bluuebunny/crossref_metadata_2025 huggingface dataset:
Specific binding of human fibrinogen to cultured human fibroblasts
Authors: DEJANA Elisabetta , VERGARA‐DAUDEN Margarita , BALCONI Giovanna , PIETRA Attilio , CHEREL Ghislaine , DONATI Maria Benedetta , LARRIEU Marie‐José , MARGUERIE Gérard
Published: March 2005

Highly purified human fibrinogen was labelled with radioactive iodine and its interaction with cultured human embryo lung fibroblasts (MRC5) was examined. The cell monolayer was incubated at 37°C with 125I‐fibrinogen in phosphate/saline buffer containing 1 mM Ca2+ and 1 mM Mg2+. A direct interaction between 125I‐fibrinogen and MRC5 was observed. The binding was time‐dependent, reached saturation at 10 min and was regulated by the density of the cell monolayer. Non‐labelled fibrinogen inhibited the interaction but unrelated proteins, including fibronectin, ovalbumin or myoglobin, did not. Monospecific Fab fragments, directed to fibrinogen, inhibited binding by 55.3% at a 50/1 molar ratio while non‐immune Fab produced a 1.5% inhibition at similar concentration. Autoradiography of the display of fibroblast‐bound 125I on a 7.5% polyacrylamide gel showed that the extract exhibited electrophoretic bands characteristic of the constitutive B β and γ chains of the fibrinogen molecule. An apparent affinity constant, Ka= 6.7 ± 0.2 × 106 M−1, was estimated from binding isotherms. After a 30‐min incubation time the interaction between 125I‐fibrinogen and the cells was completely reversible and displaceable by a large molar excess of non‐labelled fibrinogen. When compared to fibroblasts (MRC5 or W138), cultures of human embryo epithelial cells (EUE) failed to interact with 125I‐fibrinogen, providing evidence for the specificity of binding for fibroblast monolayers. Plasmin‐degradation fragments D and E, of 100000 and 50000 relative molecular mass respectively, were tested for their capacity to inhibit fibrinogen binding. At a 1/400 125I‐fibrinogen/fragment molar ratio, fragment E inhibited binding by 30% while fragment D produced a 3% inhibition only. Altogether, the results demonstrate that human fibroblasts possess specific binding sites for fibrinogen, which exhibit the characteristics of a receptor system regulated by the culture state of the cells. In addition the structural features, which are necessary for the interaction of fibrinogen with the cells, are probably located in the E domain of the molecule.
Sorbitol dehydrogenase
Authors: JEFFERY Jonathan , CEDERLUND Ella , JÖRNVALL Hans
Published: March 2005

The first primary structure for a sorbitol dehydrogenase has been determined by analysis of the tetrameric enzyme from sheep liver. The [14C]carboxymethylated protein was cleaved with CNBr and proteolytic enzymes. Peptides were purified by several methods, often utilizing exclusion chromatography for pre‐fractionation and reverse‐phase high‐performance liquid chromatography for final purification. Different methods of sequence analysis complemented each other, mainly the manual dimethylaminoazobenzene isothiocyanate method and the use of liquid‐phase sequencer degradations.

All eight major CNBr fragments were purified and form the basis of the work. Three minor CNBr fragments derived from an acid cleavage and from a partly resistant Met‐Thr bond were also obtained, as well as evidence for a contaminating homologous polypeptide. Most of the tryptic peptides were purified, including all with methionine residues, thus overlapping the CNBr fragments. Combined, all data permit the deduction of a 354‐residue amino acid sequence for the polypeptide chain of sorbitol dehydrogenase. The N terminus is acyl‐blocked, the C terminus is formed by a proline residue, tryptophan is the least common residue (two, at positions 50 and 301) and there are 10 cysteine residues, including the residue previously shown to be especially reactive (at postion 43). Similarities to ‘long’ alcohol dehydrogenases have functional implications.
Extensive variations and basic features in the alcohol dehydrogenase – sorbitol dehydrogenase family
Authors: JÖRNVALL Hans , von BAHR‐LINDSTRÖM Hedvig , JEFFERY Jonathan
Published: March 2005

Structural comparisons of sorbitol dehydrogenase with zinc‐containing ‘long’ alcohol dehydrogenases reveal distant but clear relationships. An alignment suggests 93 positional identities with horse liver alcohol dehydrogenase (25% of 374 positions) and 73 identities with yeast alcohol dehydrogenase (20%). Sorbitol dehydrogenase forms a link between these distantly related alcohol dehydrogenases and is in some regions more similar to one of them that they are to each other. 43 residues (11%) are common to all three enzymes and include a heavy over‐representation of glycine (half of all glycine residues in sorbitol dehydrogenase), showing the importance of space restrictions in protein structures.

Four regions are well conserved, two in each domain of horse liver alcohol dehydrogenase. They are two segments close to the active‐site zinc atom of the catalytic domain, and two in the central β‐pleated sheet strands of the coenzyme‐binding domain. These similarities demonstrate the general importance of internal and central building units in proteins.

Large variations affect a region adjacent to the third protein ligand to the active‐site zinc atom in horse liver alcohol dehydrogenase. Such changes at active sites of related enzymes are unusual. Other large differences concern the segment around the non‐catalytic zinc atom of horse liver alcohol dehydrogenase; three of its four cysteine ligands are absent from sorbitol dehydrogenase. Three segments with several exchanges correspond to a continuous region with superficial areas, inter‐domain contacts and inter‐subunit interactions in the catalytic domain of alcohol dehydrogenase. They may correlate with the altered quaternary structure of sorbitol dehydrogenase. Regions corresponding to top and bottom β‐strands in the coenzyme‐binding domain of the alcohol dehydrogenase are also little conserved. Within sorbitol dehydrogenase, a large segment shows an internal similarity.

The two distantly related alcohol dehydrogenases and sorbitol dehydrogenase form a triplet of enzymes illustrating basic protein relationships. They are ancestrally close enough to establish similarities, yet sufficiently divergent to illustrate changes in all but fundamental properties.
The influence of sequences adjacent to the recognition site on the cleavage of oligodeoxynucleotides by the EcoRI endonuclease
Authors: ALVES Jürgen , PINGOUD Alfred , HAUPT Wolfgang , LANGOWSKI Jörg , PETERS Frens , MAASS Günter , WOLFF Carsten
Published: March 2005

We have investigated the influence of the nucleotide sequence adjacent to the recognition site on the rate of cleavage of DNA by the restriction endonuclease EcoRI. For this purpose two decadeoxynucleotides, d(G‐G‐G‐A‐A‐T‐T‐C‐T‐T) (Ia) and d(A‐A‐G‐A‐A‐T‐T‐C‐C‐C) (Ib) were synthesized. The duplex Ia · Ib is cleaved by EcoRI preferentially in the dA‐rich strand (approximately 10 times over the dG‐rich strand). The individual nucleotides Ia and Ib are also cleaved by EcoRI, Ib at a higher rate than Ia and both at a lower rate than Ia · Ib. The temperature dependence of the reaction rate shows that only double‐stranded oligodeoxynucleotides are substrates for the EcoRI endonuclease. We have, furthermore, synthesized oligomers of d(G‐G‐A‐A‐T‐T‐C‐C), which contain two, three and four EcoRI sites, respectively. These oligodeoxynucleotides are preferentially cleaved at the sites next to the 5′ end, where the recognition site is only flanked by one dG · dC base pair, in contrast to the other sites which are flanked by three such pairs. These data indicate that sequences adjacent to the recognition site influence the rate of cleavage: dA · dT base pairs enhance and dG · dC base pairs slow down the hydrolytic activity of the EcoRI endonuclease.
Interaction of human α1‐antichymotrypsin with chymotrypsin
Authors: LAINE Anne , DAVRIL Monique , HAYEM Annette
Published: March 2005

Human α1‐antichymotrypsin reacts with bovine chymotrypsin to form an equimolar complex and this reaction is accompanied by the formation of a free, modified form of the inhibitor. Time‐course studies, performed on mixtures containing an excess of native inhibitor and kept at 0°C or at 25°C, show that the equimolar complex dissociates spontaneously; this dissociation results in the release of inactive modified α1‐antichymotrypsin and of some active enzyme, which is able to recycle with active inhibitor in excess. When all the native inhibitor is used up, the released active enzyme degrades the remaining intact complex into intermediate forms. At the endpoint of the reaction only inactive modified inhibitor and some active chymotrypsin remain. Immunochemical data indicate that, in the complex, a steric hindrance of the antigenic determinants of the inhibitor prevents the formation of the precipitate with specific antiserum. Inactive modified inhibitor, which has dissociated from the complex, has retained antigenic determinants of the native α1‐antichymotrypsin.
The role of Ca2+ uptake in the response of human platelets to adrenaline and to 1‐O‐alkyl‐2‐acetyl‐sn‐glycero‐3‐phosphocholine (platelet‐activating factor)
Authors: CLARE Katherine A. , SCRUTTON Michael C.
Published: March 2005

Thrombin induces Ca2+ uptake into both stirred and unstirred human platelets in the presence or absence of acetylsalicylate. This Ca2+ uptake is closely correlated with adenine nucleotide secretion in accord with previous observations [Massini, P. and Lüscher, E. F. (1974) Biochim. Biophys. Acta 372, 109–121] but a low level of secretion is observed in the absence of significant Ca2+ uptake.

1‐O‐Alkyl‐2‐acetyl‐sn‐glycero‐3‐phosphocholine (1‐0‐alkylAcGEPC) also induces Ca2+ uptake into both stirred and unstirred human platelets in the presence and absence of acetylsalicylate. Aggregation and adenine nucleotide secretion induced by 1‐O‐alkylAcGEPC can be observed in the absence of added fibrinogen, but addition of fibrinogen causes a very marked shift to the left in the dose/response curves for aggregation and Ca2+ uptake induced by 1‐O‐alkylAcGEPC. In the absence of added fibrinogen a close correlation is observed between Ca2+ uptake and adenine nucleotide secretion induced by 1‐O‐alkylAcGEPC. In the presence of added fibrinogen significant aggregation can be observed in the absence of detectable Ca2+ uptake.

Adrenaline induces Ca2+ uptake only into stirred human platelets in the presence of added fibrinogen. Blockade of secretion, e.g. by addition of acetylsalicylate, also prevents Ca2+ uptake.

Addition of adrenaline fails to cause breakdown of phosphatidylinositol or phosphatidylcholine in acetylsalicylate‐treated platelets under conditions where such a response is observed on addition of thrombin.

We conclude that Ca2+ uptake into human platelets induced by thrombin, 1‐O‐alkylAcGEPC and adrenaline is closely associated with the secretory response and in some circumstances, e.g. stimulation by thrombin, is clearly a consequence of this latter response. Previous reports of Ca2+ uptake as an initiating event in the response of human platelets to adrenaline [Owen, N. E., Feinberg, H. and Le Breton, G. C. (1981) Am J. Physiol. 239, H483–488] have not been confirmed in this study.
Protonmotive force in giant mitochondria
Authors: CAMPO Maria Luisa , TEDESCHI Henry
Published: March 2005

The accompanying communication [Eur. J. Biochem. 141 (1984) 1–4] indicates that the microinjection of the pH fluorescent indicator pyranine (8‐hydroxy‐1,2,6‐pyrenesulfonate) into giant mitochondria or mitoplasts does not affect their ability to carry out oxidative phosphorylation. The dye can therefore be used as a quantitative indicator of internal mitochondrial pH. We found that activation of metabolism in rotenone‐inhibited giant mitochondria by the addition of succinate produces an internal pH change corresponding to a pH shift of 0.3 to the alkaline range, approximately the same value found previously for conventional rat liver mitochondria.
Calmodulin‐dependent protein phosphorylation and calcium uptake in rat‐liver microsomes
Authors: FAMULSKI Konrad Stanislaw , CARAFOLI Ernesto
Published: March 2005

A 20‐kDa protein becomes phosphorylated in a process stimulated by Ca2+ and calmodulin in the light microsomal fraction of rat liver homogenate. The uptake of Ca2+ in light microsomal fraction is also calmodulin‐stimulated. The stimulation of Ca2+ transport is associated with the operation of a protein phosphorylation system dependent on Ca2+ and calmodulin. Transport is inhibited by a protein phosphatase which is stimulated by Ca2+ and calmodulin. It is proposed that the phosphorylation of the 20‐kDa protein, which is stimulated by Ca2+ and calmodulin, plays a role in the regulation of the microsomal Ca2+, Mg2+‐ATPase.
Pseudo‐catalytic degradation of hydrogen peroxide in the lactoperoxidase/H2O2/iodide system
Authors: HUWILER Martin , KOHLER Heinz
Published: March 2005

Non‐stoichiometric (excessive) consumption of hydrogen peroxide (H2O2), which was observed in various lactoperoxidase‐catalysed reactions, was tested in the lactoperoxidase/H2O2/iodide system. In preliminary experiments the suitability of the system was tested with special regard to the triiodide (I−3) absorption and the I2/I−3 equilibrium. Triiodide equilibrium concentrations evaluated theoretically and experimentally were compared after adding a known amount of iodine (I2) to solutions containing variable I− concentrations. A close fit of the two methods was only obtained if experiments were carried out in pure aqueous or 0.001 M H2SO4 medium. The presence of various anions, e.g. OH− and Cl−, led to a measurable decrease in I−3 and I2 equilibrium concentrations. These ions are able to displace competitively I− in forming association products with I+ and I2. When I+ and I2 were generated enzymatically by lactoperoxidase and hydrogen peroxide, additional interactions with H2O2 were observed. Depending on the enzyme and iodide concentrations, variable amounts of H2O2 disappeared non‐productively. Due to its ambivalent redox reactivity, part of the H2O2 is not reduced to H2O in the enzyme‐catalysed generation of iodine, but undergoes oxidation to O2 by an oxidized iodine compound. This suggests a pseudocatalatic side reaction which can competitively interfere with the I2 I−3 generation or (and) the iodination reaction.
The primary structure of teichuronic acid in Bacillus subtilis AHU 1031
Authors: YONEYAMA Toshie , ARAKI Yoshio , ITO Eiji
Published: March 2005

Structural studies were carried out on the acidic polysaccharide fraction obtained from lysozyme digest of the cell walls of Bacillus subtilis AHU 1031. The polysaccharide fraction contained N‐acetylmannosaminuronic acid (ManNAcA), N‐acetylglucosamine (GlcNAc), glucose, glycerol and phosphorus in a molar ratio of 2:2:4:1:1, together with glycopeptide components. The results of analyses involving Smith degradation, chromium trioxide oxidation, methylation and proton magnetic resonance spectroscopy led to the conclusion that the backbone chain of the polysaccharide has the repeating unit → 6)Glc(α1 → 3/4)ManNAcA(β1 → 4)GlcNAc(β1 →. About 50% of the N‐acetylglucosamine residues in the backbone chain seem to be substituted at C‐3 by the glycosidic branches, glycerol phospho‐6‐glucose, while the other half seem to be substituted by glucose.
Isolation of a homogeneous glucosidase II from pig kidney microsomes
Authors: BRADA Daniela , DUBACH Ulrich C.
Published: March 2005

The processing of the oligosaccharide precursor chain, (GlcNAc)2(Man)9(Gle)3, of N‐glycosylated glyco‐proteins starts with the action of glucosidase I which excises the terminal (α1–2)‐linked glucose residue. Glucosidase II removes the two inner (α1–3)‐linked glucose residues.

We have purified glucosidase II to homogeneity from pig kidney microsomes. The enzyme is a glycoprotein and contains a single type of subunit of molecular mass ∼ 100 kDa. The native enzyme is probably a tetramer. It cleaves glucosidic α1–3 and α1–4, but not α1–1, α1–2 or α1–6 bonds and lacks 2‐mannosidase and glucosidase I activity. The pH optimum is between 6.0 and 7.5. Specific antibodies against the native enzyme and the denatured subunit were prepared. By activity measurements and immune blotting, a similar enzyme was found in rat liver. In the fractionated rat liver, the enzyme was localized in the lumen of the endoplasmic reticulum, probably loosely bound to the inner face of the membrane. Purified Golgi fractions contained only low levels of the enzyme.
Conformational analysis of the single‐helical DNA fragment d(T‐A‐A‐T) in aqueous solution
Authors: MELLEMA Jan‐Remt , JELLEMA August K. , HAASNOOT Cornelis A. G. , VAN BOOM Jacques H. , ALTONA Cornelis
Published: March 2005

Proton NMR studies at 300 MHz and 500 MHz were carried out on the tetranucleoside trisphosphate d(T‐A‐A‐T). The thermodynamics of the three stacking interactions, derived from chemical shift versus temperature profiles, were used to extrapolate the observed coupling constants, measured at a range of temperatures, to values appropriate to the fully stacked forms of the molecule.

The data were interpreted in terms of N and S pseudorotational ranges [Altona, C. and Sundaralingham, M. (1972) J. Am. Chem. Soc. 94, 8205–8212]. It is shown that the stacked state of the molecule cannot be described by one conformer, but consists of one major structure (60%) in which all sugar rings have S‐type geometry and another structure (30%) in which residue dT(4) has an N‐type sugar. The remainder of the stacked states eonsists of one or more conformers with two or three sugar residues in the N‐type pseudorotational range. Detailed geometrical models are proposed for the major stacked conformers encountered in aqueous solution.
Na+‐dependent, α‐adrenergic mobilization of intracellular (mitochondrial) Ca2+ in brown adipocytes
Authors: CONNOLLY Eamorin , NÅNBERG Eewa , NEDERGAARD Jan
Published: March 2005

The existence and significance of a hormone‐sensitive, rapidly mobilizable intracellular pool of Ca2+ in hamster brown‐fat cells was investigated with 45Ca2+‐labelling techniques.

It was shown that such a pool existed and was probably located within the abundant mitochondria. It was rapidly mobilized by norepinephrine (median effective concentration 50 nM) through α‐adrenergic mechanisms. The mobilization of Ca2+ from the intracellular stores (mitochondria) required the presence of extracellular Na+, but not of Ca2+, K+ or Mg2+.

It is concluded that the experiments are in agreement with a hypothesis linking the mobilization of intracellular Ca2+ pools with an α‐adrenergically‐induced increase in plasma membrane Na+ permeability (observed as a membrane depolarization), and a subsequent activation of the mitochondrial Na+/Ca2+ exchange, leading to mobilization of mitochondrial Ca2+ and the mediation of α‐adrenergic effects as a result of an elevated cytosolic Ca2+ level.
Biliary transport of glutathione disulfide studied with isolated rat‐liver canalicular‐membrane vesicles
Authors: AKERBOOM Theo , INOUE Masayasu , SIES Helmut , KINNE Rolf , ARIAS Irwin M.
Published: March 2005

Canalicular plasma membrane vesicles isolated from rat liver (right side out) were used to study glutathione disulfide (GSSG) transport. GSSG is transported into an osmotically sensitive compartment. Extrapolation to an external osmolarity of infinity indicates 35% binding after 10 min of incubation, e.g. as mixed disulfides between glutathione and SH‐groups in membrane proteins.

Glutathione disulfide uptake occurred with equal rates in the presence of KCl or NaCl. After equilibration, no significant GSSG concentration gradient across the canalicular membrane was found. The temperature dependence of uptake is consistent with a carrier‐mediated transport for GSSG (Q10× 1.6) and seems to exclude simple diffusion Concentration dependence of GSSG uptake shows Michaelis‐Menten kinetics at concentrations up to 1 mM; a Km of 0.4 mM and a V of 1.1 nmol × min−1× mg protein−1 is calculated. At higher concentrations a linear dependence is observed without saturation kinetics.

It is concluded that transport of glutathione disulfide is mediated by a carrier present in the canalicular membrane of the hepatocyte.
Cytoplasmic mRNA‐protein complexes of chicken muscle cells and their role in protein synthesis
Authors: BAG Jnanankur
Published: March 2005

Irradiation of chicken muscle cells with ultraviolet light (254 nm) to cross‐link RNA and protein moieties was used to examine the polypeptide complements of cytoplasmic mRNA‐protein complexes(mRNP). The polypeptides of translationally active mRNP complexes released from polysomes were compared to the repressed nonpolysomal cytoplasmic (free) mRNP complexes. In general, all of the polypeptides present in free mRNPs were also found in the polysomal mRNPs. In contrast to polysomal mRNPS, polypeptides of Mr 28000, 32000, 46000, 65000 and 150000 were either absent or present in relatively smaller quantities in free mRNP complexes. On the other hand, the relative proportion of polypeptides of Mr 130000 and 43000 was higher in free mRNPs than in polysomal mRNP complexes.

To examine the role of cytoplasmic mRNP complexes in protein synthesis or mRNA metabolism, the changes in these complexes were studied following (a) inhibition of mRNA synthesis and (b) heat‐shock treatment to alter the pattern of protein synthesis. Actinomycin D was used to inhibit mRNA synthesis in chick myotubes. The possibility of newly synthesized polypeptides of cytoplasmic mRNP complexes being assembled into these complexes in the absence of mRNA synthesis was examined. These studies showed that the polypeptides of both free and polysomal mRNP complexes can bind to pre‐existing mRNAs, therefore suggesting that polypeptides of mRNP complexes can be exchanged with a pool of RNA‐binding proteins. In free mRNP complexes, this exchange of polypeptides is significantly slower than in the polysomal mRNP complexes.

Heat‐shock treatment of chicken myotubes induces the synthesis of three polypeptides of Mr= 81000, 65000 and 25000 (heat‐shock polypeptides). Whether this altered pattern of protein synthesis following heat‐shock treatment could affect the polypeptide composition of translationally active polysomal mRNPs was examined. The results of these studies show that, compared to normal cells, more newly synthesized polypeptides were assembled into polysomal mRNPs following heat‐shock treatment. A [35S]methionine‐labeled polypeptide of Mr= 80000 was detected in mRNPs of heat‐shocked cells, but not of normal cells. This polypeptide was, however, detected by AgNO3 staining of the unlabeled polypeptide of mRNP complexes of normal cells. These results, therefore, suggest that the assembly of newly synthesized 80000‐Mr polypeptide to polysomal mRNPs was enhanced following induction of new heat‐shock mRNAs.

The results of these studies reported here have been discussed in relation to the concept that free mRNP complexes are inefficiently translated in vivo. Failure to bind to regulatory protein(s) due to competition among mRNAs may be responsible for their inability to participate in protein synthesis.
Rabbit pepsinogens
Authors: KAGEYAMA Takashi , TAKAHASHI Kenji
Published: March 2005

Six pepsinogens were purified from the gastric mucosa of adult rabbits by chromatographies on DEAE‐cellulose, DEAE‐Sephacel and Sephadex G‐150. They were designated as pepsinogen I, pepsinogens II‐1, II‐2, II‐3, and II‐4, and pepsinogen III, based on their chromatographic behaviors.

Each pepsinogen was converted to pepsin at pH 2.0 and 14 °C. The resulting pepsins had roughly similar enzymatic properties. However, activation processes appeared to differ significantly among them. Pepsinogen I was converted to an active intermediate form by sequentially releasing the NH2‐terminal 25 or 26 residues. This active form was fairly stable and no further conversion occurred on further incubation for several hours. Pepsinogens II were converted to pepsins partly sequentially through intermediate forms and partly directly releasing the NH2‐terminal 44 residues as a single intact polypeptide. Pepsinogen III appeared to be converted to pepsin directly without forming intermediate species.

The amino acid sequences of the activation segments of these pepsinogens were determined together with the sequences in the NH2‐terminal regions of the resulting pepsins. Five distinct sequences were identified, indicating that most of these pepsinogens are the products of different genes.
Structural organization of the 5′ region of the human thyroglobulin gene
Authors: TARGOVNIK Hector M. , POHL Viviane , CHRISTOPHE Daniel , CABRER Bartolomé , BROCAS Huguette , VASSART Gilbert
Published: March 2005

Sequence analyses of bovine and human thyroglobulin (Tg) cDNA have demonstrated that the 5′ region of the mRNA encodes a domain responsible for thyroid hormone synthesis and exhibits striking internal repetition. Knowledge of the organization of the corresponding chromosomal DNA region would provide insight as to how such a structure has evolved.

A human genomic DNA library was screened by hybridization in situ, using a bovine Tg cDNA probe corresponding to 2.8 × 103 base pairs at the 5′ end of the mRNA. Out of 3 × 105 phage plaques, four were scored as positive and yielded three different phages containing thyroglobulin sequences. Selected human Tg cDNA probes were used to order the phages and to identify overlapping regions. Electron microscopy of hybrids between human Tg mRNA and the phage DNA was performed to determine the intron/exon organization of this region. The following conclusions were reached. (a) About 4 × 104 base pairs corresponding to the 5′ region of the gene have been isolated as three overlapping recombinant phages. (b) The three phages cover altogether 2.9 × 103 base pairs of exonic sequence at the 5′ end of the mRNA. (c) Out of the 11 exons identified in this region, 9 were of a size similar to that of the 3′ exons characterized previously (≤ 200 base pairs); exons 9 (1.12 × 103 base pairs) and 10 (0.56 × 103 base pairs) were exceptions to this rule. (d) The phage nearest the 5′ end contains about 9 × 103 base pairs of sequence located upstream from the gene.

The avalability of clones covering the region upstream from the thyroglobin gene will provide the basis for the identification of sequences involved in its transcriptional control by thyroid‐stimulating hormone (thyrotropin).
NMR studies of electron transfer mechanisms in a protein with interacting redox centres: Desulfovibrio gigas cytochrome c3
Authors: SANTOS Helena , MOURA José J. G. , MOURA Isabel , LeGALL Jean , XAVIER Antonio V.
Published: March 2005

The proton NMR spectra of the tetrahaem cytochrome c3 from Desulfovibrio gigas were examined while varying the pH and the redox potential. The analysis of the NMR reoxidation pattern was based on a model for the electron distribution between the four haems that takes into account haem‐haem redox interactions. The intramolecular electron exchange is fast on the NMR time scale (larger than 105 s−1). The NMR data concerning the pH dependence of the chemical shift of haem methyl resonances in different oxidation steps and resonance intensities are not compatible with a non‐interacting model and can be explained assuming a redox interaction between the haems. A complete analysis at pH*= 7.2 and 9.6, shows that the haem‐haem interacting potentials cover a range from ‐50mV to +60 mV. The midpoint redox potentials of some of the haems, as well as some of their interacting potentials, are pH‐dependent. The physiological relevance of the modulation of the haem midpoint redox potentials by both the pH and the redox potential of the solution is discussed.
Yeast RNA polymerase III
Authors: GABRIELSEN Odd Stokke , ANDERSEN Kjersti Elisabeth , ØYEN Tordis Beate
Published: March 2005

The chromatographic, catalytic and DNA‐binding properties of yeast RNA polymerase III are highly affected by both concentration and type of salt. The type of anion is an especially important modulating factor for the enzymological properties of the enzyme. When acetate or sulfate anions are substituted for chloride anions, RNA polymerase III exhibits a higher affinity for DEAE‐Sephadex A25, becomes able to transcribe DNA at relatively high ionic strength and shows a significant increase in the binding strength to DNA. A quantitative analysis of the binding of the enzyme to single‐stranded DNA shows that the number of ionic contacts in the complex is not affected by the type of anion, but the nonionic contribution to the binding constant is significantly increased when acetate is substituted for chloride.
Cobalt(II) as an NMR probe for the investigation of the coordination sites of conalbumin
Authors: BERTINI Ivano , LUCHINAT Claudio , MESSORI Luigi , SCOZZAFAVA Andrea
Published: March 2005

The bis and mono cobalt(II) derivatives of conalbumin in the presence of bicarbonate have been prepared. The 1H NMR spectra have been recorded at 60 MHz. Well‐shaped isotropically shifted signals in the range – 100to + 100ppm have been observed for both the mono and bis cobalt(II) derivatives; besides the intensity, all the isotropically shifted signals for the two derivatives are superimposable, indicating that the two sites are so similar as to be indistinguishable within the resolution of the technique. With the aid of T1 measurements, the signals have been assigned to the protons of two histidine and two tyrosinate ligands. The spectra are consistent with six‐coordinated, high‐spin cobalt(II) chromophores with two trans tyrosinate moieties at each site.
Occurrence of S‐adenosylhomocysteine hydrolase in prokaryote cells
Authors: SHIMIZU Sakayu , SHIOZAKI Shozo , OHSHIRO Takashi , YAMADA Hideaki
Published: March 2005

The occurrence of S‐adenosylhomocysteine hydrolase (EC 3.3.1.1) was found in a variety of prokaryotes. These prokaryotes did not exhibit any activities of S‐adenosylhomocysteine nucleosidase (EC 3.2.2.9) and S‐ribosyl‐homocysteine hydrolase (EC 3.3.1.3), which had been the generally accepted prokaryote enzymes for the regeneration of free homocysteine from S‐adenosylhomocysteine in the activated methyl cycle. In these prokaryotes S‐adenosylhomocysteine hydrolase was suggested to be the only enzyme functioning for the regeneration of free homocysteine by enzymological and immunochemical studies.

S‐Adenosylhomocysteine hydrolase was purified and crystallized from cells of a prokaryote, Alcaligenes faecalis. The purified enzyme was found to be homogenous on ultracentrifugation and gel electrophoresis. Its relative molecular mass is approximately 280000 and it is composed of six identical subunits with a Mr of approximately 48000. The NH2‐terminal and COOH‐terminal amino acids are lysine and tyrosine respectively. The enzyme contains 6 ma1 NAD/mol. Some nucleosides, such as formycin A, nebularine, adenosine N1 oxide and so on, are able to substitute for adenosine yielding the corresponding S‐nucleosidylhomocysteine congeners. Modification of the 5′‐hydroxymethyl group in adenosine leads to the most potent inhibition of the thioether formation of homocysteine with adenosine.

The enzyme from A. faecalis has some immunological similarities to other prokaryote S‐adenosylhomocysteine hydrolases, but is different from the enzymes of animal sources.
Partial purification and properties of isocitrate dehydrogenase kinase/phosphatase from Escherichia coli ML308
Authors: NIMMO Gillian A. , BORTHWICK Andrew C. , HOLMS W. Henry , NIMMO Hugh G.
Published: March 2005

Isocitrate dehydrogenase kinase and isocitrate dehydrogenase phosphatase were purified over 1000‐fold from Escherichia coli ML308 by a procedure involving fractionation with (NH4)2SO4 and chromatography on DEAE‐cellulose, blue‐dextran‐Sepharose and Sephadex G150.

The kinase and phosphatase activities copurified, in agreement with the observation [Laporte, D. C. and Koshland, D. E. (1982) Nature (Lond.) 300, 458–460] that a single protein bears both activities.

Isocitrate dehydrogenase kinase catalysed the phosphorylation of homogeneous active isocitrate dehydrogenase with a stoichiometry of just under one phosphate group incorporated per subunit. This almost completely inactivated the dehydrogenase. There was a good correlation between phosphorylation and inactivation.

Analysis of a partial acid hydrolysate of phosphorylated isocitrate dehydrogenase showed that the only phosphoamino acid present was phosphoserine.

Isocitrate dehydrogenase phosphatase catalysed the release of 32P from 32P‐phosphorylated isocitrate dehydrogenase; it required either ADP or ATP for activity. In the presence of ADP, or ATP plus an inhibitor of the kinase, the phosphatase catalysed full reactivation of isocitrate dehydrogenase and there was a good correlation between reactivation and the release of phosphate. In the presence of ATP alone the phosphatase catalysed the release of 32P from phosphorylated isocitrate dehydrogenase but the activity of the dehydrogenase remained low, indicating that the kinase and phosphatase were active simultaneously in these conditions.

The active and inactive forms of isocitrate dehydrogenase can be resolved by non‐denaturing gel electrophoresis; the two forms of the enzyme were interconverted by phosphorylation and dephosphorylation in vitro. The extent of the interconversion correlated well with the changes in isocitrate dehydrogenase activity.
Solubilization of the nitrendipine receptor from skeletal muscle transverse tubule membranes Interactions with specific inhibitors of the voltage‐dependent Ca2+ channel
Authors: BORSOTTO Marc , NORMAN Robert I. , FOSSET Michel , LAZDUNSKI Michel
Published: March 2005

The nitrendipine receptor associated with the voltage‐dependent calcium channel from rabbit skeletal muscle transverse tubule membranes has been solubilized by detergent extraction. A highly stable solubilized receptor preparation was obtained using 3‐[(3‐cholamidopropyl)dimethyl‐ammonio]‐1‐propanesulfonate as detergent with phospholipids or glycerol present as stabilizing agents. Binding of [3H]nitrendipine to the solubilized receptor was reversible and saturable. At 4°C the equilibrium dissociation constant of the [3H]nitrendipine receptor complex was 7±3nM and was close to that determined from the rate constants of association (k1= 1.3 105 M−1 s−1) and dissociation (k−1= 1.10 × 10−3 s−1) of 8.4nM. The nitrendipine concentration that gave a half‐maximal inhibition of [3H]nitrendipine binding to the solubilized receptor was 10 nM, which was similar to the values for the dissociation constant determined for the radiolabelled ligand. [3H]Nitrendipine binding to its solubilized receptor was also inhibited by other antiarrythmic drugs, such as bepridil and verapamil, and enhanced by d‐cis‐diltiazem. Since these drugs are apparent non‐competitive inhibitors of [3H]nitrendipine binding it was concluded that these different binding sites are tightly coupled. Sucrose density sedimentation of solubilized nitrendipine receptor resulted in the separation of three [3H]nitrendipine binding activities with apparent sedimentation coefficients of 11.4 S. 14.4 S and 21S.
Purification and properties of three cytosolic ribonucleases of mouse liver
Authors: MAVROTHALASSITIS George J. , GEORGATSOS John G.
Published: March 2005

The ribonucleolytic activity of mouse liver cytosol is due to at least three different enzymes, whose purification is reported. Two of these enzymes, an alkaline and a neutral RNase, have specificities practically identical with that of pancreatic RNase. The third enzyme, an acid RNase, is highly specific for NpU bonds where N is A, G or C and also cleaves ApG bonds provided they are part of a GpApG sequence and preferentially a GpApGpA repeat.
Immunochemistry, genuine size and tissue localization of collagen VI
Authors: VON DER MARK Helga , AUMAILLEY Monique , WICK Georg , FLEISCHMAJER Raul , TIMPL Rupert
Published: March 2005

Collagen VI was solubilized with pepsin from human placenta and used for preparing rabbit antisera. Major antigenic determinants were located in the central region of the antigen including triple‐helical and globular structures. Antisera prepared against a constituent chain showed preferential reactions with unfolded structures. Antibodies were purified by affinity chromatography and failed to cross‐react with other collagen types I–V and with fibronectin. These antibodies demonstrated intracellular and extracellular collagen VI in fibroblast and smooth muscle cell cultures. Immunoblotting identified a disulfide‐bonded constituent chain about twice as large as those of the pepsin fragments in both cell cultures and tissue extracts. Rotary shadowing electron microscopy indicated that the increase in mass is due to larger globular domains present at both ends of collagen VI monomers.

Indirect immunofluorescence demonstrated a wide occurrence of collagen VI in connective tissue particularly of large vessels, kidney, skin, liver and muscle. Collagen VI is apparently not a typical constituent of cartilage or of basement membranes. Ultrastructural studies using the immunoferritin technique showed collagen VI along thin filaments or in amorphous regions of aortic media or placenta but not in association with thick, cross‐striated collagen fibrils or elastin. This supports previous suggestions that collagen VI is a constituent of microfibrillar structures of the body.
Control of glycogen synthase phosphorylation in isolated rat hepatocytes by epinephrine, vasopressin and glucagon
Authors: CIUDAD Carlos , CAMICI Marcella , AHMAD Zafeer , WANG Yuhuan , DePAOLI‐ROACH Anna A. , ROACH Peter J.
Published: March 2005

Isolated rat hepatocytes were incubated in a medium containing 0.1 mM [32P]phosphate (0.1 mCi/ml) before exposure to epinephrine, glucagon or vasopressin. 32P‐labeled glycogen synthase was purified from extracts of control or hormone‐treated cells by the use of specific antibodies raised to rabbit skeletal muscle glycogen synthase. Analysis of the immunoprecipitates by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate indicated that a single 32P‐labeled polypeptide, apparent Mr 88000, was removed specifically by the antibodies and corresponded to glycogen synthase. Similar electrophoretic analysis of CNBr fragments prepared from the immunoprecipitate revealed that 32P was distributed between two fragments, of apparent Mr 14000 (CB‐1) and 28000 (CB‐2). Epinephrine, vasopressin or glucagon increased the 32P content of the glycogen synthase subunit. CB‐2 phosphorylation was increased by all three hormones while CB‐1 was most affected by epinephrine and vasopressin. These effects correlated with a decrease in glycogen synthase activity. From studies using rat liver glycogen synthase, purified by conventional methods and phosphorylated in vitro by individual protein kinases, it was found that electrophoretically similar CNBr fragments could be obtained. However, neither cyclic‐AMP‐dependent protein kinase nor three different Ca2+ ‐dependent enzymes (phosphorylase kinase, calmodulin‐dependent protein kinase, and protein kinase C) were effective in phosphorylating CB‐2. The protein kinases most effective towards CB‐2 were the Ca2+ and cyclic‐nucleotide‐independent enzymes casein kinase II (PC0.7) and FA/GSK‐3. The results demonstrate that rat liver glycogen synthase undergoes multiple phosphorylation in whole cells and that stimulation of cells by glycogenolytic hormones can modify the phosphorylation of at least two distinct sites in the enzyme. The specificity of the hormones, however, cannot be explained simply by the direct action of any known protein kinase dependent on cyclic nucleotide or Ca2+. Therefore, either control of other protein kinases, such as FA/GSK‐3, is involved or phosphatase activity is regulated, or both.
The composition of the pyruvate dehydrogenase complex from Azotobacter vinelandii
Authors: BOSMA Hans J. , DE KOK Arie , WESTPHAL Adrie H. , VEEGER Cees
Published: March 2005

An improved purification procedure of the pyruvate dehydrogenase complex of Azotobacter vinelandii is described. This procedure minimizes losses of components and results in the isolation of the pure complex with a specific activity of 15–19 U/mg and an overall yield of 40%.

The chain ratio of the three components was determined by covalent modification of the lysine residues with trinitrobenzene sulfonic acid, followed by separation of the components on sodium dodecyl sulfate gels. These determinations yielded an average chain ratio of 1.3:1:0.5 for E1:E2:E3 respectively. Based on E2 this corresponds with a minimum molecular mass of approximately 216 kDa. Because the molecular mass of the complex has been determined previously to be 800 ± 50 kDa, it is concluded that the complex as isolated from A. vinelandii is based on a tetramer of E2 chains.

The complex can be resolved into its individual components, which can be recombined to yield a fully active complex. Titration of E2E3 subcomplexes with E1 resulted in maximum complex activity at an E1/E2 ratio of 1.5–1.6. Similar titrations of E1E2 subcomplexes with E3 resulted in maximum activity at an E3/E2 ratio of 0.45–0.55. From these experiments it is concluded that the complex has maximum activity with a composition of three E1 dimers, one E2 tetramer and one E3 dimer. With excess of either E1 or E3 a decrease in activity is observed which indicates competition between these components for binding sites on E2.

As shown before [Bosma, H. J., de Kok, A., Markwijk, B. W., and Veeger, C. (1984) Eur. J. Biochem. 140, 273–280], the isolated E2 component is composed of 32 peptide chains of 66 kDa each. Upon addition of E1 or E3, E2 dissociates into tetramers. Dissociation is complete upon the addition of four E1 dimers of four E3 dimers per E2 tetramer. Addition of E1 to saturated E2E3 subcomplex or E3 to saturated E1E2 subcomplex did not result in extra binding but rather in displacement of bound E3 or E1 respectively. It is therefore concluded that the binding sites of E1 and E3 to the E2 chains are either identical or so closely spaced that steric hindrance prevents simultaneous binding of both components.

A model is presented based on the cubic structure of the isolated E2 component. In this model the 32 E2 peptide chains are arranged in tetramers in the corners of the cube. This model is discussed in connection with the existing model for the Escherichia coli complex.
Anion‐induced increases in the affinity of colcemid binding to tubulin
Authors: RAY Kunal , BHATTACHARYYA Bhabatarak , BISWAS Birendra B.
Published: March 2005

Colcemid binds tubulin rapidly and reversibly in contrast to colchicine which binds tubulin relatively slowly and essentially irreversibly. At 37°C the association rate constant for colcemid binding is 1.88 × 106 M−1 h−1, about 10 times higher than that for colchicine; this is reflected in the activation energies for binding which are 51.4 kJ/mol for colcemid and 84.8 kJ/mol for colchicine. Scatchard analysis indicates two binding sites on tubulin having different affinities for colcemid. The high‐affinity site (Ka= 0.7 × 105 M−1 at 37°C) is sensitive to temperature and binds both colchicine and colcemid and hence they are mutually competitive inhibitors. The low‐affinity site (Kb= 1.2 × 104 M−1) is rather insensitive to temperature and binds only colcemid. Like colchicine, 0.6 mol of colcemid are bound/mol of tubulin dimer (at the high‐affinity site) and the reaction is entropy driven (163 J K−1 mol−1).

Similar to colchicine, colcemid binding to tubulin is stimulated by certain anions (viz. sulfate and tartrate) but by a different mechanism. Colcemid binding affinity at the lower‐affinity site of tubulin is increased in the presence of ammonium sulfate. Interestingly, the lower‐affinity site on tubulin for colcemid, even when converted to higher affinity in presence of ammonium sulfate, is not recognized by colchicine.

We conclude that tubulin possesses two binding sites, one of which specifically recognized the groups present on the B‐ring of colchicine molecule and is effected by the ammonium sulfate, whereas the higher‐affinity site, which could accommodate both colchicine and colcemid, possibly recognized the A and C ring of colchicine.
Proteinase‐treated photoreceptor discs
Authors: BAYRAMASHVILI Dmitry I. , DRACHEV Alexander L. , DRACHEV Lel A. , KAULEN Andrey D. , KUDELIN Andrey B. , MARTYNOV Vladimir I. , SKULACHEV Vladimir P.
Published: March 2005

Photoreceptor discs from rod outer segments of cattle retina were treated with (a) papain, (b) thermolysin or (c) trypsin, the procedures resulting in the cleavage of the rhodopsin polypeptide chain between (a) 323 and 324, 236 and 237, 241 and 242, (b) 327 and 328, 240 and 241, or (c) 339 and 340 amino acid residues, respectively.

In all the cases, partially digested rhodopsins proved to be competent in generating photoelectric potential and increasing membrane conductance of the discs adsorbed onto phospholipid‐impregnated collodion film. The kinetics of generation and dissipation of photopotential as well as of formation of metarhodopsin II and of the light‐induced rhodopsin protonation were found to be the same in the partially digested preparations and in the intact one.

Incubation of papain‐treated or thermolysin‐treated discs at pH 6.0 induced formation of inside‐out vesicles which, when incorporated into the collodion film, generated an oppositely directed photopotential. Treatment of such vesicles with papain gave rise to further cleavages of the polypeptide localized between 30 and 31, 186 and 187 amino acid residues. One more proteinase‐sensitive site, localized between 104 and 105 residues, has been discovered in the inside‐out vesicles treated with thermolysin. This fact consistent with the scheme of the ‘seven column’ arrangement of the visual rhodopsin [Ovchinnikov, Yu. A. (1982) FEBS Lett. 148, 179–191].

Rhodopsin, when treated with papain on both sides, was deprived of sixty amino acid residues being split in two sites in the middle part of the polypeptide, but was still active as a photoelectric energy transducer.

The main specific feature inherent in the photoelectric response of the papain‐treated or thermolysin‐treated rhodopsin and absent from the native protein is that the former survives addition of long trains of saturating flashes when the response of the intact preparation becomes negligible. This effect was shown to be due to conversion of partially digested rhodopsin to a photolytic product that at room temperature lived for minutes even in the presence of NH2OH. A 532‐nm laser flash effectively converted this product back to rhodopsin.