bluuebunny/crossref_metadata_2025 huggingface dataset:
Evidence for Sodium Dodecyl Sulfate/Protein Complexes Adopting a Necklace Structure
Authors: Samsó Montserrat , Daban Joan‐Ramon , Hansen Steen , Jones Gareth R.
Published: March 2005

Structural analysis by cryo‐electron microscopy and small‐angle X‐ray scattering of ten sodium dodecyl sulfate/protein complexes in 25 mM Tris/HCl, 0.192 M glycine, pH 8.3, showed necklace‐like structures of spherical micelles dispersed along the unfolded peptide chain. The micelles of most SDS/protein complexes had a constant diameter (≈ 6.2nm), slightly larger than pure SDS micelles (≈ 5.7 nm), all micelles possessing a degree of surface roughness. The micelle‐associated polypeptide is mostly situated at the interface of the sulfate head groups and hydrocarbon core, intruding into the core rather than outward from the surface. Proteins with a molecular mass less than about 20 kDa formed complexes with a single SDS micelle. Multi‐micellar SDS/protein complexes had centre‐to‐centre intermicellar distances in the range 7.0–12.0 nm. Our findings on the constancy of micellar size, number of micelles/complex, and the relationship between the degree of occupancy of micelles and a polypeptide's molecular mass, have enabled us to speculate on the correlation between the electrophoretic mobility of a polypeptide in SDS/PAGE and its molecular mass. The anomalous electrophoretic behaviour observed for the sodium dodecyl sulfate/histone H5 complex is accounted for by the large micelle of its complex.
The Mutation Asp69→Ser Affects the Chaperone‐Like Activity of αA‐Crystallin
Authors: Smulders Ronald H. P. H. , Merck Karin B. , Aendekerk John , Horwitz Joseph , Takemoto Larry , Slingsby Christine , Bloemendal Hans , De Jong Wilfried W.
Published: March 2005

α‐Crystallins are members of the family of small heat‐shock proteins. The conformation and mode of action of these ‘junior chaperones’ are unknown. To investigate the structure and chaperone‐like activity, four mutants of bovine αA‐crystallin were generated by site‐directed mutagenesis. In comparison with wild‐type αA‐crystallin, the D69S mutant, in which a highly conserved charged residue has been replaced, forms larger multimers and displays a threefold reduced heat‐protection capacity. The conformation and thermal stability of this mutant are not noticeably affected. Three other mutations, replacing hydrophobic by uncharged hydrophilic residues, were aimed at disturbing hydrophobic intersubunit interactions. None of these mutations resulted in major structural perturbations and only minor differences in heat‐protective capacity were observed. Although it is assumed that small heat‐shock proteins interact with denaturing proteins via their hydrophobic surfaces, this study clearly shows that charged residues in α‐crystallin can also influence the efficiency of substrate binding.
Abnormally High pKa of an Active‐Site Glutamic Acid Residue in Bacillus Circulans Xylanase
Authors: Davoodi Jamshid , Wakarchuk Warren W. , Campbell Robert L. , Carey Paul R. , Surewicz Witold K.
Published: March 2005

The active site of Bacillus circulans xylanase (1,4‐β‐D‐xylanohydrolase, EC 3.2.1.8) contains two glutamic acid residues, Glu78 and Glu172, which are crucial for the catalytic activity of the enzyme. Fourier‐transform infrared spectroscopy was used to determine the ionization state of these residues as a function of pH. For the wild‐type enzyme, titration of one of the carboxylate groups occurs at pH 6.8. This titration is absent in the Glu78→Gln and Glu172→Gln variants of the enzyme. This, together with crystallographic data, indicates that Glu172 has an abnormally high pKa of 6.8, caused largely by electrostatic interactions of this residue with the proximal Glu78. Differential scanning calorimetry experiments with the wild‐type xylanase and a number of its mutants have shown that the presence of two nearby carboxyl groups results in a pH‐dependent destabilization of the protein structure.
Structure of the N‐Linked Oligosaccharides from Tridacnin, a Lectin Found in the Haemolymph of the Giant Clam Hippopus Hippopus
Authors: Puanglarp Narongsak , Oxley David , Currie Graeme J. , Bacic Antony , Craik David J. , Yellowlees David
Published: March 2005

Tridacnin, a glycoprotein lectin, was isolated from the symbiotic marine clam Hippopus hippopus and the structure of its major N‐glycan chains determined. Tridacnin contains only N‐linked glycans which were quantitatively cleaved by peptide‐N4‐(N‐acetyl‐β‐glucosaminyl)asparagine amidase F. Following purification by anion‐exchange HPLC, the structures of the oligosaccharides were established using a combination of electrospray ionisation mass spectrometry, 1H‐NMR spectroscopy and linkage analysis. The N‐glycans are primarily of the oligomannose type but, in addition, some contain a novel 6‐O‐Me group on the terminal mannose residue of the chain. The N‐glycan chains had the following structures.
Purification from Placenta, Amino Acid Sequence, Structure Comparisons and cDNA Cloning of Human Glutaredoxin
Authors: Padilla C. Alicia , Martínez‐Galisteo Emilia , Bárcena J. Antonio , Spyrou Giannis , Holmgren Arne
Published: March 2005

Glutaredoxin is generally a glutathione‐dependent hydrogen donor for ribonucleotide reductase and also catalyses general glutathione (GSH)‐disulfide‐oxidoreduction reactions in the presence of NADPH and glutathione reductase. A Glutaredoxin from human placenta was purified to homogeneity, as judged by SDS/PAGE and IEF (12 kDa). Purification was monitored by the activity with hydroxyethyl disulfide as a substrate. Values of pI for glutaredoxin were obtained by IEF; the pI of the protein shifted from 7.3 in its fully reduced state to 9.0 in the oxidized state after treatment with excess hydroxyethyl disulfide. The glutaredoxin preparation showed GSH‐dependent hydrogen‐donor activity with recombinant mouse ribonucleotide reductase, it exhibited dehydroascorbate reductase activity as well as hydroxyethyl‐disulfide‐reducing activity. The amino acid sequence (residues 3–104) of glutaredoxin was determined by peptide sequencing and residues 1, 2 and 105 by cDNA sequence analysis. The glutaredoxin sequence comprised the classical active site for glutaredoxins ‐Cys22‐Pro‐Tyr‐Cys25‐ and three additional half‐cystine residues; two of these in positions 78 and 82. The sequence was similar to other known mammalian glutaredoxins (about 80% identities), with important differences such as one additional Cys residue (Cys7) and no Met residue. The sequence of human glutaredoxin was compared to that of Escherichia coli glutaredoxin with known three‐dimensional structure in solution to indentify conserved residues and predict a structure from alignment. In particular the GSH‐binding site of glutaredoxin was conserved between all molecules. A cDNA that encodes the entire glutaredoxin gene (grx) and flanking sequences was isolated from a human spleen cDNA library. The nucleotide sequence of this cDNA (0.8 kb) was determined, including the complete grx gene.
The C‐Terminal Domain of α‐Spectrin is Structurally Related to Calmodulin
Authors: Travé Gilles , Pastore Annalisa , Hyvönen Marko , Saraste Matti
Published: March 2005

An alignment of amino acid sequences suggests that the spectrin domain, which contains two EF‐hand calcium‐binding motifs, is structurally related to calmodulin. It is possible to align approximately 160 residues at the C‐terminus of α‐spectrin with the entire calmodulin sequence. We have expressed this domain in Escherichia coli and purified it. Circular dichroic and nuclear magnetic resonance spectroscopy show that the protein is folded and mostly helical. The conformation of the protein, as monitored spectroscopically, is sensitive to calcium at 0.1–1.0 mM. Equilibrium dialysis shows that there are two binding sites within this domain, with affinities in the 0.5 mM range. The domain can be split into N‐terminal and C‐terminal halves which fold independently. Only the N‐terminal subdomain binds calcium. These data suggest that the C‐terminus of α‐spectrin has a domain with a calmodulin fold and two calcium‐binding sites. Sequence alignments suggest that the related domains in α‐actinin, and possibly in dystrophin, may share the same calmodulin‐like structure. However, only non‐muscle α‐actinins appear to have one or two EF‐hand(s) with the calcium‐binding consensus sequence, and a strict consensus is not found in the muscle α‐actinins or dystrophins.
Metabolic Pathway for Biosynthesis of Poly (3‐Hydroxybutyrate‐co‐4‐Hydroxybutyrate) from 4‐Hydroxybutyrate by Alcaligenes eutrophus
Authors: Valentin Henry E. , Zwingmann Gundula , Schönebaum Andreas , Steinbüchel Alexander
Published: March 2005

Various aerobic Gram‐negative bacteria have been examined for their ability to use 4‐hydroxybutyrate and 1,4‐butanediol as carbon source for growth. Alcaligenes eutrophus strains H16, HF39, PHB−4 and Pseudomonas denitrificans‘Morris’ were not able to grow with 1,4‐butanediol or 4‐hydroxybutyrate. From A. eutrophus HF39 spontaneous primary mutants (e. g. SK4040) were isolated which grew on 4‐hydroxybutyrate with doubling times of approximately 3 h. Tn5::mob mutagenesis of mutant SK4040 led to the isolation of two phenotypically different classes of secondary mutants which were affected in the utilization of 4‐hydroxybutyrate. Mutants exhibiting the phenotype 4‐hydroxybutyrate‐negative did not grow with 4‐hydroxybutyrate, and mutants exhibiting the phenotype 4‐hydroxybutyrate‐leaky grew at a significantly lower rate with 4‐hydroxybutyrate. Hybridization experiments led to the identification of a 10‐kbp genomic EcoRI fragment of A. eutrophus SK4040, which was altered in mutants with the phenotype 4‐hydroxybutyrate‐negative, and of two 1‐kbp and 4.5‐kbp genomic EcoRI fragments, which were altered in mutants with the phenotype 4‐hydroxybutyrate‐leaky. This 10‐kbp EcoRI fragment was cloned from A. eutrophus SK4040, and conjugative transfer of a pVDZ'2 hybrid plasmid to A. eutrophus H16 conferred the ability to grow with 4‐hydroxybutyrate to the wild type. DNA‐sequence analysis of this fragment, enzymic analysis of the wild type and of mutants of A. eutrophus as well as of recombinant strains of Escherichia coli led to the identification of a structural gene encoding for a 4‐hydroxybutyrate dehydrogenase which was affected by transposon mutagenesis in five of six available 4‐hydroxybutyrate‐negative mutants. Enzymic studies also provided evidence for the presence of an active succinate‐semialdehyde dehydrogenase in 4‐hydroxybutyrate‐grown cells. This indicated that degradation of 4‐hydroxybutyrate occurs via succinate semialdehyde and succinate and that the latter is degraded by the citric acid cycle. NMR studies of poly(3‐hydroxybutyrate‐co‐4‐hydroxybutyrate) accumulated from 4‐hydroxy [1‐13C]butyrate or 4‐hydroxy[2‐13C]butyrate as substrate gave no evidence for a direct conversion of 4‐hydroxybutyrate into 3‐hydroxybutyrate and therefore supported the results of enzymic analysis.
Affinity Labeling of a Sulfhydryl Group in the Cardiacglycoside Receptor Site of Na+/K+‐ATPase by N‐Hydroxysuccinimidyl Derivatives of Digoxigenin
Authors: Antolovic Roberto , Linder Dietmar , Hahnen Josef , Schoner Wilhelm
Published: March 2005

Na+/K+‐ATPase from pig kidney is inactivated by protein‐reactive N‐hydroxysuccinimidyl derivatives of digoxigenin. Like digoxigenin, its protein‐reactive derivatives N‐hydroxysuccinimidyl digoxigenin‐3‐methylcarbonyl‐ɛ‐aminocaproate (HDMA), 3‐amino‐3‐deoxydigoxigenin hemisuccinimide succinimidyl ester (ADHS), 3‐iodoacetylamino‐3‐deoxydigoxigenin (IAD) and digoxigenin‐3‐O‐succinyl‐[2‐(N‐maleimido)]ethylamide (DSME) inhibited the sodium pump in the presence of Na+, Mg2+ and ATP. At 37°C, half‐maximal inhibition of Na+/K+‐ATPase was seen by HDMA at 0.47 μM, by ADHS at 5.8 μM, by IAD at 8 μM and by DSME at 94 μM. Thus, all compounds bind to the cardiac steroid receptor site of Na+/K+‐ATPase. Affinity labeling of the α subunit by ‘front door’ or ‘back door’ phosphorylation was only seen with HDMA or ADHS in the range 0.1 μM. Excess of ouabain protected against affinity labeling. All the other protein‐reactive derivatives of digoxigenin labeled the enzyme independent of the formation of a phosphointermediate at much higher concentrations. This labeling was not suppressed by an excess of ouabain.

Tryptic hydrolysis of the HDMA‐modified Na+/K+‐ATPase gave peptides of the apparent molecular masses 20, 12.5 and 11.2 kDa. The 11.2‐kDa and 12.5‐kDa peptides started amino‐terminally with Asp68, and the 20‐kDa peptide with Asp24. Thus, the HDMA‐labeled peptides originate from the cardioactive steroid‐binding site formed by the first and second transmembrane helix. N‐Hydroxysuccinimidyl esters such as HDMA are normally thought to modify lysine and arginine residues covalently. Since such residues do not exist in the putative cardiac glycoside‐binding site, the possibility of a thioester formation of the digoxigenin derivatives HDMA and ADHS with Cys104 in the H1 transmembrane domain was tested. In fact, hydroxylaminolysis led to the release of the covalently bound HDMA, and the formation of a free sulfhydryl group. This could be labeled by [2‐14C]ICH2COOH. We therefore propose, consistent with a recent conclusion from a site‐directed mutagenesis experiment [Canessa, C. M., Horisberger, J.‐D., Louvard, D. & Rossier, B. C. (1992) EMBO J. 11, 1681–1687], that a cysteine residue (probably Cys104) participates in the structure and function of the cardiac glycoside binding.
An Increase in Phosphorylation of Microtubule‐associated Protein 2 Accompanies Dendrite Extension During the Differentiation of Cultured Hippocampal Neurones
Authors: Díez‐Guerra F. Javier , Avila Jesús
Published: March 2005

Hippocampal neurones, from embryonic rats, were cultured for different times and the extension of dendrite‐like processes was analysed morphologically and by immunofluorescence, using microtubule‐associated protein 2 (MAP2) as a marker. Simultaneously, the changes in phosphorylation in MAP2 were analyzed and a correlation between dendrite sprouting and an increase in MAP2 phosphorylation was found. Phospho‐MAP2 was cleaved by Staphylococcus aureus V8 protease limited proteolysis and its phosphopeptide pattern was compared to that obtained with two protein kinases (calcium/calmodulin‐dependent kinase and protein kinase C) in vitro. An involvement of calcium/calmodulin‐dependent protein kinase in the phosphorylation of MAP2, occurring simultaneously with dendrite extension during neuronal differentiation in vitro, is suggested.
Probing Protein Structure by Solvent Perturbation of NMR Spectra
Authors: Improta Sabina , Molinari Henriette , Pastore Annalisa , Consonni Roberto , Zetta Lucia
Published: March 2005

We have characterized the high‐temperature molten globule state of bovine α‐lactalbumin by a combined use of photochemically induced dynamic nuclear polarization and nitroxide surface perturbation. Both techniques are extremely well suited to follow the progressive increase of exposed surfaces in the native state and the appearance of partially or completely unfolded species. Our results suggest that the molten globule state obtained at high temperature and pH 7, and the state obtained at pH 2 are not only thermodynamically but also structurally very similar.
Characterization of High‐density Apolipoprotein Particles A‐I and A‐I:A‐II Isolated from Humans with Cholesteryl Ester Transfer Protein Deficiency
Authors: Duverger Nicolas , Rader Daniel , Ikewaki Katsunori , Nishiwaki Masato , Sakamoto Takuya , Ishikawa Toshitsugu , Nagano Makoto , Nakamura Haruo , Brewer H. Bryan
Published: March 2005

The cholesteryl ester transfer protein (CETP) plays an important role in metabolism of high‐density lipoprotein and reverse cholesterol transport in humans. The two major classes of high‐density lipoprotein particles are those containing apolipoprotein A‐I (LpA‐I) and those containing both apoA‐I and apoA‐II (LpA‐I:A‐II). We isolated and characterized the apoA‐I‐containing lipoprotein particles from three subjects with homozygous CETP deficiency (CETP‐D) and compared the results with those from normolipidemic control subjects. Plasma concentrations of apoA‐I in both LpA‐I and LpA‐I:A‐II were significantly elevated in CETP‐D subjects. Both LpA‐I and LpA‐I:A‐II from these subjects were larger and contained more cholesteryl ester per particle than control particles. In CETP‐D, subpopulations of LpA‐I and LpA‐I:A‐II with an unusually large size (Stokes diameters 13.8 nm and 12.6 nm, respectively) not detected in normal subjects were isolated. The molar ratio of apoA‐I to apoA‐II in LpA‐I:A‐II isolated from CETP‐D subjects was higher (mean 2.4) than those of controls (mean 1.4). ApoE was primarily associated with LpA‐1:A‐II in CETP‐D subjects. A subclass of LpA‐I with pre‐β migration on agarose electrophoresis was increased in CETP‐D subjects. Both LpA‐I and LpA‐I:A‐II from CETP‐D subjects bound with higher affinity but less capacity to HepG2 cells compared with control particles, and were internalized to a lesser extent than control particles. These data suggest that the absence of CETP in humans significantly affects the plasma concentration, size, composition, and cellular interaction of both major classes of apoA‐I‐containing lipoprotein particles.
Periplasmic Sulphide Dehydrogenase (Sud) from Wolinella succinogenes: Isolation, Nucleotide Sequence of the sud Gene and Its Expression in Escherichia coli
Authors: Kreis‐Kleinschmidt Vera , Fahrenholz Falk , Kojro Elzbieta , Kröger Achim
Published: March 2005

Wolinella succinogenes contains a periplasmic sulphide dehydrogenase when grown with formate and polysulphide as catabolic substrates. The isolated enzyme catalyzes the reduction of dimethylnaphthoqui‐none with sulphide at high values of both apparent Km and turnover number. The active enzyme consists of two identical subunits (14 kDa) and amounts to approximately 1% of the soluble cell protein. Prosthetic groups such as flavin, haem or molybdenum are missing. The corresponding gene (sud) encodes a signal peptide together with the mature subunit that consists of 129 amino acid residues including one single cysteine. The sud gene is expressed from a plasmid in Escherichia coli. The resulting enzyme catalyzes sulphide oxidation with dimethylnaphthoquinone and is located in the periplasm of E. coli.
Molecular Cloning and Sequence of two Novel P‐type Adenosinetriphosphatases from Plasmodium falciparum
Authors: Trottein François , Cowman Alan F.
Published: March 2005

We have identified two novel P‐type ATPase genes from Plasmodium falciparum and report the full‐length nucleotide and derived amino acid sequence of the ATPase2 gene from P. falciparum (PfATPase2). PfATPase2 is phylogenetically remote from the different members of prokaryotic and mammalian P‐type ATPases but shares features with a putative membrane‐spanning Ca2+ ATPase involved in ribosome function in yeast. PfATPase2 is expressed during the intraerythrocytic life cycle of the parasite and appears to be required in the late stages of its asexual development. We also present the partial sequence of another malarial gene displaying sequence similarity with the family of P‐type transporting ATPases (PfATPase4). We have analysed the organisation of the genes encoding the P‐type ATPases of P. falciparum and show that they are a highly dispersed gene family.
1H and 13C NMR Assignments and Structural Aspects of a Ferrocytochrome c‐551 from the Purple Phototrophic Bacterium Ectothiorhodospira halophila
Authors: Bersch Beate , Brutscher Bernhard , Meyer Terrance E. , Marion Dominique
Published: March 2005

Two‐dimensional nuclear magnetic resonance was used to assign the 1H and 13C resonances of ferrocytochrome c‐551 from Ectothiorhodospira halophila, a halophilic phototrophic purple bacterium. This 78‐residue protein belongs to a small subgroup of class I cytochromes c together with the analogous cyto‐chromes c ‐551 from E. halochloris and E. abdelmalekii. A nearly complete assignment of 13C resonances was obtained at natural abundance using a gradient‐enhanced 1H‐detected heteronuclear single quantum coherence experiment (HSQC). This was found to be extremely useful for the unambigous assignment of side chain protons. The secondary structure of the protein was determined from analyses of short‐ and medium‐range nuclear Overhauser enhancements (NOE), amide proton exchange and 13Cα chemical shifts. Three helices could be identified which are well conserved among the class I cytochromes c. There is some evidence for two other regions of less well defined helical structure. From a preliminary analysis of long‐range NOE it is shown that in the E. halophila cytochrome c‐551 the general cytochrome c fold is well conserved, including the three conserved helices (residues 2–8, 41–50, 63–76), the regions around the heme ligands (Cysl4‐Ser15‐Ser16‐Cys17‐His18, Met55) and the Ω loop (residues 18–28). In addition, three variable segments of the protein are discussed in detail, one of those including a cis ‐proline, a feature so far unique in the cytochrome c family. Structural alignments of the E. halophila cytochrome c‐551 with two other Pseudomonas cytochrome c5 homologs (Azotobacter vinelandii cytochrome c5 and Chlorobium limicola cytochrome c‐555) are provided which are based on sequence similarities and secondary structure alignments.
Analysis of the vhoGAC and vhtGAC Operons from Methanosarcina mazei Strain Gö1, Both Encoding a Membrane‐bound Hydrogenase and a Cytochrome b
Authors: Deppenmeier Uwe , Blaut Michael , Lentes Sabine , Herzberg Christina , Gottschalk Gerhard
Published: March 2005

DNA encompassing the structural genes of two membrane‐bound hydrogenases from Methanosarcina mazei Gö1 was cloned and sequenced. The genes, arranged in the order vhoG and vhoA as well as vhtG and vhtA, were identified as those encoding the small and the large subunits of the NiFe hydrogenases [Deppenmeier, U., Blaut, M., Schmidt, B. & Gottschalk, G. (1992) Arch. Microbiol. 157, 505–511]. Northern‐blot analysis revealed that the structural genes formed part of two operons, both containing one additional open reading frame (vhoC and vhtC) which codes for a cytochrome b. This conclusion was drawn from the homology of the deduced N‐terminal amino acid sequences of vhoC and vhtC and the N‐terminus of a 27‐kDa cytochrome isolated from Ms. mazei C16. VhoC and VhtC contain four tentative hydrophobic segments which might span the cytoplasmic membrane. Hydropathy plots suggest that His23 and His50 are involved in heme coordination. The comparison of the sequencing data of vhoG and vhtG with the experimentally determined N‐terminus of the small subunit indicate the presence of a 48‐amino‐acid leader peptide in front of the polypeptides. VhoA and VhtA contained the conserved sequence DPCXXC in the C‐terminal region, which excludes the presence of a selenocysteine residue in these hydrogenases. Promoter sequences were found upstream of vhoG and vhtG, respectively. Downstream of vhoC, a putative terminator sequence was identified. Alignments of the deduced amino acid sequences of the gene clusters vhoGAC and vhtGAC showed 92–97% identity. Only the C‐termini of VhoC and VhtC were not similar.
Kinetic Properties and Ligand Binding of the Eleven‐subunit Cytochrome‐c Oxidase from Saccharomyces cerevisiae Isolated with a Novel Large‐Scale Purification Method
Authors: Geier Birgitta M. , Schägger Hermann , Ortwein Claus , Link Thomas A. , Hagen Wilfred R. , Brandt Ulrich , Von Jagow Gebhard
Published: March 2005

A novel, large‐scale method for the purification of cytochrome‐c oxidase from the yeast Saccharomyces cerevisiae is described. The isolation procedure gave highly pure and active enzyme at high yields. The purified enzyme exhibited a heme a/protein ratio of 9.1 nmol/mg and revealed twelve protein bands after Tricine/SDS/PAGE. N‐terminal sequencing showed that eleven of the corresponding proteins were identical to those recently described by Taanman and Capaldi [Taanman, J.‐W. & Capaldi, R. A. (1992) J. Biol. Chem. 267, 22481–2248]. 15 of the N‐terminal residues of the 12th band were identical to subunit VIII indicating that this band represents a dimer of subunit VIII (Mr 5364). We conclude that subunit XII postulated by Taanman and Capaldi is the subunit VIII dimer and that cytochrome‐c oxidase contains eleven rather than twelve subunits.

We obtained the complete sequence of subunit VIa by Edman degradation. The protein contains more than 25% of charged amino acids and hydropathy analysis predicts one membrane‐spanning helix.

The purified enzyme had a turnover number of 1500 s−1 and the ionic‐strength dependence of the Km value for cytochrome‐c was similar to that described for other preparations of cytochrome‐c oxidase. This was also true for the cyanide‐binding characteristics of the preparation. When the enzyme was isolated in the presence of chloride, more than 90% of the preparation showed fast cyanide‐binding kinetics and was resistant to formate incubation, indicating that chloride was bound to the binuclear center. When the enzyme was isolated in the absence of chloride, approximately 70% of the preparation was in the fast form. This high content of fast enzyme was also reflected in the characteristics of optical and EPR spectra for cytochrome‐c oxidase purified with our method.
Processing of the 2S Storage Protein Pronapin in Brassica napus and in Transformed Tobacco
Authors: Murén Eva , Ek Bo , Rask Lars
Published: March 2005

The 2S protein napin in Brassica napus is synthesized as a proprotein from which an N‐terminal and an internal propeptide are removed. In order to investigate the mechanism of 2S storage‐protein processing, N‐terminal sequences were determined for the light and heavy chains of all major napin isoforms isolated. Mutants of a napin gene, with deletions of different portions of the propeptides, were transformed into tobacco and napin protein was isolated. Napin light and heavy chains were separated and their N‐terminal amino acid sequences determined. Further, the C‐terminal residues of one napin isoform isolated from B. napus and one mutant napin isolated from tobacco were deduced from molecular‐mass determinations of the constituent chains. Analyses suggested that the two propeptides are exposed at the surface of the proprotein. The light chain is processed to the correct length independent of the amino acid sequence in the N‐terminal propeptide and the processing site. The internal propeptide is attacked by endoproteases. Aminopeptidases and carboxypeptidases then digest portions of the propeptide to the extent allowed by the primary and the three‐dimensional structures, often resulting in 2S protein chains with partly frayed ends.
A Fluorescent Interleukin‐8 Receptor Probe Produced by Targetted Labelling at the Amino Terminus
Authors: Alouani Sami , Gaertner Hubert F. , Mermod Jean‐Jacques , Power Christine A. , Bacon Kevin B. , Wells Timothy N. C. , Proudfoot Amanda E. I.
Published: March 2005

Interleukin‐8 is the most extensively characterised member of the structurally related chemotactic and pro‐inflammatory proteins collectively called chemokines. It binds to two closely related members of the seven transmembrane chemokine receptor family found on a variety of leukocyte cell types. In order to study the interaction of interleukin‐8 with its receptors, and their distribution, we have produced a fluorescently labelled protein as an alternative to the radioactive 125I‐interleukin‐8 ligand. Interleukin‐8 is naturally produced as two forms, a 72‐residue polypeptide by monocytes and a 77‐residue form produced by endothelial cells which has an extension of five amino acids at the amino terminal. Both forms are active at nanomolar concentrations, implying that chemical modification to the amino terminus of the 72‐residue form will not destroy activity. The 72‐residue interleukin‐8 sequence starts with a serine residue, which can be oxidised under mild conditions to give a reactive glyoxylyl function which is then reacted with a nucleophilic fluorescein derivative. The site‐specifically labelled protein was easily isolated by reverse‐phase HPLC. The dissociation constant of the fluorescently labelled interleukin‐8 from its receptors on neutrophils was measured by displacement of 125I‐interleukin‐8 and found to be 10 nM compared to 1 nM for the unmodified protein. The modified protein is highly active in in vitro bioassays using human neutrophils, giving an EC50 of 7 nM in chemotaxis and an EC50 of 0.62 nM for shape change. The binding of the fluorescent protein to neutrophils can also be measured by fluorescent automatic cell sorter (FACS) analysis, and can be competed by unlabelled interleukin‐8. The amino‐terminal modification of interleukin‐8 has produced a reagent which is useful for the quantification of interleukin‐8 receptor expression, and will also be useful in monitoring the fate of the ligand after receptor binding.
Amino Acid Phosphatase Activity of Alkaline Phosphatase
Authors: Shinozaki Tetsuya , Watanabe Hideomi , Arita Satoru , Chigira Masaki
Published: March 2005

Alkaline phosphatase (ALP) hydrolyzed phosvitin and amino acid phosphates demonstrating non‐isotropy at different pH. Orthovanadate, a protein phosphatase inhibitor, more specifically inhibited the serine and tyrosine phosphatase activities of ALP than that of threonine phosphatase at concentrations >0.1 mM or 0.01 mM, respectively. Calyculin A and okadaic acid at increased concentrations increased ALP amino acid phosphatase activity. Bisphosphonates, such as disodium‐1‐hydroxy‐1‐aminopropylidine‐1,1‐diphosphonate (APD) and ethane‐1‐hydroxy‐1,1‐diphosphonate (HEBP), at increased concentrations, inhibited ALP amino acid phosphatase activity. These results suggest that ALP may function as a protein phosphatase. In terms of protein kinase inhibitors, N‐[2‐(metylamino)ethyl]‐5‐isoquinolinesulfonamide, N‐(6‐aminoheyxl)‐5‐chloro‐1‐naphthalenesulfomide hydrochloride and 4′,5,7‐trihydroxyisoflavone had little effect on ALP amino acid phosphatase activity. Staurosporine slightly enhanced ALP serine and threonine phosphatase activities at a concentration of 0.1 mM. These results suggest that protein phosphatase activity does not depend on the protein kinase activity of ALP, since duality between the former and the latter is not supported. ALP may function less as a protein kinase than as a protein phosphatase. The coupling mechanism of phosphate dynamics may be regulated indirectly.
Role of Tyrosine Kinase in the Regulation of Myogenin Expression
Authors: Hashimoto Naohiro , Ogashiwa Masayo , Iwashita Shintaro
Published: March 2005

Using an affinity‐purified anti‐myogenin antibody, three stages of mouse myoblast C2C12 cells during myogenesis could be identified: proliferating myoblasts as myogenin‐negative mononucleated cells, differentiating myoblasts as myogenin‐positive mononucleated cells, and myotubes as myogenin‐positive multinucleated cells. We found differential effects of genistein, an inhibitor of protein‐tyrosine kinase, on myogenic cells during these three stages. Genistein severely inhibited myotube formation and myogenin production in differentiating myoblasts by inhibiting the transcription of the myogenin gene in a dose‐dependent manner. We also found that genistein inactivated mitogen‐activated protein kinase (MAP kinase) accompanied by suppression of myogenin expression. In contrast, genistein failed to inactivate MAP kinase and eliminate myogenin from myotubes. The results suggest that protein‐tyrosine kinase plays a role in the transcriptional regulation of myogenin through the MAP kinase cascade during myogenesis. Furthermore, genistein inhibited the transactivation of the myosin heavy chain gene by constitutively expressed myogenin. Therefore, it is suggested that protein‐tyrosine kinase is involved in the post‐translational regulation of myogenin as well as in transcriptional regulation during myogenesis.
Evolution of Transthyretin in Marsupials
Authors: Duan Wei , Richardson Samantha J. , Babon Jeffrey J. , Heyes Rebecca J. , Southwell Bridget R. , Harms Paul J. , Wettenhall Richard E. H. , Dziegielewska Katarzyna M. , Selwood Lynne , Bradley Adrian J. , Brack Charlotte M. , Schreiber Gerhard
Published: March 2005

The evolution of the expression and the structure of the gene for transthyretin, a thyroxine‐binding plasma protein formerly called prealbumin, was studied in three marsupial species: the South American polyprotodont Monodelphis domestica, the Australian polyprotodont Sminthopsis macroura and the Australian diprotodont Petaurus breviceps. The transthyretin gene was found to be expressed in the choroid plexus of all three species. In liver it was expressed in P. breviceps and in M. domestica, but not in S. macroura. This, together with previous studies [Richardson, S. J., Bradley, A. J., Duan, W., Wettenhall, R. E. H., Harms, P. J., Babon, J. J., Southwell, B. R., Nicol, S., Donnellan, S. C. & Schreiber, G. (1994) Am. J. Physiol. 266, R1359–R1370], suggests the independent evolution of transthyretin synthesis in the liver of the American Polyprotodonta and the Australian Diprotodonta.

The results obtained from cloning and sequencing of the cDNA for transthyretin from the three species suggested that, in the evolution of the structure of transthyretin in vertebrates, marsupial transthyretin structures are intermediate between bird/reptile and eutherian transthyretin structures. In marsupials, as in birds and reptiles, a hydrophobic tripeptide beginning with valine and ending with histidine was found in transthyretin at a position which has been identified in eutherians as the border between exon 1 and intron 1. In humans, rats and mice, the nine nucleotides, coding for this tripeptide in marsupials/reptiles/birds, are found at the 5′ end of intron 1. They are no longer present in mature transthyretin mRNA. This results in a change in character of the N‐termini of the subunits of transthyretin from hydrophobic to hydrophilic. This change might affect the accessibility of the thyroxine‐binding site in the central channel of transthyretin, since, at least in humans, the N‐termini of the subunits of transthyretin are located in the vicinity of the channel entrance [Hamilton, J. A., Steinrauf, L. K., Braden, B. C., Liepnieks, J., Benson, M. D., Holmgren, G., Sandgren, O. & Steen, L. (1993) J. Biol. Chem. 268, 2416–24241.
Porcine Aorta Smooth‐muscle Myosin Contains Three Species Made of Different Combinations of two 17‐kDa Essential Light‐chain Isoforms
Authors: Katoh Tsuyoshi , Tanahashi Kazuhiro , Hasegawa Yasushi , Morita Fumi
Published: March 2005

Porcine aorta myosin was reacted with a bifunctional cross‐linking reagent, N,N′‐o‐phenylenedimaleimide. The 17‐kDa essential light chain (LC17) in each myosin head was intramolecularly cross‐linked within a single myosin molecule. The 34‐kDa cross‐linked LC17 dimer was isolated and its peptide map, after lysylendopeptidase digestion, was obtained by reverse‐phase HPLC. Based on the amino acid compositions of peptide fragments, the N‐terminal Cys residues of LC17 subunits were assigned to be cross‐linked to each other. To study the distribution of two LC17 isoforms, LC17nm and LC17gi [Hasegawa, Y., Ueda, Y., Watanabe, M. & Morita, F. (1992) J. Biochem. 111, 798–803], aorta myosin was reacted with 5,5′‐dithiobis(2‐nitrobenzoic acid) (Nbs2). The LC17 dimer cross‐linked with Nbs2 was resolved into three distinct bands on urea/PAGE using a 4% acrylamide gel. Densitometric analysis of the three band intensities showed that three pairs of LC17 isoforms in aorta myosin are present in the ratio of LC17nm‐LC17nm/LC17nm‐LC17gi/C17gi‐LC17gi = 22:46:32. This ratio is consistent with the random combination of two LC17 isoforms with myosin heavy chains.
Substitution of Apolar Residues in the Active Site of Aspartate Aminotransferase by Histidine
Authors: Vacca Rosa Anna , Christen Philipp , Malashkevich Vladimir N. , Jansonius Johan N. , Sandmeier Erika
Published: March 2005

In an attempt to change the reaction and substrate specificity of aspartate aminotransferase, several apolar active‐site residues were substituted in turn with a histidine residue. Aspartate aminotransferase W140H of (Escherichia coli) racemizes alanine seven times faster (k′cat= 2.2×10−4 s−1) than the wild‐type enzyme, while the aminotransferase activity toward l‐alanine was sixfold decreased. X‐ray crystallo‐graphic analysis showed that the structural changes brought about by the mutation are limited to the immediate environment of H140. In contrast to the tryptophan side chain in the wild‐type structure, the imidazole ring of H140 does not form a stacking interaction with the coenzyme pyridine ring. The angle between the two ring planes is about 50°. Pyridoxamine 5′‐phosphate dissociates 50 times more rapidly from the W140H mutant than from the wild‐type enzyme. A model of the structure of the quinonoid enzyme substrate intermediate indicates that H140 might assist in the reprotonation of Cα of the amino acid substrate from the re side of the deprotonated coenzyme‐substrate adduct in competition with si‐side reprotonation by K258. In aspartate aminotransferase I17H (of chicken mitochondria), the substituted residue also lies on the re side of the coenzyme. This mutant enzyme slowly decarboxylates l‐aspartate to l‐alanine (kcat′= 8×10−5 s−1). No β‐decarboxylase activity is detectable in the wild‐type enzyme. In aspartate aminotransferase V37H (of chicken mitochondria), the mutated residue lies besides the coenzyme in the plane of the pyridine ring; no change in reaction specificity was observed. All three mutations, i.e. W140→H, I17→H and V37→H, decreased the aminotransferase activity toward aromatic amino acids by 10–100‐fold, while decreasing the activity toward dicarboxylic substrates only moderately to 20 %, 20 % and 60 % of the activity of the wild‐type enzymes, respectively. In all three mutant enzymes, the decrease in aspartate aminotransferase activity at pH values lower than 6.5 was more pronounced than in the wild‐type enzyme, apparently due to the protonation of the newly introduced histidine residues. The study shows that substitutions of single active‐site residues may result in altered reaction and substrate specificities of pyridoxal‐5′‐phosphate‐dependent enzymes.
Subcellular Distribution of Serine Acetyltransferase from Pisum sativum and Characterization of an Arabidopsis thaliana Putative Cytosolic Isoform
Authors: Ruffet Marie‐Line , Lebrun Michel , Droux Michel , Douce Roland
Published: March 2005

The intracellular compartmentation of serine acetyltransferase, a key enzyme in the l‐cysteine biosynthesis pathway, has been investigated in pea (Pisum sativum)leaves, by isolation of organelles and fractionation of protoplasts. Enzyme activity was mainly located in mitochondria (approximately 76% of total cellular activity). Significant activity was also identified in both the cytosol (14 % of total activity) and chloroplasts (10% of total activity). Three enzyme forms were separated by anion‐exchange chromatography, and each form was found to be specific for a given intracellular compartment. To obtain cDNA encoding the isoforms, functional complementation experiments were performed using an Arabidopsis thaliana expression library and an Escherichia coli mutant devoid of serine acetyltransferase activity. This strategy allowed isolation of three distinct cDNAs encoding serine acetyltransferase isoforms, as confirmed by enzyme activity measurements, genomic hybridizations, and nucleotide sequencing. The cDNA and related gene for one of the three isoforms have been characterized. The predicted amino acid sequence shows that it encodes a polypeptide of Mr 34 330 exhibiting 41% amino acid identity with the E. coli serine acetyltransferase. Since none of the general features of transit peptides could be observed in the N‐terminal region of this isoform, we assume that it is a cytosolic form.
Calorimetric Investigation of Thermal Stability and Ligand‐binding Characteristics of Disulfide‐bond‐cleaved Ribonuclease T1
Authors: Haun Matthias F , Wirth Matthias , Rüterjans Heinz
Published: March 2005

A combination of differential titration calorimetry and differential scanning calorimetry was used to study the effect of disulfide bond cleavage and reaction with iodoacetamide of ribonuclease T1 on both the binding of nucleotides and the thermal stability of the free enzyme species. Although guanosine monophosphates still bind to the active site of the modified protein the transition temperature of unfolding and the transition enthalpy decrease drastically indicating a relatively loose structure. The calorimetric data presented in this study suggest a cooperative linkage between the site of the disulfide bonds, the ligand‐binding site, and the general thermodynamic stability of the enzyme.
Horse Liver Alcohol Dehydrogenase can Accept NADP+ as Coenzyme in High Concentrations of Acetonitrile
Authors: Johansson Annika , Mosbach Klaus , Månsson Mats‐Olle
Published: March 2005

The coenzyme specificity of horse liver alcohol dehydrogenase assayed in mixtures of acetonitrile and buffer, 0.01 M Tris/HCl pH 7.4, was investigated. The enzyme could accept NADP+ as coenzyme if it was first bioimprinted with NADP+, i.e. precipitated from an aqueous buffer with 1‐propanol in the presence of NADP+, dried, and then assayed in a system with less than 10% buffer. When assayed in a system with 25% buffer, no activity with NADP+ as coenzyme was observed. The activity was measured with a coenzyme regeneration assay with cinnamoyl alcohol and octylaldehyde as substrates. Other methods to prepare a binary complex of horse liver alcohol dehydrogenase and NADP+, i.e. if the enzyme was immobilized on silica and NADP+ added afterwards or if it was deposited on Celite together with NADP+, failed to show any bio‐imprinting effect. The activity of horse liver alcohol dehydrogenase bio‐imprinted with NADP+ and assayed in acetonitrile/buffer mixtures with less than 10% buffer showed the same or higher activity than an enzyme preparation prepared by bio‐imprinting with NAD+. The hypothesis is that the conformation of the active site of horse liver alcohol dehydrogenase is modified to one that is complementary to the ligand present during precipitation and drying. This is possible because of the restricted motility of the enzyme conformation in high concentrations of organic solvent. In the presence of more than 10% buffer the mobility increases in such a way that the imposed conformation with activity towards NADP+ disappears.
Fatty Acid Binding to Bovine Serum Albumin Prevents Formation of Intermediate During Denaturation
Authors: Ahmad Nisar , Qasim M. Abul
Published: March 2005

Urea‐induced denaturation of defatted BSA and BSA containing six mol palmitic acid/mol protein (fatted BSA) has been studied by the techniques of UV difference spectroscopy and fluorescence spectroscopy. The notable differences between the two transitions are a shift in the transition of fatted BSA to a higher urea concentration and a three‐state denaturation transition in defatted BSA compared to an apparent two‐state denaturation transition in fatted BSA. The stable denaturation intermediate in defatted BSA occurs at 4.5–5.0 M urea, a urea concentration at which denaturation in fatted BSA has not yet started. These results are further supported by the difference spectral results obtained at 4.5 M urea in the two albumin preparations. The occurrence of denaturation intermediate only in defatted BSA and the presence of the two strong fatty‐acid‐binding sites in domain III lead us to conclude that this domain is relatively unstable in the absence of fatty acids and is responsible for the formation of intermediate.
Characterization of a Novel Brain Neutral Glycosphingolipid Composition in House Musk Shrew (Suncus Murinus)
Authors: Watanabe Kiyohiro , Nishiyama Masako
Published: March 2005

Glycosphingolipids were extracted from the brain of house musk shrew (Suncus murinus). Neutral glycosphingolipids were purified by QAE‐Sephadex column chromatography followed by high‐perfor‐mance liquid chromatography using an Iatrobeads column. Purified glycosphingolipids were identified by high‐performance thin‐layer chromatography, carbohydrate analysis, fast‐atom bombardment mass spectrometry and TLC immunostaining. The ganglioside pattern was almost the same as the pattern obtained for rat brain gangliosides. The brain of S. murinus, however, was unique in its neutral glyco‐sphingolipid composition; it contained gangliotriaosylceramide and gangliotetraosylceramide as the major neutral glycosphingolipids in addition to monohexosylceramides. Mass spectroscopy analysis showed that C18: 1 sphingosine and C24:O normal fatty acids were the major ceramide constituents in the glycosphin‐golipis, except in the case where a slower‐migrating monohexosylceramide that contained C24 h: 0 hy‐droxy fatty acids was observed. A day after birth, monohexosylceramide contained only normal fatty acids. The amounts of hydroxy‐fatty‐acid‐containing monohexosylceramide increased rapidly as the age of the animals increased, and the ratio of these two kinds of monohexosylceramides was reversed within five weeks. Monohexosylceramide, ganglioside, and sulfatide contents in bulbus olfactorius were almost equal in amount in contrast to the glycolipids in the cerebrum and cerebellum that contained monohexosylceramide as the major constituent. The amount of monohexosylceramide in the bulbus olfactorius was 0.3–0.4 times the values obtained for the of cerebrum and cerebellum.
The Molecular Biology of Multidomain Proteins Selected Examples
Authors: Hawkins Alastair R. , Lamb Heather K.
Published: March 2005

The aim of this review is to give an overview of the contribution molecular biology can make to an understanding of the functions and interactions within multidomain proteins. The contemporary advantages ascribed to multidomain proteins include (a) the potential for metabolite channelling and the protection of unstable intermediates; (b) the potential for interactions between domains catalysing sequential steps in a metabolic pathway, thereby giving the potential for allosteric interactions; and (c) the facility to produce enzymic activities in a fixed stoichiometric ratio. The alleged advantages in (a) and (b) however apply equally well to multi‐enzyme complexes; therefore, specific examples of these phenomena are examined in multidomain proteins to determine whether the proposed advantages are apparent. Some transcription‐regulating proteins active in the control of metabolic pathways are composed of multiple domains and their control is exerted and modulated at the molecular level by protein–DNA, protein–protein and protein–metabolite interactions. These complex recognition events place strong constraints upon the proteins involved, requiring the recognition of and interaction with different classes of cellular metabolites and macromolecules. Specific examples of transcription‐regulating proteins are examined to probe how their multidomain nature facilitates a general solution to the problem of multiple recognition events. A general unifying theme that emerges from these case studies is that a basic unitary design of modules provided by enzymes is exploited to produce multidomain proteins by a complex series of gene duplication and fusion events. Successful modules provided by enzymes are co‐opted to new function by selection apparently acting upon duplicated copies of the genes encoding the enzymes. In multidomain transcription‐regulating proteins, former enzyme modules can be recruited as molecular sensors that facilitate presumed allosteric interactions necessary for the molecular control of transcription.