B2, also exhibited only a weak inhibitory activity, it was concluded that the equatorially oriented OH group at C2 is the essential feature for recognition and strong binding by the active site of glycosidases. Based on the structure/activity relationships for the five calystegins isolated from P. alkekengi var. francheti and calystegin C1 from Morus alba, we propose that the OH group at C6 of calystegin Bi or Ci, in place of the β‐glycoside oxygen, is protonated by an acidic group in the active site of the β‐glycosidase.
Mutations at the C‐Terminal Isoleucine and Other Potential Iron Ligands of 5‐Lipoxygenase
Authors: Hammarberg Tove , Zhang Ying‐Yi , Lind Birger , Rådmark Olof , Samuelsson Bengt
Published: March 2005

The non‐heme iron centre in human 5‐lipoxygenase was studied. Recombinant enzyme was expressed in Escherichia coli, purified and assayed for iron content and enzyme activity. For non‐mutated enzyme, the iron content was 1.01±0.19 mol/mol. Deletion of the C‐terminal Ile673 resulted in an iron content of 0.03±0.07 mol/mol and undetectable lipoxygenase activity. Mutations at His367, Glu376 and Asn554 led to drastically decreased enzyme activity (<2% of non‐mutated control) but iron was still present. In addition to Glu376, eight other conserved acidic residues (Asp/Glu) in 5‐lipoxygenase were replaced, none of which was crucial for enzyme activity. We conclude that Ile673 is an iron ligand in 5‐lipoxygenase, while our results do not support that Glu376 or Asn554 have this function. The possible role of His367 as a replaceable iron ligand is discussed.
Real‐Time Monitoring of Antigen‐antibody Recognition on a Metal Oxide Surface by an Optical Grating Coupler Sensor
Authors: Bernard André , Bosshard Hans Rudolf
Published: March 2005

Real‐time monitoring of intermolecular interactions can provide a direct and rapid estimate of the affinity and kinetics of interactions between biomolecules. Optical methods based on the measurement of changes of refractive index in the immediate vicinity of a liquid‐solid interface are particularly convenient because they require no radioactive, fluorescent or other labelling of the molecules under study. In the present work we have followed the specific interaction of protein molecules on a SiO2/TiO2 surface with the help of the optical grating coupler sensor instrument BIOS‐1. This instrument allows the determination of the absolute mass of protein adsorbed to the sensor surface and, therefore, the calculation of the molar ratio of the components partaking in an intermolecular interaction. For example, about 3 ng avidin/mm2 surface area could be adsorbed. This amount closely corresponds to a monolayer composed of densely packed globular avidin molecules. A dimeric, biotinylated leucine zipper peptide was bound to this avidin layer at a molar ratio of 1:1 (1 peptide molecule/4 biotin binding sites of tetrameric avidin). An average of 1/2.6 peptides was recognized by a peptide‐specific monoclonal antibody. Even though avidin was not covalently bound to the sensor surface, the avidin‐coated chip could be used repeatedly to measure the time course of antibody binding as a function of the concentration of the antibody. From such measurements it was possible to calculate the association and dissociation rate constants assuming that the interaction of the antibody with the surface‐bound antigen can be described by a simple Langmuir binding model. The limits of the Langmuir model are discussed. The same antigen‐antibody reaction was also analyzed by a surface plasmon resonance biosensor (BIAcoreTM, Pharmacia). The results obtained with the two instruments, which register different optical phenomena and employ different surface chemistry, were in good agreement.
Adenosine Inhibits Protein Synthesis in Isolated Rat Hepatocytes
Authors: Tinton Sandrine A. , Chow Sek C. , Buc‐Calderon Pedro M. , Kass George E. N. , Orrenius Sten
Published: March 2005

Extracellularly added adenosine and ATP are potent inhibitors of protein synthesis in liver cells. In this study, the possible involvement of Ca2+ in the mechanism of inhibition of protein synthesis by adenosine was investigated. Stimulation of freshly isolated hepatocytes with adenosine or ATP, at concentrations that impaired protein synthesis, induced an increase in the cytosolic free Ca2+ concentration ([Ca2+]i). However, there was no correlation between the increase in [Ca2+]i and inhibition of radiolabelled leucine incorporation into proteins. Thus, the stimulation of hepatocytes with the V1‐receptor agonist, vasopressin, or with the nucleotide triphosphates, UTP and GTP, elicited changes in [Ca2+]i similar to those observed after ATP or adenosine addition, but did not affect protein synthesis. ATP produced near complete discharge of Ca2+ from the inositol 1,4,5‐trisphosphate‐sensitive Ca2+ pool in isolated hepatocytes, whereas adenosine only had a partial effect. Depletion of the hormone‐sensitive Ca2+ pool by adenosine was transient. In contrast, prolonged depletion of internal Ca2+ by thapsigargin resulted in the inhibition of protein synthesis in hepatocytes. However, the inhibition of radiolabelled leucine incorporation into proteins by thapsigargin was further augmented by the additional presence of adenosine. These results show that the inhibition of protein synthesis by adenosine in isolated hepatocytes is not mediated by an increase in [Ca2+]i or depletion of internal pool(s) sensitive to inositol 1,4,5‐trisphosphate or thapsigargin.
Lymphocyte p56L32 is A RNA/DNA‐Binding Protein which Interacts with Conserved Elements of the Murine L32 Ribosomal Protein mRNA
Authors: Severson William E. , Mascolo Patrice L. , White Michael W.
Published: March 2005

In previous studies of the ribosomal protein L32 mRNA, we demonstrated that a conserved polypyrimidine tract found in the 5′‐untranslated region (5′‐UTR) was required for translational regulation in vivo and that a 56‐kDa protein (p56L32) from T‐lymphocytes specifically interacts with this sequence [Kaspar, R. L., Kakegawa, T., Cranston, H., Morris, D. R. & White, M. W. (1992) J. Biol. Chem. 267, 508–514]. Here we show that p56L32 binding to the L32 5′‐UTR is complex and requires other 5′‐UTR RNA sequences in conjunction with the polypyrimidine tract. Deletion and site‐directed mutagenesis studies revealed that binding of p56L32 to the L32 5′‐UTR requires a second RNA element, GGUGGCUGCC, 15 nucleotides downstream from the polypyrimidine tract. In contrast, L32 RNA transcripts altered in this downstream element were good substrates for binding of the polypyrimidine binding proteins from HeLa nuclear extracts, indicating that these proteins have RNA‐binding specificities distinct from p56L32. Competition analysis demonstrated that p56L32 will bind to DNA as well as RNA with identical sequence specificity and similar affinity. Single or double‐stranded DNAs composed of the L32 5′‐UTR sequences were found to specifically compete with L32 RNA transcripts for p56L32 binding. The L32 5′‐UTR downstream element, GGUGGCUGCC, which is required for p56L32 binding, has previously been implicated as a transcriptional element of the L32 gene. The ability of p56L32 to bind this sequence as DNA or RNA suggests p56L32 may have a dual role in the regulation of ribosomal protein mRNA accumulation and translation.
Hybrid Bacillus amyloliquefaciens X Bacillus licheniformisα‐Amylases
Authors: Conrad Birgit , Hoang Viet , Polley Andreas , Hofemeister Jürgen
Published: March 2005

A series of 33 single and mosaic hybrid α‐amylases was constructed from the genes amyBA or amyLI, encoding the α‐amylases from Bacillus amyloliquefaciens (AmyBA) and Bacillus licheniformis (AmyLI). The hybrid proteins, consisting of the entire α‐amylase sequence with a variable portion of AmyBA or AmyLI origin, were characterized in order to find enzymes with new properties (thermostability, temperature and pH optima, and substrate specificity), and to localize the amino acid sequence regions responsible for the changes.

The thermostability of the AmyBA/AmyLI (AL‐type) hybrid proteins correlated with the position and the length of the hybrid sequence. The hybrid enzymes fell into six groups retaining, in comparison to AmyBA, a certain value of the extra‐thermostability of AmyLI or becoming more thermolabile than AmyBA. Four regions are proposed to contain thermostability determinants (TSDs). They map between amino acid residues 34–76,112–142,174–179 and 263–276 of the respective hybrid enzymes, indicating the dominance of the N‐terminal half of AmyLI for these hybrid enzymes' resistance against irreversible inactivation. Two (TSD3 and TSD4) coincide with regions I and II that had already been suggested to stabilise AmyLI [Suzuki, Y., Ito, N., Yuuki, T., Yamagata, H. & Udake, S. (1989) J. Biol. Chem. 264, 18933–18938]. The temperature dependence of activity of the AL‐type hybrid α‐amylases was compared at pH 6.4 and pH 7.6 and the hybrid enzymes of one thermostability group were found to have similar temperature responses.

A hybrid region between residues 34–76 is demonstrated to correlate with the α‐amylases' substrate specificity, i.e. either hydrolysis or accumulation of maltohexaose. This region was therefore named the G6G5 region. The exchange of internal sequences between residues 17–201 of AmyBA by the AmyLI counterpart in ALA‐type mosaic hybrid α‐amylases, with one exception (ALA99–429), unexpectedly destabilized the respective ALA‐type hybrids. Two of these hybrid α‐amylases (ALA17–151 and ALA76–151) were less thermostable than AmyBA, while others (ALA112–151, ALA112–201) were enzymically inactive. These data support specific roles of the predicted Al‐B domain portion between residues 17–201 of those Bacillusα‐amylases probably for correct folding and enzymic activity.
Two Low‐Affinity Ca2+‐Binding Sites of Gelsolin that Regulate Association with Actin
Authors: Ditsch Andrea , Wegner Albrecht
Published: March 2005

The time course of binding of actin to gelsolin or 1:1 gelsolin‐actin complex was measured at defined Ca2+ concentrations in the range 0.5–500 μM. The rate of association was fol10wed by the fluorescence increase of a fluorescent label covalently linked to actin. Free Ca2+ was determined by titration with EGTA in the presence of Fura‐2 as indicator. The experimental data were quantitatively evaluated by calculations of the kinetics of association of actin with gelsolin thereby taking into account the equilibrium of binding of Ca2+ ions to gelsolin. It was found that association of gelsolin with one actin monomer is regulated by a Ca2+‐binding site with a dissociation constant Ka1= 25 μM. Binding of the second actin monomer was found to be controlled by a Ca2+‐binding site of which the dissociation constant Kd2 was 200 μM. Mg2+ ions in the concentration range 0–1 mM did not compete with Ca2+ for binding to gelsolin. More complex interactions of gelsolin with actin such as nucleated actin polymerization were found to occur even at Ca2+ concentrations below Kdl (e.g. 10 μM) at almost maximal rates.
Conformation of MgATP Bound to 5‐phospho‐α‐d‐ribose 1‐diphosphate Synthetase by Two‐dimensional Transferred Nuclear Overhauser Effect Spectroscopy
Authors: Jarori Gotam K. , Murali Nagarajan , Switzer Robert L. , Rao B. D. Nageswara
Published: March 2005

The conformation of MgATP bound at the active site of Salmonella typhimurium 5‐phospho‐α‐d‐ribose 1‐diphosphate synthetase (PRibPP synthetase) has been investigated by two‐dimensional transferred‐NOE spectroscopy (TRNOESY). Inter‐proton NOEs of the ligand were measured in the presence of the protein at several mixing times in the range of 40–300 ms at 500 MHz and 10°C. Measurements were made at low ligand concentrations (≈1 mM) in order to avoid weak non‐specific ligand–protein interactions and to ensure that the NOE arises from the ligand bound at the active site. The inter‐proton distances were determined from the experimentally observed NOE buildup curves by comparing them with theoretical simulations obtained by using the complete relaxation matrix. These distances were used as constraints in molecular modeling and energy minimization calculations to deduce the structure of the bound ligand. PRibPP synthetase is known to appreciably aggregate so that it exists in multiple oligomeric forms in solution. The structure was determined under the assumption that the ligand assumes the same conformation on each subunit of every oligomer regardless of its size. On the basis of the rotational correlation time deduced for the enzyme‐nucleotide complexes, it is estimated that the average oligomer of PRibPP synthetase, in the sample used for the TRNOESY measurements, consists of about 30 subunits, whereas the smallest active form of the protein is a pentamer. The conformation of enzyme‐bound MgATP is described by a glycosidic torsion angle χ= 50±5° and phase angle of pseudorotation P= 114.9° corresponding to a 1T° sugar pucker. It is noteworthy that the value of the glycosidic torsion angle obtained in this pyrophosphoryl transfer enzyme complex agrees well with those obtained previously for MgATP complexes of creatine kinase, pyruvate kinase (active and ancillary sites), and arginine kinase. The sugar pucker, on the other hand, differs from one enzyme complex to another.
The Interaction between Human Papillomavirus Type 16 El and E2 Proteins is Blocked by An Antibody to the N‐Terminal Region of E2
Authors: Hibma Merilyn H. , Raj Kenneth , Ely Sarah J. , Stanley Margaret , Crawford Lionel
Published: March 2005

Replication of papillomavirus DNA requires two virally encoded proteins, El and E2. We expressed human papillomavirus (HPV) type 16 El and E2 in bacteria and showed that purified full‐length E2 protein interacted directly with El, in the absence of HPV16 DNA. It was established that the first 142 amino acids of El were not required for binding as E2 protein was able to interact with El devoid of this region. The interaction of E2 with El could be blocked by a monoclonal antibody that bound E2 in the region of amino acids 18–41 of E2 whereas a monoclonal antibody reactive with a nearby part of the molecule (amino acids 2–17) only partially blocked this interaction. These results suggest that a region in the N‐terminus of E2 around amino acids 18–41 is a site of interaction with the E1 protein.
Selection and Characterization of Mammalian Cell Lines with Stable Over‐Expression of Human Pituitary Receptors for Gonadoliberin
Authors: Beckers Thomas , Marheineke Kathrin , Reiländer Helmut , Hilgard Peter
Published: March 2005

The cDNA encoding the receptor for gonadoliberin (GnRH or LH‐RH) was isolated from a human pituitary cDNA library and heterologously expressed in the murine fibroblast cell line LTK−. By using a dicistronic expression strategy utilizing the internal ribosomal‐entry‐site sequence of poliovirus, single cell clones with stable and high expression of human gonadoliberin receptors were selected. In radioligand saturation‐binding experiments, the gonadoliberin antagonist Cetrorelix showed high‐affinity binding to the heterologously expressed human gonadoliberin receptor with a Kd of 0.1 nM. The pharmacological profile using 125I‐Cetrorelix as radioligand and the authentic gonadoliberin or agonistic and antagonistic derivatives as competitors, showed a distinct rank order of binding potencies. Superagonistic gonadoliberin derivatives had more than ten‐times higher binding affinities in comparison to gonadoliberin with a Kd of 3.47 nM. The gonadoliberin receptor expressed in stably transfected LTK− cells coupled to the inositol phosphate signal‐transduction pathway. Gonadoliberin stimulated the synthesis of inositol 1,4,5‐trisphosphate in a dose‐dependent way with an EC50 of 5 nM. This stimulatory effect of gonadoliberin was completely antagonized by Cetrorelix in equimolar concentrations, demonstrating the high potency of this competitive receptor antagonist. In growth‐arrested cells, a transient expression of the c‐fos proto‐oncogene was induced by gonadoliberin or [D‐Trp6]gonadoliberin, showing that the gonadoliberin receptor couples to a putative mitogenic signal‐transduction pathway in this heterologous cell system.
Molecular Dynamics Study of the Binding of Elsamicin A to DNA
Authors: Alhambra Cristóbal , Luque Francisco J. , Portugal José , Orozco Modesto
Published: March 2005

Molecular mechanics (MM) and dynamics (MD) were used to examine the conformational flexibility of elsamicin A, an antitumour antibiotic, in aqueous solution. Furthermore, MM and MD simulations were performed to obtain the first three‐dimensional model of the elsamicin‐A–DNA complex. Comparison of MD simulations of free and DNA‐bound elsamicin A provided insight into the mechanism of DNA cleavage. Moreover, the drug‐DNA complexes were analyzed to investigate specific drug–DNA interactions, as well as the role of the solvent water molecules in the drug‐DNA binding. On the basis of the results derived from these simulations, several issues related to the sequence‐selective binding of elsamicin A to DNA are discussed.
Chemical Structures of the Core Region of Campylobacter Jejuni O:3 Lipopolysaccharide and an Associated Polysaccharide
Authors: Aspinall Gerald O. , Lynch Christina M. , Pang Henrianna , Shaver Robert T. , Moran Anthony P.
Published: March 2005

The complete structure for the core oligosaccharide region of the water‐insoluble low‐Mr lipopolysaccharide of Campylobacter jejuni serotype O:3 from phenol/water extraction of bacterial cells was assigned through studies on derivatives of the liberated oligosaccharide. Structure determinations were performed using 1H‐NMR and 31P‐NMR spectroscopies, methylation analysis supported by fast‐atom‐bombardment mass spectrometry, and Smith degradation experiments. It was concluded that the complete chains in the core oligosaccharide had the following structure in which a proportion of the terminal residues were phosphorylated:
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From a similar series of experiments, it was concluded that an associated polysaccharide, which was isolated from the water phase of the phenol/water extracts, had the following repeating unit in which a proportion of the previously unknown L‐glycero ‐D‐ido ‐heptose (L‐α‐D‐ido ‐Hep) residues were present as 3‐hydroxypropanoyl esters, and were not covalently linked to the lipopolysaccharide:
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