bluuebunny/crossref_metadata_2025 huggingface dataset:
Modulation of membrane fluidity in living protoplasts of Nicotiana plumbaginifolia by catalytic hydrogenation
Authors: VIGH László , JOÓ Ferenc , CSÉPLÓ Ágnes
Published: March 2005

A homogeneous water‐soluble Ru catalyst, has been incorporated into mesophyll protoplasts isolated from Nicotiana plumbaginifolia leaves. In the presence of hydrogen gas this complex causes an extensive loss of unsaturated fatty acid bonds and a concomitant increase in microviscosity of the cellular membranes. Although the gradual reduction of the level of unsaturation, per se, is accompanied by considerable cell damage, there is an optimum reaction time where approximately 50% of the protoplasts are still living and about 20% of the double bounds initially present in fatty acyl residues have undergone hydrogenation. The possible mechanism of the self regulatory process competing with the hydrogenation in the early stages of the reaction is also discussed.
Affinity labeling of rabbit muscle fructose‐1,6‐bisphosphate aldolase with 5′‐[p‐(fluorosulfonyl)benzoyl]‐1, N6‐ethenoadenosine
Authors: PALCZEWSKI Krysztof , HARGRAVE Paul A. , FOLTA Ewa J. , KOCHMAN Marian
Published: March 2005

Aldolase contains one tight binding site and one weak binding site per subunit for ATP [Kasprzak, A. and Kochman, M. (1980) Eur. J. Biochem. 104, 443–450]. The reaction of the ATP analog 5′‐[p‐(fluorosulfonyl)benzoyl]‐1, N6‐ethenoadenosine with rabbit aldolase A results in linear inactivation of enzyme with respect to covalent linkage of fluorescent label. The enzyme is completely protected against modification in the presence of saturating covalent binding (k2= 0.033 min−1) is preceded by a fast reversible binding step (Ki= 6.8 mM).

Chemical modification of aldolase leads to formation of stable Nɛ(4‐carboxybenzenesulfonyl‐lysine (Cbs‐Lys) and O‐(4‐carboxybenzenesulfonyl‐tyrosine (Cbs‐Tyr) derivatives. Almost all Cbs‐Lys was found in the N‐terminal CNBr peptide (CN‐1), whereas Cbs‐Tyr was present both in the N‐terminal (CN‐1) and C‐terminal (CN‐2) peptide. From carboxypeptidase digestion and tryptic peptide analysis, Cbs‐Lys was localized in position 107, a small part of Cbs‐Tyr was detected in position 84, and the majority of Cbs‐Tyr was found in the C‐terminal position Tyr‐363.

We conclude that the covalent binding of the ATP analog occurs at the mononucleotide tight‐binding site of aldolase and is associated with modification of Lys‐107 and Tyr‐363. This conclusion is based on the measurements of enzymatic activity loss as a function of ATP analog incorporation as well as on previous data. It is postulated that Lys‐107, which is the C‐6 phosphate binding site for fructose‐1,6‐P2, is in close proximity to the functionally important Tyr‐363. The rather small extent of modification of Tyr‐84 (0.15 mol/subunit), is due either to nonspecific protein modification or labeling of the weak mononucleotide binding site.
Direct assessment of β‐adrenergic receptors in intact rat adipocytes by binding of [3H]CGP 12177
Authors: LACASA Daniéle , AGLI Brigitte , GIUDICELLI Yves
Published: March 2005

The characteristics of the binding of the hydrophilic β‐adrenergic antagonist [3H]CGP 12177 to intact rat adipocytes were studied at 37°C and 6°C. At both temperatures and at 90% saturation, the non‐specific binding was less than 30% of the total binding.

At 37°C, specific [3H]CGP 12177 binding was rapid, reversible of high affinity (1.8 ± 0.4 nM) and saturable. The number of specific binding sites per adipocyte increased with the fat cell size (about 35000 and 115000 sites per cell in adipocytes with diameters of 60 μm and 88 μm, respectively) but remained constant when expressed per unit fat cell surface area. Displacement of [3H]CGP 12177 bound to adipocytes by unselective and selective β‐antagonists was stereospecific, had the same characteristics as those found in adipocyte membranes and showed a heterogeneous specificity for β1 and β2 adrenergic subtypes. In contrast, β agonist competition curves, Which modeled to two affinity‐states of binding, showed high‐affinity‐state Kd values for agonists 10–25‐times higher than those found in membranes under the same experimental conditions.

At 6°C, although the number and affinity of the specific binding sites for [3H]CGP 12177 were the same as those found at 37°C, the Kd value for (–)‐isoproterenol binding to the high affinity state of these sites (3.0±0.5 nM) was 25‐times lower than at 37°C and similar to the value found in membrane preparations (1.5–4 nM).

These results show that the [3H]CGP 12177 specific binding sites detected on intact adipocytes represent the physiological β‐adrenergic receptors. Moreover, this study extends to the adipocyte the validity of the model recently proposed for other cell lines, according to which in intact cells, but not in membranes, agonist‐binding promotes a rapid and temperature‐dependent conformational change of the β‐adrenergic receptors, leading to a progressive loss of capacity of agonists to form a high‐affinity complex.
Rat α1‐microglobulin
Authors: ÅERSTRÖM Bo , LANDIN Britta
Published: March 2005

Rat α1‐microglobulin was isolated from the urine of rats treated with sodium chromate, and was purified by the use of gel chromatography, affinity chromatography on concanavalin‐A–Sepharose and ion‐exchange chromatography. The protein was heterogeneous in charge, had a tendency to form dimers, and was associated with a brown‐coloured chromophore. The size of the protein (25 kDa) was similar to guinea pig α1‐microglobulin but smaller than the human protein, when measured with sodium dodecyl sulfate/polyacrylamide gel electrophoresis. Immunological cross‐reaction with human and guinea pig α1‐microglobulin was demonstrated. The concentration of α1‐microglobulin in rat serum was 16.4 mg/l (SD = 8.5 mg/l, n= 13) and rat serum α1‐microglobulin was eluted from a gel chromatography column at two different positions corresponding to monomeric α1‐microglobulin and IgA. The latter α1‐microglobulin activity could be absorbed by anti‐IgA serum. Rat α1‐microglobulin and albumin were continuously released into the medium of rat hepatocyte monolayers, and α1‐microglobulin was isolated from the medium by the use of immunoprecipitation with anti‐(α1‐microglobulin). Tritiated leucine, added to the medium, was incorporated into the protein, suggesting a de novo synthesis of α1‐microglobulin by the hepatocytes. The size of hepatic α1‐microglobulin was similar to that of purified urinary rat α1‐microglobulin, when determined with sodium dodecyl sulfate/polyacrylamide gel electrophoresis.
Nucleotide sequence of yeast gene CPA1 encoding the small subunit of arginine‐pathway carbamoyl‐phosphate synthetase
Authors: WERNER Michel , FELLER André , PIÉRARD André
Published: March 2005

A yeast DNA fragment carrying the gene CPA1 encoding the small subunit of the arginine pathway carbamoylphosphate synthetase has been sequenced. Only one continuous coding sequence on this fragment was long enough to account for the presumed molecular mass of CPA1 protein product. It codes for a polypeptide of 411 amino acids having a relative molecular mass, Mr, of 45358 and showing extensive homology with the product of carA, the homologou Escherichia coli gene.

CPA1 and carA products are glutamine amidotransferases which bind glutamine and transfer its amide group to the large subunits where it is used for the synthesis of carbamoyl‐phosphate. A comparison of the amino acid sequences of CPA1 polypeptide with the glutamine amidotransferase domains of anthranilate and p‐aminobenzoate synthetases from various sources has revealed the presence in each of these sequences of three highly conserved regions of 8, 11 and 6 amino acids respectively. The 11‐residue oligopeptide contains a cysteine which is considered as the active‐site residue involved in the binding of glutamine. The distances (number of amino acid residues) which separate these homology regions are accurately conserved in these various enzymes. These observations provide support for the hypothesis that these synthetases have arisen by the combination of a common ancestral glutamine amidotransferase subunit with distinct ammonia‐dependent synthetases. Little homology was detected with the amide transfer domain of glutamine phosphoribosyldiphosphate amidotransferase which may be the result of a convergent evolutionary process.

The flanking regions of gene CPA1 have been sequenced, 803 base pairs being determined on the 5′ side and 382 on the 3′ side. Several features of the 5′‐upstream region of CPA1 potentially related to the control of its expression have been noticed including the presence of two copies of the consensus sequence d(T‐G‐A‐C‐T‐C) previously identified in several genes subject to the general control of amino acid biosynthesis.
Strain‐dependent variation in ribosomal DNA arrangement in Absidia glauca
Authors: WÖSTEMEYER Johannes
Published: March 2005

Restriction analysis of total DNA from the zygomycete Absidia glauca reveals a pattern of prominent bands on a homogeneous background. By Southern blot analysis with 32P‐end‐labelled ribosomal RNA most of these bands could unequivocally be identified as repetitive copies of ribosomal DNA. There are marked differences in restriction patterns of rDNA between all seven strains tested, even of strains belonging to mating type pairs, presumably isolated from the same location. By using purified rRNAs as probes in hybridization experiments, evidence is presented that 5S rRNA is part of the ribosomal repetitive unit. A more detailed analysis of one strain pair [A. glauca CBS 100.48 (+)/101.48 (−)] provided evidence that the (+) strain, in addition to one rDNA repeat unit common to both strains, contains a second one, derived from the common form by a small deletion.
ATP‐dependent Ca2+ transport in vesicles isolated from the bile canalicular region of the hepatocyte plasma membrane
Authors: BACHS Oriol , FAMULSKI Konrad S. , MIRABELLI Francesca , CARAFOLI Ernesto
Published: March 2005

Three plasma membrane subfractions have been isolated and characterized from rat liver cells. The high affinity Ca2+‐stimulated ATPase is highly enriched in the bile canalicular subfraction. Taking into account cross‐contamination by the blood sinusoidal and lateral membranes it is suggested that the high‐affinity Ca2+ ‐ATPase is located exclusively in this fraction. The high‐affinity Ca2+‐ATPase is coupled to Ca2+ transport, is calmodulin‐insensitive, sensitive to vanadate under appropriate experimental conditions and is strongly inhibited by La3+. In the presence of Ca2+ and ATP the ATPase forms a phosphorylated intermediate of molecular mass about 200 kDa.
Induction of ornithine decarboxylase in guinea‐pig lymphocytes
Authors: OTANI Shuzo , MATSUI Isao , KURAMOTO Ayako , MORISAWA Seiji
Published: March 2005

Calmodulin antagonists, N‐(6‐aminohexyl)‐5‐chloro‐1‐naphthalenesulfonamide (W‐7), N‐(6‐aminohexyl)‐1‐naphthalenesulfonamide (W‐5) and trifluoperazine inhibited ornithine decarboxylase induction in lymphocytes activated with phytohemagglutinin or inophore A23187. W‐7, a more potent calmodulin antagonist than W‐5, suppressed ornithine decarboxylase induction in a higher extent than did W‐5. These results suggest that calmodulin may play an important role in ornithine decarboxylase induction in the activated lymphocytes. However, the extent of ornithine decarboxylase induction was greater in cells pretreated with Clostridium phospholipase C and then incubated with ionophore A23187 than in cells incubated with ionophore A23187 without the pretreatment. Moreover, combined treatment of cells with ionophore A23187 and tumor promotor, phorbol 12‐myristate 13‐acetate, caused synergistic induction of ornithine decarboxylase activity. These results, taken together, suggest that both activations of Ca2+‐activated phospholipid‐dependent protein kinase by diacylglycerol and of calmodulin‐dependent function resulted from an elevation of cytosolic Ca2+ concentration may operate in the induction of ornithine decarboxylase in the activated lymphocytes.
Identification of the major human sialoglycoprotein from red cells, glycophorin AM, as the receptor for Escherichia coli IH 11165 and characterization of the receptor site
Authors: JOKINEN Mikko , EHNHOLM Christian , VÄISÄNEN‐RHEN Vuokko , KORHONEN Timo , PIPKORN Rüdiger , KALKKINEN Nisse , GAHMBERG Carl G.
Published: March 2005

The pyelonephritogenic Escherichia coli strain 1H 11165 specifically agglutinates human erythrocytes carrying the M blood group antigen. The polymorphic forms of this antigen, M and N, are located in the NH2‐terminal region of the major human red‐cell sialoglycoprotein, glycophorin A. Radioactively labeled glycophorin A from M cells specifically bound to the bacteria. Purified glycophorin AM, but not glycophorin AN, efficiently inhibited for binding. Mild periodate treatment oxidized the NH2‐terminal serine in glycophorin AM and this resulted in loss of binding to the bacteria. High concentrations of serine and alkali‐labile oligosaccharides derived from glycophorin AM inhibited the binding, whereas the synthetic M‐specific NH2‐terminal pentapeptide Ser‐Ser‐Thr‐Thr‐Gly did not. Neuraminidase treatment of glycophorin AM did not destroy the binding. The most efficient inhibition of binding was observed with the N‐terminal glyco‐octapeptide obtained from glycophorin AM by CNBr cleavage. This peptide contains both the essential serine residue and the alkali‐labile oligosaccharides, which both are recognized by the bacterium.
Sequence of the 5′‐end quarter of the human‐thyroglobulin messenger ribonucleic acid and of its deduced amino‐acid sequence
Authors: MALTHIÉRY Yves , LISSITZKY Serge
Published: March 2005

Thyroglobulin, the dimeric glycoprotein (19 S, 2 × 330 kDa), specific to the thyroid gland, is the support for thyroid hormone synthesis. Elucidation of the mechanism for thyroid hormone synthesis requires the knowledge of the primary sequence of the protein. In this paper the sequence of the first coding 2190 nucleotides from the 5′ end of the human mRNA is presented. This was obtained by sequencing two previously described overlapping clones and by construction and sequencing of a single‐stranded cDNA corresponding to the 5′ end of the mRNA. The nucleotide sequence represents a quarter of the human thyroglobulin mRNA, from which a polypeptide sequence of 730 amino acids at the NH2‐terminal end of the monomer has been deduced. This sequence shows a repetition of five highly conserved motifs each of approximately 50 amino acids, the analysis of which allowed us to establish a consensus sequence. We have also demonstrated (a) the hormonogenic tyrosine residue recently described in the mature protein, which is located four amino acids after the NH2‐terminal Asn; (b) a prepeptide signal of thyroglobulin secretion comprising 19 amino acids preceding the Asn residue, the NH2‐terminal residue of the mature protein and (c) a six‐signal tripeptide (Asn‐Xaa‐Thr or Ser) of N‐glycosylation of the chain.
Biosynthesis of algal pheromones
Authors: BOLAND Wilhelm , MERTES Karin
Published: March 2005

Several cyclic and alicyclic C11 hydrocarbons have been shown to act as gamete releasing and/or attracting pheromones during sexual reproduction of brown algae (Phaeophyceae). The same compounds are also found in the essential oils of various plants, of which the occurrence of the cycloheptadiene‐pheromone ectocarpene in Senecio isatideus (Compositae) is noteworthy. Administration of [3H]dodeca‐3,6,9‐trienoic acid to cuttings of this plant leads to incorporation of radioactivity into ectocarpene. Double‐bond‐deuterated nona‐3,6‐dienoic acid is converted to fucoserratene, the pheromone of several Fucales, which is certainly not present among the hydrocarbons of Senecio. This proves that the pool of medium‐chain, multiply unsaturated fatty acids includes precursors of all types of highly unsaturated hydrocarbons. Appropriately labelled (deuterium markers) fatty acid homologues were synthesized and applied to Senecio plantlets to unravel the mechanistic aspects. The results strongly suggest radical initiation of the pheromone biosynthesis by abstraction of a single hydrogen from a 1, 4‐pentadienyl segment of the fatty acid followed by oxidation to the corresponding cation. This causes fragmentation of the reactive intermediate into an olefine and carbon dioxide by neighbouring‐group participation of the flanking double bonds. A tentative biosynthetic scheme is deduced from the experimental results which also sets the stereochemistry of the algal pheromones into a uniform mechanistic concept.
Dioxygen‐activating iron center in putidamonooxin
Authors: TWILFER Hans , BERNHARDT Frithjof‐Hans , GERSONDE Klaus
Published: March 2005

The mononuclear non‐haem iron center is the dioxygen‐binding site of putidamonooxin which is the dioxygen‐activating component of the 4‐methoxybenzoate monooxygenase. Replacement of dioxygen by nitrosyl leads to the formation of a rather stable Fe3+· NO− complex which is characterized by electron spin resonance (ESR) at g · 4 and g · 2. The ESR features can be composed by two spectral components which are characterized by different tetragonal distortions of the axial symmetry.

Binding of 4‐hydroxybenzoate, which is the product of the enzymatic reaction, leads to the formation of an ESR spectrum with pure axial symmetry. After binding of 4‐methoxybenzoate, i.e. the physiological substrate of the monooxygenase, only one spectral component, i.e. that with a small tetragonal distortion, is observed. Binding of substrate analogues, like 4‐aminobenzoate and 4‐trifluoromethylbenzoate, leads to a spectral heterogeneity with variable amounts of the ESR component with a large tetragonal distortion. Benzoate induces an ESR spectrum with only that spectral component with large tetragonal distortion.

The iron‐depleted substrate‐free form of the enzyme, ligated with NO, also shows ESR heterogeneity, i.e. both spectral components overlap, with 60% of the component with large tetragonal distortion. Binding of 4‐methoxybenzoate leads to the occurrence of a pure spectrum, i.e. with small tetragonal distortion, whereas binding of benzoate leads to a pure spectrum with large tetragonal distortion. Thus, the structural heterogeneity is removed by binding of both the ligand (NO) and substrate. The Fe3+· NO− complex is discussed as an analogue of the native oxy complex Fe3+· O−2.
Preparation and characterization of NADP derivatives alkylated at 2′‐phosphate and 6‐amino groups
Authors: OKUDA Keiko , SUNTINANALERTS Prasert , MIYOSHI Shinsuke , URABE Itaru , YAMADA Yasuhiro , OKADA Hirosuke
Published: March 2005

Reaction of NADP with 3‐propiolactone at pH 6 gave new NADP derivatives carboxyethylated at the 2′‐phosphate or 6‐amino group, or both: 2′‐O‐(2‐carboxyethyl)phosphono‐NAD (I), N6‐(2‐carboxyethyl)‐NADP (II), and 2′‐O‐(2‐carboxyethyl)phosphono‐N6‐(2‐carboxyethyl)‐NAD (III). Their structures were assigned on the basis of ultraviolet, 1H‐NMR and 31P‐NMR spectra, and also treatment with nucleotide pyrophosphatase or alkaline phosphatase.

Carbodiimide‐promoted reaction of derivative I with 1,2‐diaminoethane gave 2′‐O‐[N‐(2‐aminoethyl)carbamoylethyl]phosphono‐NAD (IV); derivative III gave 2′‐O[N‐(2‐aminoethyl)carbamoylethyl]phosphono‐N6‐[N‐(2‐aminoethyl)carbamoylethyl]‐NAD (VI). The same reaction of derivative II, on the other hand, gave a mixture of N6‐[N‐[N‐(2‐aminoethyl)carbamoylethyl]‐NADP (Va) and its 3′‐phosphate isomer (Vb). The mixture was converted to Va via the 2′,3′‐cyclic derivative (Vc). Their structures were assigned on the basis of ultraviolet and 1H‐NMR spectra, and also treatment with alkaline phosphatase or 3′‐nucleotidase. All the NADP derivatives obtained in this work could be reduced with yeast glucose‐6‐phosphate dehydrogenase.
Thyrotropin modifies the synthesis of actin and other proteins during thyroid cell culture
Authors: PASSAREIRO Heloisa , ROGER Pierre P. , LAMY Francoise , LECOCQ Raymond , DUMONT Jacques E. , NUNEZ Jacques
Published: March 2005

Primary cultures of dog thyroid cells have been used to study the effects of thyrotropin on the synthesis of proteins. The cells were cultured for 4 days in serum‐free and thyrotropin‐free conditions. Thyrotropin was then added for varying periods of time (6–96h). In the absence of thyrotropin, the cells have an elongated flattened aspect. Exposure to thyrotropin for 6–24 h produces retraction and rounding up of cells whereas cells incubated with thyrotropin for longer periods of time have an epithelial cuboidal shape. After varying periods of culture the cells were labelled with [35S]methionine for 6 h and then analyzed by one‐and two‐dimensional gel electrophoresis, followed by autoradiography. The results were as follows.

After exposure to thyrotropin for 32 h and 48 h, the synthesis of about 18 proteins was increased while that of about 14 others was decreased. After 6 h the labelling of three and five of these proteins was already increased or decreased, respectively.

Some of the proteins whose synthesis is modified in the presence of thyrotropin were identified. Actin synthesis was markedly decreased with a maximum 24–48 h after the addition of thyrotropin. A modification in the ratio between α and β tubulins was also observed together with very large changes in a group of proteins having both the relative molecular mass (30000–40000) and the isoelectric points of tropomyosins.

Forskolin and cholera toxin caused the same qualitative and quantitative changes as thyrotropin; this suggests that the regulation by thyrotropin of the synthesis of several thyroid cell proteins is mediated by cAMP.

In conclusion, the data obtained in this work might help to explain the molecular mechanisms by which thyrotropin (and cAMP) triggers the changes in cell shape which occur during thyroid cell culture. They also indicate that one of the main effects of thyrotropin takes place at the level of several proteins which belong to the cytoskeleton and which are involved in the definition of the cytostructure of the thyroid cells.
Electron transfer reactions in the soluble methane monooxygenase of Methylococcus capsulatus (Bath)
Authors: LUND John , WOODLAND Marc P. , DALTON Howard
Published: March 2005

Aerobic stopped‐flow experiments have confirmed that component C is the methane monooxygenase component responsible for interaction with NADH. Reduction of component C by NADH is not the rate‐limiting step for component C in the methane monooxygenase reaction.

Removal and reconstitution of the redox centres of component C suggests a correlation between the presence of the FAD and Fe2S2 redox centres and NADH: acceptor reductase activity and methane monooxygenase activity respectively, consistent with the order of electron flow: NADH → FAD → Fe2S2→ component A. This order suggests that component C functions as a 2e−1/1e−1 transformase, splitting electron pairs from NADH for transfer to component A via the one‐electron‐carrying Fe2S2 centre.

Electron transfer has been demonstrated between the reductase component, component C and the oxygenase component, component A, of the methane monooxygenase complex from Methylococcus capsulatus (Bath) by three separate methods. This intermolecular electron transfer step is not rate‐determining for the methane monooxygenase reaction.

Intermolecular electron transfer was independent of component B, the third component of the methane monooxygenase. Component B is required to switch the oxidase activity of component A to methane monooxygenase activity, suggesting that the role of component B is to couple substrate oxidation to electron transfer, via the methane monooxygenase components.
Phosphatidylserine and ornithine‐containing lipids of Bordetella, hemagglutinins of lipoamino acid structure, and their control in biomembranes
Authors: KAWAI Yohko , SUZUKI Kenji , HAGIWARA Toshikatsu
Published: March 2005

From the observation by light and electron microscopy, it was proved that phosphatidylserine agglutinates human erythrocytes by the same mechanism as that of ornithine‐containing lipids of Bordetella described previusly. The proposed mechanism was that two erythrocytes were bound through some liposomes of the lipoamino acids by hydrophobic and ionic interaction, and hydrogen bonding, between the lipoamino acids and the lipids or proteins in the membranes of erythrocytes. Hemagglutinating activity of phosphatidylserine might be controlled so as not to be expressed in biomembranes, on the basis of the finding that the liposomes constituted of phosphatidylserine and more than equal quantities of phosphatidylcholine did not exhibit hemagglutinating activity.
A 1H and 31P NMR study of cis‐Pt(NH3)2 [d(CpGpG)‐N7 (2),N7 (3)] The influence of a 5′‐terminal cytosine, on the structure of the cis‐Pt(NH3)2 [d(GpG)‐N7,N7] instrastrand cross‐link
Authors: DEN HARTOG Jeroen H. J. , ALTONA Cornelis , VANDER MAREL Gijs A. , REEDIJK Jan
Published: March 2005

Proton NMR studies at 300 MHz and 500 MHz have been carried out on the trinucleoside bisphosphate d(CpGpG) and on cis‐Pt(NH3)2[d(CpGpG)‐N7 (2),N7 (3)] [abbreviated as d(CpGpGp) ·cisPt]. For the Pt adduct, 13C and 31P NMR was also used for characterizing the oligonucleotide.

d(CpGpG) appears to revert to a B‐DNA‐type single helix at lower temperatures. The relatively small concentration dependence of the proton chemical shifts, in comparison with shifts due to intramolecular stacking effects, indicates that the compound is essentially single‐stranded.

In d(CpGpGp) ·cisPt, the first nucleoside, C(1), stacks well on top of the second, G(2), despite the N conformation of the G(2) sugar ring. The platinated GpG part in this trimer adopts largely the same structure as in cis‐Pt(NH3)2[d(GpGpG)‐N7 (1),N7 (2)] [den Hartog, J. H. J., et al. (1982) Nucleic Acids Res. 10, 4715–4730]. Main differences however, are changes in H8 chemical shifts and a 0.6‐ppm downfield shift of the third nucleotide phosphorus, P(3), in d(CpGpGp) ·cisPt with respect to P(2) in d(GpG) ·cisPt. The latter shift change is likely to be induced by a structural alteration, caused by stacking of C(1) on top of G(2). Also, the large chemical shift differences between the two H8 protons in d(NpGpG) ·cisPt fragments is discussed; the deviation from a mirror symmetry of the two guanine bases seems to be the main origin of this effect. The chemical shift changes, observed in the proton and phosphorus NMR chemical shift temperature and chemical shift pH profiles have been explained in terms of stack‐destack equilibria changes.
The crystal and molecular structure of the third domain of silver pheasant ovomucoid (OMSVP3)
Authors: BODE Wolfram , EPP Otto , HUBER Robert , LASKOWSKI Michael , ARDELT Wojciech
Published: March 2005

OMSVP3 and OMTKY3 (third domains of silver pheasant and turkey ovomucoid inhibitor) are Kazal‐type serine proteinase inhibitors. They have been isomorphously crystallized in the monoclinic space group C2 with cell dimensions of a= 4.429 nm, b= 2.115 nm, c= 4.405 nm, β= 107°. The asymmetric unit contains one molecule corresponding to an extremely low volume per unit molecular mass of 0.0017 nm3/Da. Data collection was only possible for the OMSVP3 crystals. Orientation and position of the OMSVP3 molecules in the monoclinic unit cells were determined using Patterson search methods and the known structure of the third domain of Japanese quail ovomucoid (OMJPQ3) [Papamokos, E., Weber, E., Bode, W., Huber, R., Empie, M. W., Kato, I. and Laskowski, M., Jr (1982) J. Mol. Biol. 158, 515–537]. The OMSVP3 structure has been refined by restrained crystallographic refinement yielding a final R value of 0.199 for data to 0.15 nm resolution. Conformation and hydrogen‐bonding pattern of OMSVP3 and OMJPQ3 are very similar. Large deviations occur at the NH2 terminus owing to different crystal packing, and at the C terminus of the central helix, representing an intrinsic property and resulting from amino acid substitutions far away from this site. The deviation of OMSVP3 from OMTKY3 complexed with the Streptomyces griseus protease B is very small [Fujinaga, M., Read, R. J., Sielecki, A., Ardelt, W., Laskowski, M., Jr and James, M. N. G. (1982)Proc. Natl Acad. Sci. USA, 79, 4868–4872].
Identification of the Ah receptor in selected mammalian species and induction of aryl hydrocarbon hydroxylase
Authors: DENISON Michael S. , WILKINSON Christopher F.
Published: March 2005

The Ah receptor protein, important in the mechanism of induction of aryl hydrocarbon hydroxylase activity, has been identified and partially characterized in hepatic cytosolic preparations from rat, BALB/c mouse, gerbil, hamster, rabbit, ferret and guinea‐pig by means of sucrose density centrifugation analysis and hydroxyapatite binding assays. Using 2,3,7,8‐tetrachloro[3H]dibenzo‐p‐dioxin (TCDD) as the ligand, total specific binding capacities ranged over 74–691 fmol [3H]TCDD/mg cytosolic protein and apparent dissociation constats ranged over 0.30–7.8 nM. There was no quantitative correlation between the concentration of cytosolic Ah receptors and the 3‐methylcholanthrene‐mediated induction of aryl hydrocarbon hydroxylase activity in the species studied. Competitive binding studies with a series of monohydroxylated benzo[a]pyrene derivatives suggested the importance of electronic character in their ability to bind to the Ah receptor and to compete with TCDD for specific binding sites on the receptor.
Synthetic and natural Escherichia coli free lipid A express identical endotoxic activities
Authors: GALANOS Chris , LÜDERITZ Otto , RIETSCHEL Ernst Th. , WESTPHAL Otto , BRADE Helmut , BRADE Lore , FREUDENBERG Marina , SCHADE U. , IMOTO Masahiro , YOSHIMURA Hiroyuki , KUSUMOTO Shoichi , SHIBA Tetsuo
Published: March 2005

The recently chemically synthesized Escherichia coli lipid A and the natural free lipid A of E. coli were compared with respect to their endotoxic activities in the following test systems: lethal toxicity, pyrogenicity, local Shwartzman reactivity, Limulus amoebocyte lysate gelation capacity, tumour necrotizing activity, B cell mitogenicity, induction of prostaglandin synthesis in macrophages, and antigenic specificity. It was found that synthetic and natural free lipid A exhibit identical activities and are indistinguishable in all tests.
The sequence of 967 amino acids at the carboxyl‐end of rat thyroglobulin
Authors: DI LAURO Roberto , OBICI Silvana , CONDLIFFE Donald , URSINI Valeria Matilde , MUSTI Annamaria , MOSCATELLI Claudio , AVVEDIMENTO Vittorio Enrico
Published: March 2005

The entire rat thyroglobulin mRNA sequence (about 8500 nucleotides) has been cloned in five recombinant plasmids containing overlapping cDNA inserts. The 3′ end of the mRNA is precisely defined by the poly (A) tail found in the furthest 3′ end clone. Evidence that most of the 5′ end is cloned come from size considerations and from a primer extension experiment. At the 3′ end of the mRNA only one long open reading frame is present in the sequence of 3018 nucleotides that has been established. In the deduced protein sequence we have localized two thyroxine‐forming sites in a region containing a high concentration of tyrosine residues.
Essential arginyl residues in the H+‐translocating ATPase of plasma membrane from the yeast Schizosaccharomyces pombe
Authors: DI PIETRO Attilio , GOFFEAU André
Published: March 2005

The H+‐translocating adenosine‐5′‐triphosphatase (ATPase) purified from the yeast Schizosaccharomyces pombe is inactivated upon incubation with the arginine modifier 2,3‐butanedione. The inactivation of the enzyme is maximal at pH values above 8.5. The modified enzyme is reactivated when incubated in the absence of borate after removal of 2,3‐butanedione. The extent of inactivation is half maximal at 10 mM 2,3‐butanedione for an incubation of 30 min at 30°C at pH 7.0. Under the same conditions, the time‐dependence of inactivation is biphasic in a semi‐logarithmic plot with half‐lifes of 10.9 min and 65.9 min. Incubation with 2,3‐butanedione lowering markedly the maximal rate of ATPase activity does not modify the Km for MgATP. These data suggest that two classes of arginyl residues play essential role in the plasma membrane ATPase activity.

Magnesium adenosine 5′‐triphosphate (MgATP) and magnesium adenosine 5′‐diphosphate (MgADP), the specific substrate and product, protect partially against enzyme inactivation by 2,3‐butanedione. Free ATP or MgGTP which are not enzyme substrates do not protect. Free magnesium, another effector of enzyme activity, exhibits partial protection at magnesium concentrations up to 0.5 mM, while increased inactivation is observed at higher Mg2+ concentrations. These protections indicate either the existence of at least one reactive arginyl in the substrate binding site or a general change of enzyme conformation induced by MgATP, MgADP or free magnesium.
Total number and differentiation of nucleotide binding sites on mitochondrial F1‐ATPase
Authors: WEBER Joachim , LÜCKEN Uwe , SCHÄFER Günter
Published: March 2005

In this study 3′‐O‐[3‐(4‐azido‐2‐nitrophenyl)propionyl]‐ADP was used as a photoaffinity analog for nucleotide binding sites on nucleotide‐depleted F1‐ATPase. Catalytic and binding properties of the labeled enzyme were investigated. The analog behaves as a competitive inhibitor in the dark (Ki= 50 μM). Photoirradiation of F1 in the presence of the analog leads to inactivation depending linearly on the incorporation of label. Complete inactivation is achieved at a stoichiometry of 3 mol/mol F1. The label is distributed between α and β subunits in a ratio of 30%:70%. Although three sites were blocked covalently by photolabeling, three reversible sites of much higher affinity than the labeled sites were preserved. Mild alkaline treatment of photoinactivated enzyme leads to almost complete reactivation which is due to hydrolysis of the 3′‐ester bond and release of the ADP moiety from the covalently bound analog. The conclusions drawn are as follows.

The total number of sites which can be simultaneously occupied by nucleotides on F1 is six.

Adopting the finding [Grubmeyer, C. & Penefsky, H. S. (1981) J. Biol. Chem. 256, 3718–3727] that the high‐affinity sites are the catalytic ones which can be covalently labeled by 3′‐O‐[5‐azidonaphthoyl(1)]‐ADP [Lübben, M., Lücken, U., Weber, J. & Schäfer, G. (1984) Eur. J. Biochem. 143, 483–490], it appears likely that azidonitrophenylpropionyl‐ADP is a specific photolabel for the lower‐affinity sites on nucleotide‐depleted F1. This means that both types of sites can be differentiated by specific photoaffinity analogs.

The labeled low‐affinity sites interact with the catalytic sites, abolishing enzyme turnover, when steadily occupied by ADP kept in place by the covalently linking residue, which by itself has no inhibitory effect on the enzyme.
The Muntjak satellite IA sequence is composed of 31‐base‐pair internal repeats that are highly homologous to the 31‐base‐pair subrepeats of the bovine satellite 1.715
Authors: BOGENBERGER Jakob M. , NEUMAIER Peter S. , FITTLER Friedrich
Published: March 2005

The nucleotide sequence of a cloned Muntjak satellite IA repeat unit (Muntiacus muntjak vaginalis) was determined. The repeat is 807 base pairs (bp) long. By introducing minor deletions and insertions, the whole sequence of the satellite can be arranged in 27 subrepeats of 31 bp length. Although diverged relative to each other, all subrepeats show a homology of more than 53% with the common consensus sequence. In 29 out of the 31 bp the consensus sequence of the Muntjak satellite subrepeat is identical to the 31‐bp subrepeat of the bovine satellite 1.715. This suggests that both satellites are derived from a common ancestral sequence. The results have interesting implications for the evolution of the two satellites.
Characterisation of rat 9‐kDa cholecalcin (CaBP) messenger RNA using a complementary DNA
Authors: PERRET Christine , DESPLAN Claude , BREHIER Arlette , THOMASSET Monique
Published: March 2005

The rat posesses two cholecalciferol‐induced calcium‐binding proteins, the cholecalcins (CaBP). The 9‐kDa CaBP is mainly concentrated in the duodenum while 28‐kDa CaBP is located in the kidney and cerebellum. The mRNA encoding 9‐kDa CaBP has been characterised using the cloned cDNA, pC109, synthesised from rat duodenal 9‐kDa CaBP mRNA [Desplan et al. (1983) J. Biol. Chem. 258, 13 502–13 505]. Nucleotide sequence analysis of this cDNA shows the presence of two stop codons, TGA and TAG, at positions 207 and 271, respectively, of the 3′ untranslated region. The cDNA‐hybridised mRNA, isolated from rat duodenum, directs the cell‐free synthesis of two proteins precipitable by antisera to 9‐kDa intestinal CaBP. A major protein comigrates with 9‐kDa CaBP whereas a minor product corresponds to a protein which is larger by 2000 Da. The minor protein appears to result from read‐through of the ‘leaky’ UGA stop signal. No protein band which was immunoprecipitable with 28‐kDa CaBP antiserum was detected when cDNA‐hybridised mRNA from rat kidney and cerebellum was translated in a cell‐free system. Northern blots show that the cDNA pC109 sequence hybridises to a homogeneous mRNA species 500–600 nucleotides long from rat duodenum. Larger mRNA species encoding 28‐kDa CaBP are undetectable in rat kidney and cerebellum even under low stringency conditions. All these findings demonstrate that there is no cross‐hybridisation between 9‐kDa and 28‐kDa CaBP mRNAs. Southern blot analysis of rat genomic DNA, that shows only one homologous 9‐kDa gene, is consistent with these findings. Thus, all our data indicate that there are distinct genes coding for each rat cholecalcin.
A‐form to A′‐form conformational switch of double helices in rat liver 5S and 5.8S rRNA
Authors: MÜLLER Jürgen Joachim , MISSELWITZ Rolf , ZIRWER Dietrich , DAMASCHUN Gregor , WELFLE Heinz
Published: March 2005

The wide‐angle X‐ray scattering of rat liver 5S rRNA and 5.8S rRNA molecules showed significant differences in the positions of the scattering maxima when dissolved in Mg2+‐containing Tris/HCI buffer or in Mg2+‐depleted buffer. A comparison of the experimental curves with theoretical curves calculated from atomic coordinates of double‐helical models proved a switch from A form to A' form of the double‐helical regions within the molecules by changing the buffer conditions. This result was supported by circular dichroic measurements. The A to A′ transition may have important consequences for RNA‐protein interactions.
Isolation of cardiac myosin light‐chain isotypes by chromatofocusing
Authors: VINCENT Neil D. , CUMMINS Peter
Published: March 2005

Cardiac myosin light chain isotypes have been resolved using chromatofocusing, a new preparative column chromatographic technique. The method relies on production of narrow‐range, shallow and stable pH gradients using ion‐exchange resins and buffers with even buffering capacity over the required pH range.

Light chains were resolved in order of decreasing isoelectric point in the pH range 5.2–4.5. Gradients of ΔpH = 0.004–0.006/ml elution volume were achieved which were capable of resolving light chains with isoelectric point differences of only 0.03.

Analytical isoelectric focusing of light chains in polyacrylamide gels could be used to predict the results of preparative chromatofocusing for method development.

Chromatofocusing was capable of resolving human and bovine cardiac light chain 1 and 2 subunits, atrial (ALC) and ventricular (VLC) light chain isotypes and homologous VLC‐2 and VLC‐2* light chains.

The technique was used to purify and resolve the human foetal ventricular light chain 1 (FLC‐1) from adult ventricular light chain 1 (VLC‐1) present in foetal ventricles and the atrial light chain 1 (ALC‐1) in adult atria.

Comparative peptide mapping studies and amino acid analyses were carried out on FLC‐1 and ALC‐1. No differences were detected between FLC‐1 and ALC‐1 using three different proteases and amino acid compositions were similar with the exception of glycine content. The studies indicate that FLC‐1 and ALC‐1 are homologous, and possibly identical, light chains.

Comparison of human FLC‐1/ALC‐1 with VLC‐1 suggested marked structural and chemical differences in these light chain isotypes, in particular in the contents of methionine, proline, lysine and alanine residues. Differences in the contents of these residues were also apparent in the corresponding bovine atrial and ventricular light chains [Wikman‐Coffelt, J. & Srivastava, S. (1979) FEBS Lett. 106, 207–212]. The latter three residues are known to be rich in the N‐termini of cardiac and skeletal light chain 1 isotypes, an area that has been implicated in actin binding, suggesting that atrial and ventricular light chains may differ functionally in this region.
On the mechanism of flavin modification during inactivation of flavocytochrome b2 from baker's yeast by acetylenic substrates
Authors: POMPON Denis , LEDERER Florence
Published: March 2005

The reaction of 2‐hydroxy‐3‐butynoate, a suicide substrate, with flavocytochrome b2 [F. Lederer (1974) Eur. J. Biochem. 88, 393–399] has been reinvestigated. It is shown that no inactivation occurs under anaerobic conditions. In the presence of ferricyanide, the partition ratio between oxidation and inactivation is 3200. Ketobutynoate has no effect on oxidized flavocytochrome b2. But it inactivates the reduced enzyme, while undergoing catalytic reduction to hydroxybutynoate. The partition ratio between reduction and inactivation is 5.

Inactivation followed by borohydride reduction was carried out in parallel with lactate oxidase and flavocytochrome b2. The decomposition products of the initial adduct formed between flavin and inactivator were isolated and characterized. One of them (compound II) was obtained from both enzymes and is identical to the one previously isolated from hydroxybutynoate‐inactivated lactate oxidase [Schonbrunn et al. (1976) Biochemistry 15, 1798–1807]. Its decarboxylated derivative (compound I) was also formed. Another major adduct, compound III, was isolated only from flavocytochrome b2. Its structure and the conditions in which it appears suggest it is formed from the same primary adduct as compounds I and II, but by a different decomposition mode, on the enzyme itself.

Altogether these results strengthen the idea that inactivation is caused by reaction between oxidized flavin and an allenic carbanion, the isomerisation product of a normal reaction intermediate. It is proposed that differences in the rate‐determining step of the redox reaction explain the differences in the inactivation process which are observed between flavocytochrome b2, lactate oxidase and hydroxyacid oxidase.
On porcine pancreatic α‐amylase action: kinetic evidence for the binding of two maltooligosaccharide molecules (maltose, maltotriose and o‐nitrophenylmaltoside) by inhibition studies
Authors: SEIGNER Christiane , PRODANOV Eugenio , MARCHIS‐MOUREN Guy
Published: March 2005

Hydrolysis of small substrates (maltose, maltotriose and o‐nitrophenylmaltoside) catalysed by porcine pancreatic α‐amylase was studied from a kinetic viewpoint over a wide range of substrate concentrations. Nonlinear double‐reciprocal plots are obtained at high maltose, maltotriose and o‐nitrophenylmaltoside concentrations indicating typical substrate inhibition. These results are consistent with the successive binding of two molecules of substrate per enzyme molecule with dissociation constants Ks1 and Ks2. The Hill plot, log [ν/(V‐ν)] versus log [S], is clearly biphasic and allows the dissociation constants of the ES1 and ES2 complexes to be calculated.

Maltose and maltotriose are inhibitors of the amylase‐catalysed amylose and o‐nitrophenylmaltoside hydrolysis. The inhibition is of the competitive type. The (apparent) inhibition constant Kappi varies with the inhibitor concentration. These results are also consistent with the successive binding of at least two molecules of maltose or maltotriose per amylase molecule with the dissociation constants Ki1 and Ki2. These inhibition studies show that small substrates and large polymeric ones are hydrolysed at the same catalytic site(s).

The values of the dissociation constants Ks1 and Ki1 of the maltose‐amylase complexes are identical. According to the five‐subsite energy profile previously determined, at low concentration, maltose (as substrate and as inhibitor) binds to the same two sites (4, 5) or (3, 4), maltotriose (as substrate and as inhibitor) and o‐nitrophenyl‐maltoside (as substrate) bind to the same three subsites (3, 4, 5). The dissociation constants Ks2 and Ki2 determined at high substrate and inhibitor concentration are consistent with the binding of the second ligand molecule at a single subsite. The binding mode of the second molecule of maltose (substrate) and o‐nitrophenylmaltoside remains uncertain, very likely because of the inaccuracy due to simplifications in the calculations of the subsite binding energies. No binding site(s) outside the catalytic one has been taken into account in this model.
Effect of avian osteopetrosis virus infection on cells and their collagen synthesis in vitro
Authors: TUDERMAN Leena , FRANKLIN Richard M.
Published: March 2005

Primary avian tendon fibroblasts and calvarial osteoblasts were infected with the avian osteopetrosis virus MAV.2‐O, in vitro. The infected tendon cells could be cloned in soft agar and kept in culture for at least 25 passages, a number not reached by uncloned infected cells. In contrast to many other virus‐transformed fibroblasts, these cells continued making collagen and fibronectin, and there were no gross morphological changes as observed in the light microscope. Changes were seen in their cytoskeletal structure, however, as observed by immunofluorescence. The cloned cells were not tumorigenic in nude mice, nor had they an altered pattern of protein phosphorylation.

MAV.2‐O‐infected fibroblasts and the cloned cells synthesized 2–3 times more collagen type I, the main product of their biosynthetic machinery, than control cells. The proportion of the total cellular RNA consisting of specific mRNAs for the precursor of collagen, procollagen pro‐α1 and pro‐α2 chains, was higher in the infected cells than in normal fibroblasts. Southern blotting experiments indicated that there was no rearrangement of the collagen genes after infection with this virus. Furthermore, large viral DNA fragments were not integrated into the immediate vicinity of the 5′ end of the α2‐collagen gene.