bluuebunny/crossref_metadata_2025 huggingface dataset:
The roles of ADP2− and Mg2+ in control steps of phosphoglycerate kinase
Authors: GRAHAM Heidi C. , WILLIAMS Robert J. P.
Published: March 2005

1H‐NMR measurements were made of solutions of yeast phosphoglycerate kinase containing the nucleotide, ADP, and Mg2+ in varying concentrations in order to investigate the affect that the metal ion has on the mode of ADP binding to the enzyme. A preliminary study of adenosine binding to phosphoglycerate kinase was made in order to be sure of the nature of the adenine site. From the change in chemical shifts of the ‘basic patch’ histidine resonances (His62, 167 and 170), the nucleotide C8‐H, C2‐H and C1′‐H resonances and resonances 40 and 41 (assigned to Thr373 and Thr375 in the hydrophobic, i.e. catalytic, site), it is apparent that there are at least two ADP binding sites on the enzyme: one at the hydrophobic (catalytic) site and one at the electrostatic site. A comparison of the results for ADP and ATP reveals differences due to the differential binding of the phosphate groups. The presence of Mg2+ results in further differences being observed.

The data suggest that the primary binding site of ADP, in the absence of Mg2+, involves electrostatic interactions between the diphosphate chain of the substrate and the ‘basic patch’ region of the N‐terminal domain. In the presence of ≥ 1:1 ratio of Mg2+/ADP, however, the primary binding site involves predominantly hydrophobic interactions between the adenosine moiety and the catalytic site, with secondary binding occurring at the electrostatic site. Addition of Mg2+, therefore, tends to reduce the affinity of the electrostatic site (presumably by competing for ADP).

It is suggested that α‐helix XII, including residues 372, 373 and 375, moves differentially on binding ADP, Mg ADP, ATP or Mg ATP, consistent with Mg2+ assisting the transfer of the γ‐phosphate of ATP to 3‐phosphoglycerate during catalysis.
The structure of the O‐specific polysaccharide chain of Proteus penneri strain 16 lipopolysaccharide
Authors: VINOGRADOV Eugeny V. , SIDORCZYK Zygmunt , SWIERZKO Anna , ROŻALSKI Antoni , DAEVA Elena D. , SHASHKOV Alexander S. , KNIREL Yuriy A. , KOCHETKOV Nikolay K.
Published: March 2005

O‐Specific polysaccharide was obtained by mild acid degradation of Proteus penneri strain 16 lipopolysaccharide and found to contain d‐glucose, d‐glucuronic acid, 2‐acetamido‐2‐deoxy‐d‐glucose, and 3,6‐dideoxy‐3‐[(R)‐3‐hydroxybutyramido]‐d‐galactose in the ratio of 2:1:1:1 as well as a small proportion of O‐acetyl groups. On the basis of one‐dimensional 1H‐NMR13C‐NMR and NOE spectroscopy, two‐dimensional homonuclear‐shift‐correlated spectroscopy with one‐step and two‐step relayed coherence transfer and heteronuclear 1H/13C NMR shift‐correlated spectroscopy, it was concluded that the O‐specific polysaccharide of P. penneri strain 16 has the following structure:

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This structure was confirmed by methylation analysis and structural analysis of a linear tetrasaccharide fragment prepared by cleavage of the polysaccharide with anhydrous hydrogen fluoride followed by conversion of the α‐tetrosyl fluoride obtained in to the corresponding free oligosaccharide and alditol. O‐Acetyl groups were tentatively located at position 3 of the glucuronic acid residue and at position 4 of the 6‐substituted glucose residue, the degree of acetylation being less than 20% of the total.

Cross‐reactions of P. penneri strain 16 anti‐(O‐specific polysaccharide) antiserum with lipopolysaccharides from several other Proteus strains and the role of 3,6‐dideoxy‐3‐(R)‐3‐hydroxybutyramido‐d‐galactose in the serological specificity of P. penneri strain 16 are discussed.
Isolation of the ATP synthase subunit 6 and sequence of the mitochondrial ATP6 gene of the yeast Candida parapsilosis
Authors: GUÉLIN Emmanuel , GUÉRIN Martine , VELOURS Jean
Published: March 2005

The mitochondrially translated product called subunit 6 was extracted from the yeast Candida parapsilosis mitochondria using an organic solvent mixture and purified by reverse‐phase HPLC. The partial N‐terminal sequence of subunit 6 reveals a post‐translational cleavage site as in Saccharomyces cerevisiae. The structural mitochondrial gene ATP6 was isolated form a mitochondrial DNA library using the oligonucleotide probe procedure. The gene and the surrounding regions were cloned into M13tg130 and M13tg131 phage vectors. The insert contained an open reading frame 738‐bp encoding a 246‐amino‐acid polypeptide. Mature subunit 6 contains 243 amino acid residues and the predicted molecular mass is 26511 Da. The subunit shows 52% similarity with ATP synthase subunit 6 of the yeast S. cerevisiae. Comparison between protein and DNA sequences shows that the CUN codon family codes for a leucine in C. parapsilosis mitochondria.
Labile conformation of type 2 Cu2+ centres in human ceruloplasmin
Authors: RYLKOV Vladimir V. , TARASIEV Michael Yu. , MOSHKOV Kirill A.
Published: March 2005

The investigation of human ceruloplasmin by spectral methods (EPR and spectrophotometry) demonstrated that type 2 Cu2+‐containing centres occur not in one, but in two stable forms, differing in EPR and optical spectra. The differential optical spectra of these forms were recorded and the differences in molar absorption coefficients determined.

By the EPR method, it was shown that both forms of these centres exist in the blood serum of control donors, as well as in the serum of patients. The relative content of these forms depends on the organism physiological state or on the presence of some pathological condition.

The ferroxidase activity of ceruloplasmin against hemoglobin was proved spectrophotometrically. The involvement of other serum proteins in this process cannot be ruled out. The conformational state of ceruloplasmin molecules plays an essential role in its oxidase activity.
Purification and properties of the H+‐nicotinamide nucleotide transhydrogenase from Rhodobacter capsulatus
Authors: LEVER Timothy M. , PALMER Tracy , CUNNINGHAM Ian J. , COTTON Nicholas P. J. , JACKSON J. Baz
Published: March 2005

H+‐transhydrogenase from Rhodobacter capsulatus is an integral membrane protein which, unlike the enzyme from Rhodospirillum rubrum, does not require the presence of a water‐soluble component for activity.

The enzyme from Rb. capsulatus was solubilised in Triton X‐100 and subjected to ion‐exchange, hydroxyapatite and then gel‐exclusion column chromatography. SDS/PAGE of the purified enzyme revealed the presence of two polypeptides with apparent Mr 53000 and 48000. Other minor components which were stained on the electrophoresis gels or which were revealed on Western blots exposed to antibodies raised to total membrane proteins, were probably contaminants.

Antibodies raised to the 53‐kDa and 48‐kDa polypeptides cross‐reacted with equivalent polypeptides in Western blots of solubilised membranes from Rb. capsulatus, Rhodobacter sphaeroides and Rhs. rubrum. The significance of this finding is discussed in the context of the hypothesis [Fisher, R. R. & Earle, S. R. (1982) The pyridine nucleotide coenzymes, pp. 279–324, Academic Press, New York] that the soluble component associated with H+‐transhydrogenase from Rhs. rubrum is an integral part of the catalytic machinery. Antibodies against the 48‐kDa and 53‐kDa polypeptides of the Rb. capsulatus enzyme cross‐reacted with equivalent polypeptides in solubilised membranes of Escherichia coli.

The dependence of the rate of H− transfer by purified H+ ‐transhydrogenase on the nucleotide substrate concentrations under steady‐state conditions, the effects of inhibition by nucleotide products and the inhibition by 2′‐AMP and by 5‐′AMP suggest that the reaction proceeds by the random addition of substrates to the enzyme with the formation of a ternary complex.

In conflict with this conclusion, the reduction of acetylpyridine adenine dinucleotide (AcPdAD +) by NADH in the absence of NADP+ by bacterial membranes was earlier taken as evidence for the existence of a reduced enzyme intermediate [Fisher, R. R. & Earle, S. R. (1982) The pyridine nucleotide coenzymes, pp. 279–324, Academic Press, New York]. However, it is shown here that although chromatophore membranes of Rb. capsulatus catalysed the reduction of AcPdAD+ by NADH, the reaction was not associated with the purified H + ‐transhydrogenase. Moreover, in contrast with the true transhydrogenase reaction, the reconstitution of AcPdAD+ reduction by NADH (in the absence of NADP+) in washed membranes of Rhs. rubrum with partially purified transhydrogenase factor, was only additive.
The iron‐sulfur clusters in the two related forms of mitochondrial NADH: ubiquinone oxidoreductase made by Neurospora crassa
Authors: WANG Da‐Cheng , MEINHARDT Steven W. , SACKMANN Uwe , WEISS Hanns , OHNISHI Tomoko
Published: March 2005

Two related forms of the respiratory‐chain complex, NADH: ubiquinone oxidoreductase (Complex I) are synthesized in the mitochondria of Neurospora crassa. Normally growing cells make a large, piericidin‐A‐sensitive form, which consists of some 23 different nuclear‐ and 6–7 mitochondrially encoded subunits. Cells grown in the presence of chloramphenicol make a small, piericidin‐A‐insensitive form which consists of only ∼ 13 nuclear‐encoded subunits. The subunits of the small form are either identical or similar to nuclear‐encoded subunits of the large form. The iron‐sulfur clusters in these two forms of Complex I are characterized by redox potentiometry and EPR spectroscopy. The large form of Complex I contains four EPR‐detectable iron‐sulfur clusters, N1, N2, N3 and N4, with the spin concentration of the individual clusters equivalent to the flavin concentration, similar to the mammalian counterparts. The small Complex I contains clusters N1, N3 and N4, but it is devoid of cluster N2. A model of the electron‐transfer route through the large form of Complex I has been derived from these findings and an evolutionary pathway which leads to the emergence of large Complex I is discussed.
Different patterns of expression of β‐tubulin genes in Tetrahymena pyriformis during reciliation
Authors: SOARES Helena , CYRNE Luisa , BARAHONA Isabel , RODRIGUES‐POUSADA Claudina
Published: March 2005

The ciliate Tetrahymena pyriformis contains one α‐tubulin (αTT) and two β‐tubulin (βTT1 and βTT2) genes. The specific expression of these genes was investigated by Northern blot hybridization using oligonucleotide probes complementary to βTT1 and βTT2 genes and the coding region of the α‐tubulin gene. The three genes are expressed producing 1.8‐kb mRNAs but the level of βTT1 mRNA is much higher than that of βTT2 mRNA. During cilia regeneration, we found that the expression patterns of the αTT and βTT1 genes are similar whereas that of the βTT2 gene is different. The αTT and βTT1 transcripts reached higher values between 60–120 min after the onset of reciliation than in exponentially growing cells, while βTT2 transcripts were maintained at low levels during the whole period. The differences in the amounts of steady‐state populations of the both β‐tubulin mRNAs do not correspond to the copy number per haploid genome. These differences could result from the fact that the promoter region of βTT2 may contain highly structured sequences which would affect the binding of the respective trans‐acting factor(s).

The apparent transcription rate revealed a significant increase at 15 min of reciliation which could be responsible for the high levels of αTT and βTT1 transcripts in the cytoplasm between 60–120 min of reciliation. This coordinated response to cilia regeneration of the αTT and βTT1 tubulin genes is also a relevant aspect of our findings. Several conserved motifs found in their promoter regions led us to think that some of them may function as cis‐elements in the specific binding of nuclear protein factor(s).
Identification and cloning of yeast phosphofructokinase 2
Authors: KRETSCHMER Matthias , TEMPST Paul , FRAENKEL Dan G.
Published: March 2005

Fructose‐6‐phosphate 2‐kinase (‘phosphofructokinase 2’) was purified from a strain of Saccharomyces cerevisiae lacking fructose‐6‐phosphate 1‐kinase. After chromatography on DEAE‐Sephacel, Sephacryl blue, CM‐Sephadex and rechromatography on CM‐Sephadex with fructose‐6‐phosphate elution, the specific activity was 1.6 U/mg protein.

Although the latter value is high for fructose‐6‐phosphate 2‐kinase, as was the purification factor of 3 × 104, staining with Coomassie blue showed the fraction to still contain many proteins. Incubation with [γ‐32P]ATP and the catalytic subunit of cAMP‐dependent protein kinase gave a further increase in specific activity and labeling of, only, 96‐kDa and 93‐kDa polypeptides. Antiserum raised against these polypeptides recognized them in an immunoblot and could be used to remove the enzyme activity from crude extracts.

Tryptic peptide profiles were obtained from about 10 pmol of the 96‐kDa and 93‐kDa polypeptides. The profiles were similar and sequencing allowed construction of mixed probes and identification of a putative single structural gene. Returned to yeast on a multicopy plasmid, phosphofructokinase 2 activity was considerably above the wild‐type level, as was polypeptide revealed by immunoblotting.
The purine‐2‐deoxyribonucleosidase from Crithidia luculiae
Authors: STEENKAMP Daniel J.
Published: March 2005

Crude extracts of Crithidia luciliae catalysed a deoxyribosyl transfer from purine deoxynucleosides to free purine bases. Fractionation of a 0—80% (NH4)2SO4 fraction from C. luciliae on DEAE‐cellulose resulted in the separation of three nucleosidase activities. Two of these were ribonucleosidases, one specific for inosine, uridine and xanthosine and the other for inosine and guanosine, whereas the third activity was specific for purine deoxyribonucleosides. This pattern is similar to that found in Leishmania donovani. Significant deoxyribosyltransferase activity was, however, associated with the purine‐2′‐deoxyribonucleosidase from C. luciliae. The purine‐2′‐deoxyribonucleosidase was purified to homogeneity by a six‐step procedure involving (NH4)2SO4 fractionation and chromatography on DEAE‐cellulose, hydroxyapatite, Sephadex G‐75, and a chromatofocusing resin. The purified enzyme migrated as a single band of 17 kDa on SDS/polyacrylamide gel electrophoresis. The enzyme catalysed the hydrolysis of deoxyinosine, deoxyguanosine and deoxyadenosine with Km values of 80 ± 10.5 μM, 20.7 ± 3.2 μM and 17.3 ± 5.3 μM, respectively, and V values for these substrates in the ratio 1:0.5:0.39. The pH optimum for deoxyribosyl transfer from deoxyinosine to guanine was at pH 7.7, while deoxyinosine hydrolysis in the presence of guanine was optimal in the range pH 6–7. During the synthesis of deoxyinosine from hypoxanthine and deoxyadenosine two products were formed. One of these coeluted with deoxyinosine on HPLC, while the second was tentatively identified as the positional isomer, 7‐(β‐d‐2′‐deoxyribofuranosyl)hypoxanthine.
Molecular cloning of the gene for plant proliferating‐cell nuclear antigen and expression of this gene during the cell cycle in synchronized cultures of Catharanthus roseus cells
Authors: KODAMA Hiroaki , ITO Masaki , OHNISHI Naoto , SUZUKA Iwao , KOMAMINE Atsushi
Published: March 2005

A cDNA library was screened for plant proliferating‐cell nuclear antigen (PCNA) from Catharanthus roseus (periwinkle). A λ gt11 cDNA library was constructed using poly(A)‐rich RNA isolated from the cells in the S phase. A cDNA clone for PCNA was isolated by using a rice genomic clone, pCJ‐1, which contains PCNA‐related gene sequences. The cDNA contains an open reading frame of 804 nucleotides, encoding a protein of 268 amino acids with a molecular mass of 29765 Da. When conservative substitutions were included, a high degree of similarity (about 85%) was observed between the predicted amino acid sequence of periwinkle PCNA and that of human PCNA. Expression of mRNA for periwinkle PCNA was undetectable or very weak in quiescent cells, such as phosphate‐starved cells, auxin‐starved cells and cells in the stationary phase. In the synchronous progression of the cell cycle induced by the addition of phosphate or auxin, the active accumulation of periwinkle PCNA mRNA was observed preferentially in the S phase. When an inhibitor of DNA synthesis, aphidicolin, was added to the cells at the G1 phase, an increase in the level of PCNA mRNA was observed. The partial inhibition of protein synthesis at the G1 phase by a protein inhibitor, anisomycin, caused the arrest of cells in the G1 phase. No increase of the level of periwinkle PCNA mRNA was observed in cells arrested at the G1 phase by the inhibition of protein synthesis. These results indicate that the induction of mRNA for periwinkle PCNA occurred independently of the initiation of DNA replication, but that synthesis of certain proteins at the G1 phase was required for the induction of periwinkle PCNA mRNA at the S phase.
Covalent structure of two novel neutrophile leucocyte‐derived proteins of porcine and human origin
Authors: FLODGAARD Hans , ØSTERGAARD Erik , BAYNE Stephen , SVENDSEN Allan , THOMSEN Johannes , ENGELS Michael , WOLLMER Axel
Published: March 2005

The covalent structures of two, novel, neutrophile, leucocyte‐derived, strongly basic proteins of porcine and human origin have been determined by microsequencing in combination with time‐of‐flight plasma desorption mass spectrometry. The porcine protein primary structure of 219 amino acid residues was shown to contain 6 cysteine residues, 2 putative carbohydrate sites and 14% basic residues. The human protein contained 221 amino acid residues of which 8 were cysteine, 4 putative carbohydrate sites and 12% basic. A 47% direct sequence similarity to human neutrophile elastase was found, but due to mutations of two of the three amino acids in the catalytic triad, proteolytic activity is absent. Modelling and alignment studies unveil a close relationship of both proteins to the serine protease family, the greatest similarity being to those serine proteases present in granules from peripheral blood cells. Both proteins have been shown to be chemotactically active for monocytes and fibroblasts in vitro.
Eukaryotic protein synthesis initiation factor 2
Authors: KUDLICKI Wieslaw , PICKING William D. , KRAMER Gisela , HARDESTY Boyd , SMAILOV Salim K. , MUKHAMEDZHANOV Bektas G. , LEE Anatoly V. , ISKAKOV Bulat K.
Published: March 2005

Polyproanthocyanidin (PPA), a phenolic polymer isolated from the plant Alhagi kirgisorum S. was found to interact strongly with eukaryotic initiation factor 2 (eIF‐2), thereby inhibiting reactions involving this protein. When added to a rabbit reticulocyte lysate system, PPA blocks in vitro translation and it appears to selectively bind and precipitate a relatively small number of proteins including eIF‐2 and regulin. The phosphorylation of purified regulin and eIF‐2 by casein kinase II (CK II) and the heme‐sensitive eIF‐2α kinase, respectively, was also inhibited by the polyphenolic compound. The natural fluorescence of PPA was utilized to compare its interaction with eIF‐2 and regulin to that with other natural and synthetic polypeptides.
Analysis of the spc ribosomal protein operon of Thermus aquaticus
Authors: JAHN Olaf , HARTMANN Roland K. , ERDMANN Volker A.
Published: March 2005

The gene region of Thermus aquaticus corresponding to the distal portion of the S10 operon and to the 5′‐portion of the Escherichia coli spc operon was cloned, using the E. coli gene for the ribosomal protein L5 as hybridization probe. The gene arrangement was found to be identical to E. coli, i.e. S17, L14, L24, L5, S14, S8 and L6. Stop and start regions of contiguous cistrons overlap, except for the S14–S8 intergenic region, whose size (67 bases) even exceeds the corresponding spacer regions in E. coli and Bacillus subtilis. A G + C content of 94% in third positions of codons was found in the ribosomal protein genes of T. aquaticus analyzed here. The stop codon of gene S17 (the last gene of the S10 operon in E. coli) and the start codon of gene L14 (the first gene of the spc operon in E. coli) overlap in T. aquaticus, thus leaving no space to accomodate an intergenic promoter preceding spc‐operon‐encoded genes in T. aquaticus. A possible promoter, localized within the S17 coding region, yielded only weak resistance (20 μg/ml) to chloramphenicol in E. coli and therefore could be largely excluded as the main promoter for spc‐operon‐encoded genes. We failed to detect a structure resembling the protein S8 translational repressor site, located at the beginning of the L5 gene in E. coli, in the corresponding region or any other region in the cloned T. aquaticus spc DNA.
N‐Acetylgucosaminono‐1,5‐lactone oxime and the corresponding (phenylcarbamoyl)oxime
Authors: HORSCH Markus , HOESCH Lienhard , VASELLA Andrea , RAST Dora M.
Published: March 2005

Using N‐acetylglucosaminono‐1,5‐lactone (1) as the reference, the inhibitory activity of its (formal) derivatives N‐acetylglucosaminono‐1,5‐lactone oxime (2) and N‐acetylglucosaminono‐1,5‐lactone O‐(phenylcarbamoyl)‐oxime (3) was tested against β‐N‐acetylglucosaminidase of different origins (animal, plant, fungus). Displaying inhibition constants of 0.45 μM and 0.62 μM, for the animal and plant enzyme, respectively, the simple oxime 2 was about equally potent as the parent lactone 1, and 50–400 times more efficient than two recently described new β‐N‐acetylglucosaminidase inhibitors. The (phenylcarbamoyl)oxime 3 performed even better, particularly with the fungal enzyme (Ki= 40 nM), i.e. was about 350 times more potent than the lactone. In all cases competitive inhibition was observed with 4‐nitrophenyl‐β‐N‐acetylglucosaminide as the substrate. With Ki/Km ratios up to 3300 for 2 and 13600 for 3, the mode of action of these novel inhibitors is probably that of transition state mimicry. Suggestions are made for their use as a tool in biological research.
Amino acid sequence of an extracellular, phosphate‐starvation‐induced ribonuclease from cultured tomato (Lycopersicon esculentum) cells
Authors: JOST Wolfgang , BAK Henk , GLUND Konrad , TERPSTRA Peter , BEINTEMA Jaap J.
Published: March 2005

The primary structure of an extracellular ribonuclease (RNase LE) from Pi‐depleted media of cultured cells of Lycopersicon esculentum L. cv. Lukullus has been determined. This was carried out by analysis of peptides isolated after enzymatic and chemical cleavage of the reduced and S‐ethylpyridylated protein.

RNase LE consists of 205 amino acid residues and has a molecular mass of 22666 Da and an isoelectric point of 4.24. The enzyme contains 10 half‐cystines. There are no potential N‐glycosylation sites in the sequence.

The sequence of RNase LE is homologous with those of self‐incompatibility proteins of several higher plant species and with those of a number of fungal RNases. The sequence similarity with the family of self‐incompatility proteins is greater than with the fungal RNases, suggesting that the self‐incompatibility proteins arose from ancestral RNase by gene duplication after the divergence of higher plants and fungi. Two pentapeptide sequences, i.e. HGLWP and KHGTC (or KHGSC), are present at identical positions in all the aligned proteins, suggesting that they contribute to the active site.
Anion binding at the active site of trypanosomal triosephosphate isomerase
Authors: VERLINDE Christophe L. M. J. , NOBLE Martin E. M. , KALK Kor H. , GROENDIJK Hillie , WIERENGA Rik K. , HOL Wim G. J.
Published: March 2005

The three‐dimensional structure of triosephosphate isomerase complexed with the competitive inhibitor HPO42− was determined by X‐ray crystallography to a resolution of 0.24 nm. A comparison with the native crystal structure, where SO42− is bound, revealed five changes: (a) a 0.10‐nm shift of the anion‐binding site; (b) a further closing of the flexible loop of the enzyme; (c) a ‘swinging in’ of the side chain of the catalytic Glu, that is χ1 changes from (+) to (−) synclinal; (d) an altered water structure; (e) a disappearance of the conformational heterogeneity at the C‐terminus of strand β7. Some of these changes may be related to the different hydrogen‐bond pattern about the two different anions. However, the distance of 0.10 nm between the sulphur and phosphorus positions is unexpected and remains intriguing.
Cross reactivity of monoclonal antibodies and cDNA hybridization suggest evolutionary relationships between cytochrome c oxidase subunits VIa and VIc and between VIIa and VIIb
Authors: SCHNEYDER Bettina , MELL Oliver , ANTHONY Georges , KADENBACH Bernhard
Published: March 2005

Monoclonal antibodies to subunits of bovine heart cytochrome c oxidase were prepared by immunizing mice with the isolated enzyme. The majority of antibody‐producing cell lines were found to react with two different subunits of similar molecular mass, as shown by Western blotting and ELISA titrations with the HPLC‐purified subunits. The affinities of the monoclonal antibodies to the subunits were determined by ELISA titrations with increasing concentrations of NH4SCN. Two monoclonal antibodies with a low affinity to subunit VIa had a high affinity to subunit VIc, whereas two other antibodies showed the same affinity to subunits VIIa and VIIb. The same affinity of monoclonal antibodies suggested an evolutionary relationship of subunits VIIa and VIIb, which was further supported by reactivity of these antibodies to subunits VIIa and VIIb of cytochrome c oxidase from different species and tissues. Also the evolutionary relationship between subunit VIa and VIc was shown by hybridization at low stringency of cDNAs for rat cytochrome c oxidase subunits VIc and VIa–h (heart‐type), after amplification by the polymerase chain reaction, with a probe of VIa–l (liver‐type).
The conformational effects of N‐glycosylation on the tailpiece from serum IgM
Authors: WORMALD Mark R. , WOOTEN E. Wrenn , BAZZO Renzo , EDGE Christopher J. , FEINSTEIN Arnold , RADEMACHER Thomas W. , DWEK Raymond A.
Published: March 2005

1H‐NMR spectroscopy has been used to study the conformation and dynamics of the isolated tailpiece from human serum immunoglobulin M, a 22‐residue peptide containing a single asparagine glycosylation site. The peptide is isolated as a set of glycoforms, varying only in the sequence of the oligosaccharide attached at the glycosylation site. The oligosaccharides present have the general formula (Man)n(GlcNAc)2, with 45% having n= 6, 45% having n= 8 and 10% having n= 7 and/or 9. They have been identified and their NMR parameters compared to those found for the isolated oligosaccharides in free solution. The conformation and dynamics of the peptide component have also been studied, using NOE data and hydrogen‐exchange experiments, and the results compared to those obtained from the aglycosyl peptide of the same sequence. The presence of the peptide is found to have no measurable effect on the conformation of the oligosaccharides. However, the presence of oligosaccharide causes a decrease in the conformational mobility of the backbone and sidechains of the peptide in the region of the glycosylation site. This is proposed to result from interactions between the oligosaccharide core and the amino acid side chains. Further, the conformation of the N‐glycosidic linkage has been shown to be both rigid and planar. Thus, the conformational space available to an N‐linked oligosaccharide in a glycoprotein relative to the protein may depend to a large extent upon the flexibility of the asparagine side chain. Various roles for the different glycoforms of the tail peptide are discussed.
Calcium‐dependent binding of basement membrane protein BM‐40 (osteonectin, SPARC) to basement membrane collagen type IV
Authors: MAYER Ulrike , AUMAILLEY Monique , MANN Karlheinz , TIMPL Rupert , ENGEL Jürgen
Published: March 2005

Basement membrane protein BM‐40, prepared from the mouse Engelbreth‐Holm‐Swarm tumor, was used in native, denatured and proteolytically processed form for binding to various extracellular matrix proteins. BM‐40 and its derivatives were also characterized by CD spectroscopy, calcium binding and epitope analysis. Of several basement membrane proteins tested only collagen IV showed a distinct and calcium‐dependent binding of BM‐40 in an immobilized ligand assay. This interaction was specific as shown by a low activity of other collagen types (I, III, V, VI) in direct binding and competition assays. The binding was reduced or abolished by metal‐ionchelating or chaotropic agents, high salt and reduction of disulfide bonds in BM‐40. Fragment studies indicated that domains III (α‐helix) and/or IV (EF hand) of BM‐40 possess the binding site(s) for collagen IV, while the N‐terminal domains I and II provide the major antigenic determinants. A major BM‐40‐binding site on collagen IV was dependent on a triple‐helical conformation and could be localized to a pepsin fragment from the central portion of the triple‐helical domain, in agreement with electron microscopic visualization of BM‐40 – collagen‐IV complexes.
Isolation and structural characterization of novel neutral oligosaccharide‐alditols from respiratory‐mucus glycoproteins of a patient suffering from bronchiectasis
Authors: KLEIN André , CARNOY Christophe , LAMBLIN Geneviève , ROUSSEL Philippe , van KUIK J. Albert , de WAARD Pieter , VLIEGENTHART Johannes F. G.
Published: March 2005

The carbohydrate chains of the respiratory‐mucus glycoproteins of a patient (blood group O) suffering from bronchiectasis due to Kartagener's syndrome, were released by alkaline borohydride treatment of a pronase digest. Neutral oligosaccharides were obtained after ion‐exchange chromatography and were subsequently separated utilizing gel filtration, HPLC on normal‐phase alkylamine‐bonded silica and reverse‐phase HPLC, into 46 fractions. From these fractions oligosaccharide‐alditols have been characterized by employing 500‐MHz 1H‐NMR spectroscopy, in conjunction with fast‐atom‐bombardment mass spectroscopy, methylation analysis and sugar analysis.

Here 11 novel oligosaccharide structures are described. Five of them have the common element Fucα(1 → 2)Galβ(1 → 3)GlcNAcβ(1 → 3)Galβ(1 → 4)GlcNAcβ(1 → 6)GalNAc‐ol, they are:
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Another six possess Galβ(1→4)GlcNAcβ(1→3)Galβ(1→4)GlcNAcβ(1→6)GalNAc‐ol as common element, they are:
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Alanyl‐tRNA synthetase from Escherichia coli, Bombyx mori and Ratus ratus
Authors: DIGNAM John David , DIGNAM Susan S. , BRUMLEY Lizabeth L.
Published: March 2005

Alanyl‐tRNA synthetase from Escherichia coli, Bombyx mori and rat were examined with respect to the following functional and structural properties: the effect of substrates on sensitivity to proteolysis, secondary structure as determined by circular dichroism, amino acid composition and, in the case of the rat and insect enzymes, partial amino acid sequence determination on a 60‐kDa C‐terminal tryptic fragment. Digestion of the enzyme from all three sources with trypsin resulted in significant decline in aminoacyl‐tRNA synthetase activity with little effect on pyrophosphate‐exchange activity. In each case the presence of alanine and ATP together, but not separately, reduced the rate of digestion by trypsin; the largest effect was observed with the enzyme from rat liver. Trypsin digestion generated fragments of 47 kDa and 40 kDa with all three enzymes, but detection of significant quantities of the 47‐kDa fragment from the rat enzyme required the presence of ATP and alanine. Trypsin digestion produced a fragment of 60 kDa with all three enzymes, but detection of significant quantities of this fragment with the bacterial enzyme required the presence of ATP and alanine. Limited sequence analysis of the 60‐kDa fragment from the insect and rat enzymes indicated that trypsin cleaved both proteins at the same site to generate this species. Similar effects of substrates were observed when the enzymes were digested with chymotrypsin suggesting that the effects of substrates on protease sensitivity were not unique to trypsin. Circular dichroism spectra obtained for the three enzymes were qualitatively and quantitatively similar. There is some similarity in amino acid composition between the rat and insect enzymes.
Activation of reconstituted Escherichia coli outer‐membrane phospholipase A by membrane‐perturbing peptides results in an increased reactivity towards the affinity label hexadecanesulfonyl fluoride
Authors: HORREVOETS Anton J. G. , FRANCKE Christof , VERHEIJ Hubertus M. , de HAAS Gerard H.
Published: March 2005

The activity of the Escherichia coli outer‐membrane phospholipase (OM PLA) is strictly regulated in its natural habitat, the E. coli outer membrane. OM PLA can be reconstituted in phospholipid bilayers, resulting in low specific activity of the enzyme compared to its activity on mixed lipid/detergent micelles. The enzyme can be activated by the addition to these vesicles of the membrane‐perturbing peptides polymyxin B, melittin or cardiotoxin resulting in hydrolysis of mainly the sn‐1 ester bond of the phospholipids as is also observed in vivo. We used the affinity label hexadecanesulfonyl fluoride to probe the influence of lipid environment on the activity of OM PLA. In detergent and substrate micelles, the rate constant for the sulfonylation of the active‐center serine of the purified OM PLA by the affinity label hexadecanesulfonyl fluoride depends on amphiphile concentration. We have reported a similar influence of amphiphile concentration on the activity of the enzyme [Horrevoets, A. J. G. et al. (1989) Biochemistry 28, 1139–1147]. Analysis of the rates of inactivation of OM PLA by hexadecanesulfonyl fluoride in vesicles composed of various phospholipids indicated that activation of the enzyme by membrane‐perturbing peptides can be accurately quantified with this affinity label. Our results show that the affinity label hexadecanesulfonyl fluoride can be used to monitor the state of activation of OM PLA in different lipid environments, including non‐hydrolyzable substrate analogues. Implications for the in vivo situation are discussed.
A green 2,4‐pentadienoyl‐CoA reductase from Clostridium aminovalericum
Authors: EIKMANNS Ulrich , BUCKEL Wolfgang
Published: March 2005

2,4‐Pentadienoyl‐CoA reductase from Clostridium aminovalericum was purified to homogeneity (170–182 kDa). PAGE in the presence of SDS revealed a single band (44 kDa) indicating a homotetrameric structure. The native enzyme had a green colour and contained 0.4 mol FAD/subunit. Its unusual ultraviolet/visible‐spectrum showed absorption maxima at 270, 402 and 715 nm as well as shoulders at 278, 360, 450 and 500 nm. Removal of the prosthetic group at pH 2 in the presence of salt and charcoal yielded a colourless and completely inactive apoenzyme, which could be reconstituted with FAD (not with FMN) to an active holoenzyme showing a normal flavoprotein spectrum (peaks at 369 nm and 436 nm). Thereby the FAD content increased to 0.9 mol/subunit with a concomitant rise in activity to 200% of the original value. Anaerobic reduction of the green enzyme by dithionite and reoxidation by air afforded a green preparation with a spectrum similar to that of the native enzyme. Addition of excess FAD to the green reductase also increased the activity by a factor of two.

The green enzyme catalysed the oxidation of (E)‐3‐pentenoyl‐CoA or (E)‐3‐hexenoyl‐CoA to 2,4‐pentadienoyl‐CoA or 2,4‐hexenoyl‐CoA, respectively. 2‐Pentenoyl‐CoA or 4‐pentenoyl‐CoA were not oxidised. Meldola blue (8‐dimethylamino‐2,3‐benzophenoxazine) and 2‐(4‐iodophenyl)‐3‐(4‐nitrophenyl)‐5‐phenyltetrazolium chloride (V= 26 nkat/mg protein) or ferricenium hexafluorophosphate (V= 1900 nkat/mg), but not NAD(P), served as electron acceptors. Reduction of 2,4‐pentadienoyl‐CoA (V= 370 nkat/mg) was observed with reduced benzyl viologen, but not with NAD(P)H as an electron donor. Although the enzyme had some pentenoyl‐CoA ‐isomerase activity (1.2 nkat/mg), the only product of the reduction was 3‐pentenoyl‐CoA rather than 2‐pentenoyl‐CoA.
Complete sequence of Sipunculus nudus erythrocyte histone H2B and its gene
Authors: KMIÉCIK Daniel , BÉLAÏCHE Denise , SAUTIÈRE Pierre , LOUCHEUX‐LEFEBVRE Marie‐Henriette , KERCKAERT Jean‐Pierre
Published: March 2005

The complete amino acid sequence (122 residues) of histone H2B from erythrocytes of the marine worm Sipunculus nudus, has been established from sequence analysis of peptides generated by highly specific cleavage of the protein and from the nucleotide sequence of the encoding gene. The isolation of the H2B gene was facilitated by using a highly specific nucleotide probe, devised from amino acids 58–68 of the protein. The presence of an N,N‐dimethylproline residue at the amino‐terminus of the protein was established from data provided by mass spectrometry and NMR spectroscopy. This unusual post‐translational modification of histone H2B generates a stable positive charge which could strongly interact with the linker DNA.
Interaction of mithramycin with DNA
Authors: DEMICHELI Cynthia , ALBERTINI Jean‐Paul , GARNIER‐SUILLEROT Arlette
Published: March 2005

The CD and fluorescence properties of mithramycin have been used to follow its complexation to cations such as Mg2+ and Zn2+ and the binding of these complexes to DNA. At low concentration (< 2 μM) in aqueous solution, mithramycin is always in the dimeric state, the conformation of the dimer being either a right‐handed screw when the dimer is neutral, or a left‐handed screw when the dimer is negatively charged. In the deprotonated state the dimer can bind one cation forming a complex [M2+ (Mit−)2] which has a right‐handed screw conformation. The stability constants of the complex at 37°C in 0.1 M KCl are 4 × 105 and 1 × 106 for Mg2+ and Zn2+, respectively. The complex in the right‐handed screw conformation binds DNA. In this case the stability constants of the complex [M2+ (Mit−)2] increase and are 3.6 × 106 and 1.2 × 107 for Mg2+ and Zn2+, respectively.
Human complement factor H
Authors: SCHWAEBLE Wilhelm , SCHWAIGER Helga , BROOIMANS Ricardo A. , BARBIERI Antonia , MÖST Johannes , HIRSCH‐KAUFFMANN Monica , TIEFENTHALER Martin , LAPPIN David F. , DAHA Mohamed R. , WHALEY Keith , DIERICH Manfred P.
Published: March 2005

Using cDNA clones H‐19 and H‐46, we have shown previously that three different mRNA species (4.3 kb, 1.8 kb and 1.4 kb) for complement factor H are expressed constitutively in human liver. Here we report data suggesting that the expression of these different factor‐H mRNA species is regulated by tissue‐specific control mechanisms.

Total RNA and poly(A)‐enriched RNA from various human tissues (heart, lung, temporal cortex, kidney, spleen, bone marrow and muscle) various cell lines (HepG2, HepG3, HepG4, Hep3B, H‐4, Jurkat, Molt4, H‐9, KHos24Os, A‐431, U937, Mono Mac 6 and Raji) and from primary cultures of peripheral blood monocytes, fibroblasts and human umbilical vein endothelial cells (HUVEC) were investigated for the expression of factor‐H mRNA. In RNA preparations from extrahepatic tissue, factor‐H mRNA was only detected in biopsies from the lung. Using 20 μg total RNA isolated from all 13 cell lines it was not possible to detect any factor‐H mRNA, while mRNA for factor H was expressed in monocytes, HUVEC and fibroblasts.

When expressed in extrahepatic tissues, only the 4.3‐kb and the 1.8‐kb mRNA species were detected, while the 1.4‐kb mRNA is expressed abundantly in liver. Interferon‐γ did not induce the expression of factor‐H mRNA in any of the cell lines tested. On the other hand, tumour necrosis factor‐α induced the expression of the 4.3‐kb mRNA species in U937 cells.

In HUVEC and fibroblasts the relative quantities of the 4.3‐kb and the 1.8‐kb mRNA species and the regulatory effects of interferon‐γ, interleukin‐1, dexamethasone and retinoic acid on their expression showed significant tissue specificity.
The cytosolic and glycosomal isoenzymes of glyceraldehyde‐3‐phosphate dehydrogenase in Trypanosoma brucei have a distant evolutionary relationship
Authors: MICHELS Paul A. M. , MARCHAND Martine , KOHL Linda , ALLERT Sylvie , WIERENGA Rik K. , OPPERDOES Fred R.
Published: March 2005

Trypanosoma brucei contains two isoenzymes for glyceraldehyde‐3‐phosphate dehydrogenase: one enzyme resides in a microbody‐like organelle, the glycosome; the other is found in the cytosol. Previously we have reported the characterization of the gene for the glycosomal enzyme [Michels, P. A. M., Poliszczak, A., Osinga, K. A., Misset, O., Van Beeumen, J., Wierenga, R. K., Borst, P. & Opperdoes, F. R. (1986) EMBO J. 5, 1049–1056]. Here we describe the cloning and analysis of the gene that codes for the cytosolic isoenzyme. The gene encodes a polypeptide of 330 amino acids, with a calculated molecular mass of 35440 Da. The two isoenzymes are only 55% identical. The cytosolic glyceraldehyde‐3‐phosphate dehydrogenase differs from the glycosomal enzyme in the following respects: (a) its subunit molecular mass is 3.4 kDa smaller due to the absence of insertions and a small C‐terminal extension which are unique to the glycosomal protein; (b) the cytosolic enzyme has a lower pI (7.9, as compared to 9.3 for the glycosomal isoenzyme), which is due to a reduction in the excess of positively charged amino acids (the calculated net charges of the polypeptides are + 2 and + 11, respectively).

We have compared the amino acid sequences of the two T. brucei glyceraldehyde‐3‐phosphate dehydrogenases, with 24 available sequences of the corresponding enzyme of other organisms from various phylogenetic groups. On the basis of this comparison an evolutionary tree was constructed. This analysis strongly supports the theory that T. brucei diverged early in evolution from the main eukaryotic branch of the phylogenetic tree. Further, two separate branches for the lineages leading to Trypanosoma are inferred from the amino acid sequences, suggesting that the genes for the two glyceraldehyde‐3‐phosphate dehydrogenases of the trypanosome are distantly related and must have been acquired independently by the trypanosomal ancestor. The branching determined with the glycosomal enzyme precedes that found with the cytosolic enzyme. The available data do not allow us to decide which of the two genes originally belonged to the trypanosome lineage and which entered the cell later by horizontal gene transfer.
Structural analysis of the lipid A component of Campylobacter jejuni CCUG 10936 (serotype O:2) lipopolysaccharide
Authors: MORAN Anthony P. , ZÄHRINGER Ulrich , SEYDEL Ulrich , SCHOLZ Dieter , STÜTZ Peter , RIETSCHEL Ernst Th.
Published: March 2005

The chemical structure of Campylobacter jejuni CCUG 10936 lipid A was elucidated. The hydrophilic backbone of the lipid A was shown to consist of three (1 → 6)‐linked bisphosphorylated hexosamine disaccharides. Neglecting the phosphorylation pattern, a D‐glucosamine (2‐amino‐2‐deoxy‐D‐glucose) disaccharide [β‐D‐glucosaminyl‐(1 → 6)‐D‐glucosamine], a hybrid disaccharide of 2,3‐diamino‐2,3‐dideoxy‐D‐glucose and D‐glucosamine [2,3‐diamino‐2,3‐dideoxy‐β‐D‐glucopyranosyl‐(1 → 6)‐D‐glucosamine], and a 2,3‐diamino‐2,3‐dideoxy‐D‐glucose disaccharide were present in a molar ratio of 1:6:1.2. Although the backbones are bisphosphorylated, heterogeneity exists in the substitution of the polar head groups. Phosphorylethanolamine is α‐glycosidically bound to the reducing sugar residue of the backbone, though C‐1 is also non‐stoichiometrically substituted by diphos‐phorylethanolamine. Position 4′ of the non‐reducing sugar residue carries an ester‐bound phosphate group or is non‐stoichiometrically substituted by diphosphorylethanolamine. By methylation analysis it was shown that position 6′ is the attachment site for the polysaccharide moiety in lipopolysaccharide. These backbone species carry up to six molecules of ester‐ and amide‐bound fatty acids. Four molecules of (R)‐3‐hydroxytetradecanoic acid are linked directly to the lipid A backbone (at positions 2, 3, 2′, and 3′). Laser desorption mass spectrometry showed that both (R)‐3‐hydroxytetradecanoic acids linked to the non‐reducing sugar unit carry, at their 3‐hydroxyl group, either two molecules of hexadecanoic acid or one molecule of tetradecanoic and one of hexadecanoic acid. It also suggested that the (R)‐3‐(tetradecanoyloxy)‐tetradecanoic acid was attached at position 2′, whereas (R)‐3‐(hexadecanoyloxy)‐tetradecanoic acid was attached at position 3′, or at positions 2′ and 3′. Therefore, the occurrence of three backbone disaccharides differing in amino sugar composition and presence of a hybrid disaccharide differentiate the lipid A of this C. jejuni strain from enterobacterial and other lipids A described previously.
Studies on the intracellular distributions of soluble epoxide hydrolase and of catalase by digitonin‐permeabilization of hepatocytes isolated from control and clofibrate‐treated mice
Authors: MESSING ERIKSSON Anna , ZETTERQVIST Mary‐Ann , LUNDGREN Bo , ANDERSSON Karin , BEIJE Brita , DePIERRE Joseph W.
Published: March 2005

Digitonin permeabilization of hepatocytes from control and clofibrate‐treated (0.5% by mass, 10 days) male C57bl/6 mice was used to study the intracellular distributions of soluble (‘cytosolic’) epoxide hydrolase and of catalase. The following conclusions were drawn. (1) About 60% of the total soluble epoxide hydrolase activity in control mouse hepatocytes is situated in the cytosol. (2) The rest is not mitochondrial, but probably peroxisomal. (3) Of the total catalase activity in control mouse hepatocytes, 5–10% is found in the cytosol. (4) Treatment of mice with clofibrate increases the total hepatocyte activity of soluble epoxide hydrolase 4‐fold, but does not influence the relative distribution of this enzyme between cytosol and peroxisomes. (5) The total catalase activity is increased 3.5‐fold by clofibrate treatment and 15–35% of this activity is shifted from the peroxisomes to the cytosol.
Behavior of cysteine mutants of human lysozyme in de novo synthesis and in vivo secretion
Authors: OMURA Fumihiko , TANIYAMA Yoshio , KIKUCHI Masakazu
Published: March 2005

To investigate the mechanism of disulfide‐bond‐coupled de novo folding of human lysozyme, we have constructed 23 mutant enzymes in which cysteine residue(s) were replaced by alanine(s). The mutant genes were translated in vitro in a system composed of rabbit reticulocyte lysate, canine pancreatic microsomal vesicles and oxidized glutathione. This system allows the formation of intramolecular disulfide bonds in translation products translocated into the microsomal lumen. The mobilities of the translation products were analyzed by SDS/PAGE in nonreducing conditions. Some mutant lysozymes were found to form a compact conformation with native‐like mobility in the presence of SDS. The de novo formation of the SDS‐resistant compact conformation of each mutant correlated well with its efficiency of secretion by Saccharomyces cerevisiae. Our results suggest that the de novo synthesized products reflect the conformational states in vivo to some extent, and that the formation of SDS‐resistant compact conformation can be regarded as a necessary condition for allowing lysozyme to be secreted. In addition, the analysis of a mutant C116A (Cys116 → Ala) under different oxidative conditions suggests two distinct pathways for the disulfide‐bond‐coupled formation of the compact conformation.
Metal affinity precipitation of proteins carrying genetically attached polyhistidine affinity tails
Authors: LILIUS Gösta , PERSSON Mats , BÜLOW Leif , MOSBACH Klaus
Published: March 2005

In this study, galactose dehydrogenase (EC 1.1.1.48) was chosen as a prototype target protein to investigate the capability of metal affinity precipitation to facilitate the purification of genetically engineered proteins. A DNA fragment encoding five histidine residues was fused to the 3′‐terminal end of the galactose dehydrogenase gene from Pseudomonas fluorescens and thereafter expressed in Escherichia coli. The additional five histidines functioned as an affinity tail and the modified enzyme could be purified using metal affinity precipitation when the metal‐chelate complex with ethylene glycol‐bis‐(β‐aminoethyl ether) N,N,N′,N′‐tetra‐acetic acid, EGTA(Zn)2, was added to the protein solution. The affinity tail could also be applied for the purification of the fusion protein utilising immobilised metal affinity chromatography. After purification, the pentahistidine affinity tail could be removed enzymatically by carboxypeptidase A. Furthermore, growth rate experiments demonstrated that the expression of the metal‐binding affinity tail in E. coli cells enhanced the tolerance to zinc ions when added to the growth medium.