bluuebunny/crossref_metadata_2025_split huggingface dataset:
Export of a Protein into the Outer Membrane of Escherichia coli K12
Authors: BREMER Erhard , COLE Stewart T. , HINDENNACH Ingrid , HENNING UIf , BECK Ewald , KURZ Christina , SCHALLER Heinz 
Published: March 2005

The cloned ompA gene encoding the major outer membrane protein OmpA of Eschevichiu coli has been shortened in vitro by exonuclease digestion from the end corresponding to the CO2H terminus of the protein. Nine derivatives were identified which still possessed sub:tantial parts of the ompA gene and one was constructed which had suffered a small deletion early in the gene. Gene fragments encoding NH2‐terminal OmpA sequences of 45, 133, 193, and 227 residues of the 325 amino acids of OmpA were examined in detail at the DNA level and for OmpA protein fragments synthesized. The latter two fragments were incorporated into the outer membrane and all known functions of the OmpA protein were expressed whereas the fragment with 133 OmpA‐specific residues was not stably incorporated into this membrane.In all cases where OmpA functions were observed, an OmpA‐specific polypeptide of Mr24000 was found in cell envelopes, regardless of the size of the residual ompA sequences and of the fused coding sequences in the vector DNA. Pulse‐label experiments revealed larger initial translation products, most of which were degraded to the protein of Mr 24000. The 133‐residue OmpA fragment was also detected but proved to be entirely unstable.It is argued that the OmpA protein consists of two domains and that the NH2‐terminal moiety from residues 1 to about 180 represents the membrane domain of the polypeptide. Therefore, the loss of about 50,possibly less, CO2H‐terminal residues from this domain suffices to interfere with stable incorporation into the outer membrane.
High‐Resolution 1H‐NMR Spectroscopy of Free and Glycosidically Linked O‐ Acetylated Sialic Acids
Authors: HAVERKAMP Johan , VAN HALBEEK Herman , DORLAND Lambertus , VLIEGENTHART Johannes F. G. , PFEIL Reinhard , SCHAUER Roland 
Published: March 2005

A number of naturally occurring and synthetic, partially O‐acetylated derivatives of N‐acetylneuraminic and N‐glycoloylneuraminic acids have been investigated by 360‐MHz 1H‐NMR spectroscopy. O‐Acetylation causes strong downfield shifts for the resonances of neighbouring sugar‐skeleton protons. The chemical shifts of these resonances, together with their characteristic multiplet shapes, can be used for localisation of the position of O‐acetyl substituents in the molecule. The number of such substituents in the molecule can be inferred from the number of acetyl‐methyl singlets, which also have characteristic resonance positions.This method for determination of the number and position of O‐acetyl substituents in sialic acid residues is very powerful for structural analysis of underivatized carbohydrate chains derived from glycoconjugates. This is demonstrated for the oligosaccharide N‐acetyl‐4‐O‐acetylneuraminosyl‐(α2→3)‐lactose isolated from echidna milk
Effect of Thyroid Hormones and Cyclic AMP on Some Lipogenic Enzymes of the Fat Cell
Authors: CORREZE Claude , BERRICHE Salahedine , TAMAYO Libardo , NUNEZ Jacques 
Published: March 2005

Thyroid hormones exert two types of effect on the lipogenic pathway of the fat cell of the young rat: one which is independent of cyclic AMP, and another which is under cyclic nucleotide control:

Thyroidectomy leads to an enhanced conversion of glucose to glucose 6‐phosphate by the hexokinase system. The activity of hexokinase I is increased twofold in the fat cell from hypothyroid rats. The oxidation of glucose 6‐phosphate by the pentose phosphate pathway is also increased after thyroidectomy. The V of both glucose 6‐phosphate and gluconate‐6‐phosphate dehydrogenases is doubled (56 mU × min−1× mg−1 to 114 mU × min−1× mg−1) whereas the Km, is unchanged (19 μM). The activities of ATP citrate lyase and of acetyl‐CoA carboxylase are higher in the adipocytes from hypothyroid rats. The activity of fatty acid synthetase is also stimulated, but to a lesser extent. The apparent Km of acetyl‐CoA carboxylase is 0.14 mM and 0.06 mM and the V is 17.8 nmol xmin−1 xmg‐′ and 27.8 nmolxmin−1 xmg protein−1 in the adipocytes from normal and hypothyroid rats respectively. In contrast, for ATP citrate lyase the V only is doubled (62 mU × min−1× mg−1 to 111 mUxmin−1xmg−1).
When fat cells from normal or hypothyroid rat were incubated in the presence of methylxanthines, dibutyryladenosine 3′,5′‐monophosphate, isoproterenol or epinephrine the activities of the hexokinase system and of glucose‐6‐phosphate and gluconate‐6‐phosphate dehydrogenases remained unchanged. In contrast, dibutyryl‐ adenosine 3′,5′‐monophosphate inhibited, by 60 ‐ 80%, the activities of ATP citrate lyase and acetyl‐CoA carboxylase in both cell types. In adipocytes prepared from normal rats, isoproterenol and epinephrine inhibited the activities of these two latter enzymes by 30 ‐ 60% while isobutylmethylxanthine potentiated their effect. In contrast, with fat cells from hypothyroid rats, isoproterenol and epinephrine had no effect upon the activities of ATP citrate lyase and acetyl‐CoA carboxylase, whereas a combination of epinephrine and ibobutylmethylxanthine was clearly inhibitory.

In conclusion, thyroid hormones regulate lipogenesis in the fat cell of the young rat firstly because they control the accumulation of the substrates of this pathway, glucose 6‐phosphate and NADPH, by changing the concentrations of the hexokinase system and of the rate‐limiting enzymes of the pentose phosphate pathway. Secondly, because they have a specific effect on the amount of the enzymes (ATP citrate lyase and acetyl‐CoA carboxylase) which channel acetyl‐CoA towards long chain fatty acid synthesis; and thirdly because they allow, in response to the lipolytic hormones, the accumulation of cyclic AMP which is responsible for the inactivation of these two latter enzymes.
Isolation and Characterization of New Collagens from Chick Cartilage
Authors: VON DER MARK Klaus , VAN MENXEL Marion , WIEDEMANN Hanna 
Published: March 2005

Three unique collagen chains were isolated from chick sternal cartilage following pepsin solubilization of total cartilage collagens and removal of the predominant type II collagen by fractional salt precipitation. Native molecules containing 1α,2α and 3α chains precipitated between 0.7 M and 1.2 M NaCl at acidic pH and could be purified by chromatography on carboxymethyl‐cellulose and agarose columns. Although similar to mammalian 1α, 2α and 3α chains, differences in the mobilities on sodium dodecylsulfate gel electrophoresis, CNBr peptidc profiles and amino acid composition were found. The lα and 2α chains resemble, but are structurally distinct from, the chick αl(V) and α2(V) chains. The 3α chain appears to be closely related to the αl(II) chain, although some differences in the cyanogen bromide peptides suggest that they might be different gene products.In addition, two collagenous fragments of Mr140000(M1)and 35000 (M2) were found which precipitated at 2.0 M NaCl at acidic pH. Both fragments contain interchain disulfide bonds. The larger fragment was reducible to subunits of approximate M, 120000,48000, 28000 and 11000. The smaller fragment gave rise to peptides of Mr about 12000 and 10000 after reduction. By the technique of rotary shadowing the native, unreduced larger fragment M1 appeared as a slender rod‐like molecule with a distinct bend approximately 40 nm from one end. We interpret this finding as indicative of a focal amino acid sequence irregularity, disrupting the triple‐helical conformation.
Na+ Channels with Binding Sites of High and Low Affinity for Tetrodotoxin in Different Excitable and Non‐excitable Cells
Authors: LOMBET Alain , FRELIN Christian , RENAUD Jean‐François , LAZDUNSKI Michel 
Published: March 2005

The properties of interaction of tetrodotoxin with its receptor site on the voltage‐sensitive Na+ channel were analysed in two ways: (a) by titrating Na+ channels with a tetrodotoxin derivative, [3H]ethylenediamine‐ tetrodotoxin; (b) by studying the physiological properties of interaction of the toxin with its receptor from 22Na flux measurements.Using a variety of cell types in culture, three different kinds of situations were observed.1. Cells like N1E 115 neuroblastoma, CCI 39 fibroblasts, embryonic chick cardiomyocytes and chick skeletal myotubes only have one family of Na+ channels with high‐affinity binding sites (in the nanomolar range) for tetrodotoxin. These Na+ channels are the same ones as those that are activated by the alkaloid and polypeptide toxins that accelerate 22Na+ influx.2. C9 cells have Na+ channels with low‐affinity binding sites for tetrodotoxin. These Na+ channels are also he ones that are activated by alkaloid and polypeptide toxins (the median inhibitory concentration for tetrodotoxin inhibition of 22Na+ influx through these Na+ channels is 300 nM).3. Rat myotubes that have differentiated in culture in the absence of neuronal influence have both high‐affinity binding sites (in the nanomolar range) detected with the tritiated tetrodotoxin derivative and low‐affinity binding sites (in the micromolar range) detected by 22Na+ flux experiments. Only low‐affinity binding sites correspond to Na+ channels that can be activated with alkaloid and polypeptide toxins.
The Interaction of Sulfate with Carbonic Anhydrase
Authors: SIMONSSON Ingvar , LINDSKOG Sven 
Published: March 2005

In the absence of sulfate the pH/rate profile for the 4‐nitrophenyl acetate hydrolase activity of bovine carbonic anhydrase is complex. The results fit with a microscopic ionization scheme involving two electrostatically interacting groups. The activity depends on the concentration of the basic form of one of these groups. Proton NMR spectra show that the active site residue, His‐64, has titration behaviour corresponding to that of the second group of the ionization scheme.
The addition of increasing concentrations of Na2SO4 gradually converts the pH/rate profile to that of a simple titration curve. A pKa of 6.9 is found at 50 mM Na2SO4. Concomitantly the titration curve of His‐64 changes. The results fit with the microscopic ionization scheme if it is assumed that significant SO2−4binding occurs only when both His‐64 and the activity‐linked group are protonated.
At constant pH, sulfate behaves as if it inhibits the enzyme only partially. However, data are presented suggesting that the enzyme‐sulfate complex is inactive, but the binding of SO2−4depends strongly on ionic strength. Thus, above about 25 mM sulfate any further increase of the sulfate concentration is nearly compensated by a decrease of the binding affinity. The binding of a monovalent anion, C1−, also depends on ionic strength, but this dependence is much less prominent than for SO2‐4.
Sulfate shows non‐competitive behaviour with respect to CO2 and appears to compete with HCO−3. Iodide and sulfate appear to be mutually competitive. The low pH limit of Ki for I−is 3 mM and about 0.1 mM in the presence of 50 mM Na2SO4 and in the absence of sulfate respectively.
In the absence of sulfate the Co(II)‐substituted bovine enzyme shows a complex pH/rate profile similar to that of the native enzyme. Changes of the optical spectrum parallel those of the activity. Relaxation rates (T−11)of water protons in the presence of Co(II) enzyme indicate that the Co(II)ion might have a H2O ligand at low pH in sulfate‐free solutions.
The effects on sulfate of human carbonic anhydrase II (or C) are similar to those on the bovine enzyme. The effects of sulfate on the human 1 (or B) enzyme are smaller.
Studies on the Specificity of the Binding Site of Vicia graminea Anti‐N Lectin
Authors: DUK Maria , LISOWSKA Elwira , KORDOWICZ Maria , WASNIOWSKA Kazimiera 
Published: March 2005

Incubation of polyacrylamide gels after electrophoresis of glycoproteins with Vicia graminea lectin, labeled with radioactive iodine, showed that the lectin was bound strongly to blood‐group N and Ss human sialoglycoproteins, weakly to the major sialoglycoprotein of horse red cell membranes, and was not bound to blood‐group M sialoglycoprotein.Inhibition of binding of the labeled lectin to blood‐group NN erythrocytes by untreated and modified human and horse erythrocyte glycoproteins and their structurally defined glycopeptide fragments was studied. The glycoproteins and glycopeptides were modified by desialylation, N‐acetylation, Edman degradation or by a combination of two or more of these procedures.Desialylation and N‐acetylation incrcased thc inhibitory activity of N and M glycoproteins and glycopeptideb. The lectin was inhibited by the NH2‐terminal M and N glycopeptides of various length of the polypeptide chain, and was not inhibited by the internal tryptic glycopeptide. All modified N glycopeptides (terminated with a leucine residue) were distinctly stronger inhibitors of the lectin than respective M glycopeptides (terminated with a berine residue). In each of these two groups the most active were tryptic and pronase asialoglycopeptides with blocked amino groups. The M and N tryptic asialoglycopeptides were inactivated by Edman degradation and reactivated by the subsequent N‐acetylation.Horse erythrocyte glycoprotein and its pronase glycopeptide were relatively weak inhibitors of V. graminea lectin, their activity was increased by desialylation, but was not affected by N‐acetylation.Comparison of the structures of glycopeptides reacting and not reacting with V.graminea lectin suggested the following tentative conclusions. (a) V.graminea lectin can react with glycopeptides which have clusters of unsubdiluted or sialylated Gal(βl‐3)GalNAc‐ chains located at adjacent amino acid residues. (b) Reaction of glycopeptides with the lectin is also dependent on other (e.g. amino acid) residues present at the lectin‐binding site, namely, it is enhanced by a hydrophobic residue and is weakened by a free amino group.
Gluconeogenesis in vitro
Authors: MÖRIKOFER‐ZWEZ Stéphanie , STOECKLIN Franziska B. , WALTER Paul 
Published: March 2005

A cell‐free system prepared from rat liver containing cytosol and mitochondria as well as a number of cofactors at near physiological concentrations was shown to form glucose 6‐phosphate from malate + 3‐phosphoglycerate at a rate of 1.11± 0.09 μmol. min‐1. g liver−1 (mean ± SEM, n= 9, 30°C). At least 70% of glucose 6‐phos‐ phate formed was derived from malate as calculated from experiments with [U‐14C]malate. The indicated rates were measured between 10 min and 30 min incubation time when the system was near steady state with respect to the lactate/pyruvate ratio and to most of the gluconeogenic intermediates.In the absence of mitochondria, the rate of formation of glucose 6‐phosphate from malate was about seven times lower than in their presence. A comparison between incubations carried out in presence or absence of mitochondria revealed that mitochondria decreased the lactate/pyruvate ratio and increased the ratio of (ATP + ITP)/(ADP + IDP). It could be shown that under the present incubation conditions, formation of glucose 6‐phosphate was closely linked to the ratio of (ATP + ITP)/(ADP + IDP) whereas changing redox ratios had little influence on the gluconeogenic rate.
The Role of the Components δ and y of the DNA‐Dependent RNA Polymerase of Lactobacillus curvatus in Promotor Selection
Authors: GIERL Alfons , ZILLIG Wolfram , STETTER Karl Otto 
Published: March 2005

An average of 0.44 molecule each of the initiation factor δ and the RNA polymerase binding protein y and 0.54 molecule of the subunit y per molecule of Lactobacillus curvatus DNA‐dependent RNA polymerase have been found in the cell.Free factor y displaces δ from free holo enzyme, Eδ. The formation of a binary complex from Ey, free δ, and poly[d(A‐T)], leads to immediate release of factor y. The release of the δ factor occurs upon the transition of the binary to a ternary complex.A mixture of E and δ forms binary complexes with all T7 DNA HpaII restriction fragments. In contrast a mix‐ ture of Ey and δ binds selectively to promoter‐containing DNA fragments, indicating that the stimulatory effect of y on transcription is due to an increase in the rate of promoter selection.The same RNA products are synthesised by Eδ and by Ey plus δ with T7 DNA as template. Thus the non‐ specific complexes formed by Eδ and T7 DNA are nonproductive.On the basis of these findings we propose a model for the transcription cycle in Lactobacillus curvatus.
Identification by Affinity Labeling of Potential Sites in 23‐S rRNA Interacting with the 3′ End of tRNA
Authors: LEITNER Moshe , WILCHEK Meir , ZAMIR Ada 
Published: March 2005

A derivative of tRNAPhe carrying at its 3′ end a photolyzable group was chemically synthesized by coupling p‐azidobenzoylglycylhydrazide to periodate‐oxidized tRNA. The reaction converts the 3′‐terminal ribofuranoside residue into a six‐membered ring. The binding ofp‐azidobenzoylglycylhydrazide‐t RNA to 70‐S ribosomes resembles the binding of Phe‐tRNA in its requirement for poly(U) and Mg2+dependence. Irradiation at wavelengths < 300 nm of complexes of p‐azidobenzoylglycylhydrazide‐tRNA and 70‐S ribosomes results in the covalent binding of approximately 20% of the reversibly bound tRNA to the ribosomes. The labeling occurs predominantly at a single site in 23‐S rRNA within the sequence included in the 18‐S fragment.To identify the modified sequence, an RNase A digest of 23‐S rRNA was fractionated on a hydrophobic matrix (Porapak Q) and adsorbed and non‐adsorbed fractions were compared by fingerprint analysis. A relatively intense spot was observed in the expected region in the fingerprint of the adosrbed fraction. The presence of an unusual nucleotide in the sequence of this oligonucleotide was demonstrated by a mobility‐shift analysis of a partial nuclease P1 digest. The sequence derived is G★‐A‐A‐G‐C (the asterisk denoting the site of modification). Sequences at six positions in th e 23‐S rRNA are compatible with the sequence of the oligonucleotide determined. Of the six positions five are located within nucleotides 1467‐ 1887. This region of approximately 400 nucleotides is likely to include the sequence interacting with the 3′ end of tRNA and possibly forming part of the peptidyl‐ transferase center.
Covalent Addition of H2O, Enzyme Substrates and Activators to Pyrrolo‐quinoline Quinone, the Coenzyme of Quinoproteins
Authors: DEKKER Robert H. , DUINE Johannis A. , FRANK Johannes , VERWIEL Jzn. P. Eugéne J. , WESTERLING Jaap 
Published: March 2005

The fluorescence excitation and the absorption spectrum of 2,7,9‐tricarboxy‐1H‐pyrrolo [2,3‐f]quinoline‐ 4,s‐dione (pyrrolo‐quinoline quinone, PQQ), measured in the pH range 7.0‐ 10.0, are quite different. However. when the temperature of the solution is lowered, the shape and maxima of these spectra become more similar. 1H‐NMR in 2H2/0 revealed a temperature‐dependent equilibrium between PQQ and a hydrated form. Evidence is presented that low temperature favours the formation of the fluorescing species which is PQQ, hydrated at the C‐5 position.Even further hydration is possible since absorption, fluorescence and NMR spectroscopy of PQQ in borate buffer pH 10.0 reveal additional hydration at the C‐4 position, pointing to a dihydrate.PQQ also reacts with quinoprotein enzyme substrates and activators. Spectroscopic measurements showed the existence of 5‐alkoxy‐5‐hydroxy‐PQQ and 5‐amino‐5‐hydroxy‐PQQ in the presence of alcohols and 2 M NH4CI, pH 9.0, respectively. In the latter case, the existence of 5‐imino‐PQQ could also be demonstrated. Addition compounds with amines appear to be unstable. The amines become probably oxidized because pyrrolo‐quinoline quinol (PQQH2) was found as the reaction product. On the other hand, an addition compound containing an imino bond could be isolated after addition of urea to a PQQ solution.Spectral characteristics of PQQ and its addition compounds are presented since these data are necessary for the spectral analysis of quinoproteins and the quantitative estimation of coenzyme.
Isolation and Properties of Proteoglycans from Bovine Aorta
Authors: SCHMIDT Annette , PRAGER Martin , SELMKE Peter , BUDDECKE Eckhart 
Published: March 2005

Calf arterial tissue, preincubated under organ culture conditions for 24 h in the presence of [35S]sulfate in combination with either [3H]glucosamine or [3H]mannose, yielded 81.8% of total uronic acids, 83% of incor‐ porated [35S]sulfate and 66.8 % of incorporated [3H]glucosamine on extraction with 4 M guanidinium chloride in the presence of protease inhibitors.Two proteoglycans (A and B) differing in amino acid and glycosaminoglycan composition were purified by sequential fractionation of the guanidinium chloride extract on Sepharose 4B CL, caesium chloride density gradient centrifugation under dissociative conditions and ion‐exchange chromatography.Gel filtration of the guanidinium chloride extract resulted in the separation of two [35S]proteoglycan‐con‐ taining fractions, eluted with the void volume (fraction A) and at Kav= 0.33 (fraction B).Proteoglycan A was obtained from the Sepharose fraction A as a proteoglycan complex by a dissociative gradient. On ion‐exchange chromatography in the presence of 6 M urea the proteoglycan complex dissociated into proteoglycan A (Mr 813000) and hyaluronate (M, 81 000). Proteoglycan A contained 3‐4 chondroitin 4/6‐sulfate side chains (Mr 35000) and 10.4% protein.Proteoglycan B was purified from the Sepharose fraction B by a dissociative gradient and subsequent ion‐ exchange chromatography of the bottom fraction. Proteoglycan B was characterized as a proteoglycan monomer (M r 190000) containing 20.3 % protein with 3 ‐ 4 chondroitin sulfate/dermatan sulfate chains (chondroitin sulfate 53 %, dermatan sulfate 46 %) attached. The specific 35S radioactivity of proteoglycan B was three‐times higher than that of proteoglycan A.[3H]Mannose‐labelling of the isolated proteoglycans A and B indicated that both proteoglycans contained glycoprotein‐type oligosaccharides.Pulse‐chase experiments with [35S]sulfate as label suggested that both proteoglycans A and B are primarily produced as monomers retarded by the Sepharose gel and that proteoglycan A is successively converted to a proteoglycan complex on interaction with hyaluronate.