bluuebunny/crossref_metadata_2025 huggingface dataset:
Binding of inorganic phosphate to the cadmium‐induced dimeric form of metallothionein from rabbit liver
Authors: PALUMAA Peep , VAŠÁK Milan
Published: March 2005

Recently we have demonstrated that the exposure of monomeric Cd7‐metallothionein (MT) to Cd(II) ions in potassium phosphate buffer results in the nonoxidative formation of MT dimers containing approximately two additional Cd(II) ions/monomer subunit [Palumaa, P., Mackay, E. and Vašák, M. (1992) Biochemistry 31, 2181–2186]. In this study, we demonstrate that inorganic phosphate participates in the Cd‐induced dimerization of MT. In the absence of phosphate, Cd‐ induced oligomerization of MT still takes place, but a substantially lower apparent yield of the dimeric form and an additional peak of MT tetramers were detected in gel‐filtration experiments. Arsenate exhibits a similar effect to that of phosphate, whereas a number of other anions, i.e. F−, NO3−, SO42−, ClO4−, BO3−, SCN−, HCOO− and CH3COO− had no effect on Cd‐induced oligomerization of MT. Studies on the pH dependence of MT dimerization indicate that the dianionic form of phosphate is involved in this process. Equilibrium–dialysis experiments using potassium [32P]phosphate established binding of two molecules of phosphate to the dimeric MT form with a dissociation constant, Kd, of 23 ± 3 μM (20 mM Tris/HCl and 0.1 M K Cl, pH 8.0 at 25°C), whereas binding of phosphate was not observed with the monomeric Cd7‐MT. The nonovalent nature of phosphate binding to the Cd‐induced MT dimers has been demonstrated. The presented data provide the first evidence for the binding of a nonmetallic cellular component to MT.
The distribution of charged amino acids in mitochondrial inner‐membrane proteins suggests different modes of membrane integration for nuclearly and rnitochondrially encoded proteins
Authors: GAVEL Ylva , von HEIJNE Gunnar
Published: March 2005

We have analyzed the amino acid distribution in seven nuclearly encoded and five mitochondrially encoded inner membrane proteins with experimentally well characterized topologies. The mitochondrially encoded proteins conform to the ‘positive inside’ rule, i.e. they have many more positively charged residues in their non‐translocated as compared to translocated domains. However, most of the nuclearly encoded proteins do not show such a bias but instead have a surprisingly skewed distribution of Glu residues with an almost ten times higher frequency in the intermembrane space than in the matrix domains. These findings suggest that some, but possibly not all, nuclearly encoded inner membrane proteins may insert into the membrane by a mechanism that does not depend on the distribution of positively charged amino acids.
Purification and characterization of protein PC, a component of glycine reductase from Eubacterium acidaminophilum
Authors: SCHRÄDER Thomas , ANDREESEN Jan R.
Published: March 2005

Protein PC of the glycine reductase from Eubacterium acidaminophilum was purified to homogeneity by chromatography on phenyl‐Sepharose and Sepharose S. The apparent molecular mass of the native protein, which showed an associating/dissociating behaviour, was about 420 kDa. Sodium dodecyl sulfate/polyacrylamide gel electrophoresis of protein PC revealed two protein bands corresponding to 48 and 57 kDa, indicating an α4β4 composition. The smaller subunit was identified as an acetyl‐group‐transferring protein, the 57‐kDa protein was hydrophobic. N‐terminal amino acid sequences were determined for both subunits. Antibodies raised against the 48‐kDa subunit showed cross‐reactions with extracts of E. acidaminophilum grown on different substrates and with extracts from other glycine‐utilizing anaerobic bacteria such as Clostridium purinolyticum, C. sticklandii, and C. sporogenes. The respective protein from the former two organisms corresponded in molecular mass. When protein PA was chemically carboxymethylated by iodo[2‐14C]acetate and incubated with protein PC, acetyl phosphate was a reaction product, thus establishing it as the product of the glycine reductase reaction by using homogeneous preparations of these two proteins from E. acidaminophilum.
Differential expression of the two methyl‐coenzyme M reductases in Methanobacterium thermoautotrophicum as determined immunochemically via isoenzyme‐specific antisera
Authors: BONACKER Lutz G. , BAUDNER Siegfried , THAUER Rudolf K.
Published: March 2005

Methanobacterium thermoautotrophicum contains two isoenzymes of methyl‐coenzyme M reductase (MCR), MCR I and MCR II, which catalyze the methane‐forming step and which together represent more than 10% of the cellular protein. We describe here the preparation of isoenzymespecific antisera against the two MCR isoenzymes and their use in the quantitative immunochemical determination of the two isoenzymes in the methanogen. The relative and absolute cellular concentration of the two proteins is shown to be strongly affected by growth conditions such as the temperature, pH, and substrate concentration. Conditions were found yielding cells which contained essentially only MCR I or MCR II. Using antisera against MCR I and MCR II, MCR from other methanogens were immunochemically compared. Evidence is presented that Methanobacterium wolfei also contains two isoenzymes of MCR.
An anti‐peptide antibody that recognizes the dopamine D2 receptor from bovine striatum
Authors: PLUG Maria J. , DIJK Jan , MAASSEN J. Antonie , MÖLLER Wim
Published: March 2005

The bovine dopamine D2 receptor was purified by wheat‐germ‐agglutinin‐Sepharose chromatography and affinity chromatography, using the D2‐receptor‐specific agonist N‐0434. Purification yields a preparation with a major protein band of 95 kDa. In order to ascertain the identity of this protein polyclonal antibodies against the dopamine D2 receptor have been raised using synthetic peptides based on the predicted amino acid sequence of the cloned D2 receptor. For the initial screening of these antibodies, three fusion proteins consisting of β‐galactosidase and receptor fragments were constructed. One antiserum reacted strongly with the corresponding D2 receptor fusion protein, both on Western blots and in immunoprecipitation experiments. In each case, recognition was inhibited by competition with free peptide. On Western blots of partially purified receptor preparations from bovine striatum, the antiserum specifically recognized a 95‐kDa glycoprotein. From similar preparations, the antiserum precipitated a substantial proportion of active D2 receptor, as determined by a decrease in [3H]spiperone binding in the supernatant. Active receptor could be released from the immunoprecipitate by addition of free peptide. Immunocytochemical analysis of cells transiently transfected with DNA coding for the D2 receptor showed specific staining of transfected cells. The antibody raised against a sequence in the third intracellular loop is able to shift the affinity of the receptor for dopamine from high to low, indicating that the antiserum may be interfering with receptor—GTP‐binding‐protein interactions.
α‐d‐Galactose‐bearing glycoproteins on the surface of stimulated murine peritoneal macrophages
Authors: PETRYNIAK Jerzy , HUARD Thomas K. , GOLDSTEIN Irwin J.
Published: March 2005

Two glycoproteins were isolated from lysates of thioglycollate‐stimulated, murine peritoneal macrophages by affinity chromatography on immobilized Griffonia simplicifolia I lectin and by preparative SDS/PAGE. The glycoproteins were readily labeled on the surface of intact macrophages with 3H and 125I. The labeled glycoproteins migrated as broad bands of molecular mass 92–109 kDa and 115–125 kDa. The mobility of the glycoproteins decreased only slightly after reduction with dithiothreitol, indicating the absence of intersubunit disulfide bridges. The 92‐kDa and 115‐kDa glycoproteins had pI 5.2–5.4 and pI≤ 4, respectively. Digestion of both glycoproteins with α‐galactosidase released 23% of their 3H content and abolished their ability to bind to the G. simplicifolia I lectin, showing that they contain terminal α‐d‐galactosyl groups. After reduction with 2‐mercaptoethanol, each glycoprotein fraction was sensitive to N‐glycanase; the 115‐kDa glycoproteins produced a smear with the front at approximately 67 kDa, whereas the 92‐kDa glycoprotein gave two bands of 61 kDa and 75 kDa. Unreduced glycoproteins were insensitive to N‐glycanase, suggesting the presence of intramolecular disulfide bonds. Although each glycoprotein fraction was sensitive to endoglycosidase H, this enzyme produced only slight changes in molecular mass when compared with N‐glycanase. From these results as well as from the specificity of the enzymes involved, it is concluded that each glycoprotein fraction contains complex‐type oligosaccharides and a small amount of high‐mannose and/or hybrid‐type oligosaccharides. While each glycoprotein fraction was bound to Datura stramonium lectin, they failed to react with anti‐[i‐(Den)] serum and their digestion with endo‐β‐galactosidase did not cause a band shift in SDS/PAGE. Taken together, these results suggest the presence of N‐acetyllactosamine units which are not arrayed in linear form but occur as single units, bound either to C2 and C6, or to C2 and C4, or both, of outer mannosyl residues on complex‐type oligosaccharides. The glycoprotein(s) fraction precipitated with anti‐[I (Step)] serum, suggesting the presence of branched lactosaminoglycans. Digestion of both glycoprotein fractions with a mixture of sialidase and O‐glycanase did not alter their mobility in SDS/PAGE, suggesting a lack or low content of O‐linked trisaccharides and tetrasaccharides. Each glycoprotein fraction was bound specifically to Sambucus nigra and Maackia amurensis immobilized lectins, indicating the presence of sialic acid linked α2,6 to subterminal d‐galactose or N‐acetylgalactosamine residues, and α2,3 to N‐acetyllactosamine residues, respectively.
Evolutionary optimization of the catalytic efficiency of enzymes
Authors: PETTERSSON Gösta
Published: March 2005

The rate equation for a generalized Michaelian type of enzymic reaction mechanism has been analyzed in order to establish how the mechanism should be kinetically designed in order to optimize the catalytic efficiency of the enzyme for a given average magnitude of true and apparent first‐order rate constants in the mechanism at given concentrations of enzyme, substrate and product.

As long as on‐velocity constants for substrate and product binding to the enzyme have not reached the limiting value for a diffusion‐controlled association process, the optimal state of enzyme operation will be characterized by forward (true and apparent) first‐order rate constants of equal magnitude and reverse rate constants of equal magnitude. The drop in free energy driving the catalysed reaction will occur to an equal extent for each reaction step in the mechanism. All internal equilibrium constants will be of equal magnitude and reflect only the closeness of the catalysed reaction to equilibrium conditions.

When magnitudes of on‐velocity constants for substrate and product binding have reached their upper limits, the optimal kinetic design of the reaction mechanism becomes more complex and has to be established by numerical methods. Numerical solutions, calculated for triosephosphate isomerase, indicate that this particular enzyme may or may not be considered to exhibit close to maximal efficiency, depending on what value is assigned to the upper limit for a ligand association rate constant.

Arguments are presented to show that no useful information on the evolutionary optimization of the catalytic efficiency of enzymes can be obtained by previously taken approaches that are based on the application of linear free‐energy relationships for rate and equilibrium constants in the reaction mechanism.
cDNA cloning of rice lipoxygenase L‐2 and characterization using an active enzyme expressed from the cDNA in Escherichia coli
Authors: OHTA Hiroyuki , SHIRANO Yumiko , TANAKA Kunisuke , MORITA Yuhei , SHIBATA Daisuke
Published: March 2005

A full‐length cDNA of rice lipoxygenase L‐2 was cloned from 3‐day old seedlings. The identity of the clone was determined by amino acid sequencing of selected peptides of the purified enzyme and immunological characterization of an active enzyme that was produced from the cDNA in Escherichia coli by cultivation at 15°C. The nucleotide sequence showed a strong bias toward G and C in the selection of nucleotides, especially at the third position of the codons(93%G/C). The complete amino acid sequence of the enzyme was deduced from the nucleotide sequence. The molecular mass of the enzyme was calculated to be 96657 Da based on 865 amino acids. The amino acid sequence shares similarity with those of dicot lipoxygenases throughout the enzyme at a level of 50%. A hydropathy profile calculated from the amino acid sequence resembled those of dicot lipoxygenases, suggesting conservation of the secondary structure of these enzymes. The active enzyme, expressed in Escherichia coli, was charaterized for pH dependence of the enzyme activity, intramolecular specificity, heat stability and Km. The enzyme had the same properties as the L‐2 enzyme that was isolated from seedlings, but differed from the lipoxygenase L‐3 isolated from mature plants.
The closely related homomeric and heterodimeric mannose‐binding lectins from garlic are encoded by one‐domain and two‐domain lectin genes, respectively
Authors: VAN DAMME Els J. M. , SMEETS Koen , TORREKENS Sophie , VAN LEUVEN Fred , GOLDSTEIN Irwin J. , PEUMANS Willy J.
Published: March 2005

Lectin cDNA clones for two different lectins from garlic (Allium sativum L.) bulbs, ASAI and ASAII (ASA, Allium sativum agglutinin), were isolated and characterized. the first lectin, ASAI, is a heterodimer composed of two different subunits of 11.5 kDa and 12.5 kDa. It is translated from an mRNA of 1400 nucleotides encoding a polypeptide of 306 amino acids with two very similar domains. N‐terminal sequenceing of the two polypeptides of the mature lectin confirmed that both subunits are derived from the same precursor and that each corresponds to one of the two domains in the sequence.

In contrast to ASAI, the second garlic lectin, ASAII, is a homodimer of two identical 12‐kDa subunits. It is translated from an mRNA of approximately 800 nucleotides encoding a polypeptide of 154 amino acids. Interestingly, the coding region of the ASII cDNA clones is almost identical to that of the second domain of the ASAI cDNA clones.
Crystal structure of NAD‐dependent formate dehydrogenase
Authors: LAMZIN Victor S. , ALESHIN Alexander E. , STROKOPYTOV Boris V. , YUKHNEVICH Maria G. , POPOV Vladimir O. , HARUTYUNYAN Emil H. , WILSON Keith S.
Published: March 2005

The ternary complex of NAD‐dependent formate dehydrogenase (FDH) from the methylotrophic bacterium Pseudomonas sp. 101 (enzyme‐NAD‐azide) has been crystallised in the space group P212121 with cell dimensions a = 11.60 nm, b= 11.33 nm, c= 6.34 nm. There is 1 dimeric molecule/asymmetric unit. An electron density map was calculated using phases from multiple isomorphous replacement at 0.30 nm resolution. Four heavy atom derivatives were used. The map was improved by solvent flattening and molecular averaging. The atomic model, including 2 × 393 amino acid residues, was refined by the CORELS and PROLSQ packages using data between 1.0 nm and 0.30 nm excluding structure factors less than 1 σ. The current R factor is 27.1% and the root mean square deviation from ideal bond lengths is 4.2 pm. The FDH subunit is folded into a globular two‐domain (coenzyme and catalytic) structure and the active centre and NAD binding site are situated at the domain interface. The β sheet in the FDH coenzyme binding domain contains an additional β strand compared to other dehydrogenases. The difference in quaternary structure between FDH and the other dehydrogenases means that FDH constitutes a new subfamily of NAD‐dependent dehydrogenases: namely the P‐oriented dimer. The FDH nucleotide binding region of the structure is aligned with the three dimensional structures of four other dehydrogenases and the conserved residues are discussed. The amino acid residues which contribute to the active centre and which make contact with NAD have been identified.
NAD‐linked, factor‐dependent formaldehyde dehydrogenase or trimeric, zinc‐containing, long‐chain alcohol dehydrogenase from Amycolatopsis methanolica
Authors: van OPHEM Peter W. , Van BEEUMEN Jozef , DUINE Johannis A.
Published: March 2005

NAD‐linked, factor‐dependent formaldehyde dehydrogenase (FD‐FA1DH) of the Gram‐positive methylotrophic bacterium, Amycolatopsis methanolica, was purified to homogeneity. It is a trimeric enzyme with identical subunits (molecular mass 40 kDa) containing 6 atoms Zn/enzyme molecule. The factor is a heat‐stable, low‐molecular‐mass compound, which showed retention on an Aminex HPX‐87H column. Inactivation of the factor occurred during manipulation, but activity could be restored by incubation with dithiothreitol. The identity of the factor is still unknown. It could not be replaced by thiol compounds or cofactors known to be involved in metabolism of C1 compounds. Of the aldehydes tested, only formaldehyde was a substrate. However, the enzyme showed also activity with higher aliphatic alcohols and the presence of the factor was not required for this reaction. Methanol was not a substrate, but high concentrations of it could replace the factor in the conversion of formaldehyde. Presumably, a hemiacetal of formaldehyde is the genuine substrate, which, in the case of methanol, acts as a factor leading to methylformate as the product. This view is supported by the fact that formate could only be detected in the reaction mixture after acidification. Inhibition studies revealed that the enzyme contains a reactive thiol group, being protected by the binding of NAD against attack by heavy‐metal ions and aldehydes. Studies on the effect of the order of addition of coenzyme and substrate suggested that optimal catalysis required NAD as the first binding component. Substrate specificity and the induction pattern clearly indicate a role of the enzyme in formaldehyde oxidation. However, since FD‐FA1DH was also found in A. methanolica grown on n‐butanol, but not on ethanol, it may have a role in the oxidation of higher aliphatic alcohols as well. FD‐FA1DH and the factor from A. methanolica are very similar to a combination already described for Rhodococcus erythropolis [Eggeling, L. & Sahm, H. (1985) Eur. J. Biochem. 150, 129–134].

NAD‐linked, glutathione‐dependent formaldehyde dehydrogenase (GD‐FA1DH) resembles FD‐FA1DH in many respects. Since glutathione has so far not been detected in Gram‐positive bacteria, FD‐FA1DH could be the counterpart of this enzyme in Gram‐positive bacteria. Alignment of the N‐terminal sequence (31 residues) of FD‐FA1DH with that of GD‐FA1DH from rat liver indeed showed similarity (30% identical positions). However, comparable similarity was found with class I alcohol dehydrogenase from this organism and with cytosolic alcohol dehydrogenase from Saccharomyces cerevisiae, isozyme 1. Therefore, it is concluded that the trimeric FD‐FA1DH is related to the dimeric/tetrameric, zinc‐containing, long‐chain alcohol dehydrogenases.
Gadolinium ion inhibits loss of metabolites induced by osmotic shock and large stretch‐activated channels in bacteria
Authors: BERRIER Catherine , COULOMBE Alain , SZABO Ildiko , ZORATTI Mario , GHAZI Alexandre
Published: March 2005

Bacteria subjected to a hypotonic osmotic shock lose internal ions and also metabolites, without lysis of the cells. We show that the presence in the shock medium, at submillimolar concentrations, of the ion gadolinium, recently shown to block stretch‐activated channels in Xenopus oocytes [Yang, X.‐C. & Sachs, F. (1989) Science 243 1068–1071], was sufficient to inhibit shock‐induced release of metabolites such as lactose and ATP in Escherichia coli and ATP in Streptococcus faecalis. Moreover, gadolinium was observed, in patch‐clamp experiments, to inhibit the giant stretch‐activated channels of E. coli, S. faecalis and Bacillus subtillis. taken together, these data suggest that stretch‐activated channels are localized in the cytoplasmic membrane of Gram‐negative and Gram‐positive bacteria, where they control the efflux of osmotic solutes, thus probably playing a major role in the response to hypotonic osmotic shock.
Demonstration that a human 26S proteolytic complex consists of a proteasome and multiple associated protein components and hydrolyzes ATP and ubiquitin‐ligated proteins by closely linked mechanisms
Authors: KANAYAMA Hiro‐omi , TAMURA Tomohiro , UGAI Shinichi , KAGAWA Susumu , TANAHASHI Nobuyuki , YOSHIMURA Tetsuro , TANAKA Keiji , ICHIHARA Akira
Published: March 2005

It is known that two types of high‐molecular‐mass protease complexes are present in the cytosol of mammalian cells; a 20S latent multicatalytic proteinase named the proteasome, and a large proteolytic complex with an apparent sedimentation coefficient of 26S that catalyzes ATP‐dependent breakdown of proteins conjugated with ubiquitin. In this work, we first demonstrated that a low concentration of SDS was required for activation of the latent proteasome, whereas the 26S complex degraded substrates for proteasomes in the absence of SDS. Moreover, the 26S complex was greatly stabilized in the presence of 2 mM ATP and 20% glycerol. Based on these characteristics, we next devised a novel procedure for purification of the 26S proteolytic complexes from human kidney, In this procedure, the proteolytic complexes were precipitated from cytoplasmic extracts by ultracentrifugation for 5 h at 105000 × g, and the large 26S complexes were clearly separated from the 20S proteasomes by molecular‐sieve chromatography on a Biogel A‐1.5 m column. The 26S enzyme was then purified to apparent homogeneity by successive chromatographies on hydroxyapatite and Q Sepharose, then by glycerol density‐gradient centrifugation. Electrophoretic and immunochemical analyses showed that the purified human 26S complex consisted of multiple subunits of proteasomes with molecular masses of 21–31 kDa and 13–15 protein components ranging in molecular mass over 35–110 kDa, which were directly associated with the proteasome. The purified 26S proteolytic complex degraded 125I‐labeled lysozyme‐ubiquitin conjugates in an ATP‐dependent manner. The 26S enzyme also showed high ATPase activity, which was copurified with the complex. Vanadate and hemin strongly inhibited not only ATP cleavage, but also ATP‐dependent breakdown of ubiquitinligated proteins, suggesting that the 26S complex hydrolyzes ATP and ubiquitinated proteins by closely linked mechanisms. These findings indicate that the 26S complex consists of a proteasome with proteolytic function and multiple other components including an ATPase that regulates energy‐dependent, ubiquitin‐mediated protein degradation.
The role of Lys272 in the pyridoxal 5‐phosphate active site of Synechococcus glutamate‐1‐semialdehyde aminotransferase
Authors: GRIMM Bernhard , SMITH Marvin A. , von WETTSTEIN Diter
Published: March 2005

Glutamate‐1‐semialdehyde (GSA) aminotransferase catalyzes transfer of the C2 amino group of glutamate 1‐semialdehyde to the C1 position to yield the tetrapyrrole precursor 5‐aminolevulinate. Based on spectrophotometric and steady‐state data, GSA aminotransferase is a B6‐containing enzyme which uses a ping‐pong bi‐bi mechanism described for other aminotransferases. A putative activesite lysine at position 272 of Synechococcus GSA aminotransferase was replaced by Arg, Ile or Glu, and genes encoding the corresponding of three site directed mutants were expressed in Escherichia coli. The catalytic competence of the resulting enzymes was determined. The similarity of the absorbance spectra of pyridoxal‐P‐treated forms of Lys272 → Arg, Lys272 → Ile, Lys272 → Glu with free pyridoxal‐P indicates that enzyme‐bound pyridoxal‐P does not form an internal aldimine in these three site‐directed mutants. Whereas Lys272 → Ile and Lys272 → Glu form only stable ketimines and aldimines with GSA and its analogues, addition of these compounds to the pyridoxamine‐P and pyridoxal‐P forms of Lys272 → Arg induces slow spectral changes, indicating the catalysis of a half‐reaction with GSA, 4,5‐dioxovalerate and 4,5‐diaminovalerate. 5‐Aminolevulinate apparently binds with both coenzyme forms of Lys272 → Arg, however significant tautomeric rearrangement is only observed with the pyridoxal‐P form. It is suggested that Lys272 is the covalent pyridoxal‐P‐binding site and that this catalytically active lysine residue channels the overall transamination reaction towards 5‐aminolevulinate. The second‐half reaction (4,5‐diaminovalerate ⇌ 5‐aminolevulinate) is possibly supported by the formation of an internal aldimine which correctly positions the C4 amino group of 4,5‐diaminovalerate relative to the enzyme‐bound pyridoxal‐P.
Fluorescence studies of the binding of bacteriophage M13 gene V mutant proteins to polynucleotides
Authors: STASSEN Alphons P. M. , HARMSEN Ben J. M. , SCHOENMAKERS John G. G. , HILBERS Cornelis W. , KONINGS Ruud N. H.
Published: March 2005

This investigation describes how the binding characteristics of the single‐stranded DNA‐binding protein encoded by gene V of bacteriophage M13, are affected by single‐site amino acid substitutions. The series of mutant proteins tested includes mutations in the purported monomer‐monomer interaction region as well as mutations in the DNA‐binding domain at positions which are thought to be functionally involved in monomer‐monomer interaction or single‐stranded DNA binding. The characteristics of the binding of the mutant proteins to the homopolynucleotides poly(dA), poly(dU) and poly(dT), were studied by means of fluorescence‐titration experiments. The binding stoichiometry and fluorescence quenching of the mutant proteins are equal to, or lower than, the wild‐type gene V protein values. In addition, all proteins measured bind in a more‐or‐less co‐operative manner to single‐stranded DNA. The binding affinities for poly(dA) decrease in the following order: Y61H > wild‐type > F68L and R16H > Y41F and Y41H > F73L > R21C > Y34H > G18D/Y56H. Possible explanations for the observed differences are discussed. The conservation of binding affinity, also for mutations in the single‐stranded DNA‐binding domain, suggests that the binding to homopoly‐nucleotides is largely non‐specific.
Complete cDNA sequence of chicken vigilin, a novel protein with amplified and evolutionary conserved domains
Authors: SCHMIDT Cornelia , HENKEL Barbara , PÖSCHL Ernst , ZORBAS Haralabos , PURSCHKE Werner G. , GLOE Torsten R. , MÜLLER Peter K.
Published: March 2005

The complete cDNA (4375 bp), coding for a new protein called vigilin, was isolated from chicken chondrocytes. The cDNA shows an open reading frame of 1270 amino acids which are organized in 14 tandemly repeated homologous domains. Each domain consists of two subdomains, one with a conserved sequence motif of 35 amino acids (subdomain A) and another one with a presumptive α‐helical structure of 21–33 amino acids (subdomain B). 149 amino acids at the N‐terminus and 71 amino acids at the C‐terminus of vigilin do not show the characteristic domain structure. No sequence characteristic of a signal peptide has been found, which argues for an intracellular localisation of vigilin. Vigilin is highly expressed in freshly isolated chicken chondrocytes but little in chondrocytes after prolonged time in culture. Vigilin mRNA exists in two size species, 4.4 kb and 6.5 kb in length due to the usage of different polyadenylation sites.

Comparison of the vigilin sequence with data bases showed a remarkable similarity to protein HX from Saccharomyces cerevisiae [Delahodde, A., Becam, A. M., Perea, J. & Jacq, C. (1986) Nucleic Acids Res. 14, 9213–9214]. The yeast protein consists of eight homologous domains with 11 conserved amino acid residues within a set of 35 amino acids. The N‐terminal and C‐terminal regions of vigilin and protein HX do not reveal any sequence similarity. These results, together with the demonstration of the characteristic vigilin sequence motif in a human cDNA clone, suggest that the repeats represent evolutionary conserved autonomous domains within a family of proteins found in yeast, chicken and man.
Linear and cyclic peptide analogues of the polypeptide cardiac stimulant, anthopleurin‐A
Authors: GOULD Alison R. , MABBUTT Bridget C. , LLEWELLYN Lyndon E. , GOSS Neil H. , NORTON Raymond S.
Published: March 2005

A loop corresponding to residues 8–17 in the polypeptide cardiac stimulant anthopleurin‐A is known to be important for the cardiostimulant activity of this molecule. To investigate the activity and possible conformations of this loop in isolation, two synthetic peptides have been studied. The first corresponds to residues 6–20 of anthopleurin‐A with Cys6 replaced by Thr, and the second to residues 6–21 of anthopleurin‐A, with Thr21 replaced by Cys. The introduction of an additional cysteine in the latter peptide enabled an intramolecular disulfide to be formed between the N‐ and C‐terminal residues. Both linear peptides and the disulfide‐containing analogue lack the cardiostimulant and Na+‐channel binding activity of the parent molecule, anthopleurin‐A, indicating that although the loop is important for the function of anthopleurin‐A, other regions of the molecule must also be involved in activity. Assignments of the 1H‐NMR spectra of both peptides are presented, and their pH and temperature dependences investigated. The results show that the amide protons of Gly5 and Asn11 (corresponding to Gly10 and Asn16 in anthopleurin‐A) sample hydrogen‐bonded conformations in solution. Based on these NMR data, two regions of non‐random structure, encompassing residues 2–5 and 8–11, respectively, are proposed, and the possible involvement of such structures in the activity of anthopleurin‐A is discussed.
Substance‐P‐related and neurokinin‐A‐related peptides from the brain of the cod and trout
Authors: JENSEN Jörgen , CONLON J. Michael
Published: March 2005

An extract of the brain of the rainbow trout, Oncorhynchus mykiss contained high concentrations of both neurokinin A‐like immunoreactivity (corresponding to 90 pmol mammalian neurokinin A/g wet tissue) and substance‐P‐like immunoreactivity (corresponding to 50 pmol mammalian substance P/g wet tissue) measured by radioimmunoassay using antisera directed against the C‐terminal regions of the mammalian peptides. In contrast, an extract of the Atlantic cod, Gadus morhua contained only neurokinin‐A‐like immunoreactivity (151 pmol/g). This apparent paradox was resolved by determination of the primary structures of the fish tachykinins. Trout substance P (Lys‐Pro‐Arg‐Pro‐His‐Gln‐Phe‐Phe‐Gly‐Leu‐MetNH2) has the same amino acid sequence in its C‐terminal region as that in the corresponding region of mammalian substance P. Cod substance P (Lys‐Pro‐Arg‐Pro‐Gln‐Gln‐Phe‐Ile‐Gly‐Leu‐MetNH2), however, contains a substitution at position 8 (Phe → Ile) that abolishes reactivity with the antiserum to substance P but permits reactivity with the antiserum to neurokinin A. The amino acid sequence of cod and trout neurokinin A is the same (His‐Lys‐Ile‐Asn‐Ser‐Phe‐Val‐Gly‐Leu‐MetNH2) and shows two substitutions (Thr3 → Ile and Asp4 → Asn) compared with mammalian neurokinin A. The data indicate that nervous tissue of teleost fish contain tachykinins that are analogous to the peptides found in mammalian tissues.
13C and proton NMR studies of horse cytochrome c
Authors: SANTOS Helena , TURNER David L.
Published: March 2005

The CHn groups in the aliphatic side chains of horse cytochrome c have been characterized according to the chemical shifts of both 13C‐NMR and 1H‐NMR signals, their temperature dependence and the number of attached protons, n. The primary assignments of resonances from the 55 side‐chain methyl and the 27 methine groups were obtained directly for the oxidised and the reduced forms. Specific assignments of the 13C resonances were obtained through shift‐correlation experiments and comparison with earlier 1H‐NMR studies, by further measurements of proton‐proton interactions, or by elimination.

Comparison of the paramagnetic shifts of carbon and protons indicates a small redox‐related change of conformation in the vicinity of Trp59 and a significant expansion of the protein over 30–50°C.
Substitution of ASP193 to ASN at the active site of ribulose‐1,5‐bisphosphate carboxylase results in conformational changes
Authors: SÖDERLIND Eskil , SCHNEIDER Gunter , GUTTERIDGE Steven
Published: March 2005

The crystal structure of a mutant of ribulose bisphosphate carboxylase/oxygenase from Rhodospirillium rubrum, where Asp193, one of the ligands of the magnesium ion at the activator site, is replaced by Asn, has been determined to a nominal resolution of 0.26 nm. The mutation of Asp to Asn induces both local and global conformation changes as follows. The side chain of Asn193 moves away from the active site and interacts with main‐chain oxygen of residue 165, located in the neighbouring strand β1 of the α/β barrel. The side chain of Lys166, which forms a salt bridge with Asp193 in the wild‐type enzyme, interacts with Asn54 from the second subunit and creates a new subunit‐subunit interaction. Another new subunit‐subunit interaction is formed, more than 1.2 nm away from the site of the mutation. In the mutant enzyme, the side chain of Asp263 interacts with the side chain of Thr106 from the second subunit. Asp193 is not part of a subunit‐subunit interface area or an allosteric regulatory site. Nevertheless, replacement of this residue by Asn results, unexpectedly, in a difference in the packing of the two subunits, which can be described as a slight rotation of one of the subunits relative to the second. The observed structural changes at the active site of the enzyme provide a molecular explanation for the differing behaviour of the Asp193 → Asn mutant with respect to activation.
Bundling of microtubules by synapsin 1
Authors: BENNETT Alison F. , BAINES Anthony J.
Published: March 2005

Synapsin 1 is a nerve terminal phosphoprotein whose role seems to encompass the linking of small synaptic vesicles to the cytoskeleton. Synapsin 1 can join small synaptic vesicles to neuronal spectrin, microfilaments and microtubules; it can also bundle microtubules and microfilaments. In this paper, the mode of interaction between synapsin 1 and microtubules has been investigated. Bundling is shown to be highly cooperative: the apparent Hill coefficient is 3.06 ± 0.3, and bundling is half‐maximal at 0.63 ± 0.02 μM. Bundling occurs either when whole synapsin 1 preparations (containing monomers and oligomers) or when monomeric synapsin 1 is added to microtubules. However, it is not clear that synapsin 1 remains monomeric in the presence of microtubules. Synapsin 1‐microtubule mixtures contain two types of filament. One type is characterised by microtubules often with synapsin 1 bound to their surface. The other type is composed of filaments of diameter 15 ± 5 nm. This filament type is granular and made up in part of 14‐nm‐diameter particles. These dimensions are consistent with their being made up of polymerised synapsin 1. It is possible that microtubules induce the polymerisation of synapsin 1. Synapsin 1 had independent tubulin binding sites in the N‐terminal head domain and in the C‐terminal tail domain. Whole synapsin 1 can interact with tubulin after it has been digested to remove the tubulin C terminus (des‐C‐terminal tubulin). The interaction of des‐C‐terminal tubulin with synapsin 1 appears to be via the head domain, since 125I‐des‐C‐terminal tubulin only shows specific binding to the head domain on gel blots. By contrast intact tubulin binds to both head and tail domains. Binding to the tail domain can be inhibited by a synthetic peptide representing the microtubule‐associated protein 2 (MAP2) binding site of class II β tubulin. These results suggest a model for microtubule bundling by synapsin 1 in which independent sites in the head and tail domains of synapsin 1 cross‐link microtubules by interactions with two distinct sites in tubulin.
Halothane metabolism
Authors: HUWYLER Jörg , JEDLITSCHKY Gabriele , KEPPLER Dietrich , GUT Josef
Published: March 2005

ω‐Oxidation of leukotrienes is the initial step of hepatic degradation and thus inactivation of these proinflammatory mediators. ω‐Oxidation is followed by β‐oxidation of leukotrienes from the ω‐end. After exposure of rats to a single dose of the anesthetic agent halothane, a transient decrease in leukotriene ω‐oxidation was induced both in vivo and in vitro. In untreated rats, 44.1 ± 6.0% of N‐[3H]acetylleukotriene E4 injected intravenously was recovered unchanged in bile collected for 60 min in vivo; 46.5 ± 3.0% was recovered as ω‐/β‐oxidation products, of which 24.7 ± 4.5% were associated with β‐oxidation products only (mean ± SEM; n= 5). In rats receiving a single dose of halothane 18 h before the experiment, recovery of unchanged N‐[3H]acetylleukotriene E4 was significantly increased to 79.8 ± 4.8%, while the fraction of ω‐/β‐oxidation products decreased to 9.0 ± 1.7% (n= 5); 90 h after exposure to halothane, N‐[3H]acetylleukotriene E4 recovery decreased to 30.0 ± 3.0% and ω‐/β‐oxidation products amounted to 49.1 ± 3.8%; the fraction of β‐oxidation products was significantly increased to 43.1 ± 3.4% (n= 5). Ten days after exposure of rats to halothane, the recoveries of N‐[3H]acetylleukotriene E4, of ω‐/β‐oxidation products, and of β‐oxidation products alone, returned to almost normal values. Microsomal fractions obtained from rat hepatocytes catalyzed the NADPH‐ and O2‐dependent leukotriene ω‐oxidation in vitro. The formation of ω‐hydroxy‐metabolites of leukotriene B4, leukotriene E4, and N‐acetylleukotriene E4 was decreased by 50% in microsomal fractions obtained from rats 18 h and 90 h after halothane treatment, and returned back to control levels in microsomal fractions obtained 10 days after halothane treatment. The Km value of leukotriene B4ω‐oxidation revealed no significant change in enzyme affinity towards leukotriene B4; in contrast, as reflected by the reduction of the Vmax value by 65%, a decrease in the amount of the active enzyme in microsomes obtained from rats 18 h after halothane treatment was observed. Halothane‐metabolism‐dependent trifluoroacetylation of hepatic proteins may mediate this process. Thus, the time course of the density on immunoblots of trifluoroacetylated protein adducts paralleled that of the transient decrease in leukotriene ω‐oxidation. In contrast to its ω‐oxidation, leukotriene B4 synthesis from 5‐hydroperoxyeicosatetraenoate was not inhibited in hepatocyte homogenates obtained from rats pretreated with halothane. The data suggest that metabolism of halothane causes a transient derangement of hepatic leukotriene homeostasis in vivo.
Protein‐tyrosine phosphorylations induced by concanavalin A and N‐formyl‐methionyl‐leucyl‐phenylalanine in human neutrophils
Authors: OHTA Shinji , INAZU Tetsuya , TANIGUCHI Takanobu , NAKAGAWARA Gizo , YAMAMURA Hirohei
Published: March 2005

The ability of the lectin concanavalin A (ConA) and N‐formyl‐methionyl‐leucyl‐phenylalanine (fMLF) to induce protein‐tyrosine phosphorylation in human neutrophils was examined by immunoblot analysis. ConA caused an increase in tyrosine phosphorylation of protein bands with apparent molecular masses of 120, 80, 76, 66 and 40 kDa; on the other hand, fMLF caused an increase in those of only 80‐kDa and 40‐kDa proteins. These protein‐tyrosine phosphorylations were time‐ and dose‐dependent. The tyrosine phosphorylation of 40‐kDa protein induced by fMLF was suppressed but that by ConA was not suppressed by pertussis toxin pretreatment. At the same time, pertussis toxin pretreatment also inhibited lysozyme release and aggregation of neutrophils induced by fMLF but did not inhibit those responses induced by ConA. These results suggest that the tyrosine phosphorylation of 40‐kDa protein may be involved in a part of neutrophil activation and be regulated via pleiotropic signal transduction pathways. In addition, immunoblot analysis employing antibodies against microtubule‐associated protein 2 (MAP2) kinase suggested that this tyrosine‐phosphorylated 40‐kDa protein might be the MAP2 kinase.
The legumin precursor from white lupin seed
Authors: DURANTI Marcello , GUERRIERI Nicoletta , CERLETTI Paolo , VECCHIO Giuseppe
Published: March 2005

The precursors of the legumin‐like storage protein from developing white lupin seeds (35 days after flowering) are trimers composed of protomers of Mr, 72 000 or 67 000. Some subunits of these oligomers contain processed precursor polypeptides, namely α polypeptides of either 52 000 or 44 000 linked through disulphide bonds to a β polypeptide of 21 000, typical of the mature legumin. The prolegumin is glycosylated.

Legumin oligomers purified from the same seeds are both trimers and hexamers; some of their subunits are still made of precursor polypeptides. The hexamer contains less precursor polypeptide than the trimer. A low level or absence of precursor appears to be a condition of hexamer assembly. The heterogenous prolegumin and legumin oligomers represent intermediates in the processing of the prolegumin to mature legumin.

Hydrophobic‐interaction chromatography on TSK‐phenyl‐5PW and titration with the hydro‐phobic probe 8‐anilino‐1‐naphthalenesulphonate indicate that the legumin is less hydrophobic than the prolegumin. This is attributed to structural rearrangements at processing of the propolypeptide, made evident by the behaviour in CD and by the second‐derivative ultraviolet spectra of the two proteins.

The total protein extract of developing cotyledons at 40 days after flowering contains endopeptidases, similar to those existing in the resting seeds, which cause a limited cascade degradation of the prolegumin and legumin.
Complete down‐regulation of low‐density‐lipoprotein‐receptor activity in the human hepatoma cell line Hep G2 by β‐migrating very‐low‐density lipoprotein and non‐lipoprotein cholesterol
Authors: KAMPS Jan A. A. M. , van BERKEL Theo J. C.
Published: March 2005

Regulation of low‐density‐lipoprotein‐receptor activity by low‐density lipoprotein (LDL), cholesteryl‐ester‐rich β‐migrating very‐low‐density lipoprotein (β‐VLDL) and non‐lipoprotein cholesterol was investigated in the human hepatoma cell line Hep G2.

Competition studies indicate that LDL and β‐VLDL are bound to the same recognition site, tentatively the LDL receptor. The regulatory response of the LDL receptor upon prolonged incubation with LDL or β‐VLDL was, however, markedly different. 22 h preincubation of Hep G2 cells with excess LDL caused a partial down regulation to 31% of the initial level of the high‐affinity association of LDL and 26% of the high‐affinity degradation of LDL, while with β‐VLDL a complete down regulation of the LDL‐receptor activity is observed. Preincubation of Hep G2 cells with β‐VLDL for 22 h led to a fourfold increase in intracellular cholesterol esters and a twofold increase in acylcoA:cholesterol acyltransferase activity. With LDL, the amount of intracellular cholesterol esters is increased 1.6‐fold. The more effective down regulation of LDL receptors by β‐VLDL as compared to LDL can be explained by the more effective intracellular cholesterol delivery with β‐VLDL than with LDL. Preincubation of Hep G2 cells for 22 h with acetylated LDL hardly influenced the LDL‐receptor activity. Non‐lipoprotein cholesterol, however, caused a complete down regulation of LDL‐receptor activity at even lower extracellular cholesterol concentrations than with β‐VLDL. The complete down regulation of LDL receptors by non‐lipoprotein cholesterol is not accompanied by a significant increase in acyl‐coA:cholesterol acyltransferase activity, while the intracellular cholesterol ester concentration is only increased 1.6‐fold. It is suggested that the effectiveness of non‐lipoprotein cholesterol to regulate LDL receptors is caused by its efficiency to reach the sterol regulatory site. The inability of LDL to down regulate its receptor completely can thus be explained by the inability of LDL to deliver cholesterol adequately at the intracellular regulatory site of the LDL receptor.

The observed complete down regulation of the LDL receptor by β‐VLDL may be responsible for the cholesterol‐rich‐diet induced, complete down regulation of LDL‐receptor‐mediated clearance of LDL in vivo.
Disulfide arrangement of human insulin‐like growth factor I derived from yeast and plasma
Authors: AXELSSON Kent , JOHANSSON Stig , EKETORP Gunilla , ZAZZI Henric , HEMMENDORF Barbro , GELLERFORS Pär
Published: March 2005

The disulfide arrangement of yeast derived human insulin‐like growth factor I (yIGF‐I) was determined using a combination of Staphylococcus aureus V8 protease mapping, fast‐atom‐bombardment mass spectrometry as well as amino acid sequence and composition analysis. Three disulfide bridges were found between the following cysteine residues: Cys6‐Cys48, Cys47‐Cys52 and Cys18‐Cys61. IGF‐I isolated from human plasma (pIGF‐I) was found to have an identical dilsufide configuration. A yeast‐derived isomeric form of IGF‐I (yisoIGF‐I) exhibited an altered disulfide arrangement: Cys6‐Cys47, Cys48‐Cys52 and Cys18‐Cys61. Radioreceptor analysis of pIGF‐I and yIGF‐I showed high specific activity, 20 000 U/mg. However, yisoIGF‐I demonstrated a severely reduced ability to bind to the IGF‐I receptor (19%) and was less potent in provoking a mitogenic response in Balb/C 3T3 fibroblasts (50% at doses 10–100 ng/ml). The data demonstrate the importance of correct disulfide arrangement in IGF‐I for full biological activity.