bluuebunny/crossref_metadata_2025 huggingface dataset:
Non‐destructive Detection of Methionine Sulfoxide in the Resilium of a Surf Clam by Solid‐state 13C‐NMR Spectroscopy
Authors: KIKUCHI Yasuo , TAMIYA Nobuo , NOZAWA Tsunenori , HATANO Masahiro
Published: March 2005

Methionine sulfoxide was detected in the resilium (internal hinge ligament) of a surf clam by high‐resolution solid‐state 13C‐NMR spectroscopy involving cross‐polarization and magic angle spinning, using no chemical procedure. The results support the previous report [Kikuchi, Y. and Tamiya, N. (1981) J. Biochem. (Tokyo) 89, 1975‐1976] on a high content of methionine sulfoxide observed by chemical methods in the resilium protein of surf clam species.
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.
Biosynthesis of Dog Fibrinogen
Authors: KUDRYK Bohdan , OKADA Masahisa , REDMAN Colvin M. , BLOMBÄCK Birger
Published: March 2005

Dogs were injected with 3H‐labeled L‐amino acids and the incorporation of radioactivity into fibrinogen, its component polypeptide chains and various well characterized fibrinogen fragments, was determined in newly secreted plasma fibrinogen and in nascent fibrinogen isolated from the rough endoplasmic reticulum of hepatocytes. At 15‐20 min after the administration of 3H‐labeled l‐amino acids, radioactive fibrinogen was secreted onto the blood and the specific radioactivities of the Aα, Bβ and y chains were approximately the same at all times of secretion up to 2 h. Newly secreted fibrinogen was almost entirely in its phosphorylated form. At 15‐18 min, a time at which maximal incorporation of radioactivity had occurred in proteins, fibrinogen was isolated from a deoxycholate‐soluble fraction of the rough endoplasmic reticulum by affinity chromatography using columns of fibrin monomer as well as those prepared from monospecific antibodies to dog fibrinogen and to the fibrinogen chains. The nascent fibrinogen thus isolated was further characterized by analyses of thrombin‐ released fibrinopeptides A and B and their tryptic peptides. Furthermore, the nascent fibrinogen was cleaved with cyanogen bromide, the NH2‐terminal disulfide knot was prepared and its component chain fragments as well as the tryptic peptides derived from these fragments were isolated. The specific radioactivities of the various fibrinogen fragments were determined and it is concluded that all three fibrinogen chains are synthesized to the same extent and that, within the rough endoplasmic reticulum, nascent fibrinogen has already been fully disulfide‐bonded and exists as a dimer. In contrast to plasma fibrinogen, the Aa chain of nascent fibrinogen within the rough endoplasmic reticulum is not phosphorylated and the Bβ chain is not sulfated.
Rotational Motion of Cytochrome c Derivatives Bound to Membranes Measured by Fluorescence and Phosphorescence Anisotropy
Authors: DIXIT B. P. Sudha N. , WARING Alan J. , WELLS Kenneth O. , WONG Patricia S. , WOODROW George V. , VANDERKOOI Jane M.
Published: March 2005

Molecular motion of metal‐free and metal‐substituted cytochrome c derivatives was examined using the anisotropy of emissions from the singlet and the triplet states. The anisotropy of fluorescence provides a means to study the motion of cytochrome c in the nanosecond time scale, since the fluorescence lifetime of metal‐free cytochrome c is around 10 ns. We find that the anisotropy of fluorescence of metal‐free cytochrome c when bound to mitochondria does not decay, but when bound to phospholipids has a small component which decays independently of the rotation of the whole molecule. The use of phosphorescence extends the time scale for study into the millisecond regime, since the lifetime of the excited triplet state of zinc cytochrome c, as measured by triplet‐triplet absorption and phosphorescence emission is ∼ 9 ms for free zinc cytochrome c and 7 ms for mitochondrial membrane‐bound zinc cytochrome c at room temperature. The decay of anisotropy of phosphorescence emission of mitochondrial membrane‐bound zinc cytochrome c is clearly biphasic; the fast component corresponds to a rotational relaxation time of 300 μs and the slow component with relaxation time of ∼ 6 ms. The slow component appears to be due to the rotation of the entire mitochondrion, whereas the fast component was interpreted to be due to the rotation of cytochrome c in a cone about single axis perpendicular to the plane of the membrane surface.
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].
Recognition Site for Phosphorus‐Containing Compounds and Other Negatively Charged Solutes on the PhoE Protein Pore of the Outer Membrane of Escherichia coli K12
Authors: OVERBEEKE Nico , LUGTENBERG Ben
Published: March 2005

Experiments are described which were designed to obtain insight into the question why Escherichia coli K12 induces the PhoE protein pore in its outer membrane under conditions of phosphate limitation. Growth experiments showed that the presence of PhoE protein is advantageous for the growth rate of cells growing in a medium with large linear polyphosphates, but not with Pi, as the only phosphorus source. Further experiments, in which rates of permeation of β‐lactam antibiotics through the outer membrane were determined, showed that the most likely explanation for this result is that the PhoE protein pore is a relatively efficient channel for negatively charged solutes in general. Comparison of the rates of permeation of the two β‐lactam antibiotics cephaloridine and cefsulodin through PhoE protein pores and through OmpF protein pores in vivo suggested that the relatively efficient permeation of cefsulodin through PhoE protein pores must be attributed to the negative charge of a sulphate residue which is present in cefsulodin but absent in cephaloridine. Subsequent experiments showed that the rate of uptake of cefsulodin through PhoE protein pores, but not through OmpF protein pores, was reversibly inhibited by linear polyphosphates whereas phosphorylated compounds and other negatively charged solutes were also found to be inhibitory. The largest inhibition was observed with solutes containing several phosphate residues. Dixon plots showed that inhibition by Pi and linear polyphosphates was of the competitive type. The results are interpreted in terms of a recognition site on the PhoE protein pore for phosphorus‐containing solutes and for other negatively charged compounds.
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 Nocardicin A with the Penicillin‐Binding Proteins of Escherichia coli in Intact Cells and in Purified Cell Envelopes
Authors: BERENGUER José , DE PEDRO Miguel Angel , VÁZQUEZ David V.
Published: March 2005

This study deals with the interaction of nocardicin A with Escherichia coli penicillin‐binding proteins. Competition experiments with two different isotopically labelled β‐lactams indicated that nocardicin A interacts with penicillin‐binding proteins 1a, 1b, 2 and 4 in intact cells. Binding of nocardicin A to the penicillin‐binding proteins was abolished, or greatly reduced, when the assays were carried out with purified cell envelopes. Important differences between the binding patterns of benzyl [14C]penicillin to intact cells and to purified cell envelopes were also observed. These results suggest that the interaction of β‐lactam antibiotics with their target proteins depends to a very great extent on the state of the cell envelope as a whole.
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.
Binding‐Mechanism and Sensitivity in Gamete Chemotaxis of the Phaeophyte Cutleria multifida
Authors: BOLAND Wilhelm , TERLINDEN Rolf , JAENICKE Lothar , MÜLLER Dieter G.
Published: March 2005

Female gametes of the marine brown alga Cutleria multifida discharge multifidene into the water to attract the swimming males. The physicochemical principle of interaction between semiochemical and membrane receptor site is seen in mutual but complementary induced dipoles. Halogenated derivatives of multifidene with intact overall structure and stereochemistry but altered electronic properties were synthesized and assayed as lures. A direct correlation was observed between increasing polarizability and biological activity, thus proving the proposed mechanism. The low threshold concentration of this communication system (4 × 109 molecules/ml), together with a detailed knowledge of the gamete's architecture, swimming velocity and stimulus response time, indicates that very few, may be even single, molecules are able to trigger the sensomotoric chain. The rigidity of the natural signal molecule was recognized as an important element in inducing conformational fitting in the ligand/receptor complex.
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.
Biosynthesis of Mitochondrial Porin and Insertion into the Outer Mitochondrial Membrane of Neuruspora crassa
Authors: FREITAG Helmut , JANES Michael , NEUPERT Walter
Published: March 2005

Mitochondrial porin, the major protein of the outer mitochondrial membrane is synthesized by free cytoplasmic polysomes. The apparent molecular weight of the porin synthesized in homologous or heterologous cell‐free systems is the same as that of the mature porin. Transfer in vitro of mitochondrial porin from the cytosolic fraction into the outer membrane of mitochondria could be demonstrated. Before membrane insertion, mitochondrial porin is highly sensitive to added proteinase; afterwards it is strongly protected. Binding of the precursor form to mitochondria occurs at 4°C and appears to precede insertion into the membrane. Unlike transfer of many precursor proteins into or across the inner mitochondrial membrane, assembly of the porin is not dependent on an electrical potential across the inner membrane.
Location and Nucleotide Sequence of frdB, the Gene Coding for the Iron‐Sulphur Protein Subunit of the Fumarate Reductase of Escherichia coli
Authors: COLE Stewart T. , GRUNDSTRÖM Thomas , JAURIN Bengtåke , ROBINSON John J. , WEINER Joel H.
Published: March 2005

The frdB gene, encoding the iron‐sulphur protein subunit of fumarate reductase, has been located and its complete nucleotide sequence determined. The identity of the gene was confirmed by protein chemical studies and determination of the NH2‐terminal sequence of the FrdB protein. The frdB gene is situated distal to and partially overlapped by frdA which codes for the flavoprotein subunit of the reductase. Its reading frame contains 244 codons and predicts a protein of Mr 27092. In composition, the FrdB protein is strikingly similar to the corresponding subunit of the related flavoenzyme, succinate dehydrogenase. Analysis of the protein's primary structure revealed several features characteristic of iron‐sulphur 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.
Nucleotide Sequence of Cloned cDNA Coding for Mouse ɛ Casein
Authors: HENNIGHAUSEN Lothar G. , STEUDLE Annelotte , SIPPEL Albrecht E.
Published: March 2005

We isolated cDNA clones, which correspond to the mRNA coding for the smallest of the seven mouse caseins. From the nucleotide sequence of the cDNA we deduced the amino acid sequence of the protein, which we named ɛ casein. Mouse ɛ casein and cow αs2 casein show amino acid sequence homologies in the N‐terminal region of the mature protein. The signal peptide of mouse ɛ casein shows in length and sequence remarkable homology to the signal peptides of the calcium‐precipitable caseins of other species. In accordance with this group of caseins mouse ɛ casein contains in the sequence‐Ser‐Ser‐Glu‐Glu‐ a site for potential multiple phosphorylation.
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.
Chemical Cross‐Linking of Elongation Factor G to both Subunits of the 70‐S Ribosomes from Escherichia coli
Authors: SKÖLD Sven‐Erik
Published: March 2005

Ribosomal proteins situated at or near the binding site of elongation factor G (EF‐G) on the Escherichia coli ribosome have been identified by use of the bifunctional, cleavable cross‐linker, dimethyl‐4,9‐diaza‐5,8‐dioxo‐6,7‐dihydroxy‐dodecanebisimidate. Five different bimolecular EF‐G · ribosomal‐protein complexes were isolated electrophoretically. The ribosomal proteins found in each of these complexes were identified as the 50‐S‐subunit proteins L6, L7/L12 and L14 and the 30‐S‐subunit proteins S12 and S19. In the presence of thiostrepton, which prevents binding of EF‐G to the ribosome, there was a considerable decrease in the yield of each of these cross‐linked complexes. The data suggest that EF‐G is bound close to the ribosomal subunit interface.
Sites of Biosynthesis of Carotenoids in Capsicum Chromoplasts
Authors: CAMARA Bilal , BARDAT Françoise , MONÉGER René
Published: March 2005

Capsicum chromoplasts incubated with isopentenyl diphosphate actively synthesize carotenoids. The enzymes involved in these reactions are compartmentalized: the stroma is the site of phytoene synthesis, the first colourless carotenoid, while desaturation and cyclization of the latter leading to coloured carotenoids, occur in the membrane fraction (chromoplast envelope + achlorophyll lamellae derived in part from the inner envelope membrane). Phytoene synthetase could be used as a marker of chromoplast stroma.
Proteolytic Maturation of the Turnip‐Yellow‐Mosaic‐Virus Polyprotein Coded in vitro Occurs by Internal Catalysis
Authors: MORCH Marie‐Dominique , ZAGÓRSKI Wlodzimierz , HAENNI Anne‐Lise
Published: March 2005

The genomic RNA of turnip yellow mosaic virus is translated in vitro into two major high‐molecular‐weight proteins, the larger of which (Mr 195000) undergoes post‐translational cleavage. The mechanism of formation of the primary cleavage products (Mr 120000 and Mr 78000) of the 195000‐Mr protein has been examined. The fact that cleavage partly occurs at a rate insensitive to dilution of the 195000‐Mr protein is suggestive of an intramolecular mechanism of proteolytic maturation.
Turnover of Adenosine Triphosphatase from Rat Liver MitochondriaM
Authors: PÉREZ‐PASTOR Ernesto , WALLACE Ruth , GRISOLÍA Santiago
Published: March 2005

The half‐life of mitochondrial adenosine triphosphatase and the relative rate constants of protein degradation for several fractions of rat liver have been measured by the double‐isotope technique. It has been shown that the apparent turnover rates of some mitochondrial enzymes, far apart in size, such as carbamoyl phosphate synthetase, glutamate dehydrogenase and malate dehydrogenase, are not related to molecular weight or to size of subunits. In view of the possibility that mitochondrial proteins are degraded by different mechanisms, it was of interest to determine the half‐life of a protein tightly bound to the inner membrane such as adenosine triphosphatase. The rate constants of degradation for rats fed a basal diet and injected at three‐day intervals with isotopic leucine were: homogenate, kd= 0.195 days−1; mitochondria, kd= 0.135 days−1; cytosol, kd= 0.140 days−1; microsomes, kd= 0.28 days−1; ATPase, kd= 0.275 days−1. The rate constants of the cellular fractions of liver of rats fed a high protein diet did not change or showed a small increase, compared with those of animals fed the basal diet, while those from rats on the protein‐free diet showed a decrease. The rate constant for adenosine triphosphatase showed an increase with high‐protein and a decrease with protein‐free diet. A procedure for the purification of ATPase from a single liver of a rat is described.
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).
High‐Affinity Binding of ADP and of ADP Analogues to Mitochondrial F1‐ATPase
Authors: TIEDGE Henri , LÜCKEN Uwe , WEBER Joachim , SCHÄFER Gunter
Published: March 2005

Nucleotide‐depleted F1‐ATPase was prepared from beef heart mitochodria. By use of fluorescence techniques and isotope binding analyses, we investigated the occupation of the high‐affinity binding sites on F1 by ADP and the ADP analogues 3′‐O‐(1‐naphthoyl)adenosine diphosphate (N‐ADP) and 3′‐O‐[1‐(5–dimethylamino)‐naphthoyl]adenosine diphosphate (DMAN‐ADP). F1‐ATPase was found to exhibit three binding sites for ADP (Kd= 50 nM for one site; Kd= 3 μM for the remaining two sites), two binding sites for N‐ADP (Kd= 20–50 nM for both of the sites), and three binding sites for DMAN‐ADP (Kd= 50 nM for all of the sites). Since the adenine nucleotides under consideration are bound to the same class of sites, the binding data can be explained best on the basis of the hypothesis that the binding process is anticooperative with ADP, whereas the analogues are able to overcome anticooperativity partially (N‐ADP) or completely (DMAN‐ADP). This binding model is consistent with the view that the exchangeable tight sites are involved directly in the catalytical process of ATP‐synthesis in oxidative phosphorylation.
The Polypeptide Components from Light‐Harvesting Pigment‐Protein Complex II (B800–850) of Rhodopseudomonas capsulata
Authors: TADROS Monier H. , ZUBER Herbert , DREWS Gerhart
Published: March 2005

A new procedure for isolation, purification and identification of the three polypeptides of the membrane‐bound light‐harvesting complex II (B800–850) of Rhodopseudomonas capsulata has been developed.

The polypeptides were extracted from crude intracytoplasmic membranes with chloroform/methanol/ammonium acetate and separated by chromatography on Sephadex LH60. The peak fractions were transferred to solvents of different polarity and separated by gel filtration or ion‐exchange chromatography. The three major polypeptides isolated by this two‐step chromatography were found to be homogenous and identical with the three polypeptides of the light‐harvesting complex II, as judged by amino acid analysis and N‐terminal sequence determination.

Contaminating minor polypeptides, of which the functions are unknown, were different from the polypeptides of the B800–850 complex studied by the same criteria.
The Role of Phosphate in the Regulation of the Independent Calcium‐Efflux Pathway of Liver Mitochondria
Authors: ZOCCARATO Franco , NICHOLLS David
Published: March 2005

The rate of spontaneous efflux of Ca from liver mitochondria incubated in the absence of ATP and Mg increases with time and is associated with a synchronous collapse of membrane potential and with Pi efflux. In the presence of Mg and ATP the ruthenium‐red‐induced Ca efflux does not change with time. The activity of the Ca efflux pathway in Pi‐depleted mitochondria is 15‐fold greater than in mitochondria equilibrated with 3.3 mM Pi. 50% inhibition is caused by 0.3 mM Pi. The membrane potential is not affected by changes in Pi concentration, although the steady‐state extra‐mitochondrial free Ca concentration reflects the alterations in efflux rate. In the presence of Pi, the ruthenium‐red‐induced efflux rate is independent of the total matrix Ca content; however in Pi‐depleted mitochondria, with acetate substituting as permeant anion, the efflux rate increases with total matrix Ca content. The lowered efflux rate in the presence of Pi is not due to a limitation in the rate of dissociation of the matrix Ca‐phosphate complex. The efflux pathway is activated by a lowered membrane potential, but the relative effect of Pi is retained. Under the present conditions Na slightly inhibits the efflux rate. The lack of an effect of total matrix Ca content on the efflux rate in the presence of Pi is used as the basis of a highly accurate determination of the activity of the Ca uniporter as a function of external free Ca concentration.
Effect of Lysosomes on Rat‐Liver Catalase
Authors: MAINFERME Francis , WATTIAUX Robert
Published: March 2005

The electrophoretic behaviour of rat‐liver catalase in polyacrylamide gel depends on the subcellular fraction the enzyme was isolated from. Catalase extracted from the mitochondrial fraction is more anodic than catalase recovered from the unsedimentable fraction of the homogenate. The difference disappears if the sedimentable enzyme is extracted from purified peroxisomes. On the other hand, when treated with a lysosomal extract, catalase present in the unsedimentable fraction or in purified peroxisomes, behaves like the enzyme isolated from the mitochondrial fraction. Factors that influence that effect of lysosomes on catalase indicate that it is due to a proteolysis by an enzyme like cathepsin B. These results suggest that catalase isolated from the mitochondrial fraction differs from catalase isolated from peroxisomes or from the unsedimentable fraction, because it has been subjected to a proteolysis caused by lysosomes present in the mitochondrial fraction, during the extraction procedure. As a matter of fact, catalase isolated from the mitochondrial fraction is endowed with a lower molecular weight than catalase extracted from purified peroxisomes or from the unsedimentable fraction. This structural modification does not apparently affect the stability and the catalytic power of the enzyme.
Chemical Characterization of a Rabbit Leukocytic Pyrogen
Authors: PACÁK František , SIEGERT Rudolf , Heipel Heide , Schrödter Kornelia , Brandl Franz
Published: March 2005

Crude pyrogen was obtained by stimulating 1.4 × 1012 peritoneal‐exudate leukocytes from rabbits in vitro with Escherichia coli endotoxin. A pyrogen molecule was isolated by ammonium‐sulfate precipitation, polyacrylamide gel electrophoresis at pH 5.5 and 8.9, and gel filtration on Bio‐Gel P‐100. These procedures yielded a pyrogenic fraction which was electrophoretically and immunologically homogeneous. The pyrogenicity was 50 ng/kg of rabbit. This leukocyte pyrogen consisted of a protein with a molecular weight of approximately 14000. Amino‐acid analysis showed high amounts of glutamic acid and glycine; methionine and tryptophan were present in small quantities. Arginine was found to be the N‐terminal amino acid. The molecule contained about 0.8% neutral sugar and 0.7% lipids. Its approximate isoelectric point in citrate/phosphate buffer was 4.2. The activity was lost by lyophilization and storage at ‐18°C for six months. The data presented here are the first detailed characterization of a rabbit leukocyte pyrogen. The properties in common to leukocytic pyrogen and interleukin 1 are discussed.
Purification and Characterization of Acetoacetyl‐CoA Synthetase from Zoogloea ramigera I‐16‐M
Authors: FUKUI Tetsuya , ITO Masaki , TOMITA Kenkichi
Published: March 2005

Acetoacetyl‐CoA synthetase was purified to electrophoretic homogeneity from Zoogloea ramigera I‐16‐M, a poly(3‐hydroxybutyrate)‐accumulating bacterium, which lacks 3‐ketoacid CoA‐transferase. The purified enzyme had a specific activity of 52.2 μmol acetoacetyl‐CoA formed min−1 mg protein−1, which constituted a 680‐fold purification compared to the crude extract, with a 5.1% yield. The enzyme absolutely required ATP, CoA, a monovalent cation (K+, Rb+, Cs+ or NH+4) and a divalent cation (Mg2+, Mn2+, Ca2+ or Ni2+) for the activation of acetoacetate, yielding acetoacetyl‐CoA, AMP and pyrophosphate in equimolar amounts. The pH optimum of the enzyme reaction was 8.4. The molecular weight of the enzyme was approximately 70000 as estimated by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, and 72000 by Sephadex G‐200 gel filtration. The enzyme was active only on acetoacetate and to a lesser extent on l(+)‐3‐hydroxybutyrate, and the Km values for acetoacetate, l(+)‐3‐hydroxybutyrate, ATP and CoA were 7.6 × 10−5 M, 1.4 × 10−3 M, 3.3 × 10−5 M and 9.1 × 10−5 M respectively.
The Binding Sites for Large and Small High‐Mobility‐Group (HMG) Proteins
Authors: SCHRÖTER Hennrik , BODE Jürgen
Published: March 2005

Studies in vitro of binding high‐mobility‐group (HMG) proteins to nucleosomal particles that differ in their DNA contents reflect several aspects pertinent to their function in vivo. Two molecules of HMG 14 or 17 are accomodated by particles with 140 or 180 base pairs of DNA whereas HMG 1 or 2 are only bound by the larger specimens irrespective of the presence of HMG 14/17. It is concluded that one molecule of HMG 1 or 2 binds to the 40 base pairs of linker DNA whereas the HMG 14 or 17 molecules associate with the nucleosomal core. At physiological ionic strength, HMG 14 binding is cooperative, probably by triggering a conformational change in the nucleosomal particle. The phenomenon has been studied by two independent techniques. Besides the common gel‐electrophoretic system, a centrifugation assay is described, which permits the derivation of a Hill coefficient nH= 1.3 and dissociation constants in the range of 30–90 nM at 0.15 M NaCl, pH 6.8.
Bile‐Salt‐Binding Polypeptides in Plasma Membranes of Hepatocytes Revealed by Photoaffinity Labelling
Authors: KRAMER Werner , BICKEL Ulrich , BUSCHER Hans‐Peter , GEROK Wolfgang , KURZ Gerhart
Published: March 2005

Photoaffinity labelling of a subfraction of plasma membranes of rat liver, enriched with sinusoidal surfaces, with the sodium salts of (3β‐azido‐7α, 12α‐dihydroxy‐5β‐cholan‐24‐oyl)‐2‐amino[2‐3H(N)]ethanesuifonic acid, (7, 7‐azo‐3α, 12α‐dihydroxy‐5β‐cholan‐24‐oyl)‐2‐amino[2‐3H(N)]ethanesulfonic acid and (11ξ‐azido‐12‐oxo‐3α, 7α‐dihydroxy‐5β‐cholan‐24‐oyl)‐2‐amino[2‐3H(N)]ethanesulfonic acid resulted with each derivative in a clear covalent incorporation of radioactivity into polypeptides with the apparent molecular weights of 67000, 52000, 48000, 43000 and about 20000.

Photoaffinity labelling of a membrane subfraction predominantly composed of bile canalicular membranes by the photolabile derivatives of the conjugated bile salts also showed covalent incorporation of radioactivity into polypeptides of the same apparent molecular weights as with the subfraction enriched with the sinusoidal membranes.

The extent of photoaffinity labelling of the different membrane polypeptides is dependent upon the photolabile bile‐salt derivative used. However, with each of the photolabile derivatives the relative ratio of the labelling of the different membrane polypeptides was similar for both membrane subfractions. Provided that the uptake as well as the secretion of bile salts by hepatocytes are carrier‐mediated processes, this suggests the participation of the same polypeptides in both processes.