bluuebunny/crossref_metadata_2025_split huggingface dataset:
The open/closed conformational equilibrium of aspartate aminotransferase
Authors: PICOT Daniel , SANDMEIER Erika , THALLER Christina , VINCENT Michael G. , CHRISTEN Philipp , JANSONIUS Johan N. 
Published: March 2005

Aspartate aminotransferase undergoes major shifts in the conformational equilibrium of the protein matrix during transamination. The present study defines the two conformational states of the enzyme by crystallographic analysis, examines the conditions under which the enzyme crystallizes in each of these conformations, and correlates these conditions with the conformational behaviour of the enzyme in solution, as monitored by a fluorescent reporter group.Cocrystallization of chicken mitochondrial aspartate aminotransferase with inhibitors and covalent coenzyme‐substrate adducts yields three different crystal forms. Unliganded enzyme forms triclinic crystals of the open conformation, the structure of which has been solved (space group P1) [Ford, G. C., Eichele, G. & Jansonius, J. N. (1980) Proc. Natl Acad. Sci. USA 77, 2559–2563; Kirsch, J. F., Eichele, G., Ford, G. C., Vincent, M. G., Jansonius, J. N., Gehring, H. & Christen, P. (1984) J. Mol. Biol. 174, 487–525]. Complexes of the enzyme with dicarboxylate ligands form monoclinic or orthorhombic crystals of the closed conformation. The results of structure determinations of the latter two crystal forms at 0.44 nm resolution are described here. In the closed conformation, the small domain has undergone a rigid‐body rotation of 12–14° which closes the active‐site pocket.Shifts in the conformational equilibrium of aspartate aminotransferase in solution, as induced by substrates, substrate analogues and specific dicarboxylic inhibitors, can be monitored by changes in the relative fluorescence yield of the enzyme labelled at Cys166 with monobromotrimethylammoniobimane. The pyridoxal and pyridoxamine forms of the labelled enzyme show the same fluorescence properties, whereas in the apoenzyme the fluorescence intensity is reduced by 30%. All active‐site ligands, if added to the labelled pyridoxal enzyme at saturating concentrations, cause a decrease in the fluorescence intensity by 40–70% and a blue shift of maximally 5 nm. Comparison of the fluorescence properties of the enzyme in various functional states with the crystallographic data shows that both techniques probe the same conformational equilibrium.The conformational change that closes the active site seems to be ligand‐induced in the reaction of the pyridoxal form of the enzyme and syncatalytic in the reverse reaction with the pyridoxamine enzyme.
Separation of the poly(glycerophosphate) lipoteichoic acids of Enterococcus faecalis Kiel 27738, Enterococcus hirae ATCC 9790 and Leuconostoc mesenteroides DSM 20343 into molecular species by affinity chromatography on concanavalin A
Authors: LEOPOLD Klaus , FISCHER Werner 
Published: March 2005

This study shows for the first time microheterogeneity of 1,3‐linked poly(glycerophosphate) lipoteichoic acids. The lipoteichoic acids investigated were those of Enterococcus faecalis Kiel 27738 (I), Enterococcus hirae (Streptococcus faecium) ATCC 9790 (II), and Leuconostoc mesenteroides DMS 20343 (III). Lipoteichoic acids II and III are partially substituted by mono‐, di‐, tri‐, and tetra‐α‐D‐glucopyranosyl residues with (1 → 2) interglycosidic linkages. Lipoteichoic acid I is substituted with α‐kojibiosyl residues only. Lipoteichoic acids I and III additionally carry D‐alanine ester.Lipoteichoic acids were separated on columns of concanavalin‐A—Sepharose according to their increasing number of glycosyl substituents per chain. It was evident that all molecular species are usually glycosylated and that alanine ester and glycosyl residues occur on the same chains. The chain lengths of lipoteichoic acid I and II vary between 9–40 glycerophosphate residues, whereas those of lipoteichoic acid III appear to be uniform (33 → 2 residues). Molecular species differ in the extent of glycosylation but their content of alanyl residues is fairly constant. All lipoteichoic acids contain a small fraction (5–15%) different in composition from the bulk and most likely reflecting an early stage of biosynthesis.Two procedures for chain length determination of poly(glycerophosphate) lipoteichoic acids are described.
Primary structure of a new actin‐binding protein from human seminal plasma
Authors: SCHALLER Johann , AKIYAMA Kazuko , KIMURA Hiroshi , HESS Daniel , AFFOLTER Michael , RICKLI Egon E. 
Published: March 2005

Secretory actin‐binding protein (SABP), a glycoprotein from human seminal plasma, was isolated according to Akiyama and Kimura [Akiyama, K. & Kimura, H. (1990) Biochim. Biophys. Acta 1040, 206–210]. The complete amino acid sequence of SABP was determined with the aid of fragments generated by trypsin, Staphylococcus aureus V8 protease and pepsin. The single polypeptide chain of SABP contains 118 amino acids with a calculated Mr of 13506 and pyroglutamic acid as the N‐terminal residue. A single N‐glycosidic carbohydrate moiety is located at Asn77. The carbohydrate composition shows an unusually high amount of fucose. The arrangement of the two disulfide bonds is Cys37–Cys63 and Cys61–Cys95.Sequence comparison revealed a high degree of similarity with a 14‐kDa submandibular gland protein from mouse (45% identity and 64% similarity). SABP is identical with a prolactin‐inducible protein and a protein termed gross cystic disease fluid protein 15 (sequences translated from cDNA clones), both from human breast tissues.Although SABP was also detected in saliva, in extracts of the submandibular gland and seminal vesicles, little is known of its function.
Isolation and analysis of the human 46‐kDa mannose 6‐phosphate receptor gene
Authors: KLIER Hans‐Jürgen , von FIGURA Kurt , POHLMANN Regina 
Published: March 2005

From a genomic library in EMBL 3, two overlapping clones for the human 46‐kDa mannose 6‐phosphate receptor (MPR46) were isolated, which span the entire coding sequence. The human MPR46 gene is distributed over 12 kb and is divided into seven exons (110–1573 bp). All the intron/exon borders agree with the consensus sequences of splice junctions. Exon 1 codes for a 5′ untranslated sequence. The ATG initiation codon begins with the second nucleotide in exon 2. A signal sequence of 26 amino acid residues is followed by the extracytoplasmic (luminal) domain, which extends to exon 5. The transmembrane domain of the receptor spans exons 5 and 6 and the cytoplasmic domain is encoded by exons 6 and 7. The latter domain also codes for an extended 3′ untranslated sequence.The transcription‐initiation site was defined by primer extension. The sequence upstream of the cap site has strong promoter activity and contains structural elements characteristic of promoters found in housekeeping genes.No correlation between the genomic organization and known protein domains of the MPR46 was apparent. Moreover, the sequence of about 150 amino acids within the luminal domain of MPR46, which is homologous to the 15 repeats that constitute the luminal domain of the 300‐kDa mannose 6‐phosphate receptor (MPR300), does not correlate with intron/exon borders. MPR46 and MPR300 have therefore diverged from a common ancestral gene before introduction of the present intron sequences.
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.
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.
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.
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.
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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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.