bluuebunny/crossref_metadata_2025_split huggingface dataset:
Heterogeneity of the Glycans O‐Glycosidically Linked to the Hinge Region of Secretory Immunoglobulins from Human Milk
Authors: PIERCE‐CRETEL Annick , PAMBLANCO Mercedes , STRECKER Gérard , MONTREUIL Jean , SPIK Geneviève 
Published: July 2010

Pure secretory immunoglobulin A was isolated from human milk by fractionation in gradients of pH and (NH4)2SO4 concentration followed by gel filtration. The hinge region containing all the O‐glycosidically linked oligosaccharides was isolated en bloc after trypsin and pepsin hydrolysis and separated by gel filtration. The mixture of O‐glycosidically linked oligosaccharides contained N‐acetylneuraminic acid (NeuAC), fucose (Fuc), galactose (Gal). N‐acetylglucosamine (GlcNAc) and N‐acetylgalactosamine (GalNAc) in the molar ratio of 0.2:0.5:2.5:2:1 respectively. After β‐elimination several oligosaccharides were separated by a combination of ion‐exchange chromatography and gel‐filtration chromatography. The complete structure of four of these oligosaccharides was determined by methanolysis, methylation and mass spectrometry.The structure of the four oligosaccharides which arc linked to serine or threonine residues of the hinge region are as follows: β‐Gal‐(1→3)‐GalNAc‐ol; α‐HeuAc‐(2→3)‐β‐Gal‐(1→3)‐GalNAc‐ol; β‐Gal‐(1→3)‐[β‐GlcNAc‐(1→6)]‐GalNAc‐ol; β‐Gal‐(1→3)‐[β‐Gal‐(1→4)‐β‐GlcNAc‐(1→6)]‐GalNAc‐ol.These oligosaccharides are more complex and heterogenous than the oligosaccharides linked to serine residues of the hinge region from myeloma seram immunoglobulin A1.
Specificity of Twelve Lectins Towards Oligosaccharides and Glycopeptides Related to N‐Glycosylproteins
Authors: DEBRAY Henri , DECOUT Dominique , STRECKER Gérard , SPIK Geneviève , MONTREUIL Jean 
Published: July 2010

Glycopeptides and oligosaccharides of either the N‐acetyllactosaminic or the oligomannosidic type derived from glycoproteins containing the N‐glycosylamine linkage were used to define the specificity of different lectins (concanavalin A, Lens culinaris agglutinin, Vicia faba agglutinin, Pisum sativum agglutinin, Ricinus communis agglutinins, soybean agglutinin, wheat germ agglutinin, Solanum tuberosum agglutinin, Datura stramonium agglutinin, Lotus tetragonolobus agglutinin, Ulex europeus agglutinin) by studying the inhibition of human red blood cell agglutination by these structures. The results obtained show that lectins considered ‘identical’ in terms of monosaccharide specificity, possess the ability to recognize fine differences in more complex structures. In fact, different lectins are able to recognize different saccharidic sequences on the same glycan structure. As these sequences are likely to be common to numerous glycoproteins, including cell membrane glycoproteins, the results obtained with lectins in the study of cell surface carbohydrates have to be very carefully interpreted.Moreover, our results confirm previous data on the spatial configuration of the glycan moiety of glycoproteins deduced from the construction of molecular models; the fact that oligosaccharides bearing an α‐NeuAc‐(2→6)‐Gal unit are more powerful inhibitors than oligosaccharides bearing an α‐NeuAc‐(2→3)‐Gal unit could be related to the high rotational freedom of α‐2, 6 linkage; the observation that glycoasparagines, glycopeptides and glycoproteins possess a higher affinity for lectins than the related oligosaccharides could be explained by the fact that the glycan‐amino acid linkage leads to structures more rigid than those of the oligosaccharides themselves.
An α‐Amanitin‐Resistant DNA‐Dependent RNA Polymerise II from the Fungus Aspergillus nidulans
Authors: STUNNENBERG Hendrik G. , WENNEKES Lambertus M. J. , SPIERINGS Titus , van den BROEK Hendrikus W. J. 
Published: July 2010

An α‐amanitin‐resistant DNA‐dependent RNA polymerise II has been purified from the lower eukaryote Aspergillus nidulans to apparent homogeneity by extraction of the enzyme at low salt concentration, polymin P (polyethylene imine) fractionation, binding to ion‐exchangers and density gradient centrifugation. By this procedure 0.4 mg of RNA polymerise II can be purified over 6000‐fold from 500 g (wet weight) of starting material with a yield of 25% and a specific activity of 550 units/mg.The subunit composition has been resolved by polyacrylamide gel electrophoresis in the presence of sodium dodecylsulphate and by two‐dimensional gel electrophoresis using a non‐denaturing gel in the first dimension and a dodecylsulphate slab gel in the second dimension. The putative subunits have molecular weights of 170000, 150000, 33000, 27000, 24000, 19000, 18000 and 16000. Only one form of RNA polymerise II could be resolved by electrophoresis. The chromatographic and catalytic properties and the subunit composition of the purified RNA polymerise II are clearly different from RNA polymerise I from A. nidulans but throughout comparable with other class II enzymes. It differs from all other class II enzymes by its insensitivity towards the toxin α‐amanitin, even at concentrations up to 400 μg/ml, and appears to be unable to bind O‐[14C]methyl‐γ‐amanitin at a concentration of 10 μg/ml of the toxin.We conclude that the purified RNA polymerise from A. nidulans is a real, but exceptional, type of the class II RNA polymerises.
Murein Biosynthesis and O‐Acetylation of N‐Acetylmuramic Acid during the Cell‐Division Cycle of Proteus mirabilis
Authors: GMEINER Jobst , KROLL Hein‐Peter 
Published: July 2010

The murein of Proteus mirabilis is unique with respect to the O‐acetylation of part of its N‐acetylmuramic acid residues. Working with synchronized cells and providing radioactive N‐acetylglucosamine with the medium, the murein was pulse‐labelled in vivo for 10‐min periods at different times of the cell cycle. After isolation of murein and enzymatic cleavage with endo‐N, O‐diacetylmuramidase from Chalaropsis, the distribution of radioactivity between uncrosslinked murein subunits (monomers) and the peptide‐crossliitked dimers was analyzed. The following results were obtained.

Murein synthesized at different times during the cell cycle did not show significant variation regarding the degree of crosslinkage and the degree of O‐acetylation. However, the degree of O‐acetylation of pulse‐labelled murein was found to be about sevenfold lower than that of murein from continuously labelled asynchronous cultures.
A pulse‐chase experiment revealed that only non‐O‐acetylated murein subunits were incorporated into the growing murein sacculus, part of which became O‐acetylated subsequently. Since a certain amount of newly incorporated subunits was lost during the chase, which reappeared in the sacculus about one generation later, limited murein turnover during the cell cycle became evident.
The degree of crosslinkage of both the O‐acetylated as well as the non‐O‐acetylated newly synthesized murein increased during subsequent growth.
The labelled peptide‐crosslinked dimers were cleaved by an endopeptidase isolated from P. mirabilis. The distribution of radioactivity between the resulting non‐O‐acetylated and O‐acetylated monomers indicated that most of the mono‐O‐acetylated dimer fraction carried radioactivity exclusively in the non‐O‐acetylated subunit which was not O‐acetylated during subsequent murein biosynthesis.
Since only a small portion of the labelled mono‐O‐acetylated dimer fraction carried radioactivity in both subunits, and only at specific times of murein biosynthesis, it was concluded that thin portion of the labelled mono‐O‐acetylated dimer fraction represented an intermediate in the O‐acetylation process.From these results a defined sequence of steps of murein biosynthesis is proposed.
Purification and Characterization of DNA‐Dependent ATP Phosphohydrolases from KB Cells
Authors: DE JONG Peter J. , VAN DER VLIET Peter C. , JANSZ Hendrik S. , Jan TOMMASSEN P. M. 
Published: July 2010

DNA‐dependent ATPases have been purified from logarithmically growing KB cells by chromatography on single‐stranded DNA cellulose and phosphocellulose. Phosphocellulose resolved the DNA‐dependent ATPases into three activities designated ATPase I. II and III, respectively. From gel filtration and sedimentation analysis ATPases II and III were found to be very similar, both with calculated molecular weights of 78000. Due to the extreme lability these enzymes were not purified further. The molecular weight of ATPase I determined by gel filtration and sedimentation analysis was calculated to be 140000. ATPase I was further purified by gradient elution on ATP‐agarose, revealing two peaks of activity (IA and IB), and by sucrose gradient sedimentation. Analysis of the fractions from the sucrose gradient by sodium dodecylsulphate gele electrophoresis revealed only one broad polypeptide band co‐sedimenting with both ATPase IA and ATPase IB. This band was composed of four closely spaced polypeptides with apparent molecular weights of 66000, 68000, 70000 and 71000. Comparison of the native molecule weight (140000) with these results suggests that ATPase I is a dimer. ATPase IA and IB were indistinguishable in their structural and enzymatic properties and presumably represent the same enzyme.The purified enzyme has an apparent Km of 0. 5 mM for ATP producing ADP + Pi. A maximum activity of 2100 molecules of ATP hydrolyzed per enzyme molecular per minute was found. Hydrolysis of ATP requires the presence of divalent canons (Mg2+ > Ca2+ > Mn2+ > Co2+). A broad pH optimum (pH 6–8) was observed. The enzyme uses ATP or dATP preferentially as a substrate, while other deoxyribonucleoside or ribonucleoside triphosphates were inactive. ATPase I prefers denatured DNA as cofactor. The activity with native DNA is 40% of that with denatured DNA.
Studies on the Mechanism of Pyrophosphate‐Mediated Uptake of Iron from Transferrin by Isolated Rat‐Liver Mitochondria
Authors: KONOPKA Krystyna , ROMSLO Inge 
Published: July 2010

Respiring rat liver mitochondria accumulate iron released from transferrin by pyrophosphate. The amount of iron accumulated is 1–1. 5 nmol mg protein−1 h−1, or approximately 60% of the amount of iron mobilized from transferrin.
The uptake declines if respiration is inhibited, substrate is deleted, or the experiments are run under anaerobic conditions. Substrate depletion and respiratory inhibitorsare less inhibitory under anaerobic conditions.
More than 80% of the amount of iron accumulated by aerobic, actively respiring mitochondria can be chelated by bath ophenanthroline sulphonate, and with deuteroporphyrin included, up to 30% of the amount of iron accumulated is recovered as deuteroheme. Iron accumulated by respiration‐inhibited mitochondria under aerobic conditions is not available for heme synthesis.
With time the uptake of iron increases eightfold relative to the uptake of pyrophosphate.
The results are compatible with a model in which ferric iron is mobilized from transferrin by pyrophosphate, ferric iron pyrophosphate is bound to the mitochondria, iron is reduced, dissociates from pyrophosphate and is taken up by the mitochondria. Ferrous iron thus formed is available for heme synthesis.
The Role of Penicillin‐Binding Proteins in the Action of Cephalosporins against Escherichia coli and Salmonella typhimurium
Authors: CHASE Howard A. , FULLER Christopher , REYNOLDS Peter E. 
Published: July 2010

The binding of two radioactively‐labelled cephalosporins, LY97962 and LY121998, to the penicillin‐binding proteins of Escherichia coli and Salmonella typhimurium has been studied in detail. The concentrations of the cephalosporins that resulted in 50% saturation of the penicillin‐binding proteins were measured in growing cells as well as in preparations of disrupted cell envelopes. This technique has allowed the crypticity of an organism towards a particular β‐lactam to be measured directly, i.e. the extent to which a penicillin‐binding protein is accessible in intact cells. The cephalosporin LY97962 appeared to have no difficulty in penetrating the outer layers of the cell whereas access of cephalosporin LY121998 to the penicillin‐binding proteins appeared to be somewhat restricted.At concentrations below the minimum growth inhibitory concentration the two cephalosporins inhibited cell division and caused the cells to grow as long filaments. When growing cells were incubated with the radioactive β‐lactam antibiotics at these concentrations, the β‐lactams bound almost exclusively to penicillin‐binding protein 3. The degree of binding of the cephalosporins to protein 1 A in cells growing in the presence of the minimum growth inhibitory concentrations of antibiotics varied with the duration of the incubation. After exposure for 5 min, very little binding to protein 1 A was apparent, whereas substantial binding was observed after 60–180 min. It was only at the end of this period of growth in the presence of the cephalosporins that lysis of the cells became apparent. This provided evidence that the binding of β‐lactams to protein 1 A of gramnegative organisms is an important step in the bactericidal action of certain cephalosporins. Protein 1 B was excluded as a target for the lethal action of these cephalosporins because binding to this protein was observed only at concentrations greater than those needed to inhibit growth of the cells.Microscopical examination of the growth of S. typhimurium in the presence of cefotaxime at sub‐lytic concentrations showed that cells grew as filaments for at least six generations without lysis. Observations of the division of the filament into individual cells after removal of the antibiotic showed that the entire length of the filament was viable. It is concluded that inhibition of cell division as a result of the binding of β‐lactams to protein 3 of gram‐negative organisms is not in itself a lethal event for the cells, but requires concomitant binding of antibiotic to protein 1 A before lysis is initiated.
Regeneration of Rhodopsin and Bacteriorhodopsin
Authors: TOWNER Paul , GAERTNER Wolfgang , WALCKHOFF Baerbel , OESTERHELT Dieter , HOPF Henning 
Published: July 2010

The rate of regeneration of rhodopsin, from 11‐cis‐retinal and opsin, and bacteriorhodopsin from all‐trans‐retinal and bacterio‐opsin, in the presence or absence of compounds whose structures partially resemble retinal were measured. Some of these compounds severely slowed down the regeneration process, but did not influence the extent of regeneration. In the case of compounds with a carbonyl functional group they were not joined to the active site of the apo‐protein via a Schiff's base linkage since after treatment with NaBH4 an active apo‐protein remained. The most effective inhibitors of rhodopsin regeneration were molecules whose structure could be superimposed on 9‐cis or 11‐cis retinal up to carbon atom 11. These C13 and C15 molecules were not distinguished between aldehyde, ketone or alcohol functional groups.The regeneration of bacteriorhodopsin was not inhibited by retinal analogues with short side chains. The most effective inhibitors were the all‐trans C17‐aldehyde (β‐ionylideneacetaldehyde) or C18‐ketone (β‐ionylidenepent‐3‐ene‐2‐one) which, compared to retinal, lack two or three carbon atoms from the end of the polyene chain. The inhibition was very dependent upon the presence of the all‐trans isomer and required aldehyde or ketone as functional group; nitriles and alcohols were less effective. However, similarly to retinol, the all‐trans C17 and C18 alcohols underwent a bathochromic shift and showed fine‐structured spectra when mixed with bacterio‐opsin.
Fast Stages of Photoelectric Processes in Biological Membranes
Authors: DRACHEV Lei A. , KAULEN Andrey D. , KHITRINA Lyubov' V. , SKULACHEV Vladimir P. 
Published: July 2010

Bacteriorhodopsin‐containing fragments of Halobacterium halobium membrane (bacteriorhodopsin sheets) were incorporated into a lecithin‐impregnated collodion film, and fast stages of flash‐induced electrogenesis were measured by two electrodes separated by this film.It is found that a single turnover of bacteriorhodopsin results in an electrogenic response composed of three main stages of the following τ: the first < 200 ns, the second 15–70 μs and the third 10 ms. The second and third phases are of the same direction as an electric response to continuous illumination, whereas the first one is oppositely directed. The lts and ms stages were shown to correlate, in the first approximation, with formation and decomposition of the bacteriorhodopsin intermediate absorbing at 412 nm, respectively. Both the second and third phases of the photoelectric response are sums of at least two exponents.The third stage is specifically inhibition by La3+ ions which are also shown to decrease the rate of regeneration of the original bacteriorhodopsin absorbing at 570 nm from the intermediate absorbing at 412 nm.Acidification of the medium induces parallel inhibition of the second and third phases and of formation of the intermediate absorbing at 412 nm as if protonation of a group with pK= 3.6 were responsible for this inhibition. The first (opposite) phase survives acidification. It even increases at pH lower than 1.5. At such a low pH, one can show a good correlation of decays of photopotential and of a bacteriorhodopsin bathointermediate. The decays are biphasic (τ1= 200 μs and τ2= 2 ms), formation of both the photopotential and the bathointermediate being faster than 200 ns.At higher pH, when a three‐phase photoelectric response is revealed, decay of the formed electric potential difference gives the average τ value of about 1 s. It can be accelerated by compounds that increase ionic conductance of biomembranes.At pH below 4, fluoride is found to completely inhibit the second and third phases, so that only the first phase is observed.The results are discussed in terms of a scheme postulating that the first electrogenic phase is a result of translocation of the protonated Schiff base inside the membrane due to a light‐induced conformation change in retinal or protein. The second and third phases are explained by H+ transfer from the Schiff base to the outer membrane surface and from inner (cytoplasmic) surface of membrane to the Schiff base, respectively.
Substrate Specificity of the Nuclear Protein Kinase NII from Porcine Liver
Authors: HOPPE Jürgen , BAYDOUN Hassan 
Published: July 2010

The substrate specificity of the nuclear protein kinase NII from pig liver was studied using the purified casein variants αs1B and βA2 both in the phosphorylated and dephosphorylated form. Chymotryptic peptides of the phosphorylated casein were analyzed by ascending thin‐layer chromatography followed by thin‐layer electrophoresis. Phosphorylated peptides were detected by autoradiography. The determination of a phosphorylated site in a cluster of three serines was performed by solid phase Edman degradation of the whole βA2 casein.Phosphorylated βA2 casein was by far the best substrate. A threonine at position 41 in the amino acid sequence was found to be predominantly phosphorylated. Dephosphorylated βA2 casein became rephosphorylated at serine‐17 whereas phosphorylation at threonine‐41 was greatly reduced. In both sites acidic amino acid side chains are present at the C‐terminal side though at different positions suggesting that acidic residues in the vicinity of the serine or threonine residue to be phosphorylated play an important role in the recognition by the nuclear protein kinase NIL
The Complete Amino‐Acid Sequence of the K88 Antigen, a Fimbrial Protein from Escherichia coli
Authors: KLEMM Per 
Published: July 2010

The complete primary structure of the fimbrial protein of the K88 antigen has been elucidated. This protein, which makes up the building block for the macromolecular structure that comprises a fimbria, consists of 264 amino acid residues in a single polypeptide chain. The K88 antigen was fragmented by chemical cleavage with cyanogen bromide, and by subsequent enzymatic sub‐cleavage of resulting fragments with trypsin and chymotrypsin, and was additionally cleaved with o‐iodosobenzoic acid. Peptides were sequenced by manual Edman degradation.The carboxy‐terminal part of the molecule is remarkable in being almost devoid of charged amino acid residues and is highly hydrophobic. Furthermore, this part of the structure could have a specific function as a molecular anchor.
Self‐Stabilization of the Energy Charge in a Reconstituted Enzyme System Containing Phosphofructokinase
Authors: SCHELLENBERGER Wolfgang , ESCHRICH Klaus , HOFMANN Eberhard 
Published: July 2010

The self‐stabilization of the energy charge and of ATP was investigated in an open reconstituted enzyme system containing phosphofructokinase, pyruvate kinase, adenylate kinase, and glucose‐6‐phosphate isomerase. The experiments were performed in a stirred flow‐through reactor containing gel‐entrapped enzymes.The dynamics of the system were analyzed theoretically by a model based on the kinetic properties of the individual enzymes. The energy charge was identified as one of the essential variables of the system.According to the theoretical prediction, homoeostasis of the energy charge was observed experimentally when either the maximal activity of phosphofructokinase, the energy charge of the influx solution or the flow rate through the reaction chamber was varied. It is shown that the efficiency of stabilization of the energy charge is related to the occurrence of alternative stationary states.
Characterization of the Second Prosthetic Group in Methanol Dehydrogenase from Hyphomicrobium X
Authors: DUINE Johannis A. , FRANK Johannes , Jzn  , VERWIEL P. Eugène J. 
Published: July 2010

Procedures are described for preparing 2,7,9‐tricarboxy‐1H‐pyrrolo[2,3‐ƒlquinoline‐4,5‐diol (pyrrolo‐quinoline quinol) from 2,7,9‐tricarboxy‐1H‐pyrrolo[2,3‐ƒ]quinoline‐4, 5‐dione (pyrrolo‐quinoline quinone).When methanol dehydrogenase is denatured, two compounds are liberated which have the same properties as the quinone and quinol mentioned above. On analysing the extract by high‐performance liquid chromatography, one molecule of the quinone and one molecule of the quinol per enzyme molecule are found.Mixtures of pyrrolo‐quinoline quinone and pyrrolo‐quinoline quinol at high pH produce the semiquinone form and, under certain conditions, a diamagnetic complex. Since electron spin resonance (ESR) shows that methanol dehydrogenase contains the semiquinone and the absorption spectrum suggests the presence of a diamagnetic dimer, it is tentatively concluded that the two prosthetic group molecules in the enzyme interact with each other.NMR experiments of pyrrolo‐quinoline quinone in 2H2O demonstrate that it is partly hydrated, most prohably at the C‐5 position. Although methanol adds in the same way, it is still questionable whether the product of this addition plays a role in the mechanism of the enzymic reaction.Potentiometric titrations show a midpoint potential of the quinone/quinol couple of + 90 mV at pH 7.0 and the information of the semiquinone as an intermediate in the titration at pH 13.0.