bluuebunny/crossref_metadata_2025_split huggingface dataset:
The Major Cell Envelope Protein of Micrococcus radiodurans (R1)
Authors: BAUMEISTER Wolfgang , KARRENBERG Franck , RACHEL Reinhard , ENGEL Andreas , TEN HEGGELER Beatrice , SAXTON W. Owen 
Published: March 2005

The so‐called HPI layer, representing the major cell envelope protein of the radiotolerant bacterium Micrococcus radiodurans, is a hexagonally patterned macromolecular monolayer; it is intimately associated with the outer membraneous layer (‘backing layer’) of the bacterium and covers its surface completely. By virtue of its quite unusual resistance to chemical perturbants it can easily be isolated, e.g. by detergent extraction of whole cells.A detailed description of the morphology of the HPI layer protein is given (resolution 1.8 nm), based on electron microscopy and image processing. Infrared spectroscopy shows that the secondary structure of the protein is stable under a wide range of environmental conditions; a considerable portion (30%) of the protein exists in the form of B structure.Direct mass measurements in a scanning transmission electron microscope reveal that the HPI layer protein has a molecular mass of approximately 655 kDa. Although masked by extensive proteolytic cleavage it appears that the morphologicaI protein units are composed of six identical protomers. All evidence accumulated so far supports our view that proteolysis is not a preparation artifact, but occurs already in vivo, without affecting the physical structure of the protein.
A Novel Non‐heme Iron‐Containing Dioxygenase
Authors: MÜLLER Rudolf , SCHMITT Stefan , LINGENS Frdnz 
Published: March 2005

Previously we purified an enzyme from Phenylobacterium immobilis DSM 1986, which cleaves the catechol derivative of the herbicide Chloridazon [5‐amino‐4‐chloro‐2‐phenyl‐3(2H)‐pyridazinone] in the meta position. The enzyme, which could be crystallized, proved in Ouchterlony double‐diffusion tests to consist of a single protein species. No cross‐reaction was observed with other meta‐cleaving enzymes. Its light absorption spectrum showed a maximum at 279 nm (ɛ= 310 mM−1 cm−1), shoulders at 289 nm and 275 nm and a very weak band at around 430 nm (ɛ= 1.14 mM−1 cm−1). The amino acid analysis showed a slight excess of acidic amino acids, in agreement with the PI of 4.5.Surprisingly the enzyme per se is completely inactive, although it contains one non‐dialysable iron atom per subunit. It has to be activated by preincubation with ferrous ions or ascorbate. The enzyme activated this way is autoxidizable and returns to its non‐activated state in the presence of oxygen. During the reaction with the substrate, this inactivation seems to be enhanced about 100 times. Since this kind of activation and inactivation is not observed in other meta‐cleaving enzymes, this enzyme seems to represent a new type of a non‐heme iron dioxygenase. We tentatively propose the name Chloridazon‐catechol dioxygenase for this enzyme.
The D(V/K) Isotope Effect of the Cytochrome P‐450‐Mediated Oxidation of Ethanol and Its Biological Applications
Authors: DAMGAARD Stig E. 
Published: March 2005

Purified preparations of rat liver microsomes consist of flat discoid structures, presumably vesicles. They catalyze oxidation of ethanol to acetaldehyde in the absence of alcohol dehydrogenase and catalase with a specific activity of 1.4‐53 nmol acetaldehyde formed min−1 (nmol cytochrome P‐450)−1. The D(V/ K) isotope effect of this microsomal ethanol‐oxidizing system was 1.15. This value is clearly different from those of alcohol dehydrogenase (3.0) and catalase (1.9). Particulate aldehyde dehydrogenases present in the preparation are active at unphysiologically high concentrations of aldehyde. Coincidently, the activity of the microsomal ethanol‐ oxidizing system, was well as the D(V/K) isotope effect associated with it, was increased significantly. This observation confirms the enzymic nature of the reaction studied.In microsomes washed once in KCI, ethanol oxidation was catalyzed mainly by catalase, but also by the microsomal ethanol‐oxidizing system, as judged from the D(V / K) isotope effect upon the net reaction. In the presence of sodium azide alcohol dehydrogenase activity could be demonstrated. In hepatocytes from rat or pig, 4‐methyl pyrazole at high concentrations reduced the rate of ethanol oxidation to 3‐10%. The D(V/K) isotope effect upon the residual ethanol oxidation reflected activities of both catalase and the microsomal ethanol‐ oxidizing system. The activity of this latter system was inhibited by 4‐methyl pyrazole competitively with ethanol, Ki= 5.7 mM. When this inhibition is taken into account, the uninhibited membrane system activity in these cells amount to maximally 10% of the rate of ethanol oxidation in the absence of inhibitor.
Heterogeneity of Human Erythrocyte Band 3 Analyzed by Two‐Dimensional Gel Electrophoresis
Authors: IDEGUCHI Hiroshi , MATSUYAMA Hiroyuki , HAMASAKI Naotaka 
Published: March 2005

Human erythrocyte membrane and purified band 3 were separated initially by isoelectric focusing and then examined in a second dimension by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. Band 3 was segregated into three major bands whether the protein was contained within the membranes or was present in the isolated state. The isoelectric points of these major bands were 5.25, 5.35 and 5.70. Of chymotryptic fragments of band 3, the 60‐kDa fragment was also separated into three major bands whose PI values were 4.75, 5.10 and 5.30. The multiplicity of band 3 appears to be due to different charges carried by the peptide(s) and is not ascribed to oxidation of band 3 during its preparation.Isoelectric points of the purified 60‐kDa fragment were different from the PI values of the fragment coexisting with the complementary 35‐kDa fragment, in which case the PI values were exactly the same as those of intact band 3. This suggests that these fragments interact tightly in situ even after being cleaved by chymotrypsin, and the tight interaction must still be present during electrophoresis in the first dimension.
The Core Histones from the Slime Mold Physarum polycephalum
Authors: CHAMPAGNE Madeleine , DEMOUVEAU Marie‐France , COUPPEZ Maurice , SAUTIÉRE Pierre 
Published: March 2005

The histones of Physarum polycephalum have been isolated and fractionated using a combination of gel exclusion and ion‐exchange chromatography or differential precipitation. The four core histones were unambiguously identified by gel electrophoresis, amino acid composition and N‐terminal analysis.The molecular weight of Physarum histones H3, H2B and H4 are very close or identical to the corresponding histones from chicken erythrocytes. Physarum H2A is significantly larger than chicken erythrocyte H2A and its N‐terminal residue is alanine. From the differences in amino acid compositions and in the peptide maps between Physarum and calf H4, one can expected some changes in the amino acid sequence of Physarum H4.
Ferredoxin from Methanosarcina barkeri: Evidence for the Presence of a Three‐Iron Center
Authors: MOURA Isabel , MOURA José J. G. , HUYNH Boi‐Hanh , SANTOS Helena , LeGALL Jean , XAVIER António V. 
Published: March 2005

Methanosarcina barkeri ferredoxin was purified and characterized by electron paramagnetic resonance (EPR) and Mössbauer spectroscopy. The purification procedure included chromatographic steps on DEAE‐cellulose and gel filtration. The isolated protein is unstable under aerobic conditions. The ferredoxin exhibits charge transfer bands at 283 nm and 405 nm with an absorption ratio A405/A283= 0.73. Its molecular weight has been estimated to be 20000–22000 by gel filtration chromatography. The native ferredoxin exhibits an intense EPR signal at g= 2.02 and only a very weak g= 1.94 signal develops upon reduction with dithionite. The Mössbauer spectra of the reduced protein are characteristic of a [3 Fe‐3S] center.The combined EPR and Mössbauer studies show that M. barkeri ferredoxin contains only [3 Fe‐3S] clusters, similar to Azotobacter vinelandii Fd [Emptage, M. H., Kent, T. A., Huynh, B. H., Rawlings, J., Orme‐Johnson, W. H. & Münck, M. (1980) J. Biol. Chem. 255, 1793–1796], Desulfovibrio gigas FdII [Huynh, B. H., Moura, J. J. G., Moura, I., Kent, T. A., LeGall, J., Xavier, A. V. & Münck, E. (1980) J. Biol. Chem. 255, 3242–3244] and mitochondrial beef heart aconitase [Kent, T. A., Dreyer, J.‐L., Kennedy, M. C., Huynh, B. H., Emptage, M. H., Beinert, H. & Münck, E. (1982) Proc. Natl Acad. Sci. USA, 79, 1096–1100].
Transport of Proteins into Chloroplasts
Authors: PFISTERER Jörg , LACHMANN Petra , KLOPPSTECH Klaus 
Published: March 2005

A system has been constructed in vitro for the binding of cytoplasmically synthesized chloroplast proteins to the chloroplast envelope which precedes the uptake into the organelle in vivo.Isolated chloroplast envelopes from young pea or spinach are capable of binding the majority of proteins obtained by translation of poly(A)‐containing RNA from greening plants in vitro. Among the bound proteins the precursors to the light‐harvesting chlorophyll a/b apoprotein and the small subunit of ribulose‐1,5‐bisphosphate carboxylase are prominent. Binding is an intrinsic property of the envelope membrane and does not require energy in the form of ATP. Bound proteins remain on the surface of the envelope vesicles and can be digested by protease. Binding is complete within minutes, shows a high affinity of the reactants, and is non‐ionic in nature.Protein binding is specific for translation products of poly(A)‐containing RNA from greening plants. Precursors to chloroplast protein are bound preferentially as compared to the mature proteins. The specificity is further demonstrated by the low binding of proteins obtained by run‐off translation of polysomes.Binding of radioactive labeled proteins is subject to competition by excess unlabeled homologous proteins. Once bound, the proteins are withdrawn from competition indicating a high binding stability.All the properties found for binding of proteins to isolated envelopes are consistent with the concept of the so‐called envelope carrier hypothesis.
ATP Synthetase (F1F0) of Escherichia coli K‐12
Authors: SCHNEIDER Erwin , ALTENDORF Karlheinz 
Published: March 2005

The purified ATP synthetase complex (F1F0) from Escherichia coli was adsorbed to immobilized poly‐(l‐lysine)‐deoxycholic acid. About 0.7 mg F1F0 were bound per ml of settled gel. The hydrophilic F1 part was dissociated from the complex by treatment with 7 M urea. F0 was eluted in high yield either with deoxycholate (6 mM) or taurodeoxycholate (10 mM). About 14% of the total protein bound to the column was eluted as F0, which corresponds to 64% of the total F0, in the F1F0 complex.
The purified F0 preparation obtained was composed of three different kinds of subunits with apparent molecular weights of 24000 (a), 19000 (b) and 8300 (c), respectively as determined by sodium dodecyl sulfate gel electrophoresis.
After incorporation into liposomes and the generation of a potassium diffusion potential by valinomycin, the F0 preparation mediated H+ translocation. This H+ uptake is inhibited by either dicyclohexylcarbodiimide or purified F1 ATPase.
Incubation of F0‐containing liposomes with F1 led to the reconstitution of an ATP‐driven quenching of acridine‐dye fluorescence. The quenching was abolished by uncoupler and prevented by dicyclohexylcarbodiimide.
Interaction of β‐Lactam Antibiotics with Penicillin‐Binding Proteins from Bacillus megaterium
Authors: RODRÍGUEZ‐TÉBAR Alfredo , ROJO Fernando , VÁZQUEZ David 
Published: March 2005

The binding properties of 25 β‐lactam antibiotics to Bacillus megaterium membranes have been studied. The affinities of the antibiotics for the penicillin‐binding proteins (PBPs) are also reported. We found that PBP 4 has the highest affinity for nearly all the antibiotics studied whereas PBP 5 has the lowest affinity. Both PBP 4 and PBP 5 appear to be dispensable for the maintenance of bacterial growth and survival and appear to be dd‐carboxypeptidases. Only the β‐lactam cefmetazol bound preferentially to PBP 5 and has been used to study the inhibition of dd‐carboxypeptidase.Comparative studies with β‐lactam that simultaneously result in (a) binding to PBPs 1 and 3, (b) inhibition of cell growth and (c) lysis, stressed the importance of PBPs 1 and 3 for cell growth and survival.
Inhibition of Cell‐Free Protein Synthesis by Low‐Molecular‐Weight RNAs from Free Cytoplasmic Ribonucleoprotein Particles
Authors: KÜHN Barbara , VILLRINGER Arno , FALK Heinz , HEINRICH Peter C. 
Published: March 2005

Free cytoplasmic messenger ribonucleoprotein (mRNP) particles from rat liver were treated with EDTA and separated into two populations of RNP particles with sedimentation maxima of 20 S and 35 S respectively. The 20‐S and 35‐S RNP particles, treated with 0.5 M KCl, have protein‐to‐RNA ratios of 0.31:1 and 5.7:1 respectively. Whereas 20‐S and 35‐S RNP particles exhibit a similar protein complement of seven major polypeptides, the low‐molecular‐weight RNA components of the two particle populations are different. A characteristic set of distinct low‐molecular‐weight RNAs is found for 20‐S and 35‐S RNP particles.When the individual low‐molecular‐weight RNAs of 20‐S and 35‐S RNP particles isolated from preparative polyacrylamide gels were assayed for their capability to inhibit protein synthesis in vitro, several potent translational inhibitory RNAs were detected. In particular, the low‐molecular‐weight RNAs of 147, 203 and 263 nucleotides in length associated with the 35‐S RNP particles turned out to be strong inhibitors of protein synthesis.
Synthesis of α1‐Antitrypsin in Rat‐Liver Hepatocytes and in a Cell‐Free System
Authors: GEIGER Thomas , NORTHEMANN Wolfgang , SCHMELZER Elmon , GROSS Volker , GAUTHIER Francis , HEINRICH Peter C. 
Published: March 2005

The biosynthesis of α1‐antitrypsin, a secretory glycoprotein, has been studied in vitro in a cell‐free translation system from wheat germ and in vivo with hepatocytes. When messenger RNA from rat liver polysomes was translated in a heterologous cell‐free system a translation product was immunoprecipitated by anti‐(α1‐antitrypsin) which had a smaller molecular weight (42000) on sodium dodecyl sulfate/polyacrylamide gel electrophoresis than the authentic glycoprotein (54000) isolated from rat serum. An intact amino terminus of the α1‐antitrypsin synthesized in vitro could be demonstrated by repetitive Edman degradation.When hepatocytes, labeled with [35S]methionine, were analyzed with respect to newly synthesized α1‐antitrypsin, a protein with a molecular weight of 49000 was immunoprecipitated from a high‐speed supernatant. When the hepatocytes were treated with tunicamycin, an inhibitor of protein glycosylation, a protein of a molecular weight of 40000 was immunoprecipitated from the cells as well as from the medium. Interestingly, an α1‐antitrypsin form of an even higher molecular weight (54000) was immunoprecipitated from the hepatocyte monolayer medium. Several conclusions can be drawn from these findings. First, α1‐antitrypsin is synthesized as a larger‐molecular‐weight precursor with an extra amino‐terminal sequence of about 20 amino acids. Second, the liver form of α1‐antitrypsin is different from the α1‐antitrypsin which has been secreted, and third, the carbohydrate moieties of rat liver α1‐antitrypsin do not appear to represent an absolute requirement for the secretion process.A length of 1930 ± 170 nucleotides was estimated for the poly(A)‐rich mRNA of the precursor of rat liver α1‐antitrypsin; a non‐coding region of about 700 nucleotides must exist.Furthermore, it is shown that the major part of the poly(A)‐rich RNA for α1‐antitrypsin is found in loosely and tightly membrane‐bound polyribosomes. This finding is in agreement with the current ideas on the site of synthesis of secretory proteins.
Similarity of Acetylcholine Receptors of Denervated, Innervated and Embryonic Chicken Muscles
Authors: SUMIKAWA Katumi , MEHRABAN Fuad , DOLLY J. Oliver , BARNARD Eric A. 
Published: March 2005

Acetylcholine receptors were purified to homogeneity from chicken embryonic, adult innervated and denervated muscles, by bio‐specific chromatographies using immobilised α‐neurotoxin and lentil lectin. A minimum specific activity for the pure receptor was estimated to be 6000 nmol α‐toxin binding sites/g protein. For analysis, the receptors were radio‐iodinated or tritiated to high specific radioactivity with succinimidyl‐[2,3‐3H]propionate. All of the iodinated protein present in the purified receptor preparation reacted with antibody against the pure acetylcholine receptor from Torpedo marmorata electric organ. In the case of all three muscle types used the same oligomeric forms were obtained. The principal form has a sedimentation coefficient of about 9 S, while a minor species (∼ 5 S) was also appreciable in crude preparations of embryonic and denervated muscles.Immunization of rabbits with the homogenous receptor from chick denervated muscle produced muscle weakness characteristic of experimental autoimmune myasthenia gravis. These antisera were equally reactive towards the receptor–125I‐α‐bungarotoxin complexes from chick innervated and denervated muscles. Likewise, the electrophoretic mobilities of the receptors (9‐S form) from all three muscle types were identical, as were the isoelectric points of their complexes with 125I‐α‐bungarotoxin. Collectively, these findings and associated ones on subunit structure denote that the 9‐S receptor molecules from junctional and extra‐junctional area and embryonic stage of chicken muscle are indistinguishable by all criteria yet applied to them.
A Thermodynamic Study of Cooperative Structures in Rabbit Immunoglobulin G
Authors: TISCHENKO Vladimir M. , ZAV'YALOV Vladimir P. , MEDGYESI György A. , POTEKHIN Sergei A. , PRIVALOV Peter L. 
Published: March 2005

The intramolecular melting of rabbit immunoglobulin G and its proteolytic fragments has been studied by scanning microcalorimetry in the acidic pH region. By thermodynamic analysis of the heat capacity function the following conclusions have been made.

The Fab and Fc fragments in IgG molecules are thermodynamically practically independent subunits in the pH region studied.
The Fab fragment exhibits three cooperative transitions at melting. Two of these transitions are explained assuming that equivalent domains in this fragment are paired, forming two cooperative blocks, while the third transition seems to correspond to the disruption of contacts between these cooperative blocks.
Fc and pFc' fragments exhibit four and two cooperative transitions, respectively. These transitions can be explained only by assuming that each of the structural domains of these fragments consists of two subparts and that the pairs of equivalent subparts are merged into the cooperative blocks thus forming four cooperative blocks in the Fc fragment.
Amphiphilic Pig Intestinal Microvillus Maltase/Glucoamylase
Authors: SØRENSEN Susanne Hedeager , NORÉN Ove , SJÖSTRÖM Hans , DANIELSEN E. Michael 
Published: March 2005

Pig intestinal microvillus maltase/glucoamylase (EC 3.2.1.20) was solubilized from microvillus membranes using Triton X‐100 and was purified by a specific anti‐(maltase/glucoamylase) immunoadsorbent using elution with hypotonic solution. The specific activity was 20–35 U × mg−1 and the yield 11%. The enzyme preparations were free of other known microvillus disaccharidases and peptidases (less than 0.4%) and showed one precipitate in crossed immunoelectrophoresis.
The Triton‐solubilized maltase/glucoamylase was amphiphilic as indicated by its detergent‐binding properties. These properties were abolished by treatment with papain and was not registered for the enzyme released from the microvillus membranes by papain. The Mr of the enzyme purified after solubilization by Triton X‐100 and papain was 330000 and 210000, respectively, as determined by gel filtration. The Mr, difference indicates that small‐molecular‐weight peptide(s) binding a Triton X‐100 micelle is (are) removed by proteolytic treatment.
Maltase/glucoamylase isolated from pigs in which the pancreas had been completely disconnected from the duodenum three days before killing, migrated in polyacrylamide gel electrophoresis in dodecyl sulphate as one polypeptide chain (Mr 245000–240000). This was also the only band seen in some preparations obtained from ordinary pigs. However, in preparations from normal pigs, most frequently two additional bands (Mr, 135000 and Mr 125000) with similar intensity to the larger band were seen. These two bands could be generated by treating preparations containing solely the larger band by pancreatic proteinases.
Maltase/glucoamylase hydrolysis of maltose and starch had a broad pH optimum (pH 6–7). Using maltose as substrate the kinetics seemed to obey Michaelis‐Menten kinetics (Km 1.74 mM). The enzyme effectively cleaves off glucose residues from the nonreducing end of glucose polymers containing α‐(1→4) glucosidic bonds; α‐(1→6) bonds are also hydrolyzed but at a slow rate. The highest activities were observed for maltose and maltohexaose while the intermediate linear α(1→4) glucose polymers were hydrolyzed less effectively.
2′‐Phosphodiesterase Activity in Human Cell Lines Treated or Untreated with Human Interferon
Authors: VERHAEGEN‐LEWALLE Marline , CONTENT Jean 
Published: March 2005

2′‐Phosphodiesterase activity was investigated, by measuring either the disappearance of (2′‐5′)oligo(adenylate) or the release of 5′AMP, in several human cell lines (RSa, IFr, HEC‐1, WGAr and HeLa) possessing different sensitivities to interferon, and treated or untreated with human interferon. In various cell lines whose (2′‐5′)‐oligo(adenylate) synthetase was normally induced by interferon treatment, both kinetic studies and measurements at different enzyme concentrations indicated that 2′‐phosphodiesterase activity remained unchanged after interferon treatment. This observation was confirmed over a broad range of substrate concentrations (1–2500 nM). The activity of 2′‐phosphodiesterase was dependent on Mg(OAc)2.Our results indicate that in various human cell lines the modulation of (2′‐5′)oligo(adenylate) metabolism by interferon does not involve an increase of 2′‐phosphodiesterase activity.
Purification and Characterization of Human‐Brain Aldose Reductase
Authors: WERMUTH Bendicht , BÜRGISSER Hanspeter , BOHREN Kurt , VON WARTBURG Jean‐Pierre 
Published: March 2005

Aldose reductase (EC 1.1.1.21) from human brain has been purified to apparent homogeneity. The enzyme catalyzes the NADPH‐dependent reduction of several physiological and xenobiotic aldehydes. Isocortico‐steroids, e.g. isocortisol and isocorticosterone, are the best substrates (Km < 1 μm), followed by aromatic and arylalkylaldehydes, including biogenic aldehydes (Km= 3–15 μM). The activity towards aldoses is highest with glyceraldehyde (Km= 25 μM) and decreases with increasing number of carbon atoms of the sugar.Flavonoids, e.g. quercetin and rutin, inhibit aldose reductase (IC50= 2–5 μM). Sulfate ions, on the other hand, stimulate the enzyme activity. Thiol‐modifying reagents, e.g. 4‐hydroxymercuribenzoate and iodoacetate, cause a time‐dependent inactivation.Aldose reductase consists of a single polypeptide chain with a molecular weight of 38000 and an isoelectric point of 5.9. In the presence of thiol reagents the isoelectric point is shifted to 5.1.Antibodies against aldose reductase do not cross‐react with other carbonyl reductases, Nevertheless, the comparison of structural and enzymic properties of aldose reductase with those of other carbonyl reductases suggests a relationship between aldose reductase and aldehyde reductase (EC 1.1.1.2).