bluuebunny/crossref_metadata_2025 huggingface dataset:
Substrate‐Binding Isotherms of Spinach Chloroplastic Fructose‐1,6‐bisphosphatase and the Photoregulation of the Calvin Cycle
Authors: MEUNIER Jean‐Claude , BUC Jean , SOULIÉ Jean‐Michel , PRADEL Jacques , RICARD Jacques
Published: March 2005

Fluorescence titration experiments of chloroplastic, fructose‐1,6‐bisphosphatase by fructose bisphosphate and magnesium have been effected using the inactive dimeric, the inactive tetrameric and the active tetrameric enzyme forms.

Magnesium binding to the inactive dimeric enzyme exhibits a positive cooperativity whereas fructose 1,6‐bisphosphate exhibits no cooperativity at all. The binding of either magnesium or fructose bisphosphate to the inactive oxidized tetramer exhibits a succession of negative and positive cooperativities (mixed cooperativity). Upon reduction of the inactive tetramer by dithiothreitol and activation, the ligand binding properties of the enzyme are changed. Magnesium and fructose bisphosphate are bound to the active enzyme with a positive cooperativity.

Non‐linear least‐square fitting allows one to estimate the binding constants, and therefore the free energy of binding of either magnesium or fructose bisphosphate to the various forms of the enzyme. Whatever the state of fructose‐ 1,6‐bisphosphatase, one ligand is bound per subunit. The affinity constants of magnesium for the active or inactive enzyme forms are much greater than those of fructose bisphosphate. This suggests that the metal ion gives to the enzyme the right conformation to bind the sugar phosphate.
Electron‐Paramagnetic‐Resonance Spectroscopy of Spin‐Labelled Cowpea‐Chlorotic‐Mottle Virus
Authors: KRÜSE Jacob , HEMMINGA Marcus A.
Published: March 2005

Cowpea chlorotic mottle virus has been labelled with a maleimide spin label when the nucleoprotein particles were in the swollen state. After the spin‐labelling reaction, the virus was dialyzed to bring the nucleoprotein particles into different conformational states. The mobility of the labelled part of the protein was monitored by electron paramagnetic resonance (EPR) spectroscopy and by the saturation‐transfer EPR technique. The latter technique especially showed sensitivity to conditions that influence the RNA conformation and the RNA‐protein interaction. The shape of the EPR and saturation‐transfer EPR spectra can only be explained by an anisotropic motion of the whole spin‐labelled viral protein or the spin‐labelled part of the protein subunit in the core of the virus.
Regulation of the Synthesis of Ribulose‐1,5‐Bisphosphate Carboxylase and Its Subunits in the Flagellate Chlorogonium elongatum
Authors: BOEGE Frank , WESTHOFF Peter , ZIMMERMANN Kurt , ZETSCHE Klaus
Published: March 2005

Shifting of heterotrophically cultured cells of Chlorogonium elongatum to autotrophic culture conditions results in a more than tenfold increase in the level of the chloroplastic enzyme ribulose‐1,5‐bisphosphate carboxylase. This increase in enzyme amount is caused by an increased synthesis of the enzyme de novo under autotrophic culture conditions, whereas the rate of enzyme degradation is low and very similar under both growth conditions.

The synthesis of the enzyme is mainly under the control of two factors. Light promotes the synthesis, while acetate, the carbon source of this organisms, has a strong inhibitory effect on this process.

Since a mutant lacking chlorophyll shows a normal response to the change of culture conditions the chlorophylls can be ruled out as photoreceptors.
Relationship between Methylation of Adenine Near the 3′ End of 16‐S Ribosomal RNA and the Activity of 30‐S Ribosomal Subunits
Authors: IGARASHI Kazuei , KISHIDA Kazuo , KASHIWAGI Keiko , TATOKORO Ikuko , KAKEGAWA Tomohito , HIROSE Seiyu
Published: March 2005

The relationship between methylation of adenine near the 3′ end of 16‐S ribosomal RNA and the activity of 30‐S ribosomal subunits has been studied using 30‐S subunits from kasugamycin‐sensitive and kasugamycin‐resistant bacteria. Analysis of the proteins of 30‐S subunits by gel electrophoresis showed that the content of protein S1 in 30‐S subunits from a kasugamycin‐resistant strain was smaller than that in 30‐S subunits from the parent strain. Although polyphenylalanine‐synthetic activity of 30‐S subunits from a kasugamycin‐resistant strain previously methylated by a methylase purified from Escherichia coli Q13 was nearly equal to that of untreated 30‐S subunits, both phenylalanine‐synthetic activity and the content of protein S1 in the 30‐S particles reconstituted from 23‐S core particles and split proteins from the kasugamycin‐resistant strain increased by prior methylation of 23‐S core particles by the methylase. These results suggest that methylation of adenine near the 3′ end of 16‐S rRNA induces an increase of polypeptide‐synthetic activity by the acceleration of binding of protein S1 to S1‐depleted 30‐S subunits.
Effect of Trypsin Treatment of Rat Adipocytes on Biological Effects and Binding of Insulin and Insulin‐Like Growth Factors
Authors: ZAPF Jürgen , SCHOENLE Eugen , WALDVOGEL Margaretha , SAND Inger , FROESCH E. Rudolf
Published: March 2005

Trypsin‐treatment of isolated rat adipocytes abolishes the metabolic effects not only of insulin, but also of the insulin‐like growth factors: in trypsin‐treated cells, concentrations of these hormones that are otherwise maximally effective no longer stimulate 3‐O‐methylglucose transport and lipogenesis or inhibit epinephrine‐induced lipolysis. Concomitantly, the trypsin‐treated adipocytes no longer display specific insulin binding. In contrast, the characteristics of the binding of the insulin‐like growth factors are not grossly affected by prior trypsinization of the adipocytes.

These findings add further support to the concept that the insulin‐like growth factors act on glucose metabolism and antilipolysis via the insulin receptor of the adipocyte.
The Ribosomal Proteins of L Cells
Authors: CAZILLIS Michèle , HOUSSAIS Jean‐François
Published: March 2005

To approach the problem of the RNA‐protein and protein‐protein non‐covalent interactions in mammalian ribosomes, the behaviour of the ribosomal proteins of L cells has been studied under various conditions. Firstly the electrophoretic patterns of the ribosomal proteins extracted from KCl‐washed subunits dissociated by EDTA or puromycim have been compared. All the ribosomal proteins of the EDTA‐derived subunits are found on the puromycin‐derived subunits with the exception of two (S20, S22) not observed when puromycin is used to dissociate the subunits. Three polypeptides of the puromycin‐derived subunit, SP0, LP1, LP3, are stripped by the sole removal of Mg2+ when EDTA is used. Other additional polypeptides are found on the KC1‐washed puromycin‐derived subunits. Secondly, the dissociating effect of 0.5 M KCl on the proteins of the subunits dissociated by EDTA or puromycin has been studied. Although the EDTA‐derived subunits appear much more sensitive to KCl, nevertheless 0.5 M KCl also releases ribosomal proteins from the puromycin‐derived subunits. Other polypeptides (core proteins) are not susceptible to KCl split off. Thirdly, some ribosomal proteins are able to transfer from one type of subunit to the other; these proteins have been classified into three groups. Although the deprivation of Mg2+ enhances this phenomenon, seven pairs of putative transferable proteins are observed in subunits dissociated both by EDTA and puromycin. Lastly, a comparative study has been made between the proteins split off by KCl, the core proteins and the transferable proteins with the ribosomal polypeptides labelled in the absence of rRNA synthesis. A correlation has been observed between the proteins split off by KCI, the transferable proteins and the labelled proteins; however, none of the core proteins are transferable and the major part of them remain unlabelled. This study suggests that the transfer, splitting of and labeling of proteins are properties related to the topological location of the proteins in the ribosomal structure.
Systematic Application of Two‐Dimensional 1H Nuclear‐Magnetic‐Resonance Techniques for Studies of Proteins
Authors: NAGAYAMA Kuniaki , WÜTHRICH Kurt
Published: March 2005

This and the following paper describe the practical application of recently developed, two‐dimensional nuclear magnetic resonance techniques for studies of proteins. In the present report spin‐echo‐correlated spectroscopy and two‐dimensional J‐resolved spectroscopy are used to identify complete spin systems of non‐labile, aliphatic protons in the basic pancreatic trypsin inhibitor. Overall, 41 out of the 58 aliphatic spin systems in this protein were identified for the first time the spin systems of all the glycyl residues in a protein have been identified in the 1H NMR spectrum. Combined with the following paper, the present data yield new individual assignments for numerous amino acid residues and provide a new avenue, based on accurate measurements of spin‐spin coupling constants in the two‐dimensional J‐resolved spectra, for studying changes of static and dynamic aspects of protein conformation between single crystals and solution, or between different conditions of solvent and temperature.
Systematic Application of Two‐Dimensional 1H Nuclear‐Magnetic‐Resonance Techniques for Studies of Proteins
Authors: WAGNER Gerhard , KUMAR Anil , WÜTHRICH Kurt
Published: March 2005

This paper describes a new nuclear magnetic resonance approach for the determination of secondary structure in globular proteins. To illustrate the practical application of the new procedure, two‐dimensional correlated spectroscopy and two‐dimensional nuclear Overhauser enhancement spectroscopy were used to obtain individual assignments for all the backbone protons of the β‐sheet secondary structures in the basic pancreatic trypsin inhibitor. First, combined connectivity diagrams of these two methods recorded in both 2H2O solution and H2O solution of the inhibitor were employed to obtain sequential, individual resonance assignments for the separate strands in the β sheet. Second, a 2D nuclear Overhauser enhancement spectrum recorded with a long mixing time was used to determine how the separate, extended polypeptide strands are linked by hydrogen bonds in the sheet structures. By combination of these results with the identifications of the amino acid side‐chain resonances described in the preceding paper, the β‐sheet structures can, without reference to data on the spatial structure obtained with other techniques, be localized in the amino acid sequence. This investigation confirms results on limited regions of the β sheet in the inhibitor obtained previously with one‐dimensional nuclear magnetic resonance experiments and demonstrates that the entire β‐sheet structure seen in single crystals of the inhibitor is preserved in aqueous solution.
Cell‐Free Synthesis and Glycosylatlon of the Major Human‐Red‐Cell Sialoglycoprotein, Glycophorin A
Authors: JOKINEN Mikko , ULMANEN Ismo , ANDERSSON Leif C. , KÄÄRIÄINEN Leevi , GAHMBERG Carl G.
Published: March 2005

The human erythroid cell line, K562, synthesizes the major red cell sialoglycoprotein, glycophorin A. We have isolated an mRNA fraction which codes for glycophorin A from K562 cells and studied the synthesis of the sialoglycoprotein in a rabbit reticulocyte cell‐free system. In the absence of membranes a precursor form of glycophorin A was synthesized. This was identified using specific anti‐(glycophorin A) serum. The apparent molecular weight of the carbohydrate‐free precursor of glycophorin A wss 19500. This exceeds the molecular weight of the glycophorin A apoprotein by approximately 5000. In the presence of membranes from dog pancreas, the synthesized glycophorin A precursor was N‐glycosylated and probably also O‐glycosylated. The oligosaccharide chains remained incompleted and the glycoprotein synthesized in vitro corresponded to the glycosylated precursor of glycophorin A obtained in intact cells.
Purification and Physicochemical Properties of ATP Citrate (pro‐3S) Lyase from Lactating Rat Mammary Gland and Studies of Its Reversible Phosphorylation
Authors: GUY Paul S. , COHEN Philip , HARDIE D. Grahame
Published: March 2005

A simple procedure is described for the isolation of ATP‐citrate lyase, acetyl‐CoA carboxylase and fatty acid synthase from lactating rat mammary gland. ATP‐citrate lyase was purified 48‐fold by poly(ethyleneglycol) precipitation, ammonium sulphate precipitation and chromatography on DEAE‐Sepharose at pH 7.5. The preparation was completed in 2–3 days and 30 mg of enzyme was isolated from 10 animals, which represented a yield of 38%. The preparation was of a high degree of purity as judged by polyacrylamide gel electrophoresis and ultracentrifugal analysis. The sedimentation constant, S20,w was 13.6 ± 0.2 S, the absorption coefficient, A1%280nm measured refractometrically was 12.2 ± 0.7 and the amino acid composition was determined.

The subunit molecular weight determined by gel electrophoresis in the presence of sodium dodecyl sulphate was 116000 and the molecular weight of the native enzyme measured by sedimentation equilibrium was 473000. These experiments indicate that mammary ATP‐citrate lyase is a tetramer. The purified enzyme contained 0.5 mol of alkali‐labile phosphate covalently bound/subunit.

The phosphorylation of ATP‐citrate lyase by cyclic‐AMP‐dependent protein kinase reached a plateau when 0.7 ± 0.2 mol of phosphate were bound to a serine residue in a single tryptic peptide. The same tryptic peptide was phosphorylated by cyclic‐GMP‐dependent protein kinase, although at a much slower rate. ATP‐citrate lyase was not phosphorylated by phosphorylase kinase or by glycogen synthase kinase 3.

The phosphate introduced by cyclic‐AMP‐dependent protein kinase could be completely removed by incubation with protein phosphatase 1 or protein phosphatase 2. However the alkali‐labile phosphate present in ATP‐citrate lyase as isolated was not removed by these protein phosphatases, indicating that it is located at a distinct site(s). Phosphorylation of purified ATP‐citrate lyase by cyclic‐AMP‐dependent protein kinase did not alter the V or the apparent Km for citrate, ATP or CoA.

The properties of mammary ATP‐citrate lyase are compared to those of the rat liver enzyme and the possible role of phosphorylation in the regulation of its activity is discussed.
Factors Modulating Transcription and Translation in vitro of Ribosomal Protein S20 and Isoleucyl‐tRNA Synthetase from Escherichia coli
Authors: WIRTH Reinhard , KOHLES Veronika , BÖCK August
Published: March 2005

The DNA‐dependent protein‐synthesizing system developed by Zubay [Zubay, G. (1973) Annu. Rev. Genet. 7, 267–287] was optimized for the transcription and translation of genes from the 0.5‐min region of the Escherichia coli chromosome carried by transducing λ phages. The E. coli gene products synthesized were isoleucyl‐tRNA synthetase, ribosomal protein S20, dihydrodipicolinic acid reductase and (possibly) the two subunits of carbamoyl‐phosphate synthetase.

Formation of ribosomal protein S20 is specifically stimulated by the addition of 16‐S rRNA and not by 5‐S or 23‐S rRNA. 16‐S rRNA increases the rate of S20 synthesis, the final yield of product depends on the duration of persistence of the RNA added. Addition of 16‐S rRNA to the separate transcription and translation systems showed that it is the translation of the S20 mRNA which is enhanced. Furthermore, S20 synthesis is stimulated more than fourfold when concomitant synthesis of rRNA occurs from a plasmid carrying an rrn transcriptional unit. The results described are explained in terms of a model which suggests that ribosomal protein S20 feedback inhibits its synthesis at the translational level and that removal of S20 into ribosomal assembly (i.e. binding to 16‐S rRNA) releases inhibition. The model postulates a direct link between synthesis of ribosomal RNA and ribosomal protein and between the rates of ribosomal assembly and ribosomal protein synthesis.

The stimulatory effect of guanosine 3′‐diphosphate 5′‐diphosphate on isoleucyl‐tRNA synthetase formation and its inhibition of the synthesis of ribosomal protein S20 in vitro occurs at the level of transcription. Its relevance in vivo, however, remains to be demonstrated.

Formation of isoleucyl‐tRNA synthetase in vitro is not influenced either by the addition of a surplus of purified enzyme nor by the limitation of protein synthesis by the addition of anti‐(isoleucyl‐tRNA synthetase) serum. There is no evidence, therefore, that isoleucyl‐tRNA synthetase is autogenously regulated.
Sequences of Sixteen Phosphoserine Peptides from Ovalbumins of Eight Species
Authors: HENDERSON Janet Y. , MOIR Arthur J. G. , FOTHERGILL Linda A. , FOTHERGILL John E.
Published: March 2005

Phosphoserine peptides have been isolated by a diagonal electrophoresis method from proteolytic digests of ovalbumins from hen, grouse, turkey, golden pheasant, magpie goose, chinese goose, Aylesbury duck and fulvous whistling duck. The amino acid sequences of these peptides have been determined. There are two sites of phosphorylation in each ovalbumin, which are located in two different regions of the ovalbumin molecule. Amino acid replacements are more frequent in the site 1 sequences than in the site 2 sequences. Both site 1 and site 2 sequences contain invariant residues. Sequence variations occur near the serine residues that are phosphorylated, but the amino acid two residues C‐terminal to the phosphoserine is always glutamic acid, suggesting that this may be a recognition signal for the phosphorylating enzyme. Variations in amino acid sequence among the species are consistent with differences in the ovalbumins determined by peptide mapping and quantitative immunoprecipitation assays. A phylogenetic tree has been constructed from a comparison of the sequences of 248 residues from the eight ovalbumins.
Quaternary Structure of Bovine Cytochrome Oxidase
Authors: SARASTE Matti , PENTTILÄ Timo , WIKSTRÖM Mårten
Published: March 2005

A hydrodynamically homogeneous preparation of bovine mitochondrial cytochrome c oxidase can be obtained by anion‐exchange chromatography of alkaline‐treated enzyme, followed by a gel permeation chromatography step, which further removes some (aggregated) apoprotein. The molecular weight, Mr of the monodisperse enzyme in Triton X‐100 was found to be 210000. This complex is composed of six different polypeptides, with Mr summing up to about 110000 in toto, in a relative one‐to‐one stoichiometry. Two sets of these subunits constitute the 210000‐Mr enzyme complex. In contrast to our earlier report [Saraste, Penttilä, Coggins, and Wikström, FEBS Lett. 114 (1980) 35–38] the 210000‐Mr enzyme contains four (and not two) haems A, and therefore represents the dimer of cytochrome aa3. One of the proposed seven subunits, number III, is lacking in this enzyme preparation.
Accumulation of Histone H1° during Chemically Induced Differentiation of Murine Neuroblastoma Cells
Authors: PIELER Christian , ADOLF Guenther R. , SWETLY Peter
Published: March 2005

A basic nuclear protein with properties similar to lysine‐rich histories accumulates during differentiation in vitro of C1300 murine neuroblastoma cells (clone Neuro‐2a) induced by either n‐butyrate, dimethyl sulfoxide, hexamethylene bisacetamide or 1,6‐dibutyryl‐adenosine 3′,5′‐monophosphate. A protein with the same electrophoretic properties, present in adult mouse brain cell nuclei in amounts similar to those in the induced neuroblastoma cells, has been characterized as histone H1°. Digestion of the two proteins with Staphylococcus aureus V8 protease gives identical peptide maps (sharing no common peptides with those of histones H1A or H1B), which indicates that the induced protein qualifies as H1°. Accumulation of historic H1° in neuroblastoma cell nuclei accompanies shutoff of DNA synthesis and transgression of the cells into a G1‐phase resting state. Upon removal of n‐butyrate the cells resume proliferation and their H1° content decreases, indicating an association of H1° with the resting state during which differentiated cellular functions are expressed.
The Isolation and Characterisation of Nerve Growth Factor from the Prostate Gland of the Guinea‐Pig
Authors: CHAPMAN Carey A. , VERNON Charles A. , BANKS Barbara E. C. , WALKER John M.
Published: March 2005

Nerve growth factor from the guinea‐pig prostate gland has been completely purified‐and characterised. Its chemical and biological properties in vitro are compared with those of nerve growth factor from other sources.
Characterization of a Membrane‐Bound Biotin‐Containing Enzyme: Oxaloacetate Decarboxylase from Klebsiella aerogenes
Authors: DIMROTH Peter
Published: March 2005

Oxaloacetate decarboxylase from Klebsiella aerogenes is firmly bound to the cytoplasmic membrane, from which it can be solubilized with nonionic detergents. The solubilized enzyme behaved like‐the membrane‐bound enzyme with respect to its inhibition by avidin and to the requirement of sodium ions for catalytic activity. The decarboxylase was purified 4.5‐fold over the solubilized membrane extract by conventional means. Dodecylsulfate disc‐gel electrophoretic analysis indicated that the enzyme consists of polypeptides of a single size. The molecular weight of these polypeptides is 68000. Radioactive biotin was incorporated specifically into these polypeptide chains upon growth of the bacteria in the presence of the radioactive vitamin. Biotin as the prosthetic group of oxaloacetate decarboxylase is now firmly established. The enzyme in the absence of detergent occurs in a highly aggregated form which elutes in the exclusion volume of a Biogel A 1.5 m column.

The reported inhibition of oxaloacetate decarboxylase by citrate could not be repeated. On the other hand oxalate, 2‐oxomalonate and glyoxylate proved to be very potent inhibitors of the decarboxylase.

The stereochemical course of the oxaloacetate decarboxylation reaction was determined starting from stereospecifically labelled malates, which by malate dehydrogenase and oxaloacetate decarboxylase were converted to chiral pyruvates. The chirality of these pyruvates was analysed via their conversion to acetates and malates by determining the extent of tritium retention upon incubation of the latter with fumarase. It was found that oxaloacetate decarboxylation occurs stereospecifically with retention of configuration.
The Regulation of Glycolysis in Human Erythrocytes
Authors: ATAULLAKHANOV Fazoil I. , VITVITSKY Victor M. , ZHABOTINSKY Anatoly M. , PICHUGIN Aleksei V. , PLATONOVA Olga V. , KHOLODENKO Boris N. , EHRLICH Lev I.
Published: March 2005

According to the mathematical model, the dependence of the rate of glycolysis on the ATP concentration can be represented by the bell‐shaped curve with the descending part at the concentration of ATP close to physiological values. The existence of the descending part is a result of the strong inhibition of phosphofructokinase by ATP and hexokinase by glucose 6‐phosphate and provides stabilisation of the intracellular concentration of ATP.

Using arsenate as an uncoupler of the oxidation of glucose and ATP synthesis, the dependence of the rate of glycolysis on the ATP concentration in erythrocytes was measured. This dependence (glycolysis characteristic) is represented by a bell‐shaped curve. The normalized glycolysis characteristics are the same for all the donors investigated.

The increase of permeability of the erythrocyte membrane to monovalent cations by the polyene antibiotic levorin leads to an increase of glycolytic rate in erythrocytes (40–70%) and to a decrease in the ATP concentration (15%) and glucose 6‐phosphate (25–60%). The data obtained with levorin, and also other results on Na, K‐ATPase activation are in a good agreement with the characteristic obtained using arsenate.

In all cases, inhibition of Na+, K+‐ATPase of erythrocytes by ouabain leads to the decrease of the rate of lactate production (10–20%) and increase of the concentration of glucose 6‐phosphate (14–40%). The ATP concentrations remains almost unchanged or is only slightly increased. The rate of glucose utilisation in erythrocytes from different donors show two types of behaviour. In the first type of erythrocyte it drops proportionally to the decrease in the lactate production rate; in the second type of erythrocyte it remains unchanged.

The experimental results presented are in qualitative agreement with the predictions of the mathematical model.
Binding of the Chemoattractant Folic Acid by Dictyostelium discoideum Cells
Authors: VAN DRIEL Roel
Published: March 2005

The binding of the chemoattractant folic acid to intact Dictyostelium discoideum cells has been studied. A few hundred thousand ligand molecules can be bound per cell with a dissociation constant in the order of 3 × 10−7 M. Intact cells equilibrate rapidly with extracellular folic acid, suggesting that the binding sites are located on the outer cell surface. The folic‐acid‐binding properties do not change during early cell development induced by starvation for nutrients.

A number of putative folate‐binding proteins have been identified in a detergent‐solubilized plasma membrane fraction by affinity chromatography on folate‐Sepharose and specific elution with free folate. One of these proteins appeared to be an integral membrane protein as was deduced from its amphiphilic behaviour. It may be that among these proteins is the chemotactic receptor for folate.
Amino‐Terminal Sequence of Chicken Preproalbumin
Authors: COMPERE Sally J. , LIVELY Mark O. , MacGILLIVRAY Ross T. A.
Published: March 2005

Poly(A)‐containing RNA was isolated from chicken liver and translated in a reticulocyte lysate protein‐synthesizing system in the presence of radiolabeled amino acids. Chicken albumin was isolated from the translation products by immunoprecipitation, and subjected to automated Edman radiosequencing. Comparison with the sequence of proalbumin showed that the translation product (preproalbumin) contains an NH2‐terminal extension of 18 amino acid residues. The NH2‐terminal sequence of chicken preproalbumin was as follows: Met−18‐Lys‐Asn‐Val−15‐Thr‐Leu‐Ile‐Ser‐Phe−10‐Ile‐Phe‐Leu‐Phe‐Ser−5‐Ser‐Ala‐Thr‐Ser−1‐Arg1, where Arg1represents the NH2‐terminal residue of proalbumin. This NH2‐terminal extension is very rich in hydrophobic amino acid residues and is similar to the signal sequences found in other secreted proteins. The signal sequence of chicken preproalbumin shows considerable homology with the signal sequences of rat and bovine preproalbumins, but little homology with the signal sequences of other chicken preproteins.
Cytotoxicity Acquired by Conjugation of an Anti‐Thy1.1 Monoclonal Antibody and the Ribosome‐Inactivating Protein, Gelonin
Authors: THORPE Philip E. , BROWN Alexander N. F. , ROSS Walter C. J. , CUMBER Alan J. , DETRE Simone I. , EDWARDS D. Caird , DAVIES Anthony J. S. , STIRPE Fiorenzo
Published: March 2005

Gelonin, a plant protein which can powerfully reduce the protein‐synthetic capacity of ribosome preparations, was covalently coupled to anti‐Thy1.1 antibody. The conjugate was prepared using N‐succinimidyl‐3‐(2‐pyridyldithio)propionate which generates a disulphide linkage between the component molecules. Two conjugate fractions were obtained with Mr of 180000 and > 200000.

After its linkage to the antibody, gelonin suppressed those Thy1.1‐bearing T lymphocytes from AKR mice which will respond to phytohaemagglutinin and concanavalin A in tissue culture. The [3H]leucine incorporation with the T‐cell mitogens was inhibited by 50% with the 180000‐Mr fraction at a concentration of 0.4 nM and with the > 200000‐Mr fraction of pM. Unconjugated gelonin induced comparable reductions in T‐cell responsiveness but at concentrations of 30 nM. The conjugates exerted little or no effect upon B lymphocytes or T lymphocytes from CBA mice (Thy1.2+ve). Two Thy1.1‐expressing AKR lymphoma cell lines, AKR‐A and BW5147, were found to be sensitive to the conjugates, albeit much less so than the normal T lymphocytes. The conjugates injected in vivo significantly prolonged the life of CBA mice bearing an AKR‐A lymphoma allograft.

It is concluded that gelonin can, by its linkage to an antibody, be rendered cytotoxic with a potency to match or exceed those of the toxins abrin and ricin.
Mechanism of Uptake of l‐Arginine by Sugar‐Cane Cells
Authors: KOMOR Ewald , THOM Margaret , MARETZKI Andrew
Published: March 2005

Suspension cells of sugar cane were used as a model system for cells of higher plants to study the mechanism of l‐arginine uptake.

The uptake system is specific for the l‐arginine molecule in the fully ionized state, i.e. δ‐guanidine group and α‐amino group positively charged and carboxyl group negatively charged. This was concluded because the Km value for uptake increased strongly for: (a) l‐arginine analogues which lack the charged carboxyl group (l‐arginine methyl ester, agmatin); (b) l‐arginine analogues, which lack the charged α‐amino group (l‐arginine acid, γ‐guanidinobutyric acid); (c) l‐arginine analogues which lack the charged δ‐guanidine group or γ‐guanidinoxy group (l‐citrulline, l‐canavanine at neutral and alkaline pH‐values).

The importance of the positive charge at the δ‐guanidine group or γ‐guanidinoxy group was further documented by Km values for l‐arginine and l‐canavanine uptake at different pH values. Only at pH values where the γ‐guanidinoxy group is protonated, was there an effective uptake of l‐canavanine and effective competition of l‐canavanine with l‐arginine.

The length of the l‐arginine molecule was less important: slightly larger l‐homoarginine) or shorter analogues (l‐lysine) were taken up rather well. A spatial rearrangement at the α‐carbon (d‐arginine) was, however, not tolerated.

The uptake of l‐arginine proceeds by electrogenic uniport, there is no evidence for symport or antiport of another molecule (though l‐canavanine uptake at neutral pH value causes a transient alkalinization of the suspension medium). Charge equilibration is brought about by efflux of protons and potassium ions.
Collagen Type I Trimer Synthesis by Cultured Embryonic Mouse Molars
Authors: LESOT Hervé
Published: March 2005

Embryonic mouse tooth germs were cultured in vitro and the collagens type I, type III and type I trimer were purified and biochemically characterized. Collagen type I trimer has been identified by means of CM‐cellulose chromatography, CNBr peptide analysis, pepsin resistance and molecular sieve chromatography. Already before the odontoblasts were functional, this molecule was found to be a constituent of the dental extracellular matrix. However, the synthesis of collagen type I trimer was considerably increased when odontoblasts polarized and became functional. The incorporation of 5‐bromodeoxyuridine into the dental cells inhibited the polarization of odontoblasts as well as the amplification of collagen type I and type I trimer synthesis.
The Catalytic Mechanism of 2‐Oxoacid: Ferredoxin Oxidoreductases from Halobacterium halobium
Authors: KERSCHER Lorenz , OESTERHELT Dieter
Published: March 2005

The catalytic cycle of the 2‐oxoacid: ferredoxin oxidoreductases from Halobacterium halobium was investigated. The first step is binding of the 2‐oxoacid to the enzyme followed by decarboxylation and transfer of one electron to the [4Fe‐4S] cluster of the functional unit. The cluster is then reoxidized by ferredoxin or, in the absence of the physiological electron acceptor, by oxygen. In the resulting stable enzyme‐intermediate radical the decarboxylation product of the 2‐oxoacid remains tightly bound until reaction with coenzyme A causes formation of acyl‐CoA and concomitant transfer of the second electron to the cluster, which again is reoxidized by ferredoxin or oxygen. After purification, part of the enzyme molecules still contain the intermediate radical. Enzyme preparations either free of radical or containing enhanced amounts are obtained by treatment with coenzyme A or 2‐oxoacid, respectively. Whenever the radical is present in an enzyme molecule the respective binding site for the 2‐oxoacid is blocked.
The Primary Structure of Escherichia coli RNA Polymerase
Authors: OVCHINNIKOV Yury A. , MONASTYRSKAYA Galina S. , GUBANOV Valentin V. , GURYEV Sergey O. , CHERTOV Oleg Yu. , MODYANOV Nickolay N. , GRINKEVICH Vladimir A. , MAKAROVA Irma A. , MARCHENKO Tatjana V. , POLOVNIKOVA Irina N. , LIPKIN Valery M. , SVERDLOV Eugene D.
Published: March 2005

The combined structural study of proteins and of their corresponding genes utilizing the methods of both protein and nucleotide chemistry greatly accelerates and considerably simplifies both the nucleotide and protein structure determination and, in particular, enhances the reliability of the analysis. This approach has been successfully applied in the primary structure determination of the β and β′ subunits of Escherichia coli DNAdependent RNA polymerise and of their structural genes, yielding a continuous nucleotide, sequence (4714 base pairs) that embraces the entire rpoB gene, the initial part of the rpoC gene and the intercistronic region, together with the total amino acid sequence of the β subunit, comprising 1342 residues, and the N‐terminal sequence of the β′ subunit (176 residues).
Isolation and Partial Characterization of the Lipoprotein Families A and A‐I from High‐Density Lipoproteins of Human Serum
Authors: NORFELDT Per‐Ingvar Palle , OLOFSSON Sven‐Olof , FAGER Gunnar , BONDJERS Göran
Published: March 2005

Procedures for the isolation of two lipoprotein fractions from plasma high‐density lipoproteins (HDL), characterized by apolipoprotein A‐I and apolipoprotein A‐I together with apolipoprotein A‐II, have been elaborated. Apolipoprotein A‐I was identified as the protein moiety of one of these fractions (lipoprotein A‐I) with polyacrylamide gel electrophoresis (at basic and acidic pH, as well as in the presence of sodium dodecyl sulphate), immuno‐double‐diffusion, and amino acid analysis. Apolipoproteins A‐I and A‐II were identified as the protein moiety of the other fraction (lipoprotein A) with polyacrylamide gel electrophoresis (basic and acidic pH) and immuno‐double‐diffusion. Lipoprotein A‐I consisted of spherical particles with a diameter similar to that of HDL as judged from negative stains in the transmission electron microscope. The diameter was estimated to be 8.7 nm from gel chromatography. Lipoprotein A‐I migrated in the HDL position on crossed immunoelectrophoresis. On iso‐electric focusing lipoprotein A‐I appeared as multiple bands in the pH range 5.05–5.55. Lipo protein A‐I had the density of an HDL‐2 fraction (ϱ: 1.063–1.105). Lipoprotein A consisted of spherical particles with a diameter similar to that of HDL, as judged from negative stains in the transmission electron microscope. The diameter was estimated to be 7.9 nm from gel chromatography. The molar ratio between the A‐I and A‐II polypeptides was estimated to 1.3: 1 with electroimmunoassay and calculations from the amino acid compositions. Lipoprotein A migrated in the position of HDL on crossed immuno‐electrophoresis. On iso‐electric focusing lipoprotein A appeared as one major and two minor bands in the pH range 5.10–5.30. Lipoprotein A had the hydrated density of an HDL‐2 fraction.
A Mouse Cell Line, which Is Unprotected by Interferon against Lytic Virus Infection, Lacks Ribonuclease F Activity
Authors: EPSTEIN David A. , CZARNIECKI Christine W. , JACOBSEN Helmut , FRIEDMAN Robert M. , PANET Amos
Published: March 2005

A mouse cell line, NIH 3T3, does not respond to some of the activities of interferon. Even after treatment with high concentrations of interferon the replication of lytic viruses, such as encephalomyocarditis virus (EMCV) and vesicular stomatitis virus (VSV) is not inhibited in these cells. In contrast, interferon treatment of these same cells results in the inhibition of Moloney murine leukemia virus (MMuLV) production. We have analyzed enzymatic pathways which are induced by interferon in these cells. After interferon treatment, the level of the (2′‐5′)oligoadenylate [(2′‐5′) An] synthetase activity and the phosphorylation of the 67000‐dalton protein (P1) are enhanced in NIH 3T3 cells to approximately the same level as interferon‐sensitive mouse L‐cells. Moreover, NIH 3T3 and L‐cells contain approximately the same levels of enzymes which in activate (2′‐5′)An. Both exogenously added (2′‐5′)A3 or double‐stranded RNA (dsRNA) failed to inhibit protein synthesis in NIH 3T3 extracts even though they were potent inhibitors of L‐cell extract‐directed protein synthesis. Direct measurements of the (2′‐5′)An ‐dependent ribonuclease F (RNase F) failed to detect such activity in NIH 3T3 cells. Our results, therefore, suggest that the presence of RNase F activity is necessary for the interferon‐induced antiviral activity against EMCV and against VSV. The induction of protein kinase activity by interferon treatment of NIH 3T3 cells appears to have no direct effect on EMCV and VSV replication.
Receptor‐Mediated Gonadotropin Action in Ovary
Authors: AZHAR Salman , MENON K. M. Jairam
Published: March 2005

The role of cell‐surface sialic acid in the interaction of 125I‐labeled choriogonadotropin with gonadotropin receptors from plasma membranes of bovine corpus luteum was investigated. Pretreatment of plasma membranes with neuraminidases purified from Clostridium perfringens or Vibrio cholera enhanced the binding activity of 125I‐choriogonadotropin in a concentration‐dependent manner. C. perfringens neuraminidase (100 mU/ml) and V. cholerae neuraminidase (1.0 I.U./ml) maximally stimulated gonadotropin‐binding activity. The neuraminidase stimulation of binding was due to unmasking of gonadotropin‐binding sites and not due to a change in affinity of the receptor for 125I‐choriogonadotropin. The activity of plasma‐ membrane 5′‐nucleotidase, a glycoprotein enzyme, however, was not affected by neuraminidase treatment. The stimulatory effect of C. perfringens enzyme was abolished by co‐incubation of plasma membrane with glycoproteins, fetuin and mucin. Furthermore, the stimulatory effect of neuraminidase was due to an intrinsic property of the enzyme and was accompanied by a parallel dose‐dependent loss of plasma‐membrane‐associated sialic acid residues. Other neuraminidase preparations including that purified from Arthrobacter ureafaciens also enhanced 125I‐choriogonadotropin binding with concomitant hydrolysis of plasma‐membrane sialic acid. By contrast, enzyme purified from influenza virus did not release plasma‐membrane sialic acid and consequently failed to modulate receptor activity. Treatment of plasma membrane with neuraminidase and subsequent fractionation demonstrated that the enzyme hydrolyzed 50–60% sialic acid from glycoprotein and ganglioside components. Preincubation of plasma membrane with wheat‐germ agglutinin partially blocked the hydrolysis of glycoprotein sialic acid as well as stimulation of 125I‐choriogonadotropin‐binding activity. Incubation with other lectins, concanavalin A and peanut agglutinin, neither affected the hydrolysis of glycoprotein/ganglioside sialic acid by neuraminidase nor prevented the stimulatory action of enzyme on 125I‐choriogonadotropin‐binding activity. These studies demonstrate a possible involvement of plasma‐membrane‐associated sialic acid (sialoglycoproteins) in the activity of gonadotropin receptors.
Effect of Chloroquine and Methylamine on Endocytosis of Fluorescein‐Labelled Control IgG and of Anti‐(Plasma Membrane) IgG by Cultured Fibroblasts
Authors: SCHNEIDER Yves‐Jacques , TROUET André
Published: March 2005

We report here the effect of chloroquine and methylamine two lysosomotropic drugs, on the binding, uptake and subcellular localization of fluorescein‐labelled control immunoglobulm G (control IgG) a marker for non‐specific adsorptive endocytosis and of anti‐(plasma membrane) IgG (specific IgG), a specific ligand of cell‐surface antigens.

At 4 °C, methylamine and chloroquine inhibit the binding of control IgG to the cell surface, probably by a reversible competition. These two drugs, methylamine more than chloroquine, considerably slow down the rate at which control IgG is transferred from its binding sites on the phagosomal membrane to the lysosomal compartment; both drugs block almost completely the intralysosomal digestion of this IgG as well as the release of degradation products into the culture‐medium. They do not aftect the binding and uptake of the specific IgG. In addition, methylamine seems to inhibit partially the return to the cell surface of membrane antigens and of membrane fragments bearing 5′‐nucleotidase or binding sites for control IgG.

We conclude that important steps (binding to cell surface, delivery to lysosomes, digestion and recycling of plasma membrane) involved in the uptake and the processing of IgG by fibroblasts are inhibited by these two substances. The effects of lysosomotropic agents on the regulation and function of the endocytic pathway and of lysosomes could have many pharmacological and therapeutic implications.
Immunochemical Studies of Human Placental‐Type Variants of Alkaline Phosphatase
Authors: WEI Shirley C. , DOELLGAST George J.
Published: March 2005

Hyperimmune antisera raised against the rare FD and SD phenotypes of human placental alkaline phosphatase and the related cancer‐associated Nagao isoenzyme were exhaustively absorbed with the common SS and FF phenotype enzymes conjugated to Sepharose. The antibodies so obtained had one of three specificities, determined by testing all the common phenotypes of placental alkaline phosphatase, the rare FD, SD and S‐17 phenotypes, and five samples of purified cancer patient enzymes: (I) antibodies reactive with the S, D and I variant enzymes, but not with the F, 17 or Nagao enzymes; (II) antibodies reactive with the F, 17 and Nagao enzymes, but not with the S, D and I variant enzymes; and (III) antibodies reactive with the D, 17 and Nagao enzymes and not with the S, F or I variant enzymes. Based on these data, and on the information obtained from biochemical characterization, it is argued that the Nagao isoenzyme and the D‐variant are closely related but distinct enzymes. We propose a hypothetical scheme for the divergence of these two enzymes from a common ancestral gene product. In this scheme, an ancestral gene duplication resulted in two isoenzymes, one, of which is completely specific to the placenta, and one of which (the ‘Nagao isoenzyme’) has an unknown normal tissue localization, but is found in some cases of cancer. We use the term ‘syn‐placental’ to describe this latter isoenzyme, reflecting its close structural relationship to the placental isoenzyme.
Biosynthesis of the Light‐Harvesting Chlorophyll a/b Protein
Authors: BENNETT John
Published: March 2005

When etiolated pea seedlings were exposed to continuous light for 24 h and then returned to darkness, 38% of the chlorophyll a, 74% of the chlorophyll b and 84% of the light‐harvesting chlorophyll a/b protein that had accumulated under illumination proved to be unstable in darkness. The unstable chlorophyll displayed a half‐life of abdüt 90 min. In contrast, the α and β subunits of the chloroplast coupling factor and the large and small subunits of ribulose 1,5‐bisphosphate carboxylase continued to accumulate in darkness, although at a slower rate than in plants maintained under light.

Short‐term labelling in vivo with L‐[35S]methionine showed that leaves continued to synthesize the light‐harvesting protein and the small subunit of ribulose 1,5‐bisphosphate carboxylase for up to 48 h after transfer of plants from light to darkness. However, after long‐term labelling (16 h), the light‐harvesting chlorophyll a/b protein was found to be labelled to high specific activity only in illuminated leaves.

I conclude that the light‐harvesting chlorophyll a/b protein is subject to turnover after transfer of plants from light to darkness. The site of breakdown appears to be the photosynthetic membrane. I suggest that turnover of the protein is part of the normal physiological mechanism for co‐ordinating the accumulation of the pigment and protein components of the light‐harvesting chlorophyll a/b complex.
Lipid‐Synthesis‐Dependent Biosynthesis (or Assembly) of Major Outer‐Membrane Proteins of Escherichia coil
Authors: BOCQUET‐PAGES Catherine , LAZDUNSKI Claude , LAZDUNSKI Andrée
Published: March 2005

Upon inhibition of fatty acid synthesis in the presence of cerulenin, the uptake of 5′AMP and of other nutrients using similar pore systems can be inhibited as much as 70%. The same effect was observed upon inhibition of phospholipid synthesis after glycerol deprivation in a mutant strain defective in sn‐glycerol‐3‐phos‐phate acyltransferase. Resumption of both fatty acid synthesis and phospholipid synthesis restores a normal uptake of 5′AMP. The protein composition of the outer membranes, analyzed after pulse radiolabelling by was mainly altered in OmpF and OmpC proteins. These proteins are the main points used by most nutrients like 5′AMP. Whereas the synthesis or the assembly of OmpF protein seems to be more inhibited than that of OmpC protein after inhibition of fatty acid synthesis, the reverse case was observed after inhibition of phospholipid synthesis. No protein produced during inhibition of fatty‐acid or phospholipid synthesis is reincorporated into the outer membrane after resumption of these syntheses. These results are discussed with regard to the biosynthesis and assembly of these proteins.
Evidence for an Essential Lysine at the Active Site of l‐Histidinol : NAD+ Oxidoreductase; a Bifunctional Dehydrogenase
Authors: BÜRGER Erich , GÖRISCH Helmut
Published: March 2005

Histidinol dehydrogenase (EC 1.1.1.23) from Salmonella typhimurium is inhibited by formaldehyde and pyridoxal 5‐phosphate (pyridoxal‐P). ɛ‐Pyridoxyl‐lysine is isolated upon acid hydrolysis of pyridoxal‐P‐treated enzyme reduced by sodium borohydride. In the presence of formnylhistidinol and formylhistidine (specific ligands of the enzyme) inactivation of histidinol dehydrogenase by pyridoxal‐P is prevented. Extrapolation of the initial part of the inactivation, curve caused by pyridoxal‐P indicates that modification of two essential lysine residues results in inactivation of the dimeric enzyme. The essential lysine residues appear to participate in the reversible oxidation/reduction reaction converting histidinol to histidinal.
Vicia graminea Anti‐N Lectin: Partial Characterization of the Purified Lectin and Its Binding to Erythrocytes.
Authors: DUK Maria , LISOWSKA Elwira
Published: March 2005

Vicia graminea, lectin, purified by affinity chromatography, was homogeneous in sodium dodecylsulphate/ polyacrylamide gel electrophoresis and migrated with a velocity corresponding to a molecular weight of 125000. Heating in 0.1% sodium dodecylsulphate at 100°C caused dissociation of the lectin into subunits with an apparent molecular weight of 31000. The results of isoelectric focusing suggested non‐identity of the lectin subunits and existence of several molecular forms of the lectin. The lectin was neither dissociated nor inactivated by succinyl ation, but was irreversibly inactivated by 8 M urea. The lectin was totally bound to concanavalin‐A—Sepharose and could be eluted with α‐d‐mannopyranoside.

Binding of the 125I‐labeled Vicia gramninea lectin to untreated and desialylated human erythrocytes of blood groups M and N, horse and bovine erythrocytes was characterized. The lectin was bound specifically to sites specific for blood group N on untreated human erythrocytes with an uniform affinity, and association constant Ka= 1.5 × 108 M−1. Desialylated human NN and MM erythrocytes bound more lectin, with a distinctly higher, but non‐uniform affinity. Vicia graminea lectin bound weakly to horse erythrocytes, and the effect of their desialylation was similar to that obtained with human erythrocytes. The lectin was not bound either to untreated or to desialylated bovine erythrocytes. Binding of the labeled lectin to human NN erythrocytes was inhibited by desialylated glycoproteins of the M and N blood groups, by untreated N glycoprotein, and weakly by untreated M glycoprotein.