bluuebunny/crossref_metadata_2025 huggingface dataset:
The identification of lysosomal ganglioside sialidase in human cells
Authors: ZEIGLER Marcia , SURY Vivi , BACH Gideon
Published: March 2005

In this report we present evidence for the existence of a lysosomal ganglioside sialidase. The sialidase activity was solubilized by sonication and stimulated by cholate. The absence of ganglioside sialidase activity in sialidosis patients indicates that lysosomal sialidase is active towards gangliosides and glycoproteins. The plasma membranes were associated with two types of ganglioside sialidase activities, one was enhanced by cholate while the other was partially inhibited by this detergent.
Dynamic interactions between enzyme activity and the microstructured environment
Authors: CHOPINEAU Joël , THOMAS Daniel , LEGOY Marie‐Dominique
Published: March 2005

A new approach for the study of an enzyme's relationship with its own reaction medium has been developed. One technique of micellar enzymology is the use of pseudohomogeneous systems composed of surfactant/water/organic solvent. In such systems, the physicochemical properties and textures of the medium depend on the relative ratios of the different components. Enzymes are catalytically active in such systems and up to the present have been studied in different microenvironments, such as micelles, microemulsions and lyotropic liquid crystals. Our purpose was to develop a system in which the enzyme could, by its activity, modify one of the components in such a way that the relative ratios among them changed sufficiently to produce a transition from one phase domain to another. The three components, water (or glucose in water), octanol and octyl‐β‐D‐glucoside, form a classical ternary water/oil/surfactant system. The relevant phase diagram shows different macroheterogeneous phases and microstructured domains. The enzyme β‐D‐glucoside hydrolyses octyl‐β‐D‐glucoside to form glucose and octanol. The enzyme was found to change the relative ratios of water (or glucose in water), octanol and octyl‐β‐D‐glucoside in such a manner that the physicochemical structure of the medium was modified. At the beginning of the reaction β‐D‐glucosidase was present in a micellar solution of octyl‐β‐D‐glucoside in water. As the enzymatic reaction proceeded, the medium became biphasic. One of the two phases was the micellar solution of octylβ‐D‐glucoside in water, while the other phase was either a microemulsion or a liquid crystalline phase. In addition the enzyme, through its catalytic activity, was able to modify the physicochemical properties of the reaction medium.
Structural analysis of the carbohydrate chains of a mouse monoclonal IgM antibody
Authors: KROTKIEWSKI Hubert , NILSSON Bo , SVENSSON Sigfrid
Published: March 2005

A mouse monoclonal IgM antibody, directed against human blood group B determinant, was isolated from hybridoma culture growth medium. Chemical analysis indicated presence of N‐ and O‐linked oligosaccharides. The N‐ and O‐linked carbohydrate chains were liberated using two different conditions of reductive alkaline degradation. Structural analysis was carried out on the isolated chains using chemical analysis, 500‐MHz 1H‐NMR spectroscopy and fast‐atom‐bombardment mass spectrometry. The following composite structures of the N‐linked chains were found:

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where R = OH for biantennary structures and R = Neu5Acα2 – 3Galβ1 – 4GlcNAcβ1 – or Neu5Acα2 – 3Galβ1 – 3[Neu5Acα2 – 6]GlcNAcβ1 – for triantennary structures.

The O‐linked oligosaccharides, found in the light chains. were shown to have the structure Neu5Acα2 – 3Galβ1 – 3GalNAc. The native IgM antibody could be separated on a concanavalin‐A – Sepharose column into two subfractions, differing in the presence of a high‐mannose‐type oligosaccharide.
Antisense oligonucleotide inhibition of encephalomyocarditis virus RNA translation
Authors: SANKAR Sabita , CHEAH Keat‐Chye , PORTER Alan G.
Published: March 2005

We report the inhibition of encephalomyocarditis virus (EMCV) RNA translation in cell‐free rabbit reticulocyte lysates by antisense oligonucleotides (13–17‐base oligomers) complementary to (a) the viral 5′ non‐translated region, (b) the AUG start codon and (c) the coding sequence. Our results demonstrate that the extent of translation inhibition is dependent on the region where the complementary oligonucleotides bind. Non‐complementary and 3′ ‐non‐translated‐region‐specific oligonucleotides had no effect on translation. A significant degree of translation inhibition was obtained with oligonucleotides complementary to the viral 5′ non‐translated region and AUG initiation codon. Digestion of the oligonucleotide: RNA hybrid by R Nase H did not significantly increase translation inhibition in the case of 5′‐non‐translated‐region‐specific and initiator‐AUG‐specific oligonucleotides; in contrast, RNase H digestion was necessary for inhibition by the coding‐region‐specific oligonucleotide. We propose that (a) 5′‐non‐translated‐region‐specific oligonucleotides inhibit translation by affecting the 40S ribosome binding and/or passage to the AUG start codon, (b) AUG‐specific oligonucleotides inhibit translation initiation by inhibiting the formation of an active 80S ribosome and (c) the coding‐region‐specific oligonucleotide does not prevent protein synthesis because the translating 80S ribosome can dislodge the oligonucleotide from the EMCV RNA template.
Glycosylation of glycoproteins 52 and 65 encoded by the polycythemia‐inducing strain of Friend spleen focus‐forming virus
Authors: STRUBE Karl‐Hermann , LOTTSPEICH Friedrich , GEYER Rudolf
Published: March 2005

The primary envelope gene product of the polycythemia‐inducing strain of Friend spleen focus‐forming virus, glycoprotein 52 (gp52), as well as its processed form, glycoprotein 65 (gp65), were isolated from virus‐infected normal rat kidney cells metabolically labeled with [2‐3H]mannose. Following digestion with trypsin, glycopeptides containing individual N‐glycosylation sites were obtained by gel filtration and subsequent reversed‐phase high‐performance liquid chromatography. N‐terminal amino acid sequencing of the glycopeptides demonstrated that only asparagine residues 11 and 26, located in the N‐terminal domains of gp52 and gp65, carry carbohydrate substituents, while the potential N‐glycosylation sites in the C‐terminal portions of the molecules are not used. Carbohydrates attached were liberated by treatment with endo‐β‐N‐acetylglucosaminidase H or peptide:N‐glycosidase F and characterized by high‐performance liquid chromatography. The results demonstrated that gp52 carries similar patterns of oligomannosidic glycans in both positions. In gp65, however, asparagine residue 11 is almost exclusively substituted by complete, fucosylated N‐acetyllactosaminic oligosaccharides, whereas asparagine residue 26 carries oligomannosidic or truncated N‐acetyllactosaminic glycans.
Functional complementation of catalase‐defective peroxisomes in a methylotrophic yeast by import of the catalase A from Saccharomyces cerevisiae
Authors: HANSEN Hans , ROGGENKAMP Rainer
Published: March 2005

A mutant of the methanol‐utilizing yeast Hansenula polymorpha defective in catalase was isolated. It lacks the ability to grow on methanol as the sole source of carbon and energy due to a loss of peroxisomal function that is required for the dissimilation and assimilation of this substrate. Growth of the mutant on glucose or glycerol was not impaired. Transformation of mutant cells with the gene coding for catalase A from Saccharomyces cerevisiae [Cohen, G., Fessl, F., Traczyk, J., Rytka, J. & Ruis, H. (1985) Mol. Gen. Genet. 200, 74–79] conferred constitutive expression of catalase activity. When the gene was placed under control of the regulatory methanol oxidase promoter from H. polymorpha, high levels of activity subject to glucose repression were obtained. In both cases efficient targeting of catalase A to the heterologous peroxisomes and assembly into an active form could be demonstrated. Concomitantly, growth on methanol was restored in the transformed mutant. The results are in line with a high conservation of transport signals on peroxisomal proteins. Expression of a cytosolic catalase in H. polymorpha did not confer the ability to grow on methanol. Therefore, proper localization of the catalase activity is a prerequisite for peroxisomal function.
O‐Linked GlcNAc in serotype‐2 adenovirus fibre
Authors: CAILLET‐BOUDIN Marie‐Laure , STRECKER Gérard , MICHALSKI Jean‐Claude
Published: March 2005

Serotype‐2 adenovirus fibre is shown to possess an O‐linked GlcNAc residue and to have affinity for wheat germ agglutinin. The cytoplasmic and nuclear fibres are both glycosylated. Glycosylation seems to take place in the cytoplasm since most of the [14C]GlcN‐labelled fibre is found in this compartment, little label being associated with the microsomes. Glycosylation of the fibre was not affected by inhibitors of N‐ and O‐glycosylation.

A variation in fibre glycosylation is observed among adenovirus. Among the serotypes tested, only serotype‐5 adenovirus (another subgroup C virus) also incorporated [14C]GlcN into its fibre, but did not possess affinity for wheat‐germ agglutinin.

The GlcNAc is located in the N‐terminal two‐thirds of the fibre and more probably in the N‐terminal one‐third. The free or penton‐base‐associated fibres are similarly glycosylated. These results suggest that glycosylation is not involved in viral adsorption and in assembly with the capsid penton base. Thus, glycosylation might be a characteristic feature of subgroup C viruses.
The role of sodium ions in methanogenesis
Authors: KAESLER Bruno , SCHÖNHEIT Peter
Published: March 2005

Cell suspensions of Methanosarcina barkeri were found to oxidize formaldehyde to CO2 and 2H2 (ΔG0′= ‐ 27 kJ/mol CO2), when methanogenesis was inhibited by 2‐bromoethanesulfonate. We report here that this reaction is coupled with (a) primary electrogenic Na+ translocation at a stoichiometry of 2–3 Na+/CO2, (b) with secondary H+ translocation via a Na+/H+ antiporter and (c) with ATP synthesis driven by an electrochemical proton potential. This is concluded from the following findings.

Formaldehyde oxidation to CO2 and 2H2 was dependent on Na+ ions, 2–3 mol Na+/mol formaldehyde oxidized were extruded. Na+ translocation was inhibited by Na+ ionophores, but not affected by protonophores or Na+/H+ antiport inhibitors.

Formaldehyde oxidation was associated with the build up of a membrane potential in the order of 100 mV (inside negative), which could be dissipated by sodium ionophores rather than by protonophores.

Formaldehyde oxidation was coupled with ATP synthesis, which could be inhibited by Na+ ionophores, Na+/H+ antiport inhibitors, by protonophores and by the H+ ‐translocating‐ATP‐synthase inhibitor, dicyclohexyl‐carbodiimide.

With cell suspensions of Methanobacterium thermoautotrophicum similar results were obtained.
Thyroid hormone regulation of nuclear‐encoded mitochondrial inner membrane polypeptides of the liver
Authors: JOSTE Vigg , GOITOM Zere , NELSON B. Dean
Published: March 2005

The effects of thyroid hormone on nuclear‐encoded mitochondrial inner membrane proteins was investigated by in vitro translation of the endogenous mRNA present in a postmitochondrial fraction from the livers of rats treated in vivo with hormone. The levels of the mRNAs were estimated by quantitative immunoabsorption of the translation mixture. Total protein synthesis was increased 2.6‐fold after 4 days of in vivo hormone treatment, but only 10–15% of the polypeptides were dramatically altered (> 5‐fold). Among the most highly elevated were cytochrome c1 (> 10‐fold increase) and the Rieske iron‐sulfur protein of the cytochrome bc1 complex. Other inner membrane proteins (core protein 1, β subunit of F1 ATPase, subunit IV of cytochrome oxidase, 3‐hydroxybutyrate dehydrogenase) and non‐mitochondrial proteins (rat serum albumin, β2‐microglobulin) were not altered significantly by hormone treatment. Cytochrome c1 and the Rieske protein increased after 12 h of hormone treatment, a relatively early response in mammalian mitochondrial biogenesis. The possible significance of this response for the regulation of mitochondrial synthesis and assembly is discussed.
Purification and characterization of the thermostable ribulose‐1,5‐bisphosphate carboxylase/oxygenase from the thermophilic purple bacterium Chromatium tepidum
Authors: HEḊA Ghanshyam D. , MADIGAN Michael T.
Published: March 2005

The Calvin cycle enzyme ribulose‐bisphosphate carboxylase/oxygenase has been purified and characterized from the thermophilic and obligately anaerobic purple sulfur bacterium, Chromatium tepidum. The enzyme is an L8S8 carboxylase with a molecular mass near 550 kDa. No evidence for a second form of the enzyme lacking small subunits was obtained. C. tepidum ribulose‐bisphosphate carboxylase/oxygenase was stable to heating to temperatures of 60°C and could be readily purified in an active form at room temperature. Both carboxylase and oxygenase activities of this enzyme were Mg2+‐dependent and carboxylase activity was sensitive to the effector 6‐phosphogluconic acid. The Km for ribulose bisphosphate for the carboxylase activity of the C. tepidum enzyme was substantially higher than that observed in mesophilic Calvin cycle autotrophs. Amino acid composition and immunological analyses of C. tepidum and Chromatium vinosum ribulose‐bisphosphate carboxylases showed the enzymes to be highly related despite significant differences in heat stability. It is hypothesized that thermal stability of C. tepidum ribulose‐bisphosphate carboxylase/oxygenase is due to differences in primary structure affecting folding patterns in both the large and small subunits and is clearly not the result of any unique quaternary structure of the thermostable enzyme.
Isolation of a cDNA encoding the rat liver S‐adenosylmethionine synthetase
Authors: HORIKAWA Saburo , ISHIKAWA Mahide , OZASA Hisashi , TSUKADA Kinji
Published: March 2005

We have isolated cDNA clones encoding the rat liver S‐adenosylmethionine synthetase by means of immunological screening from a phage λgt 11 expression library containing cDNA synthesized from adult rat liver poly(A)‐RNA. The amino acid sequence deduced from the cDNA indicates that the rat liver enzyme for this protein contains 397 amino acid residues and has a molecular mass of 43697 Da. The deduced amino acid sequence of rat liver S‐adenosylmethionine synthetase was 68% similar to those of yeast S‐adenosylmethionine synthetases encoded by two unlinked genes SAM1 and SAM2. The rat liver S‐adenosylmethionine synthetase also shows 52% similarity with the deduced amino acid sequence of the MetK gene encoding the S‐adenosylmethionine synthetase in Escherichia coli.
Effect of evolution on the kinetic properties of enzymes
Authors: PETTERSSON Gösta
Published: March 2005

The likely effect of a selective pressure in the direction of higher reaction fluxes on rate parameters for enzyme reactions confirming to Michaelis‐Menten kinetics has been analyzed on the basis of relationships which take into account the changes in metabolite concentrations that must be associated with mutational changes of the kinetic properties of enzymes participating in metabolic pathways.

Arguments are presented to show that such a pressure should tend to increase kcat, whereas Km may decrease or increase depending on what stage of evolutionary development the enzyme has reached. While the early evolution of enzymes must have been associated with decreasing Km values, an increase of both kcat and Km is mandatory for enhancement of the rate performance of extensively developed enzymes which exhibit kcat/Km ratios approaching the diffusion‐control limit. The latter limit is dependent on the equilibrium constant for the catalysed reaction.

Enzymes which have reached the diffusion‐control limit for their second‐order rate performance cannot be considered as perfectly evolved catalysts, but may well undergo further development towards a higher catalytic efficiency in response to the improvement of other enzymes in the metabolic pathway with regard to the criterion of an enhanced reaction flux. Such evolution is associated with an increase of the metabolite levels in the pathway, and a simple model system is examined in order to illustrate the ultimate limits for the metabolite levels and reaction flux that may obtain.

The theoretical evidence presented lends no support to previous proposals that certain enzymes (e.g. triosephosphate isomerase), or enzymes showing certain kinetic characteristics (e.g. kcat/Km quotients approaching 109 s–1 M–1), have reached the end of their evolutionary development. A claim that any specific enzyme has reached catalytic perfection would provide the unreasonable inference that all enzymes participating in intermediary metabolism have reached catalytic perfection.
The free radical of pyruvate formate‐lyase
Authors: UNKRIG Volker , NEUGEBAUER Franz A. , KNAPPE Joachim
Published: March 2005

The first‐derivative EPR spectrum of the active from of Escherichia coli pyruvate formate‐lyase shows an asymmetric doublet with partially resolved hyperfine splittings (g= 2.0037). Isotope substitution studies demonstrated couplings of a carbon‐centered unpaired electron to a solvent‐exchangeable proton (a= 1.5 mT) and to further hydrogen nuclei (a= 0.36 and 0.57 mT). By selective incorporation of unlabelled tyrosine into 2H‐labelled enzyme protein, a tyrosyl radical structure has been ruled out. Circumstantial evidence indicates that the organic free radical, which also displays an ultraviolet absorption signal at 365 nm, is located on a standard amino acid residue of the polypeptide chain. EPR signal quantification found a stoichiometry of 1 spin per active site.

The formate analogue hypophosphite has been characterized as a specific kcat inhibitor of pyruvate formatelyase which destroys the enzyme radical. Protein‐linked 1‐hydroxyethylphosphonate was previously described as the dead‐end product after reaction of the analogue with the intermediary acetyl‐enzyme form of the catalytic cycle [W. Plaga et al. (1988) Eur. J. Biochem. 178, 445–450]. EPR spectroscopy of this system has now identified the corresponding α‐phosphoryl radical as a reaction intermediate [g= 2.0032; a(P) = 2.72 mT, a(3H) = 1.96 mT]; it showed a half‐life of about 20 min at 0°C. This finding proves that the enzyme radical is a hydrogen‐atom‐transferring coenzymic element.
Structure and some characterization of the gene for phenylalanine ammonia‐lyase from rice plants
Authors: MINAMI Ei‐Ichi , OZEKI Yoshihiro , MATSUOKA Makoto , KOIZUKA Nobuya , TANAKA Yoshiyuki
Published: March 2005

A nearly full‐length cDNA and a genomic clone were isolated that encoded the phenylalanine ammonia‐lyase (PAL) of rice plants, and the complete nucleotide sequences were determined. The gene encodes a polypeptide of 701 amino acid residues. The deduced amino acid sequence is highly similar to that of PAL from Phaseolus vulgaris deduced from an incomplete cDNA fragment. The cloned gene spans 4412 bp and consists of two exons and one intron. The site of initiation of transcription was located – 86 nucleotides (position 1) upstream from the translational initiation codon by the primer‐extension method. Sequences analogous to TATA‐box and GC‐box were found in the 5′‐upstream region from the transcriptional initiation site. Southern blot analysis showed that the PAL gene of rice plants exists as a small multi‐gene family. Within this family, the genomic clone isolated in this study was shown to be responsive by light. This also indicated that this genomic sequence functions as a gene for phenylalanine ammonia‐lyase in rice plants in vivo.
Purification and characterization of a fatty‐acid‐binding protein from the gastric mucosa of rats
Authors: KANDA Tatsuo , ISEKI Shoichi , HITOMI Masahiro , KIMURA Hideki , ODANI Shoji , KONDO Hisatake , MATSUBARA Yoichi , MUTO Terukazu , ONO Teruo
Published: March 2005

Fatty acid‐binding protein (FABP) was purified from rat gastric mucosa by successive Sephadex G‐75 chromatography, DEAE‐cellulose chromatography and HPLC on an RP‐2 (Merck) reversed‐phase column. The purified stomach FABP migrated as a single band corresponding to an apparent molecular mass of 15 kDa on SDS/PAGE.

Stomach FABP appeared to be identical with rat heart FABP, as judged from its electrophoretic mobility, amino acid composition and tryptic peptide map. In addition, the amino acid sequences of two selected tryptic peptides coincided completely with the rat heart FABP sequence deduced from that of cDNA. Stomach FABP showed immunochemical identity with rat heart FABP when tested with an antiserum against rat heart FABP. Immunohistochemically, stomach FABP was specifically stained with anti‐(rat heart FABP) serum in parietal cells of the gastric mucosa.

The results suggested that the primary structure of stomach FABP is identical with that of rat heart FABP, and showed that stomach FABP is localized in parietal cells of the gastric mucosa.
Heterogeneous assembly of nascent core histones to form nucleosomal histone octamers in mouse FM3A cells
Authors: SUGASAWA Kaoru , ISHIMI Yukio , YAMADA Masa‐atsu , HANAOKA Fumio , UI Michio
Published: March 2005

We investigated the assembly of newly synthesized histones into nucleosomal histone octamers using an isopycnic centrifugation analysis of these proteins of mouse FM3A cells that had been labeled by culture with heavy amino acids. Cross‐linked histone octamers or non‐cross‐linked core histones were separated electrophoretically from other proteins on sodium dodecyl sulfate/polyacrylamide gels before being banded to equilibrium in cesium formate/guanidinium chloride density gradients. The density of core histones rapidly came to equilibrium after culture of cells in the medium containing heavy amino acids for a time as short as 30 min, indicating that the density of histone octamers in the density‐labeled cells reflects the content of these new (dense) core histones relative to old (light) ones in the octamer assembly. Cross‐linked histone octamers from these cells were of heterogeneous densities as judged from a broad band in a density gradient. The position and width of the band was essentially unaffected, though the peak position of the density distribution within the band moved progressively from a less to a more dense area, when the time of culture was prolonged from 2 h to 16 h. It is concluded, therefore, that new and old histones are assembled into histone octamers in heterogeneous ratios in contradiction to a conservative or semi‐conservative assembly model and it is also suggested that incorporation into chromatin of new histones is not necessarily restricted to newly replicated chromatin regions.
Derailment product in NADPH‐dependent synthesis of a dihydroisocoumarin 6‐hydroxymellein by elicitor‐treated carrot cell extracts
Authors: KUROSAKI Fumiya , KIZAWA Yuji , NISHI Arasuke
Published: March 2005

Synthetic activity of 6‐hydroxymellein, the immediate precursor of carrot phytoalexin 6‐methoxymellein, from acetyl‐CoA and malonyl‐CoA was induced in carrot cell extracts when the root disks were treated with CuCl2 or oligogalacturonide elicitor. These elicitors showed specific inducing activity of phytoalexin production and did not affect fatty acid synthesis in carrot tissues which may share some common properties with 6‐hydroxymellein biosynthesis. 6‐Hydroxymellein production was an NADPH‐dependent process and, in the absence of the reagent, triacetic acid lactone was produced as a derailment product of the reaction process. This finding suggested that the reduction of the double bond at the 3,4‐position of the phytoalexin takes place during the elongation of the poly(oxomethylene) chain. This NADPH‐dependent reduction seems to occur at the triacetate stage before the condensation of the third malonyl‐CoA as the conversion of carbonyl to hydroxyl group.
The amino acid sequence of rabbit skeletal muscle glycogenin
Authors: CAMPBELL David G. , COHEN Philip
Published: March 2005

The amino acid sequence of glycogenin from rabbit skeletal muscle has been determined. The N‐acetylated protein consists of 332 amino acids and has a molecular mass of 37278 Da. The novel tyrosyl‐glucose linkage between glycogenin and glycogen [Smythe, C., Caudwell, F. B., Ferguson, M. & Cohen, P. (1988) EMBO J. 7, 2681–2686] is shown to occur at a single site, tyrosine‐194. Although glycogenin is a UDP‐Glc utilising glucosyltransferase that self‐glucosylates [Pitcher, J., Smythe, C. & Cohen, P. (1988) Eur. J. Biochem. 176, 391–395], following addition by an unknown enzyme of the first glucose to tyrosine‐194, it is not homologous to either human glycogen synthase or other UDP‐Glc‐requiring enzymes.
Amino acid sequence of the monomer subunit of the giant multisubunit hemoglobin from the earthworm Pheretima sieboldi
Authors: SUZUKI Tomohiko
Published: March 2005

The giant extracellular hemoglobin of the earthworm Pheretima sieboldi is mainly composed of two hemecontaining subunits: a monomer; chain I and a disulfide‐bonded trimer of chains II, III and IV. Both subunits can be separated easily by gel filtration under alkaline conditions. The amino acid sequence of chain I has been determined. It is composed of 141 residues, has two half‐cystine residues forming a intrachain disulfide bridge, and has a molecular mass of 16911 Da including a heme group. Heterogeneity was found at position 37 (His or Ser).

The amino acid sequence of Pheretima chain I showed 30–50% identity with those of eight heme‐containing chains of Lumbricus and Tylorrhynchus hemoglobins. The sequences of nine chains of annelid giant hemoglobins were comparéd separately in the functionally essential central exonic region and structurally essential side exonic regions, and a phylogenetic tree was constructed. The amino acid substitution rate for the central exon was found to be about 1.5 times slower than that for the side exons.
Inhibition of transmitter release by botulinum neurotoxin A
Authors: POULAIN Bernard , WADSWORTH Jonathan D. F. , MAISEY E. Anne , SHONE Clifford C. , MELLING Jack , TAUC Ladislav , DOLLY J. Oliver
Published: March 2005

The contribution of a proteolytic fragment (H2L) of botulinum neurotoxin type A (comprised of the amino‐terminal region of the heavy‐chain disulphide‐linked to the light chain) to inhibition of neurotransmitter release was investigated, using central cholinergic synapses of Aplysia, rodent nerve‐diaphragm preparations and cerebrocortical synaptosomes.

No reduction in neurotransmitter release was observed following external application to these preparations of highly purified H2L or after intracellular injection into Aplysia neurons.

The lack of activity was not the result of alteration in the light chain of H2L during preparation of the latter because (a) renaturation of this light chain with intact heavy chain produced a toxic di‐chain form and (b) simultaneous application of heavy chain and light chain from H2L inhibited transmitter release in Aplysia.

Bath application of H2L and heavy chain together inhibited release of transmitter; however, at the neuromuscular junction the potency of this mixture was much lower than that of native toxin. A similar blockade resulted when heavy chain was applied intracellularly and H2L added to the bath, demonstrating that H2L is taken up into cholinergic neurons of Aplysia. This uptake is shown to be mediated by the amino‐terminal moiety of heavy chain (H2), because bath application of light chain plus H2 led to a decrease in acetylcholine release from a neuron that had been injected with heavy chain.

A role within the neuron is implicated for a carboxy‐terminal portion of heavy chain (H1) since intracellular injection of light chain and H2 did not affect transmitter release. Although the situation is unclear in mammalian nerves, these collective findings indicate that blockade of transmitter release in Aplysia neurons requires the intracellular presence of light chain and H1 (by inference), whilst H2 contributes to the internalization step.
Purification and characterization of dihydropyrimidine dehydrogenase from pig liver
Authors: PODSCHUN Beate , WAHLER Gudrun , SCHNACKERZ Klaus D.
Published: March 2005

Dihydropyrimidine dehydrogenase was isolated from cytosolic pig liver extracts and purified 3100‐fold to apparent homogeneity. Purification made use of ammonium sulfate fractionation, precipitation with acetic acid and chromatography on DEAE‐cellulose and 2′,5′‐ADP‐Sepharose with 28% recovery of total activity. The native enzyme has a molecular mass of 206 kDa and is apparently composed of two similar, if not identical, subunits. Proteolytic cleavage reveals two fragments with apparent molecular masses of 92 kDa and 12 kDa. The C‐terminal 12‐kDa fragment seems to be extremely hydrophobic. The enzyme contains tightly associated compounds including four flavin nucleotide molecules and 32 iron atoms/206‐kDa molecule. The iron atoms are probably present in iron‐sulfur centers. The flavins released from the enzyme were identified as FAD and FMN in equal amounts. An isoelectric point of 4.65 was determined for the dehydrogenase. Apparent kinetic parameters were obtained for the substrates thymine, uracil, 5‐aminouracil, 5‐fluorouracil and NADPH.
Heat‐shock protein 83 of Leishmania mexicana amazonensis is an abundant cytoplasmic protein with a tandemly repeated genomic arrangement
Authors: SHAPIRA Michal , PINELLI Elena
Published: March 2005

The 83‐kDa heat‐shock protein HSP83 is a highly abundant protein in Leishmania amastigotes and promastigotes exposed to elevated temperature. Antibodies against this protein were obtained by immunization with a synthetic peptide derived from a conserved region. These antibodies both the denatured and the native form of the molecule and were used for immunofluorescence analysis. These experiments, together with analysis by cell fractionation, show that HSP83 is distributed in the cytoplasm of Leishmania parasites. The gene for HSP83 in Leishmania mexicana amazonensis has been cloned from a genomic library, and molecular characterization shows it is present in several copies of 4‐kb repeats arranged in tandem.
Molecular organization and clustering of cell‐wall‐bound enzymes as a source of kinetic apparent co‐operativity
Authors: DUSSERT Christophe , MULLIERT Guillermo , KELLERSHOHN Nicolas , RICARD Jacques , GIORDANI Roger , NOAT Georges , PALMARI Jacqueline , RASIGNI Monique , LLEBARIA Antoine , RASIGNI Georges
Published: March 2005

When fixed charges and enzyme molecules are not homogeneously distributed in a matrix, the degree of organization of charges, of enzyme molecules and of charges with respect to enzyme molecules modulate the enzyme reaction rate. The overall reaction velocity of the bound enzyme system may be expressed in terms of monovariate moments of the charge density distribution and of the bivariate moments of the charge and enzyme density distributions. With respect to the situation where fixed charges and enzyme molecules are randomly distributed in the matrix, the molecular organization, as expressed by the monovariate and bivariate moments results in an increase or a decrease, of the overall reaction rate, as well as in the appearance of a kinetic co‐operativity.

The degree of spatial organization of objects may be expressed quantitatively through the concept of minimal spanning tree. This concept may thus be applied to the quantification of the degree of order that may exist in the bidimensional distribution of enzyme molecules in a charged matrix.

Primary walls of isolated plant cells in sterile culture behave as a polyanion and contain different enzymes. The spatial distribution in sycamore cell walls of an acid phosphatase has been studied through the concept of minimal spanning tree and shown to be non‐randomly distributed in the polyanionic matrix, but clustered in that matrix. This spatial organization results in a modulation of the reaction rate of the cell‐wall‐bound phosphatase reaction.

Both the theoretical and experimental results presented in this study leave little doubt as to the validity of the idea thatin situthe organization of fixed charges and enzyme molecules modulate the overall dynamics of enzyme reactions.
α‐Actinin expression during avian myogenesis in vivo
Authors: KOBAYASHI Ryoji , ITOH Hideaki , TASHIMA Yohtalou
Published: March 2005

The isoforms of skeletal muscle α‐actinin present during chick embryogenesis were analyzed by two‐dimensional electrophoresis in combination with the immunoblot technique. Chicken embryonic muscles at 8–15 days contain an embryo‐specific isoform of α‐actinin. The embryonic α‐actinin isoform has a molecular mass of 112 kDa and an isoelectric point of 5.8, whereas the values for the adult isoform of α‐actinin were 100 kDa and 5.85, respectively. To characterize the two classes of α‐actinin polypeptides we have compared the two proteins by 125I‐labeled two‐dimensional peptide mapping. The embryonic isoform is highly similar to, but exhibited extensive peptide differences to, the adult isoform of α‐actinin.

The developmental sequence of the expression of the α‐actinins was also studied. In extracts of skeletal muscle from 8–10‐day‐old embryos, only the embryonic isoform was detected. In extracts from 15‐day‐old embryos, both the embryonic and the adult isoforms coexisted. However by 21 days, the embryonic isoform had disappeared and only the adult isoform was detected.

These data suggested that the embryonic and the adult isoform of α‐actinins are distinct proteins and that during skeletal myogenesis in ovo one class of α‐actinin is replaced by a new class of α‐actinin polypeptides, and that the latter is maintained into adulthood.
Conformation of two somatostatin analogues in aqueous solution
Authors: WYNANTS Chantal , TOURWE Dirk , KAZMIERSKI Wieslaw , HRUBY Victor J. , VAN BINST Georges
Published: March 2005

Cyclic‐disulfide‐containing analogues of somatostatin, [Xaa1= H or DPhe; Xaa3= Phe or Tyr; Xaa7= Cys, Me2Cys or Me2DCys; Xaa8= OH, Thr8 (OH) or Thr8NH2], were examined in aqueous solution by 1H‐NMR spectroscopy and circular dichroism.

The influence of the helical nature of the disulfide bridge and the presence of exocyclic residues on biological activity were investigated with particular care.
A new probe for affinity labelling pancreatic cholecystokinin receptor with minor modification of its structure
Authors: FOURMY Daniel , LOPEZ Patrick , POIROT Sandrine , JIMENEZ Juan , DUFRESNE Marlène , MORODER Luis , POWERS Stephen P. , VAYSSE Nicole
Published: March 2005

Biochemical studies on receptors for peptides are most often carried out on affinity‐labelled (peptide‐receptor) complexes. Necessarily, the assumption is made that a covalent (peptide‐receptor) complex behaves as the native receptor. The validity of this assumption is dependent on both the affinity‐labelling technique and the resolution of the analytical method used for biochemical characterization. We designed a new affinity‐labelling probe in order to minimize structural modifications occurring within the affinity‐labelled cholecystokinin (CCK) receptor protein. The probe was 125I‐labelled 2‐(p‐azidosalicylamido)‐1,3‐dithiopropionate‐[Thr28,Ahx3]CCK‐25‐33, (125I‐ASD‐[Thr28, Ahx31]CCK‐25‐33), the peptide moiety of which was released from its binding site by reduction. It was obtained by coupling a photoactivable chemical to [Thr28,Ahx31]CCK‐25‐33 via its N‐terminus. The resulting peptide was HPLC purified and radioiodinated in the presence of chloramine T. Binding of 125I‐ASD‐[Thr28,Ahx31]CCK‐25‐33 was time‐ and temperature‐dependent and reversible. At 25°C, a steady‐state level was reached after 60 min and half‐maximal dissociation after 38 min. Binding was inhibited by [Thr28, Ahx31]CCK‐25‐33 and L‐364‐718 antagonist with IC50 0.4 nM and 0.9 nM, respectively. Photoaffinity labelling of pancreatic plasma membranes by 125I‐ASD‐[Thr28, Ahx31]CCK‐25‐33 identified a glycoprotein of Mr 85000‐100000 which was retained on immobilized wheat germ agglutinin. Enzyme cleavage by endoproteinase Glu‐C generated a main fragment of Mr 30000‐34000. The same glycoprotein was photoaffinity labelled with 125I‐DTyr‐Gly‐[Ahx28.31, pNO2Phe33]CCK‐26‐33 (Ahx, 2‐aminohexanoic acid; pNO2Phe, p‐nitrophenylalanine) an intrinsic probe having its photolabile group sited in the binding domain of cholecystokinin. 125I‐ASD‐[Thr28,Ahx31]CCK‐25‐33 is a potentially powerful tool for biologically and biochemically studying cholecystokinin receptors.
Characterization of a high‐affinity monoclonal antibody to calcineurin whose epitope defines a new structural domain of calcineurin A
Authors: HUBBARD Michael J. , KLEE Claude B.
Published: March 2005

Monoclonal antibodies have been raised against native calcineurin using conventional in vivo immunization and hybridoma procedures. The relatively high affinity of nonimmune IgG for the two subunits of calcineurin resulted in large nonspecific binding values for immunoassays of native, dissociated and denatured calcineurin, which complicated the antibody screening.

Monoclonal aCn5, a high‐affinity IgG1 that exhibits specific binding, was characterized. Other calmodulin‐binding proteins tested were not recognized by aCn5. Simple binding properties were exhibited in solid‐phase experiments, Kd= 26 (±4) pM, but the stoichiometry was low. The loss of immunoreactivity after denaturation of calcineurin indicated that the aCn5 epitope is of the assembled topographic, not segmental, type. The epitope was located to the A subunit and affinity was unaffected by the presence of calcineurin B. The epitope remained intact after proteolytic removal of the amino‐terminal 20 residues of calcineurin A essential for phosphatase activity, and the carboxyl‐terminal inhibitory and calmodulin‐binding domains. The calmodulin‐binding peptide derived from calcineurin, cA8, was not recognized by aCn5. Addition of Ca2+, Mn2+, Ni2+, chelators or dithiothreitol did not influence the affinity of aCn5 for the holoenzyme. Phosphatase activity of calcineurin, in the presence and absence of calmodulin and after removal of the inhibitory domain, was little affected by aCn5. Thus, the aCn5 epitope defines a previously unidentified structural domain of calcineurin A located in a region of the proteolytically resistant core that is topologically distinct from the catalytic, inhibitory, calmodulin‐binding and calcineurin‐B‐binding domains, and not functionally connected with calcineurin B or the putative metal‐binding domain(s).
The nucleoskeleton and the topology of transcription
Authors: COOK Peter R.
Published: March 2005

Transcription is conventionally believed to occur by passage of a mobile polymerase along a fixed template. Evidence for this model is derived almost entirely from material prepared using hypotonic salt concentrations. Studies on subnuclear structures isolated using hypertonic conditions, and more recently using conditions closer to the physiological, suggest an alternative. Transcription occurs as the template moves past a polymerase attached to a nucleoskeleton; this skeleton is the active site of transcription. Evidence for the two models is summarised. Much of it is consistent with the polymerase being attached and not freely diffusible. Some consequences of such a model are discussed.
The mitochondrial inner‐membrane anion channel possesses two mercurial‐reactive regulatory sites
Authors: BEAVIS Andrew D.
Published: March 2005

The mitochondrial inner membrane anion channel catalyzes the electrophoretic transport of a wide variety of anions and is inhibited by matrix divalent cations and protons. In this paper, evidence is provided that mersalyl and p‐chloromercuribenzene‐sulfonate each interact with this uniporter at two distinct sites. Binding to site 1 causes a shift in the pH dependence of transport, characterized by a decrease in the pIC50 for protons from about 7.8 to about 7.3, and leads to substantial stimulation of transport in the physiological pH range. This effect is not reversed by addition of thiols such as thioglycolate. Binding of mersalyl and p‐chloromercuribenzenesulfonate to site 2 inhibits the transport of most anions including Pi, citrate, malonate, sulfate and ferrocyanide. The transport of Cl− is inhibited about 60% by mersalyl, but is not inhibited by p‐chloromercuribenzenesulfonate. These data suggest that inhibition is a steric effect dependent on the size of the anion and the size of the R group of the mercurial. This inhibition is reversed by thioglycolate. Dose/response curves indicate that mersalyl binds to site 1 as the dose increased from 7 to 13 nmol/mg, whereas it binds to site 2 as the dose is increased from 10 to 18 nmol/mg. Thus, at certain pH values both stimulatory and inhibitory phases can be seen in the same dose/response curve. It is suggested that these sites may contain thiol groups and that physiological regulators may exist which can effect changes in activity of the inner membrane anion uniporter similar to those exerted by mercurials.
rfaP mutants of Salmonella typhimurium
Authors: HELANDER Ilkka M. , VAARA Martti , SUKUPOLVI Soila , RHEN Mikael , SAARELA Sirkku , ZÄHRINGER Ulrich , MÄKELÄ P. Helena
Published: March 2005

Salmonella typhimurium rfaP mutants were isolated and characterised with respect to their sensitivity towards hydrophobic antibiotics and detergents, and their lipopolysaccharides were chemically analysed. The rfaP mutants were selected after diethylsulfate mutagenesis or as spontaneous mutants. The mutation in two independent mutants SH7770 (line LT2) and SH8551 (line TML) was mapped by cotransduction with cysE to the rfa locus. The mutants were sensitive to hydrophobic antibiotics (clindamycin, erythromycin and novobiocin) and detergents (benzalkoniumchloride and sodium dodecyl sulfate). Analysis of their lipopolysaccharides by chemical methods and by sodium dodecyl sulfate/polyacrylamide gel electrophoresis revealed that their saccharide portion was, to a large extent, of chemotype Rc with small proportions of material containing a more complete core oligosaccharide and O‐specific chains. Only 2.5 mol phosphate/mol lipopolysaccharide was found whereas the phosphate content of the lipopolysaccharide of a galE mutant strain was 4.8 mol. Thus the rfaP mutant lipopolysaccharides lacked more than two phosphate residues. Assessment of the location of phosphate groups in rfaP lipopolysaccharides revealed the presence of at least 2 mol phosphate in lipid A, indicating that the core oligosaccharide was almost devoid of phosphate. The chemical, physiological and genetic data obtained for these mutants are in full agreement with those reported earlier for rfaP mutants of Salmoneila minnesota.
Interaction of tRNA with the A and P sites of rabbit‐liver 80S ribosomes and their 40S subunits
Authors: RODNINA Marina V. , EL'SKAYA Anna V. , SEMENKOV Yuri P. , KIRILLOV Stanislav V.
Published: March 2005

The interaction between tRNA and rabbit liver 80S ribosomes and 40S subunits was studied using a nitrocellulose membrane filtration technique. Binding of the different tRNA forms (aminoacyl‐, peptidyl‐ or deacylated) to poly(U)‐programmed 40S subunits and 80S ribosomes was found to be a cooperative process. The association constants of AcPhe‐tRNAPhe for the A and P sites of 80S ribosomes and the cooperativity constant were measured at different temperature and Mg2+ concentration. The AcPhe‐tRNAPhe association constant for the P site was shown to be between 2 × 107 M−1 and 2 × 108 M−1 at 25–37°C and 5–20 mM Mg2+, while the affinity for the A site was 10–100‐fold lower. The cooperativity constant was shown to decrease with the increase of incubation temperature and the decrease of Mg2+ concentration. The affinity of AcPhe‐tRNAPhe for the A site of 80S ribosomes was shown to depend upon the codon specificity of tRNA at the P site. The cooperativity of the tRNA interaction with 80S ribosomes was suggested to be mostly contributed by the association with the 40S subunit and result from the correct codon‐anticodon pairing at the P site. The data presented imply a codon‐anticodon interaction at the P site of eukaryotic 80S ribosomes.
Isolation, characterization and immunocytochemical localization of caldesmon‐like protein from molluscan striated muscle
Authors: BARTEGI Aghleb , FATTOUM Abdellatif , DAGORN Caroline , GABRION Jacqueline , KASSAB Ridha
Published: March 2005

A 140‐kDa polypeptide present in the striated muscle of Pecten maximus and Sepia officinalis was purified to homogeneity and its main properties were investigated using biochemical and cytochemical approaches. The protein was found to be similar to chicken gizzard caldesmon. It is a heat‐stable protein. It cross‐reacts immuno‐logically with anti‐(gizzard caldesmon) antibody, binds to calmodulin‐Sepharose in a Ca2+‐dependent manner, cosediments with F‐actin filaments and acts in the absence and presence of tropomyosin as a potent inhibitor of rabbit skeletal actomyosin Mg2+‐ATPase.

The immunocytochemistry of ultrathin sections revealed, at the light microscopy resolution level, that caldesmon‐like protein is present in all types of muscles hitherto examined from invertebrates and vertebrates. However, according to the distribution and the intensity of the fluorescent reaction, we concluded that, under our experimental conditions, caldesmon is not homogeneously distributed and not located in the myofibrillar bands of striated muscles but rather in the sarcoplasmic elements, at the periphery of the fibres.
Microsomal Δ8,14‐sterol Δ14‐reductase in higher plants
Authors: TATON Maryse , BENVENISTE Pierre , RAHIER Alain
Published: March 2005

An enzymatic assay for Δ8,14‐sterol Δ14‐reductase, an enzyme involved in sterol biosynthesis, has been developed for the first time in higher plants. The properties of the microsomal enzyme have been established with respect to cofactor requirements, kinetics and substrate specificity. This enzymatic double‐bound reduction is thought to proceed through an electrophilic addition mechanism, involving a C14 putative carbonium ion high‐energy intermediate. Using this in vitro assay, ammonium and iminium analogues of this cationic intermediate were shown to be potent inhibitors of the reduction reaction. Thus, compounds of the N‐alkyl‐8‐aza‐4α,10‐dimethyl‐trans‐decal‐3β‐ol series strongly inhibited sterol reductase (I50= 0.07–4 μM) (I50/Km= 10‐4–10−3), as did the antimycotic agent 15‐azasterol (I50= 0.03 μM); all of these compounds act as reaction‐intermediate analogues of the proposed C14 carbonium ion intermediate. Moreover, the in vitro inhibition of the plant sterol reductase by a series of ammonium‐ion‐containing fungicides was demonstrated. The relative specificity of these different series of inhibitors toward cycloeucalenol‐obtusifoliol isomerase, Δ8→Δ7‐sterol isomerase and Δ8,14‐sterol Δ14‐reductase, was directly studied.