bluuebunny/crossref_metadata_2025 huggingface dataset:
Presence of hormonogenic and repetitive domains in the first 930 amino acids of bovine thyroglobulin as deduced from the cDNA sequence
Authors: MERCKEN Luc , SIMONS Marie‐Jeanne , DE MARTYNOFF Guy , SWILLENS Stéphane , VASSART Gilbert
Published: March 2005

The sequence of the first 2831 nucleotides of bovine thyroglobulin mRNA has been determined from the analysis of a cDNA clone. Following a 41‐nucleotide 5′ untranslated sequence, a single open‐reading frame encoding 930 amino acids was observed. This corresponds to the aminoterminal third of thyroglobulin, preceded by a putative signal peptide of 19 amino acids. The protein sequence was found to be essentially made of the sevenfold repetition of a 60‐amino‐acid‐long building unit, interrupted at fixed positions by unrelated segments of variable length. The presence of an internal homology within the repetitive unit itself suggests that the 5′ region of the thyroglobulin gene has evolved from the initial duplication of a relatively short sequence, followed by the serial duplication of the resulting unit.

The tyrosine residue at position five has been assigned an important hormonogenic function [Mercken, L., Simons, M.‐J. and Vassart, G. (1982) FEBS Lett. 149, 285–287]. This residue is flanked by sequence elements related to the repeated unit, suggesting that the hormonogenic domain evolved also from the basic ancestor sequence.
Stepwise unfolding of chromatin by urea
Authors: NIELSEN Peter Eigil , MATSUOKA Yukio , NORDÉN Bengt J. F.
Published: March 2005

The unfolding of chromatin by urea (0 – 7 M) was studied by means of flow linear dichroism, photoaffinity labeling and nuclease digestion. The linear dichroism results indicate that the unfolding of the DNA is accomplished through two distinct transitions at 1 – 2 M urea and 6 – 8 M urea, respectively. The photoaffinity labeling studies indicate that an opening of the nucleosome histone core occurs above 2 M urea, accompanied by general loosening of the structure. Based on the results a model for the unfolding of chromatin fibers by urea is proposed, which includes a stretching of the linker DNA (0 – 2 M urea) followed by a “loosening” of the nucleosome core, possibly to a one‐loop DNA conformation (2 – 6 M urea), and finally resulting in an almost total stretching of the DNA (> 6 M urea).
Oxygen binding to dithionite‐reduced chloroperoxidase
Authors: LAMBEIR Anne‐Marie , DUNFORD H. Brian
Published: March 2005

Both the kinetics of ferric chloroperoxidase reduction by dithionite and the binding of molecular oxygen to ferrous chloroperoxidase have been studied. The oxyferrous chloroperoxidase decays spontaneously to the ferric enzyme. In addition the corresponding rapid‐scan spectra have been recorded. The reduction reaction is caused by SO2 with a rate constant of (7.7 ± 1.0) × 104 M−1 s−1. Oxygen binding occurs with a rate constant of (5.5 ± 1.0) × 105 M−1 s−1 over the pH range 3.5–6. Oxyferrous chloroperoxidase has a Soret absorption peak at 428 nm and two partially resolved peaks at 555 nm and 588 nm. Isosbestic points occur at the following wavelengths: between ferrous and oxyferrous chloroperoxidase at 419, 545, 555 and 580 nm; between oxyferrous and ferric chloroperoxidase at 419, 487, 540, 609 and 682 nm.
The sub‐cellular localisation and regulatory properties of pyruvate carboxylase from Rhizopus arrhizus
Authors: OSMANI Stephen A. , SCRUTTON Michael C.
Published: March 2005

Cell‐free extracts of Rhizopus arrhizus contain exclusively cytosolic pyruvate carboxylase and NAD‐glutamate dehydrogenase, a single mitochondrial isoenzyme of NADP‐isocitrate dehydrogenase, and both mitochondrial and cytosolic isoenzymes of NADP‐malate dehydrogenase (decarboxylating). Other enzymes examined have sub‐cellular localisations similar to those characteristic of mammalian liver.

Purified preparations of R. arrhizus pyruvate carboxylase are subject to partial regulatory inhibition by L‐aspartate and 2‐oxoadipate. L‐Glutamate acts as a less effective analogue of L‐aspartate while 2‐oxoaglutarate is ineffective. Competition studies indicate the presence of separate inhibitory sites for L‐aspartate and 2‐oxoadipate.

Under routine assay conditions R. arrhizus pyruvate carboxylase shows significant activation by acyl derivatives of coenzyme A with long chain acyl CoA being more effective than acetyl‐CoA. This activation is no longer observed in the presence of high concentrations of pyruvate, MgATP2‐ and HCO−3.

The concentrations of L‐aspartate and 2‐oxoadipate required to give 50% inhibition ([I]0.5), and the maximal extents of inhibition, are increased by addition of acetyl‐CoA. Acetyl‐CoA increases the sigmoidal character of the relationship: initial rate/[L‐aspartate], but decreases this parameter for the relationship: initial rate/ [2‐oxoadipate].

The studies indicate that R. arrhizus possesses an entirely cytosolic pathway for the conversion of glucose to fumaric acid and that both the organisation of pyruvate metabolism and the regulation of pyruvate carboxylase differ significantly in this organism as compared to that proposed previously for Aspergillus nidulans.
ɛ‐Crystallin, a novel avian and reptilian eye lens protein
Authors: STAPEL Steven O. , ZWEERS Anneke , DODEMONT Huub J. , KAN Jaap H. , de JONG Wilfried W.
Published: March 2005

Gel filtration of Peking duck eye lens proteins reveals a component eluting just behind δ‐crystallin and comprising approximately 10% of the total soluble protein. The native Mr of this additional component is estimated to be 120000; it appears to be composed of three identical chains of Mr 38000 and pl 7.5. Circular dichroic spectroscopy showed a relatively high α‐helical content. No immunological cross‐reactivity is found with α‐, β‐, γ‐ or δ‐crystallins, and partial amino acid sequence determinations likewise failed to reveal any similarity with other known crystallins. We conclude that this protein represents another and novel family of eye lens proteins, for which we propose the designation ɛ‐crystallin. ɛ‐Crystallin is translated from a 1450‐base mRNA, which has been partially purified. ɛ‐Crystallin is found scattered among avian and reptilian taxa, but not in other vertebrates. Its rate of evolutionary change seems to be as slow as that of α‐ and β‐crystallins.
Nucleotide sequence analysis of the nuclear gene coding for manganese superoxide dismutase of yeast mitochondria, a gene previously assumed to code for the Rieske iron‐sulphur protein
Authors: MARRES Carla A. M. , VAN LOON Adolphus P. G. M. , OUDSHOORN Peter , VAN STEEG Harry , GRIVELL Leslie A. , SLATER E. C.
Published: March 2005

We have previously reported the isolation of the gene coding for a 25‐kDa polypeptide present in a purified yeast QH2:cytochrome c oxidoreductase preparation, which was thus identified as the gene for the Rieske ironsulphur protein [Van Loon et al. (1983) Gene 26, 261–272]. Subsequent DNA sequence analysis reported here reveals, however, that the encoded protein is in fact manganese superoxide dismutase, a mitochondrial matrix protein. Comparison with the known amino acid sequence of the mature protein indicates that it is synthesized with an N‐terminal extension of 27 amino acids. In common with the N‐terminal extensions of other imported mitochondrial proteins, the presequence has several basic residues but lacks negatively charged residues. The function of these positive charges and other possible topogenic sequences are discussed. Sequences 5′ of the gene contain two elements that may be homologous to the suggested regulatory sites, UAS 1 and UAS 2 in the yeast CYC1‐gene [Guarente et al. (1984) Cell 36, 503–511]. The predicted secondary structures in manganese superoxide dismutase appear to be very similar to those reported for iron superoxide dismutase, suggesting similar three‐dimensional structures. Making use of the known three‐dimensional structure of the Fe enzyme, the Mn ligands are predicted.
Quaternary structure of chloroplast F1‐ATPase in solution
Authors: WAGNER Richard , ENGELBRECHT Siegfried , ANDREO Carlos S.
Published: March 2005

The hydrodynamic properties of isolated ATPases were studied via their rotational diffusion in buffer solution. Chloroplast F1‐ATPase (CF1) and Escherichia coli F1‐ATPase (EF1) were covalently labeled with eosin‐isothiocyanate and then investigated by polarized laser spectroscopy.

The rotational correlation time in aqueous buffer of latent (five‐subunit) CF1 was 390 ns. Four‐subunit (δ‐deficient) CF1 showed the same correlation time, however, for three‐subunit (δ, ɛ‐deficient) CF1 the rotational correlation time was more than eight times larger (3200 ns).

The rotational correlation time of activated CF1 was three times larger than the one of latent CF1. These large changes in the rotational correlation times are directly related to changes in the quaternary structure of CF1 upon activation. EF1 was found to behave essentially as activated CF1.

Based on the observed rotational correlation times we concluded that the mass distributions of latent CF1 and of δ‐deficient CF1 resemble a dimeric arrangement. The structure of δ,ɛ‐deficient CF1 more likely resembles a hexagon, the mass centers of the six main subunits lie in one plane. The structure of the activated forms of CF1 can be described best as an intermediate between the dimeric arrangement of latent CF1 and an octahedron. The large changes in the quaternary structure of isolated CF1 are reversed when the activation of the enzyme is reversed.
Mössbauer investigation of the cofactor iron of putidamonooxin
Authors: BILL Eckhard , BERNHARDT Frithjof‐Hans , TRAUTWEIN Alfred Xaver , WINKLER Heiner
Published: March 2005

Mononuclear non‐heme cofactor iron of putidamonooxin has been investigated in the binary oxidized ‘enzyme · substrate’ complex and in the ternary ‘enzyme · substrate · NO’ complex via Mössbauer spectroscopy. The experimental spectra were analyzed on the basis of the spin‐Hamiltonian formalism. The resulting fine and hyperfine structure parameters are compared with literature values of similar compounds. From this comparison we conclude that in the binary complex (reduced and oxidized) the mononuclear non‐heme cofactor iron has a coordination number higher than four. Additionally, the cofactor iron shows remarkable spectral similarities with iron in protocatechuate 3,4‐dioxygenase, though the catalytic properties of the iron sites in the two proteins are different. The data obtained form the ternary “enzyme · substrate · NO” complex indicate that the cofactor iron (a) is in the ferric intermediate spin state (S= 3/2) and (b) is pentacoordinated, which means that upon NO binding to the reduced cofactor iron at least one ligand has to be released. Comparing our data with literature values suggests that the cofactor iron in the binary as well as in the ternary NO complex is not directly bound to a sulfur atom, though biochemical arguments seem to indicate the opposite.
Influence of dA · dT and d(2aminoA) · dT base pairs on the B⇄Z transition of DNA fragments
Authors: CAVAILLÈS Jean‐Aristide , NEUMANN Jean‐Michel , TRAN‐DINH Son , HUYNH‐DINH Tam , d'ESTAINTOT Bétrice Langlois , IGOLEN Jean
Published: March 2005

The Helical structures of d(C‐G‐C‐A‐m5C‐G‐T‐G‐m5C‐G), d(m5C‐G‐C‐A‐m5C‐G‐T‐G‐C‐G) and d(C‐2aminoA‐C‐G‐T‐G) were studied in aqueous solution at various salt concentrations and temperatures by 1H‐NMR spectroscopy. In 0.1 M NaCl solution only the B form was evidenced for these DNA fragments whereas in 4 M NaCl both B and Z forms, in slow exchange on the NMR time scale, were observed. Under these conditions the Z form accounted for less than 60% of the decamer conformation; conversely d(C‐G)3 hexamers containing methylated cytidines were predominantly in the Z form (>90%) [Tran‐Dinh et al. (1984) Biochemistry 23, 1362; Cavaillès et al. (1984) J. Biomol. Struct. Dyn. 1, 1347–1371]. On the other hand, d(C‐2aminoA‐C‐G‐T‐G) in which the d(2aminoA) · dT base pair forms three hydrogen bonds, was found to adopt the Z conformation in 4M NaCl solution which was not the case for d(C‐A‐C‐G‐T‐G) (unpublished results).

The present study shows that the B⇄Z transition in solution is highly sequence‐dependent and that correlation exists between the stability of the duplexes (essentially governed by the number of hydrogen bonds between complementary bases) and their ability to adopt the Z conformation.
Mitochondrial transcription and processing of transcripts during release from glucose repression in ‘resting cells’ of Saccharomyces cerevisiae
Authors: ZENNARO Elisabetta , GRIMALDI Luca , BALDACCI Giuseppe , FRONTALI Laura
Published: March 2005

Mitochondrial transcription and processing of transcripts have been investigated at different stages of release from glucose repression in resting cells of Saccharomyces cerevisiae.

Transcripts were identified by hybridization with nick‐translated or terminally labelled gene‐specific probes. This allowed the determination of the steady‐state levels of individual transcripts in the mitochondrial RNA population.

Results showed different gene‐specific patterns of response to respiratory induction: no increase in the level of transcripts (oxi2); a rapid increase in the steady‐state levels of all transcripts (cob); a very strong increase in the processing of the high‐molecular‐mass precursors (oxi3 and oli2); an increase in the level of stable circular transcripts (oxi3). As a whole the results indicate specific and differentiated effects of release from glucose repression on the expression of the different mitochondrial genes and demonstrate the importance of processing events in mitochondrial regulation.
Modification of the carbohydrate in ricin with metaperiodate — cyanoborohydride mixtures
Authors: THORPE Philip E. , DETRE Simone I. , FOXWELL Brian M. J. , BROWN Alex N. F. , SKILLETER David N. , WILSON Glynn , FORRESTER J. Anthony , STIRPE Fiorenzo
Published: March 2005

Attempts to target antibody‐ricin conjugates (immunotoxins) to designated cell types in vivo may be thwarted by their rapid clearance by hepatic reticuloendothelial cells which have receptors that recognise oligosaccharide side chains on the toxin. The B‐chain of ricin contains high mannose type oligosaccharides and the A‐chain contains a complex unit (GlcNAc)2‐Fuc‐Xyl‐(Man)4–6, all of which potentially could be recognised by the reticuloendothelial system.

Treatment of ricin with a mixture of sodium metaperiodate and sodium cyanoborohydride at pH 3.5 resulted in oxidative cleavage of the carbohydrates and reduction of the aldehyde groups thus formed to primary alcohols. By conducting the modification procedure at acidic pH, both the possibility of Schiff's base formation between the aldehyde groups and amino groups in the protein and the possibility of non‐specific oxidation of amino acids were minimised. The extent of the carbohydrate modification depended on the duration of treatment, resulting maximally in the destruction of 13 of the 18 mannose residues and of all xylose and fucose.

The toxicity of the modified toxin to cells in culture declined by up to 90% as the carbohydrate was destroyed. This was not due to a reduced ability of the B‐chain to bind to cells or of the A‐chain to inactivate ribosomes. In contrast to the in vitro results, the toxicity of the modified toxin to mice and rats was elevated by up to fourfold. The modification greatly reduced the clearance of the toxin by non‐parenchymal cells in the liver and prevented the damage to hepatic Kupffer and sinusoidal cells and to the red pulp of the spleen that is inflicted by the native toxin. The elevated toxicity to animals appears to be because the modified toxin evades the reticuloendothelial system and persists in the bloodstream for longer periods, thus resulting in lethal damage to vital tissues in the animal at lower dosage.

The results suggest that immunotoxins prepared from modified ricin would not be readily cleared by the reticuloendothelial system and so be more effective at killing their target cells.
Amino acid sequence of the non‐collagenous globular domain (NC1) of the α1 (IV) chain of basement membrane collagen as derived from complementary DNA
Authors: OBERBÄUMER Ilse , LAURENT Maryvonne , SCHWARZ Ulla , SAKURAI Yoshiki , YAMADA Yoshihiko , VOGELI Gabriel , VOSS Tilman , SIEBOLD Bernhard , GLANVILLE Robert W. , KÜHN Klaus
Published: March 2005

NC1, the C‐terminal non‐collagenous globular domain of collagen IV, represents one of the two end regions responsible for the assembly and cross‐linking of the extracellular network of basement membrane collagen. Several cDNA clones for the NC1 domain of the α1(IV) collagen chain of mouse have been isolated by using synthetic oligonucleotides as screening probes for mouse libraries. The oligonucleotides were synthesized according to known stretches of the corresponding protein sequence. Sequencing of the overlapping cDNA clones allowed the complete amino acid sequence of the NC1 domain to be deduced as well as the C‐terminal 165 amino acid residues of the triple helix. It consists of 229 amino acid residues which comprise two homologous regions with a high content of cysteine. These DNA and protein sequences are compared to the corresponding sequences of other collagens and discussed with respect to their structural and biological significance.
Isolation and characterization of cytoplasmic poly(A) polymerase from cryptobiotic gastrulae of Artemia salina
Authors: ROGGEN Erwin , SLEGERS Herman
Published: March 2005

Poly(A) polymerase has been purified to near homogeneity from the cytoplasm of Artemia salina cryptobiotic gastrulae by ion‐exchange chromatography on DEAE‐cellulose, DEAE‐Sepharose CL‐6B and phosphocellulose P11, gel filtration on CL‐Sepharose 6B, affinity chromatography on poly(A)‐Sepharose 4B and ATP‐agarose. The enzyme is fully dependent on exogeneous oligo(riboadenylic acid) and is free of any nuclease or other enzyme activities. In standard assay conditions the enzyme preparation has a specific activity of 5.6 μmol AMP · h−1· (mg protein)−1.

Sodium dodecyl sulphate/polyacrylamide gel electrophoresis reveals the presence of only two proteins with Mr 94000 and 70000. The Mr‐70000 protein has been identified as poly(A) polymerase.

The enzyme is exclusively activated by Mn2+. Addition of Ca2+, Mg2+, Zn2+, NH+4, K+ or Na+ inhibits the enzymatic reaction. The activity is specific for ATP and competitive inhibition is observed in the presence of other ribonucleoside 5′‐triphosphates. AMP incorporation is time‐dependent and is increased non‐linearly with protein and primer concentration.
The primary structure of α‐lactalbumin from camel milk
Authors: BEG Obaid Ullah , von BAHR‐LINDSTRÖM Hedvig , ZAIDI Zafar H. , JÖRNVALL Hans
Published: March 2005

The primary structure of camel α‐lactalbumin was determined by analysis of the intact protein, and of CNBr fragments and enzymatic peptides from the carboxymethylated protein chain. Results show that camel α‐lactalbumin has 123 residues and a molecular mass of 14.6 kDa. The amino acid sequence is strictly homologous to α‐lactalbumins characterized, but also exhibits extensive differences: 39 residues differ in relation to the bovine protein and only 35 residues are conserved among hitherto known α‐lactalbumins with characterized structures. All residues ascribed critical structural or functional roles are strictly invariant in the camel protein.
Coenzymic activity of NADP derivatives alkylated at 2′‐phosphate and 6‐amino groups
Authors: OKUDA Keiko , URABE Itaru , OKADA Hirosuke
Published: March 2005

Coenzymic activities of the following NADP derivatives were investigated: 2′‐O‐(2‐carboxyethyl)phosphono‐NAD (I), N6‐(2‐carboxyethyl)‐NADP (II), 2′‐O‐(2‐carboxyethyl)phosphono‐N6‐(2‐carboxyethyl)‐NAD (III), 2′‐O‐[N‐(2‐aminoethyl)carbamoylethyl]phosphono‐NAD (IV), N6‐[N‐(2‐aminoethyl)carbamoylethyl]‐NADP (Va), 2′, 3′‐cyclic NADP, and 3′‐NADP. Derivatives I and IV show the effects of modification at the 2′‐phosphate group, and derivatives II and Va show those at the 6‐amino group of NADP. As for enzymes, alcohol, isocitrate, 6‐phosphogluconate, glucose, glucose‐6‐phosphate, and glutamate dehydrogenases were used. These enzymes were grouped, and derivatives II and Va show those at the 6‐amino group of NADP. As for enzymes, alcohol, isocitrate, 6‐phosphogluconate, glucose, glucose‐6‐phosphate, and glutamate dehydrogenases were used. These enzymes were grouped on the basis of the ratio of the activities for NAD and NADP into NADP‐specific enzymes (ratio <0.01), NAD(P)‐specific enzymes (0.01 < ratio < 100), and NAD‐specific enzymes (ratio > 100).

For NADP‐specific enzymes, modifications at the 2′‐phosphate group of NADP caused great loss of cofactor activity. The relative cofactor activities (NADP = 100%) of derivatives I and IV for these enzymes were 0.5–20 and 0.01 – 0.5%, respectively. On the other hand, NAD(P)‐specific enzymes showed several types of responses to the NADP derivatives. The relative cofactor activities of I and IV for Leuconostoc mesenteroides and Bacillus stearothermophilus glucose‐6‐phosphate dehydrogenases and beef liver glutamate dehydrogenase were 60 – 200%; whereas, for B. megaterium glucose dehydrogenase and L. mesenteroides alcohol dehydrogenase, the values were 0.8–8%. For NAD‐specific enzymes, these values were 20–50%. The relative cofactor activities of 2′,3′‐cyclic NADP and 3′‐NADP were very low (less than 0.2%) except for beef liver glutamate dehydrogenase, B. stearothermophilus glucose‐6‐phosphate dehydrogenase, and horse liver alcohol dehydrogenase. Kinetic studies showed that the losses of the cofactor activity of NADP by these modifications were mainly due to the increase of the Km value. The mechanisms of coenzyme specificity of dehydrogenases are discussed.

Unlike the 2′‐phosphate group, the 6‐amino group is common to NAD and NADP, and the effects of modification at the 6‐amino group were independent of the coenzyme specificity of enzymes used for the assay. Derivatives II and Va had high relative cofactor activities (65 – 130%) for most of the enzymes except for isocitrate and glucose dehydrogenases (less than 1%) and L. mesenteroides alcohol dehydrogenase (20–60%). The cofactor activity of derivative III was generally lower than those of I and II.
Intracellular maturation and secretion of acid phosphatase of Saccharomyces cerevisiae
Authors: SCHÖNHOLZER Fred , SCHWEINGRUBER Anne‐Marie , TRACHSEL Hans , SCHWEINGRUBER M. Ernst
Published: March 2005

To elucidate intracellular maturation and secretion of acid phosphatase of Saccharomyces cerevisiae we prepared a monoclonal antibody that recognizes specifically the protein moiety of this cell surface glycoprotein. With this antibody membranes and soluble fractions of wild‐type cells, grown in low‐phosphate medium in the presence and absence of tunicamycin, were examined by the immunoblot technique. Similarly, secretory mutants, blocked at distinct steps in the secretory pathway at the restrictive temperature as well as a strain harboring several copies of the structural gene PHO5 for repressible acid phosphatase, were analyzed. The data suggest the following sequence of events in acid phosphatase maturation and secretion: three unglycosylated precursors with molecular masses of 60 kDa, 58 kDa and 56 kDa are synthesized into membranes of the endoplasmic reticulum, where these are core glycosylated in a membrane‐bound form. They appear on sodium dodecyl sulfate gels as bands with molecular masses of 76 kDa and 80 kDa. Owing to a rate‐limiting maturation step, occurring after core glycosylation, they can accumulate in a membrane‐bound form. At the Golgi apparatus outer carbohydrate chains are attached to the core and the enzyme appears in a soluble form, indicating a release of acid phosphatase from the membrane between the endoplasmic reticulum and the Golgi. Pulse‐chase experiments suggest that the time for acid phosphatase synthesis and its transport to the Golgi is about 5 min.
Further characterisation of the FAD and Fe2S2 redox centres of component C, the NADH: acceptor reductase of the soluble methane monooxygenase of Methylococcus capsulatus (Bath)
Authors: LUND John , DALTON Howard
Published: March 2005

The absorbance contributions of the FAD and Fe2S2 redox centres of component C of the soluble methane monooxygenase complex have been resolved, using mersalyl to destroy the Fe2S2 centre. The Fe2S2 seems to be very similar to that of spinach ferredoxin, by its absorbance and electron paramagnetic resonance (EPR) spectra, and the FAD semiquinone is a neutral semiquinone. Spectrophotometry near room temperature and EPR spectroscopy near liquid‐helium temperature allow the three redox couples of component C to be ordered. Component C can exist in Oe−1 (oxidised), 1e−1 (semiquinone), 2e−1 (mostly semiquinone and reduced Fe2S2), and 3e−1 forms (dihydroquinone and reduced Fe2S2), under equilibrium conditions. The ability of component C to support odd‐electron forms is consistent with its proposed role as a 2e−1/1e−1 transformase, splitting electron pairs from NADH for passage to component A in one‐electron steps. (The FAD appears to interact with NADH, and transfers single electrons to the Fe2S2, for donation to component A at a constant redox potential.)

The mid‐point potentials of component C were found using redox dyes and EPR spectroscopy and were: FAD/FAD·, Em= ‐150 mV; Fe2S2/Fe2·S2, Em=−220 mV; FAD·/FAD·, Em=−260 mV. the presence of NADH did not alter these mid‐point potentials. These mid‐point potentials are consistent with the role of component C as the NADH:component A reductase, passing electrons from NADH (Em=−320 mV) onto component A (Em=+150 m V and Em=−150 m V). The reducing power from NADH appears to be required by component A to acitvate one atom of oxygen, to insert into methane, and the reducing equivalents derived from NADH end up with the other oxygen atom, as water.
Location of the adenylylation site in T4 RNA ligase
Authors: THØGERSEN Hans C. , MORRIS Howard R. , RAND Keith N. , GAIT Michael J.
Published: March 2005

The purification of the enzyme T4 RNA ligase is described from an Escherichia coli strain, KR 54, in which the RNA ligase gene (g63) has been inserted into the plasmid pDR540 for inducible expression of g63 from the tac promoter. Adenylylation of the purified enzyme with [14C]rATP followed by digestion with chymotrypsin yielded an adenylylated peptide, the identity of which was determined by fast‐atom‐bombardment mass spectrometric analysis. The results show that the AMP residue is bound covalently to the lysine at position 99 of the RNA ligase protein sequence.
Characterization and primary structures of DNA‐binding HU‐type proteins from Rhizobiaceae
Authors: KHANAKA Hussain , LAINE Bernard , SAUTIERE Pierre , GUILLAUME Jean
Published: March 2005

The DNA‐binding HU‐type proteins from several species of Rhizobiaceae including Rhizobium meliloti, two strains of Rhizobium leguminosarum with highly different phenotypic characters and Agrobacterium tumefaciens, were characterized and their amino acid sequences were determined.

HU‐type proteins isolated from R. leguminosarum L18 and A. tumefaciens are identical and show slight differences with the R. meliloti HU‐type protein. On the other hand the R. leguminosarum L53 HU‐type protein is quite different from the proteins cited above; several amino acid substitutions encountered in this protein result in significant changes in the folding of the polypeptide chain.

The biochemical characteristics of these proteins are in good agreement with the respective position of these bacteria in the phylogeny determined by numerical taxonomy.
Primary structure of histone H2B from gonads of the starfish Asterias rubens
Authors: MARTINAGE Arlette , BRIAND Gilbert , VAN DORSSELAER Alain , TURNER Christopher H. , SAUTIERE Pierre
Published: March 2005

The complete amino acid sequence (121 residues) of histone H2B from gonads of the starfish Asterias rubens has been established from structural data obtained essentially from large fragments generated by cleavage of histone H2B at aspartyl residues and by limited hydrolysis of the dimer H2A‐H2B with mouse submaxillary gland protease.

No real sequence homology can be found between the amino‐terminal sequence (residues 1–21) of starfish and calf H2B. One non‐conservative substitution (serine‐32 in calf → lysine‐28 in starfish) leads to the presence of a cluster of eight basic residues (sequence 23 – 30) and to the disappearance of a potential site of phosphorylation.

A particular structural feature of starfish histone H2B is the presence of N‐dimethylproline at its aminoterminal end. By comparison with N‐terminal acetylation, which is commonly found in histones, N‐terminal methylation is rarely observed. At the present time the functional significance of the N‐terminal methylation as well as that of the proline‐rich nature of the amino‐terminal sequence of the starfish histone H2B remain to be defined.
cGMP‐dependent protein kinase
Authors: HOFMANN Franz , GENSHEIMER Hans‐Peter , GÖBEL Claus
Published: March 2005

cGMP‐dependent protein kinase binds 4 mol cGMP/mol enzyme to two different sites. Binding to site 1 (apparent Kd 17 nM) shows positive cooperativity and is inhibited by Mg · ATP, whereas binding to site 2 (apparent Kd 100 – 150 nM) is non‐cooperative and not affected by Mg · ATP. Autophosphorylation of the enzyme abolishes the cooperative binding to site 1 and the inhibitory effect of Mg · ATP. The association (K1) and dissociation (K−1) rate constant for site 2 and K1 for site 1 are not affected significantly by Mg · ATP or autophosphorylation. The dissociation rate from site 1 measured in the presence of 1 mM unlabelled cGMP is decreased threefold and over tenfold by Mg · ATP and autophosphorylation, respectively. In contrast, the dissociation rate from site 1 measured after a 500‐fold dilution of the enzyme‐ligand complex is 100‐fold faster than that determined in the presence of 1 mM cGMP and is only slightly influenced by Mg · ATP or autophosphorylation. Only Kd values calculated with the latter K−1 vlalues are similar to the Kd values obtained by equilibrium binding. These results suggest that autophosphorylation of cGMP‐dependent protein kinase affects mainly the binding characteristics of site 1.
Purification and characterization of the low‐molecular‐mass (type X) collagen from chick‐embryo tibial cartilage
Authors: QUARTO Natalina , CANCEDDA Ranieri , DESCALZI‐CANCEDDA Fiorella
Published: March 2005

Type X collagen, synthesized in large amount by cultured tibial chondrocytes, is deposited in vivo in the epiphyseal cartilages of 17‐day‐old chick embryo tibiae. Here we report the extraction of this collagen from these cartilages by limited pepsin digestion and its purification to electrophoretic homogeneity by salt precipitation followed by agarose gel filtration. Identity of the collagen purified from cartilage with the type X collagen synthesized by cultured chondrocytes is confirmed by comparison of the amino acid compositions. The high glycosylation extent of type X collagen is reminiscent of the glycosylation extent of pericellular collagens. The possible role of type X collagen is discussed.
Intracellular calcium fluxes in human platelets
Authors: THOMPSON Neil T. , SCRUTTON Michael C.
Published: March 2005

Fluorescence changes and secretory recretory responses have been measured on addition of various excitatory agonists to platelets loaded with the cytosolic Ca2+ probe, Quin 2 or with chlortetracycline as a probe for membraneassociated Ca2+.

When extracellular [Ca2+] is decreased to less than 0.1 μM by addition of EGTA a linear correlation is observed between the extent of increase in cytosolic [Ca2+] and the extent of mobilisation of membrane‐associated Ca2+ on stimulation by maximal doses of five excitatory agonists. A similar linear correlation between the increase in cytosolic [Ca2+] and the extent of ATP secretion is observed over the thrombin dose/response curve.

Similar EC50 values are observed for ATP secretion, the increase in cytosolic [Ca2+] and the decrease in chlortetracycline fluorescence induced by thrombin. However, the decrease in chlortetracycline fluorescence shows a sigmoidal relationship with the increase in cytosolic [Ca2+] and a hyperbolic relationship with ATP secretion over this dose/response curve.

Addition of prostaglandin D2 prior to thrombin causes parallel inhibition of the increase in cytosolic [Ca2+] and the decrease in chlortetracycline fluorescence induced by this agonist. However, addition of prostaglandin D2 after thrombin reverses the increase in cytosolic [Ca2+] induced by this agonist but fails to cause a similar reversal of the decrease in chlortetracycline fluorescence.

The data provide further evidence supporting the proposal that chlortatracycline can be used as a probe to monitor mobilisation of membrane‐associated Ca2+ but suggest that, in platelets stimulated in the effective absence of extracellular Ca2+, both Ca2+ mobilisation and Ca2+ removal can under some conditions involve sites which are not monitored by this probe.
Specific involvement of calmodulin and non‐specific effect of tropomyosin in the sensitivity to ouabain of Na+, K+‐ATPase in murine plasmocytoma cells
Authors: LELIEVRE Lionel G. , POTTER James D. , PIASCIK Michael , WALLICK Earl T. , SCHWARTZ Arnold , CHARLEMAGNE Daniele , GENY Blandine
Published: March 2005

The Kd for ouabain for inhibition of Na+, K+‐ATPase isolated from murine plasmocytoma MOPC 173 cells is 120 μM, but when isolated in the presence of EDTA, it is 100‐fold lower (1.2 μM). Simultaneous addition of muscle tropomyosin and calcium to sensitive membranes restored the original insensitivity (tropomyosin bound to the membranes in an irreversible and saturable manner).

For comparison 86Rb influx into intact cells, mediated by the Na+, K+‐pump, is half‐maximally inhibited at 50 μM ouabain.

Calcium converts the enzyme to an insensitive form. This appeared to involve calmodulin because after extraction of calmodulin with EDTA and EGTA from sensitive membranes, they could not be made insensitive by the addition of tropomyosin and Ca2+. Addition of exogenous calmodulin to these calmodulin‐depleted membranes was required, in addition to tropomyosin and Ca2+, to decrease the ouabain sensitivity.

The involvement of calmodulin was further assessed by measuring the range of Ca2+ concentrations required to convert to the insensitive form. At saturating concentrations of tropomyosin, increasing free [Ca2+] up to 3 μM led to an heterogenous population of Na+, K+‐ATPase forms. The calcium dependency was a saturable process. The shift to the insensitive form was half maximal at 0.65 + 0.11 μM free Ca2+ and was abolished by the addition of troponin I or trifluoroperazine (0.1 mM).

These results suggest that, in murine plasmocytoma cells, the intrinsic sensitivity of Na+, K+‐ATPase to ouabain might be regulated by a calmodulin‐dependent process within a submembrane contractile‐like environment.
Transport of β‐globin mRNA from nuclei of murine friend erythroleukemia cells
Authors: KINDÅS‐MÜGGE Ingela , SAUERMANN Georg
Published: March 2005

An in vitro assay system for analysis of β‐globin mRNA transport is described. Nuclei isolated from murine Friend erythroleukemia cells induced to synthesize globin mRNA, were incubated in micro‐assays. By electrophoresis and hybridization analysis, released 9‐S β‐globin mRNA was shown to be undegraded. After direct blotting, the released mRNA was quantified by hybridization with a labeled plasmid containing a β‐globin DNA restriction fragment. The inducibility of β‐globin mRNA transport corresponded to that previously reported for the release of rapidly labeled RNA in other assay systems. In contrast to the ineffectiveness of high concentrations of the sulfhydryl reagent iodoacetate, low concentrations of the oxidizing sulfhydryl reagent, o‐iodosobenzoate, inhibited the release of β‐globin mRNA from nuclei of erythroleukemia cells, as well as the release of rapidly labeled RNA from rat liver nuclei. The inhibitory effect of the oxidizing agent on β‐globin mRNA transport could be reversed by postincubation of the nuclei with the reducing agent, dithiothreitol. The potential role of disulfide bond formation on RNA transport is discussed.
The widespread distribution of α‐N‐trimethylalanine as the N‐terminal amino acid of light chains from vertebrate striated muscle myosins
Authors: HENRY Gillian D. , TRAYER Ian P. , BREWER Susan , LEVINE Barry A.
Published: March 2005

Identical tripeptides of the sequence X‐Pro‐Lys, where X is an unknown blocking group, were isolated from trypsin digests of bovine cardiac alkali light chain and the LC2 light chain of rabbit fast muscle. Chemical, electrophoretic and 1H‐NMR evidence characterized X as an unusual amino acid, α‐N‐trimethylalanine (Me3Ala), which was earlier reported as the N‐terminal amino acid of the A1 alkali light chain of rabbit fast muscle [Henry et al. (1982) FEBS Lett. 144, 11–15]. The narrow line width and chemical shift position (δ= 3.23 ppm) of the –N+ ‐(CH3) protons of Me3Ala made 1H‐NMR spectroscopy a convenient method to search for this residue in other light chains. A survey of many different light chains showed that this signal was present in all vertebrate striated muscle light chains of the A1‐type (LC1, ‘essential’ light chains) and LC2‐type (‘DTNB’‐light chains, ‘phosphorylatable’ light chains) but was absent from all invertebrate muscle and vertebrate smooth muscle light chains tested. It was also absent from the vertebrate fast‐muscle‐specific A2‐type (LC3) light chains. The spectral characteristics of these signals were consistent with their having arisen from the protons of an – N+ ‐(CH3)3 grouping. Since no ɛ‐trimethyllysine could be detected in acid hydrolysates of these proteins, it appears that Me3Ala is a general feature as the N‐terminal amino acid in these light chains.

1H‐NMR studies on bovine cardiac myosin subfragment 1 (S1) showed that the Me3Ala methyl proton signal was clearly visible and that the spectrum more closely resembled that of a rabbit S1 isoenzyme, S1(A1), than S1(A2), suggesting that the 40‐residue N‐terminal segment of the alkali light chain in cardiac S1 also possesses a high segmental mobility. Addition of actin caused the same gross changes to the cardiac S1 spectrum as noted earlier for rabbit S1(A1) [Prince et al. (1981) Eur. J. Biochem. 121, 213–219]. In particular, a marked reduction in the segmental mobility of the N‐terminal region of the alkali light chain was noted, consistent with a direct interaction of this area with actin.
Purification of GTP:α‐d‐mannose‐1‐phosphate guanyltransferase
Authors: SMOOT Jeffrey W. , SERIF George S.
Published: March 2005

The enzyme GTP:α‐d‐mannose‐1‐phosphate guanylyltransferase from porcine thyroid tissue has been purified 69 900‐fold on columns of blue‐Sepharose, DEAE‐Sepharose, phenyl‐Sepharose and agarose‐GTP affinity materials. Although it exhibits a tendency to aggregate, the enzyme travelled, upon sucrose velocity sedimentation, as a single oligomer with a molecular mass of 412 kDa. Michaelis constants were determined to be 1.0 μM, 1.0 mM, 3.5 μM and 0.4 μM for GDP‐α‐d‐mannose, pyrophosphate, GTP and mannose‐1‐phosphate, respectively. The enzyme appears to be specific for the mannose moiety but will accept an inosine replacement for guanine and a deoxyribose replacement for ribose in GTP.
Is coenzyme M bound to factor F430 in methanogenic bacteria?
Authors: HÜSTER Rudolf , GILLES Hans‐Harald , THAUER Rudolf K.
Published: March 2005

Coenzyme M (2‐mercaptoethane sulfonic acid) and factor F430 (a nickel porphinoid) are coenzymes found in methanogenic bacteria. Recently it has been proposed that in these bacteria a coenzyme MF430 also exists which plays a key role in methane formation and in which coenzyme M and F430 are bound to each other. To test this hypothesis Methanobrevibacter ruminantium, which requires coenzyme M as a vitamin, was grown in the presence of [2‐14C]CoMSH. F430 and ‘CoM’ (mixture of CoMSH and its disulfides) were quantitatively extracted from these cells and from partially purified methyl‐CoM reductase using various methods. The extracts were chromatographed on cellulose or Sephadex G‐10. Under all conditions factor F430 and ‘CoM’ were completely (>99%) separated. There was no indication for the existence of a protein‐free F430 species containing covalently bound coenzyme M in Mb. ruminantium. The results support the structure previously assigned to coenzyme F430.
The binding of vanadium (V) oligoanions to sarcoplasmic reticulum
Authors: VARGA Sandor , CSERMELY Peter , MARTONOSI Anthony
Published: March 2005

The binding of monovanadate and decavanadate anions to sarcoplasmic reticulum vesicles was measured by equilibrium sedimentation. The affinity of vanadate binding and the molar amount of vanadium (V) bound at equilibrium is much greater with decavanadate than with monovanadate. The binding data can be rationalized in terms of one binding site per ATPase molecule for monovanadate and two sites per ATPase for decavanadate.

The Ca‐ATPase crystals formed with monovanadate and with decavanadate are similar in appearance, but decavanadate is particularly effective in promoting the crystallization of Ca2+‐ATPase at low V concentration (10–100 μM) in a Ca2+‐free medium.
Chemical modifications of the Na+– H+ antiport in Escherichia coli membrane vesicles
Authors: DAMIANO Evelyne , BASSILANA Martine , LEBLANC Gérard
Published: March 2005

The effects of chemical modifications of the Na+– H+ antiport in Escherichia coli have been analyzed by studying the resulting variations of the energy‐dependent, downhill Na+ efflux from membrane vesicles. The histidyl reagent diethylpyrocarbonate (EtO)2C2O3 prevents the activation of the Na+ efflux mechanism by ΔH+ or its components. Inactivation of the antiporter by (EtO)2C2O3 is completely reversed by hydroxylamine. The data suggest that histidine residues are involved in the molecular mechanism of the Na+– H+ antiport. In contrast, no conclusive evidence suggesting participation of carboxylic, tyrosine or sulfhydryl residues in the Na+– H+ exchange reaction has been obtained.
Control of ureogenesis
Authors: MEIJER Alfred J. , LOF Cor , RAMOS Inès C. , VERHOEVEN Arthur J.
Published: March 2005

Control of urea synthesis was studied in rat hepatocytes incubated with physiological mixtures of amino acids in which arginine was replaced by equimolar amounts of ornithine. The following observations were made.

Intramitochondrial carbamoyl phosphate was always below 0.1 mM. Only when ornithine was absent and when, in addition, the concentration of amino acids was higher than four times their plasma concentration, intramitochondrial carbamoyl phosphate rose up to about 3 mM; under these conditions ammonia accumulated in the medium.

The relationship between ornithine‐cycle flux and the concentration of the cycle intermediates at varying amino acid concentration indicated that under near‐physiological conditions the ornithine‐cycle enzymes are far from being saturated with their substrates.

Moderate concentrations of norvaline had no effect on the rate of urea synthesis unless the cells were severely depleted of ornithine.

Activation of carbamoyl‐phosphate synthetase (ammonia) by addition of N‐carbamoylglutamate only slightly stimulated urea production at all amino acid concentrations. However, in the presence of the activator the curve relating ornithine‐cycle flux to the steady‐state ammonia concentration was shifted to lower concentrations of ammonia.

The intramitochondrial concentration of carbamoyl phosphate in rat liver in vivo was below 0.1 mM. This value is far below the concentration required for substantial inhibition of carbamoyl‐phosphate synthetase.

It is concluded that in vivo the function of activity changes in carbamoyl‐phosphate synthetase, via the well‐documented alterations in the intramitochondrial concentration of N‐acetylglutamate, is to buffer the intrahepatic ammonia concentration rather than to affect urea production per se. At constant concentration of ammonia the rate of urea production is entirely controlled by the activity of carbamoyl‐phosphate synthetase.
Interaction of fibronectin with C1q and collagen
Authors: INGHAM Kenneth C. , LANDWEHR Ruth , ENGEL Jürgen
Published: March 2005

By attaching native collagen and C1q to Sepharose, it was possible to test the binding of fibronectin (Fn) to the native and heat‐denatured forms of these proteins without complications due to aggregation, precipitation, or fibril formation. Binding to the native proteins occurred only at low (sub‐physiological) ionic strength whereas binding to the denatured proteins occurred even in 1 M NaCl. Thus both of these proteins possess one or more strong sites which are masked in the native state and become exposed during thermal denaturation. Fn did not bind to albumin‐Sepharose or IgG‐Sepharose either before or after heat‐denaturation. C1q bound readily to native IgG‐Sepharose but did not mediate the binding of Fn. Nor did Fn inhibit the reconstitution of C1 on antibody‐coated erythrocytes. The fluorescence polarization of fluorescein‐labeled collagen in 1 M NaCl displayed a downward transition at 38–40°C consistent with unfolding of the triple helix. In the presence of Fn, the same material displayed an upward transition at slightly lower temperature suggesting that gross unfolding is not required to expose the strong binding site(s).
NADPH oxidation catalyzed by the peroxidase/H2O2 system
Authors: VIRION Alain , MICHOT Jean Luc , DEME Danièle , POMMIER Jacques
Published: March 2005

Oxidation of NADPH catalyzed by the peroxidase/H2O2 system is known to require the presence of mediating molecules. Using either lactoperoxidase or horseradish peroxidase, we demonstrated that in the peroxidase/H2O2 system, NADPH oxidation was mediated by iodide. The oxidation product was the iodinated NADP. This product was shown to possess spectral characteristics different from those of NADP+ and NADPH, since for iodinated NADP, increased absorbance was observed in the 280‐nm region and was directly proportional to the rate of iodination. It is suggested that oxidation and iodination of NADPH proceed via a single reaction between the intermediary iodide oxidation species and NADPH. Experiments with different molecules of NADPH analogues indicated that iodination occurred in the nicotinamide part of the NADPH molecule.