bluuebunny/crossref_metadata_2025 huggingface dataset:
Limited proteolysis of tetanus toxin
Authors: KRIEGLSTEIN Kerstin G. , HENSCHEN Agnes H. , WELLER Ulrich , HABERMANN Ernst
Published: March 2005

Tetanus toxin is synthesized by Clostridium tetani as a 151‐kDa peptide chain. The primary gene product is processed post‐translationally by removal of the initiating methionine residue, formation of disulfide bridges and limited proteolysis by bacterial or exogenous proteinases. The mature toxins consist of a 52‐kDa light chain and a 98‐kDa heavy chain, linked together by a disulfide bond. Proteolytic nicking is accompanied by increased pharmacological potency. To identify the structural alterations involved, single‐chain toxin has been subjected to limited proteolysis with various enzymes. The new N‐termini have been determined by Edman degradation and the C‐termini by isolation of short C‐terminal peptide fragments and subsequent analysis of the sequence and composition. All two‐chain toxins result from proteolytic nicking within the 17‐residue segment of residues 445–461. Thus, the protease(s) of the culture broth cleave on the C‐terminal side of Glu449 and partially Ala456, giving rise to two heavy chain N‐termini. Trypsin and clostripain first attack the C‐terminal of Arg454 and later Arg448, whereas endoproteinase Arg‐C cleaves the former bond only. Chymotrypsin and endoproteinase Glu‐C each split a single peptide bond, i.e. that located after Tyr452 and Glu449, respectively. Papain gives rise to a large number of cleavages within the 17‐residue segment, the new C‐terminus being Thr445 or Asn446 and the new N‐terminus being Asp460 or Leu461. Further papain digestion leads to an additional cleavage within the heavy chain between Ser863 and Lys864. The original N‐terminal Prol and C‐terminal Aspl 1314, predicted from the nucleotide sequence, are conserved in all proteolytic digests. The pharmacological activity of the various two‐chain toxins was 5–11 times that of the single‐chain toxin, as estimated from the inhibition of [3H]noradrenaline release from rat‐brain homogenate. The present data on the processing and activation by limited proteolysis prove the existence of several active tetanus isotoxins. These data, together with our previous data on the localization of disulfide bridges and sulfhydryl groups (Krieglstein, K., Henschen, A., Weller, U. & Habermann, E. (1990) Eur. J. Biochem. 188, 39–45), provide the detailed protein chemical characterization of the tetanus isotoxins.
Aluminum interrupts the formation of alkaline‐ribonuclease‐inhibitor complex from bovine brain
Authors: CHO Sung‐Woo , KIM Geum‐Yi
Published: March 2005

The effect of aluminum on alkaline ribonuclease (RNase) and RNase inhibitor, purified from bovine brain, was investigated. Incubation of alkaline RNase with aluminum interrupted binding of RNase inhibitor to alkaline RNase. A stoichiometry of 1:1 for the binding of aluminum to brain alkaline RNase was estimated, whereas no aluminum was found to be bound to the RNase inhibitor. Aluminum‐bound alkaline RNase, however, retained a full alkaline RNase activity. None of the enzyme‐bound aluminum was dissociated by dialysis against 50 mM Hepes, pH 7.0, at 4°C for 24 h. Citrate, EDTA, NaF and apotransferrin protected the alkaline RNase against aluminum binding. Aluminum did not bind to the incubated alkaline RNase‐inhibitor complex, suggesting that aluminum might compete with RNase inhibitor for the binding site. However, the data from chemical modification and spectroscopic studies indicate that it is also highly possible that aluminum binding to the enzyme induces conformational changes at or near the inhibitor‐binding site, which subsequently interrupt binding of RNase inhibitor to alkaline RNase. These results suggest that accumulation of aluminum in brain might affect the regulation of RNA metabolism.
Lysine excretion by Corynebacterium glutamicum
Authors: BRÖER Stefan , KRÄMER Reinhard
Published: March 2005

Corynebacterium glutamicum effectively excretes lysine when the internal lysine concentration is elevated. Lysine efflux was investigated using selected mutants which are not able to regulate lysine biosynthesis by feedback inhibition. Secretion of lysine is not the consequence of unspecific permeability of the plasma membrane but is mediated by a secretion carrier which is specific for lysine. Lysine export is characterized by high activation energy and follows Michaelis‐Menten type kinetics with an internal Km of 20 mM and a Vmax of 12 nmol · min−1· mg dry cells−1. Excretion can proceed against a preexisting chemical gradient and against the electrical potential, which rules out a previously suggested pore model. Lysine excretion can also be observed in the wild‐type strain especially under conditions of peptide uptake. Its possible physiological function may be related to regulation of internal amino acid concentrations under special growth conditions.
Lysine excretion by Corynebacterium glutamicum
Authors: BRÖER Stefan , KRÄMER Reinhard
Published: March 2005

Lysine excretion in Corynebacterium glutamicum was characterized as secondary transport process. It is modulated by three forces: the membrane potential, the chemical potential of lysine, and the proton gradient. The ATP content of the cells did not correlate with the export activity. Lysine is excreted in symport with presumably two OH− ions which is not distinguishable experimentally from an antiport mechanism against two protons. The substrate‐loaded carrier is uncharged. When the external substrate concentration is low and no proton gradient present, reorientation of the positively charged, unloaded carrier is rate‐limiting. Export then depends on the membrane potential. When the external substrate is high, translocation of the loaded, uncharged carrier is rate‐limiting, and export is not modulated by the membrane potential. The lysine secretion system in C. glutamicum is shown to be well adapted to the requirements of metabolite export.
Ca2+ ionophore A23187 and thrombin inhibit the pertussis‐toxin‐induced ADP‐ribosylation of the α‐subunit of the inhibitory guanine‐nucleotide‐binding protein and other proteins in human platelets
Authors: SIESS Wolfgang , WILHELM Bernhard , AEPFELBACHER Martin , GENNITY Joseph M.
Published: March 2005

Inhibitory guanine‐nucleotide‐binding proteins (Gi proteins) are substrates for pertussis toxin and the decreased pertussis‐toxin‐dependent ADP ribosylation of Gi proteins upon prior specific hormonal stimulation of cells is thought to reflect the receptor‐mediated activation of Gi proteins, leading to their subsequent dissociation into αi and β/y subunits. In the present study, the effect of various platelet stimuli on the subsequent pertussis‐toxin‐dependent ADP ribosylation of the α subunit of Gi (Giα) in saponized platelets and platelet membranes were studied. Stimulation of intact platelets with the Ca2+ ‐ionophore A23187 or thrombin, but not phorbol 12,13‐dibutyrate, decreased the subsequent pertussis‐toxin‐dependent ADP ribosylation of Giα in saponin‐permeabilized platelets in a time‐dependent and dose‐dependent manner. Thrombin was more effective than A23187. Parallel measurements of Ca2+ mobilization and pertussis‐toxin‐dependent ADP ribosylation of Giα in platelets showed that Ca2+ mobilization could only partly account for the decrease in pertussis‐toxin‐dependent ADP ribosylation in platelets stimulated by thrombin. When the ADP‐ribosylation reaction was carried out in platelet membranes, a decrease in ADP ribosylation was still observed after stimulation of platelets with thrombin, but not with A23187. In addition to Giα, two other proteins were found to be ADP ribosylated by pertussis toxin; their ADP ribosylation was also decreased after A23187 and thrombin stimulation of platelets. The results indicate that Ca2+ mobilization can decrease the pertussis‐toxin‐dependent ADP ribosylation of Giα in saponized platelets; the decrease of pertussis‐toxin‐dependent ADP ribosylation of Giα after thrombin stimulation of platelets can only, in part, be explained by Ca2+ mobilization and involves additional mechanisms; the decrease in pertussis‐toxin‐dependent ADP ribosylation after A23187 and thrombin stimulation is not confined to Giα and involves other proteins. We conclude that the decrease in pertussis‐toxin‐dependent ADP ribosylation of Gi in thrombinstimulated platelets might not be solely caused by a specific structural change, such as dissociation of Gi. It is likely that A23187 and thrombin stimulation of platelets generates substances which interfere with the ADP‐ribosylating activity of pertussis toxin.
Complete amino acid sequence of endo‐β‐N‐acetylglucosaminidase from Flavobacterium sp.
Authors: TAKEGAWA Kaoru , MIKAMI Bunzo , IWAHARA Shojiro , MORITA Yuhei , YAMAMOTO Kenji , TOCHIKURA Tatsurokuro
Published: March 2005

The complete amino acid sequence of endo‐β‐N‐acetylglucosaminidase from Flavobacterium sp. has been determined by analysis of peptides after cleavage with lysyl endopeptidase, pepsin and chymotrypsin. The protein consists of a single polypeptide chain consisting of 267 amino acid residues and a molecular mass of 27972 Da. The sequence of Flavobacterium endo‐β‐N‐acetylglucosaminidase is very close to that of the Streptomyces enzyme (endo‐H), having 60% similarity and very similar hydropathy profiles. Similarities were also found between Flavobacterium endo‐β‐N‐acetylglucosaminidase and chitinases from Bacillus circulans, Serratia marcescens and Phaseolus vulgaris.
Rat‐liver‐type phosphofructokinase mRNA
Authors: HOTTA Kikuko , NAKAJIMA Hiromu , YAMASAKI Tomoyuki , HAMAGUCHI Tomoya , KUWAJIMA Masamichi , NOGUCHI Tamio , TANAKA Takehiko , KONO Norio , TARUI Seiichiro
Published: March 2005

We have cloned a full‐length cDNA for rat‐liver‐type phosphofructokinase. The similarities of the rat liver‐type phosphofructokinase mRNA to the human and mouse counterparts were 94% and 99% in their amino acid sequences and 88% and 94% in the nucleotide sequences of their coding regions, respectively. Rat liver‐type phosphofructokinase mRNA was expressed in all tissues examined, but its level was regulated tissue‐specifically. The nutritional and hormonal regulations of the mRNA in the liver were examined in comparison with those of two other key glycolytic enzymes, glucokinase and L‐type pyruvate kinase. The level of liver‐type phosphofructokinase mRNA was essenstially unchanged by starvation (72 h) or diabetes. The mRNA level also did not change significantly on refeeding starved rats on a high carbohydrate diet, or treating diabetic ones with insulin. These results suggested that rat liver‐type phosphofructokinase mRNA in the liver was not under control of diet or insulin, in contrast to glucokinase and L‐type pyruvate kinase.
Two lipid‐anchored cAMP‐binding proteins in the yeast Saccharomyces cerevisiae are unrelated to the R subunit of cytoplasmic protein kinase A
Authors: MÜLLER Günter , BANDLOW Wolfhard
Published: March 2005

We show that the yeast, Saccharomyces cerevisiae, contains two cAMP‐binding proteins in addition to the well‐characterized regulatory (R) subunit of cytoplasmic cAMP‐dependent protein kinase (PKA). We provide evidence that they comprise a new type of cAMP receptor, membrane‐anchored by covalently attached lipid structures. They are genetically not related to the cytoplasmic R subunit. The respective proteins can be detected in sral mutants, in which the gene for the R subunit of PKA has been disrupted and a monoclonal antibody raised against the cytoplasmic R subunit does not cross‐react with the two membrane‐bound cAMP‐binding proteins. In addition, they differ from the cytoplasmic species also with respect to their location and the peptide maps of the photoaffinity‐labeled proteins. Although they differ from one another in milecular mass and subcellular location, peptide maps of the cAMP‐binding domains resemble each other and both proteins are membrane‐anchored by lipid structures, one to the outer surface of the plasma membrane, the other to the outer surface of the inner mitochondrial membrane. Both anchors can be metabolically labeled by Etn, myo‐Ins and fatty acids. In addition, the anchor structure of the cAMP receptor from plasma membranes can be radiolabeled by GlcN and Man. After cleavage of the anchor with glycosyphosphatidylinositol‐specific phospholipase C from trypanosomes, the solubilized cAMP‐binding protein from plasma membranes reacts with antibodies which specifically recognize the cross‐reacting determinant from soluble trypanosomal coat protein, suggesting similarity of the anchors. Degradation studies also point to the glycosylphosphatidylinositol nature of the anchor from the plasma membrane, whereas the mitochondrial counterpart is less complex in that it lacks carbohydrates. The plasma membrane cAMP receptor is, in addition, modified by an N‐glycosidically linked carbohydrate side chain, responsible mainly for its higher molecular mass.
Sequence‐specific 1H‐NMR assignment and secondary structure of the Tyr41 → His mutant of the single‐stranded DNA binding protein, gene V protein, encoded by the filamentous bacteriophage M13
Authors: FOLKERS Paul J. M. , van DUYNHOVEN John P. M. , JONKER Aafke J. , HARMSEN Ben J. M. , KONINGS Ruud N. H. , HILBERS Cornelis W.
Published: March 2005

Sequence‐specific 1H‐NMR assignments are reported for the Tyr41 → His (Y41H) mutant of the single‐stranded DNA binding protein, encoded by gene V of the filamentous bacteriophage M13 (GVP). The mutant protein was chosen for this purpose because it exhibits significantly improved solubility characteristics over wild‐type GVP [Folkers et al. (1991) Eur. J. Biochem. 200, 139–148]. The secondary structure elements present in the protein are deduced from a qualitative interpretation of the nuclear Overhauser enhancement spectra and amide exchange data. The protein is entirely composed of antiparallel β‐structure. It is shown that identical structural elements are present in wild‐type GVP. Previously, we have demonstrated that the secondary structure of the β‐loop, encompassing residues 13–31 which is present in GVP in solution, deviates from that proposed for the same amino acid sequence on the basis of X‐ray diffraction data [van Duynhoven et al. (1990) FEBS Lett. 261, 1–4]. Now that we have arrived at a complete description of the secondary structure of the protein in solution, other deviations with respect to the crystallographically determined structure became apparent as well. The N‐terminal part of the protein is, in solution, part of a triple‐stranded β‐sheet while, in the crystal, it is an extended strand pointing away from the bulk of the protein dimer. One of the antiparallel β‐sheets in the protein which had been designated earlier as the complex loop has, in the solution structure, a different pairwise arrangement of the residues in its respective β‐ladders. Residues 30 and 48 are opposite to one another in the solution structure while in the crystal structure residues 32 and 48 are paired. A similar observation is made for the so‐called dyad domain of the protein of which the β‐sheet in the solution structure is shifted by one residue with respect to that of the crystal structure.
Human kidney hexosaminidase A and hexosaminidase B form a complex
Authors: PSHEZHETSKY Alexey V. , BUNEEVA Olga A. , DVORKIN Vladimir M.
Published: March 2005

The isolation and purification of human kidney hexosaminidases A and B was carried out. Regulation of the supramolecular organization and catalytic activity of hexosaminidases was investigated in the bis(2‐ethylhexyl)sulphosuccinate reversed micellar system modeling the enzyme microenvironment in the lysosomes. It was shown that hexosaminidases A and B associate forming 280–300‐kDa dimeric complexes under these conditions. At pH 4.75, the hexosaminidases A and B can be isolated from kidney tissue only in the form of this complex.
Pericytes of the brain microvasculature express γ‐glutamyl transpeptidase
Authors: FREY Andreas , MECKELEIN Barbara , WEILER‐GÜTTLER Hartmut , MÖCKEL Babette , FLACH Regina , GASSEN Hans G.
Published: March 2005

The expression of γ‐glutamyl transpeptidase (GGT) is a specific property of the brain capillary endothelium that constitutes the blood‐brain barrier. We report here the detection of GGT, not only in endothelial cells, but also in pericytes, demonstrating that a brain capillary‐specific pericyte population exists. We raised antibodies to GGT using a porcine brain microvessel GGT–protein‐A (staphylococcal protein A) fusion protein as antigen which was expressed in Escherichia coli. The immunohistochemical analysis of the subcapillary distribution of GGT in procine brain cortex and cerebellum sections by both light and electron microscopy revealed the expression of GGT in the capillary‐adjacent pericytes in addition to the GGT‐positive endothelial layer. We confirmed these data for cultured porcine brain microvascular endothelial cells and pericytes. GGT immunofluorescence could be detected in both cell types in culture. Endothelial cells exhibited a weak staining, whereas pericytes were strongly positive for GGT. Due to the high phagocytotic activity of pericytes and their location on the abluminal surface of the microvessels, we propose a possible protective or detoxifying function of GGT in cerebrovascular pericytes.
Restriction enzymes permit quantitative determination of defined chromatin structures within the chromosome
Authors: JACK Robert S. , MORITZ Peter , CREMER Sabine
Published: March 2005

Open chromatin structures, operationally defined as nuclease‐hypersensitive sites, are frequently found spanning the controlling regions of genes and they may ensure that trans‐acting factors have ready access to their genomic substrates. The rapidity and extent of induction of a gene may be dependent on the probability that its promoter is folded into an open structure. We show that restriction enzymes can be used to estimate the probability that a given promoter region is contained within a defined structure in the chromosome. In the case of the Drosophila major heat‐shock‐protein gene, we show that an individual promoter element is folded in an accessible form in at least 75% of embryonic chromosomes. This efficient maintenance of the hypersensitive region may be a necessary precondition for a rapid heat‐shock response.
Effect of N‐glycan removal on the enzymatic activity of porcine thyroid peroxidase
Authors: LONG Yannick , FRANC Jean‐Louis , KANIEWSKI Jacques , LANET Jeanne , GIRAUD Annie
Published: March 2005

Active porcine thyroid peroxidase (pTPO) has been purified either by deoxycholate extraction followed by immunoaffinity purification (pTPO A) or by trypsin/digitonin extraction followed by ion‐exchange and gelfiltration chromatography (pTPO B); pTPO A appeared as a full‐length molecule, while pTPO B appeared as peptide fragments. Purified pTPO were deglycosylated either by peptide N‐glycosidase F (PNGase F) or by endo‐β‐N‐acetylglucosaminidase H (endo H) treatment. Electrophoretic controls and affinity blotting with concanavalin A indicated that deglycosylation was not total and that pTPO was more efficiently deglycosylated by endo H than by PNGase F.

The enzymatic activity of pTPO A, checked by guaiacol and iodide oxidation, was inhibited by PNGase F and endo H deglycosylation, while that of pTPO B was not. After deglycosylation, the apparent Km of pTPO A for guaiacol and iodide increased, while the Vmax for both substrates decreased. The state of aggregation of pTPO A before and after deglycosylation was checked by sucrose density‐gradient centrifugation. Results indicated that this inhibition was not due to a loss of pTPO A solubility. These observations suggest that deglycosylation induced a modification of the tertiary structure of pTPO A which affected the active‐site domain of the enzyme.
Temperature dependence of charge recombination from the P+QA− and P+QB− states in photosynthetic reaction centers isolated from the thermophilic bacterium Chloroflexus aurantiacus
Authors: VENTUROLI Giovanni , TROTTA Massimo , FEICK Reiner , MELANDRI B. Andrea , ZANNONI Davide
Published: March 2005

The temperature dependence of charge recombination from the P+QA− and from the P+QB− states produced by a flash was studied in reaction centers isolated from the photosynthetic thermophilic bacterium Chloroflexus aurantiacus. P designates the primary electron donor; QA and QB the primary and secondary quinone electron acceptors respectively. In QB‐depleted reaction centers the rate constant (kAP) for P+QA− recombination was temperature independent between 0–50°C (17.6 ± 0.7 s−1 at pH 8 and pH 10). The same value was obtained in intact membranes in the presence of o‐phenanthroline. Upon lowering the temperature from 250 K to 160 K, kAP increased by a factor of two and remained constant down to 80 K. The overall temperature dependence of kAP was consistent with an activationless process. Ubiquinone (UQ‐3) and different types of menaquinone were used for QB reconstitution. In UQ‐3‐reconstituted reaction centers charge recombination was monoexponetial (rate constant k= 0.18 ± 0.03 s−1) and temperature independent between 5–40°C. In contrast, in menaquinone‐3‐and menaquinone‐4‐reconstituted reaction centers P+ rereduction following a flash was markedly biphasic and temperature dependent. In menaquinone‐6‐reconstituted reaction centers a minor contribution from a third kinetic phase corresponding to P+QA− charge recombination was detected. Analysis of these kinetics and of the effects of the inhibitor o‐phenanthroline at high temperature suggest that in detergent suspensions of menaquinone‐reconstituted reaction centers a redox reaction removing electrons from the quinone acceptor complex completes with charge recombination. Instability of the semiquinone anions is more pronounced when QB is a short‐chain menaquinone. From the temperature dependence of P+ decay the activation parameters for the P+QB− recombination and for the competing side oxidation of the reduced menaquinone acceptors have been derived. For both reactions the activation enthalpies and entropies change markedly with menaquinone chain length but counterbalance each other, resulting in activation free energies at ambient temperature independent of the menaquinone tail. When reaction centers are incorporated into phospholipid vesicles containing menaquinone‐8 a temperature‐dependent, monophasic, o‐phenanthroline‐sensitive recombination from the P+QB− state is observed, which is consistent with the formation of stable semiquinone anions. This result seems to indicate a proper QB functioning in the two‐subunit reaction center isolated from Chloroflexus aurantiacus when the complex is inserted into a lipid bilayer.
Protein stability and molecular adaptation to extreme conditons
Authors: JAENICKE Rainer
Published: March 2005

Proteins, due to the delicate balance of stabilizing and destabilizing interactions, are only marginally stable. Adaptation to extreme environments tends to shift the ‘mesophilic’ characteristics of proteins to the respective extremes of temperature, hydrostatic pressure, pH and salinity, such that, under the mutual physiological conditions, the molecular properties are similar regarding overall topology, flexibility and solvation. Enhanced intrinsic stability requires only minute local structural changes so that general strategies of stabilization cannot be established. Apart from mutative changes of amino‐acid sequences, extrinsic factors (or cellular components) may be involved in ‘extremophilic adaptation’.

The molecular basis of acidophilic, alkalophilic and barophilic adaptation is still obscure. Mechanisms of enhanced thermal stability involve improved packing density, as well as specific local interactions. In halophiles, water and salt binding of the intrinsically stable protein inventory is accomplished by favoring acidic over basic amino acid residues and decreased hydrophobicity.

General limits of viability are: (a) the susceptibility of the covalent structure of the polypeptide chain toward hydrolysis or hydrothermal degradation; (b) the competition of extreme solvent parameters with the weak electrostatic and hydrophobic interactions involved in protein stabilization; (c) perturbations of the folding and assembly of proteins; and (d) ‘dislocation’ of biochemical pathways due to effects of extreme conditions on the intricate network of metabolic reactions.
Characterization of glutamate‐1‐semialdehyde aminotransferase of Synechococcus
Authors: SMITH Marvin A. , KANNANGARA C. Gamini , GRIMM Bernhard , von WETTSTEIN Diter
Published: March 2005

Synechococcus glutamate‐1‐semialdehyde aminotransferase was expressed in large amounts in transformed cells of Esherichia coli. The resulting purified enzyme has an absorption spectrum characteristic of B6‐containing enzymes and could be converted to the pyridoxal‐phosphate form with excess dioxovalerate (O2 Val), and back to the pyridoxamine‐phosphate form with diaminovalerate (A2 Val). Both enzyme forms are similarly active in the conversion of glutamate 1‐semialdehyde (GSA) to 5‐aminolevulinate (ALev), suggesting that A2 Val and O2 Val are intermediates. Initial rates of Alev synthesis at various fixed concentrations of GSA followed typical Michaelis‐Menten kinetics (Km of GSA for the pyridoxamine‐phosphate form of GSA aminotransferase = 12 μM, kcat= 0.23 s−1). In submicromolar amounts A2Val stimulates ALev synthesis, and in a series of concentrations with various fixed concentrations of GSA, gives a family of parallel lines in Lineweaver‐Burk plots (Km for A2Val = 1.0 μM). On the other hand, O2Val gives competitive inhibition of the pyridoxamine‐phosphate form of GSA‐aminotransferase and mixed‐type inhibition of the pyridoxal‐phosphate form (Ki for O2Val = 1.4 mM). In general the kinetics were typical of ping‐pong bi‐bi mechanisms in which A2Val is the second substrate (intermediate) and O2Val is an alternative first substrate. There is no compelling evidence that O2Val accepts an amino group at its C5 position resulting in the direct formation of ALev, or the reverse involving the apparent formation of O2Val from ALev. These results are consistent with the hypothesis that the mechanism of GSA aminotransferase mimics that of other aminotransferases and that A2Val is the intermediate.
Structural characterization of mating pheromone precursors of the ciliate protozoan Euplotes raikovi
Authors: MICELI Cristina , La TERZA Antonella , BRADSHAW Ralph A. , LUPORINI Pierangelo
Published: March 2005

The precursors of Euplotes raikovi pheromones Er‐2 and Er‐10 have been structurally characterized from the sequences of their coding regions that were amplified and cloned using the polymerase chain reaction and oligonucleotide primers corresponding to conserved sequences of the gene for phermone Er‐1. The predicted amino acid sequences contain 75 residues distributed through three domains: signal peptide, pro segment and mature pheromone. Despite the conservation of the overall length, there is variation in the size of the pro segments and of the mature pheromones. The comparison of the sequences shows a gradient of identity from the amino to the carboxyl terminus; the signal sequences are identical (with > 95% identity in the nucleotide sequences), the pro segments more variable and the mature pheromones quite diverse. The processing site of the pro pheromones, to produce the mature forms, is apparently characterized by the unusual Xaa‐Asp sequence.
Site‐directed mutagenesis of the conserved histidine residue of phosphoenolpyruvate carboxylase
Authors: TERADA Kazutoyo , IZUI Katsura
Published: March 2005

Histidine residues have previously been suggested to be essential for the activity of phosphoenolpyruvate carboxylase as demonstrated by chemical modification of these residues. Although the location of these residues on the primary structure is not known, a comparison of nine phosphoenolpyruvate (P‐pyruvate) carboxylases sequenced recently revealed that there are only two conserved histidine residues (His138 and His579, coordinates from the E. coli enzyme). Site‐directed mutagenesis of these residues were undertaken with the E. coli P‐pyruvate carboxylase and the properties of purified mutant enzymes were investigated. Mutation of His138 to asparagine (H138N) produced a protein which did not show carboxylase activity. However, this mutant enzyme catalyzed the bicarbonate‐dependent dephosphorylation (Vmax= 1.4 μmol · min−1· mg‐1) of the P‐pyruvate. Since this reaction is due to one of the two partial reactions proposed for this enzyme, the results indicate that His138 is obligatory for the second‐step reaction, i.e. the carboxylation of the enolate form of pyruvate by carboxyphosphate. Mutation of His579 to asparagine (H579N) produced an enzyme which had 69% of the wild‐type carboxylase activity, but its affinity for P‐pyruvate was decreased by 24‐fold.
Post‐transcriptional regulation of the transferrin receptor and 4F2 antigen heavy chain mRNA during growth activation of spleen cells
Authors: TEIXEIRA Santuza , KÜHN Lukas C.
Published: March 2005

The expression of several cell surface proteins including the transferrin receptor and 4F2 antigen is induced when quiescent cells are activated and proliferate. We have studied this induction in mouse spleen cells after stimulation with 4β‐phorbol 12‐myristate 13‐acetate (PMA), the Ca2+‐ionophore, ionomycin and recombinant interleukin‐2 (IL‐2). The 4F2 antigen heavy chain and transferrin receptor mRNA were barely detectable in resting cells, but increased 60‐fold within 4 h of growth stimulation. The corresponding proteins became measurable at the cell surface after 6 h, prior to the S phase. In run‐on transcription assays the transferrin receptor gene was transcribed to almost the same extent in resting and growth‐stimulated cell populations and the 4F2 antigen heavy chain gene was induced fivefold. This suggests that post‐transcriptional control mechanisms are mainly responsible for the accumulation of the respective mRNA at the onset of cell proliferation. In the case of the transferrin receptor, the induction correlated with an activation of the mRNA‐binding iron‐regulatory factor which is known to increase the stability of the cytoplasmic transferrin receptor mRNA.
The longest 18S ribosomal RNA ever known
Authors: KWON O‐Yu , OGINO Kimihiro , ISHIKAWA Hajime
Published: March 2005

An EMBL4 recombinant phage which encodes one of the full length of the aphid ribosomal DNA has been isolated from the aphid genomic library. Determination of the complete nucleotide sequence of the aphid 18S rRNA gene revealed that it is 2469 bp with a G + C content of 59%. The aphid 18S rRNA gene studied here is the longest and has the highest G + C content among the 18S rRNA genes examined so far. Evidence provided by the S1 nuclease assay suggests that the aphid 18S rRNA gene examined in this study is not a pseudogene containing an insertion sequence. Based on the nucleotide sequence of the 18S rRNA gene, we constructed a presumed secondary‐structure model of the aphid 18S rRNA. In the aphid 18S rRNA, the eucaryote‐specific E21 and 41 region are supposed to be longer and more complex than the counterparts of other 18S rRNA.
Site‐directed mutagenesis of the dihydrolipoyl transacetylase component (E2p) of the pyruvate dehydrogenase complex from Azotobacter vinelandii
Authors: SCHULZE Egbert , WESTPHAL Adrie H. , BOUMANS Hans , de KOK Arie
Published: March 2005

Site‐directed mutagenesis was performed in the protease‐sensitive region, between the lipoyl and catalytic domains and in the catalytic domain, of the dihydrolipoyl transacetylase component (E2p) of the pyruvate dehydrogenase complex from Azotobacter vinelandii. The interaction of the mutated enzymes with the peripheral components pyruvate dehydrogenase (E1p) and lipoamide dehydrogenase (E3) was studied by gel filtration experiments, analytical ultracentrifugation and reconstitution of the pyruvate dehydrogenase complex.

Upon binding of peripheral components, the 24‐subunit core of A. vinelandii wild‐type E2p dissociates into tetramers. Four E1p or E3 dimers can bind to a tetramer. Binding is mutually exclusive, resulting in an active complex containing one E3 and three E1p dimers.

Large deletions of the protease‐sensitive region of E2p resulted in a total loss of the E1p and E3 binding. A small deletion (Δ P361‐R362) or the point mutation K367Q in the protease‐sensitive region did not influence E3 binding, but affected E1p binding strongly, although with excess E1p almost complete reconstitution was reached. For E2p with the point mutation R416D in the N‐terminal region of the catalytic domain only 16% overall activity could be measured in reconstituted complexes. This is due to a very weak E1p/E2p interaction, whereas the E3 binding was not affected. The point mutation R416D did not influence the catalytic activity of E2p, although a function for this residue in the formation of the active site was predicted from amino acid similarities with chloramphenicol acetyltransferase type III from Escherichia coli.

Deletion of the complete Ala + Pro‐rich sequence between the protease‐sensitive region and the catalytic domain did not affect the enzymological properties of E2p, nor the affinity for E1p or E3. A further deletion of 20 N‐terminal residues from the catalytic domain destroyed the E2p activity. From gel filtration experiments it was concluded that the quaternary structure was unaffected, as was E3 binding. E1p binding was lost and, in contrast to the wild‐type enzyme, no dissociation of the core upon addition of E3 was observed. This mutant enzyme possesses, like E. coli E2p, six E3 binding sites and clearly shows that interaction of E3 or E1p with the E1p sites and dissociation are linked processes.

It is concluded that the binding site for E3 is located on the N‐terminal part of the protease‐sensitive region. In contrast, the binding site for E1p consists of two regions, one located on the protease‐sensitive region and one of the catalytic domain. These regions are separated by a flexible sequence of about 20 amino acids.
Synthesis and evaluation of (+) and (−)‐2,2‐difluorocitrate as inhibitors of rat‐liver ATP‐citrate lyase and porcine‐heart aconitase
Authors: SAXTY Barbara A. , NOVELLI Riccardo , DOLLE Roland E. , KRUSE Lawrence I. , REID David G. , CAMILLERI Patrick , WELLS Timothy N. C.
Published: March 2005

The enantiomers (+) and (−)‐2,2‐difluorocitrate have been synthesized. Both are good inhibitors of ATP‐citrate lyase, showing competitive inhibition against citrate, with Kis= 0.7 μM for (+)‐2,2‐difluorocitrate and 3.2 μM for (−)‐2,2‐difluorocitrate. The inhibition patterns with either ATP or CoA as the varied substrate were uncompetitive and mixed, respectively, but with much weaker inhibition constants. Neither isomer undergoes carbon‐carbon bond cleavage as a substrate and there is no evidence of irreversible time‐dependent inactivation. When ATP‐citrate lyase is incubated with CoA and difluorocitrate, the maximal intrinsic ATPase rate is 10% of the citrate‐induced rate for the (+)‐enantiomer and 2% for the (−)‐enantiomer. 19F‐NMR studies confirm that only the (+)‐enantiomer is chemically processed.

The effects of the difluorocitrate enantiomers on the reaction catalysed by aconitase were examined. (−)‐2,2‐Difluorocitrate is a competitive inhibitor against citrate (Kis= 1.5 μM), whereas the (+)‐enantiomer is a relatively poor mixed inhibitor (Ki > 300 μM). The (−)‐enantiomer irreversibly inactivates aconitase at 1.1 min‐1· mM‐1 at 25°C and pH 7.4, whereas no irreversible inhibition is seen with the (+)‐enantiomer. Therefore, it would be expected that the (+)‐enantiomer would slow the rate of acetyl‐CoA synthesis in vivo, without inhibiting the citric acid cycle.
Combined glyceraldehyde‐3‐phosphate dehydrogenase/phosphoglycerate kinase in catecholamine‐stimulated guinea‐pig cardiac muscle
Authors: BÜNGER Rolf , MUKOHARA Naoki , KANG Young‐Hee , MALLET Robert T.
Published: March 2005

The steady‐state reactant levels of triose‐phosphate isomerase and the glyceraldehyde‐3‐phosphate dehydrogenase/phosphoglycerate kinase system were examined in guinea‐pig cardiac muscle. Key glycolytic intermediates, including glyceraldehyde 3‐phosphate were directly measured and compared with those of creatine kinase. Nonworking Langendorff hearts as well as isolated working hearts were perfused with 5 mM glucose (plus insulin) under normoxia conditions to maintain lactate dehydrogenase near‐equilibrium. The cytosolic phosphorylation potential ([ATP]/([ADP]·[Pi]) was derived from creatine kinase and the free [NAD+]/(NADH]·[H+]) ratio from lactate dehydrogenase. In Langendorff hearts glycolysis was varied from near‐zero flux (hyperkalemic cardiac arrest) to higher than normal flux (normal and maximum catecholamine stimulation). The triose‐phosphate isomerase was near‐equilibrium only in control or potassium‐arrested Langendorff hearts as well as in postischemic ‘stunned’ hearts. However, when glycolytic flux increased due to norepinephrine or due to physiological pressure‐volume work the enzyme was displaced from equilibrium.

The alternative phosphorylation ratio [ATP]‘/([ADP]’·[Pi]) was derived from the magnesium‐dependent glyceraldehyde‐3‐phosphate dehydrogenase/phosphoglycerate kinase system assigning free magnesium different values in the physiological range (0.1–2.0 mM). As predicted, [ATP]/([ADP]·[Pi]) and [ATP]'/([ADP]'·[Pi]') were in excellent aggreement when glycolysis was virtually halted by hyperkalemic arrest (flux ∼ 0.2 μmol C3· min‐1· g dry mass‐1). However, the equality between the two phosphorylation ratios was not abolished upon resumption of spontaneous beating and also not during adrenergic stimulation (flux ∼ 5–14 μmol C3· min‐1· g dry mass‐1). In contrast, when flux increased due to transition from no‐work to physiological pressure‐volume work (rate increase from ∼ 3 to 11 μmol C3· min‐1· g dry mass‐1), the two ratios were markedly different indicating disequilibrium of the glyceraldehyde‐3‐phosphate dehydrogenase/phosphoglycerate kinase. Only during adrenergic stimulation or postischemic myocardial ‘stunning,’ not due to hydraulic work load per se, glyceraldehyde‐3‐phosphate levels increased from about 4 μM to > 16 μM. Thus the guinea‐pig cardiac glyceraldehyde‐3‐phosphate dehydrogenase/phosphoglycerate kinase system can realize the potential for near‐equilibrium catalysis at significant flux provided glyceraldehyde‐3‐phosphate levels rise, e.g., due to ‘stunning’ or adrenergic hormones.
Activation of a keratinocyte phospholipase A2 by bradykinin and 4β‐phorbol 12‐myristate 13‐acetate
Authors: KAST Raimund , FÜRSTENBERGER Gerhard , MARKS Friedrich
Published: March 2005

The release of arachidonic acid from cellular phospholipids and its subsequent conversion to eicosanoids is the common early response of skin keratinocytes to a wide variety of exogenous or endogenous agonists including irritant skin mitogens such as the phorbol ester, 4β‐phorbol 12‐myristate 13‐acetate (PMA) or the inflammatory peptide bradykinin. In mouse keratinocytes labeled with [14C]arachidonic acid, both PMA and bradykinin induced the release of the fatty acid in a dose‐dependent and time‐dependent manner. Three lines of evidence indicate phospholipase A2 activity to be involved in arachidonic acid release: (a) its inhibition by mepacrine, (b) the concomitant generation of lysophosphatidylcholine from [3H]choline‐labeled cells and (c) an increase in arachidonic acid release from 14C‐labeled phosphatidylcholine in particulate fractions from PMA‐treated and bradykinin‐treated keratinocytes. Inhibition or down regulation of protein kinase C (PKC) led to a suppression of PMA‐induced but not bradykinin‐induced arachidonic acid release, indicating a critical involvement of this kinase in phorbol‐ester‐induced activation of epidermal phospholipase A2 activity. Bradykinin‐induced activation of phospholipase A2 was however, shown to be mediated by specific B2 receptors coupled to GTP‐binding proteins (G protein). In support of this mechanism it was demonstrated that the bradykinin‐induced phospholipase A2 activity was increased in the presence of non‐hydrolysable GTP but decreased upon addition of non‐hydrolysable GDP analogues. Moreover, cholera toxin stimulated both basal and bradykinin‐induced phospholipase A2 activity in a cAMP‐independent manner, whereas pertussis toxin was found to be inactive in this respect. The data suggest that epidermal phospholipase A2 activity can be stimulated by bradykinin via a B2 receptor‐G‐protein‐dependent pathway, which is independent of PKC and a PKC‐dependent pathway which is activated by phorbol esters such as PMA.
Characterization of a guanosine‐nucleotide‐binding‐protein‐coupled receptor for pituitary adenylate‐cyclase‐activating polypeptide on plasma membranes from rat brain
Authors: SCHÄFER Heiner , SCHWARZHOFF Rainer , CREUTZFELDT Werner , SCHMIDT Wolfgang E.
Published: March 2005

Pituitary adenylate‐cyclase‐activating polypeptide (PACAP), a novel brain‐gut hormone, was isolated from ovine hypothalami and represents the latest mammalian member of the secretin‐glucagon peptide family. PACAP exists in two C‐terminally amidated molecular forms, PACAP(1–27) and PACAP(1–38), comprising 27 or 38 amino acid residues, respectively. In order to identify a specific receptor for PACAP, we studied binding of 125I‐labelled PACAP(1–27) to plasma membranes from rat brain. We identified a single high‐affinity binding site (Kd, 340 pM and Bmax, 3.34 pmol/mg), specific for synthetic PACAP(1–38) and PACAP(1–27). Hormone binding was reversible and time, protein and temperature dependent. In contrast, neither the analogues PACAP(1–23), PACAP(18–38) and PACAP(3–25), nor vasoactive intestinal peptide (VIP), secretin and growth‐hormone‐releasing factor (GRF) revealed significant binding at concentrations up to 1 μM. A specific receptor protein, with an apparent molecular mass of 60 kDa, was identified by means of affinity cross‐linking with disuccinimidyl suberate (DSS) and ethylene glycol disuccinimidyl suberate (EGS). PACAP receptors are associated with a GTP‐binding protein as determined by the influence of different nucleotides on PACAP binding. PACAP‐binding activity was solubilized with the detergents 3‐[(3‐cholamidopropyl)dimethylammonio]2‐hydroxy‐1‐propane sulfonate (Chapso) or Triton X‐100 and was characterized as a high‐molecular‐mass receptor complex (400 kDa) by non‐reducing size‐exclusion chromatography on Sepharose CL‐6B. These data imply the following: high‐affinity PACAP receptors are expressed abundantly on rat‐brain plasma membranes; PACAP receptors are specific for PACAP and show no affinity for VIP, secretin and GRF; the PACAP receptor molecule has an apparent molecular mass of 60 kDa; the PACAP receptor complex is associated with a GTP‐binding protein.
Base 2661 in Escherichia coli 23S rRNA influences the binding of elongation factor Tu during protein synthesis in vivo
Authors: TAPIO Soile , ISAKSSON Leif A.
Published: March 2005

The binding of the EF‐Tu · GTP · aminocl‐tRNA ternary complex (EF, elongation factor) to the ribosome is known to be strengthened by a 2661G‐to‐C mutation tio 23S ribosomal RNA, whereas the binding to normal ribosomes is weakened if the factor is in an appropriate mutant form (Aa). In this report we describe the mutual effects by the 2661C alteration in 23S rRNA and EF‐Tu(Aa) on bacterial viability and translation efficiency in strains with normal or mutationally altered ribosomes. The rrnB(2661C) allele on a multicopy plasmid was introduced by transformation into Escherichia coli K‐12 strains, harbouging either the wild‐type or the mutant gene (tufA) for EF‐Tu as well as normal or mutant ribosomal protein S12 (rpsL). Together with wild‐type EF‐Tu, the 2661C mutant ribosomes decreased the translation elongation rate in a rpsL+ strain or a non‐restrictive rpsL224 strain. This reduction was not seen in strains which harbored EF‐Tu(Aa) instead of EF‐Tu(As) (As, wild type form). Nonsense condon suppression by tyrT(Su3) suppressor tRNA was reduced by 2661C in a rpsL224 srain in the presence of EF‐Tu(As) but not in the presence of EF‐Tu(Aa). The lethal effect obtained by the combination of 2661C and a restrictive ribosomal protein S12 mutation (rpsL282) disappeared if EF‐Tu(As) was replaced by EF‐Tu(Aa) in the strain. In such a viable strain, 2661C had no effect on either the translation elongation rate or nonsense condon suppression. Our data suggest that the G base at position 2661 in 23S rRNA is important for binding of EF‐Tu during protein snthesis in vivo. The interaction between this abse and EF‐Tu is strongly influenced by the structure of ribosomal protein S12.
Transcriptional and post‐transcriptional effects of glucose on liver phosphoenolpyruvate‐carboxykinase gene expression
Authors: MEYER Silke , HÖPPNER Wolfgang , SEITZ Hans J.
Published: March 2005

The present stdy investigates the effect fo glucose on the gene expression of the hepatic glucoregulatory enzyme, phosphoenolpyruvate carboxykinase (PPrvck). By use of hepatocytes in culture and FAO hepatoma cells it could be demonstrated that glucose suppressed the effect of dibutyrul cyclic AMP (Bt2cAMP), glucocorticoids or both, to increase PPrvck mRNA and consequently PPrvck enzyme activity. Glucose had a dual effect; it reduced PPrvck gene transcription and it accelerated the rate of Pprvck mRNA degradation.

The effect wa sspecific for glucose, as glucose‐related carbohydrates such as manose, galactose and sorbitol were without effect on PPrvck mRNA. The repressive effect of glucose was limited to certain proteins; glucose had no effect on Bt2cAMP and glucocorticoid provoked induction of tyrosine aminotransferase (TAT). Also the pattern of mRNA in vitro translation products was virtually unaffected when FAO hepatoma cells were incubated either in the presence or absence of glucose, demonstrating gthe specificity of the effect of glucose on gene expression of selected proteins.

In FAO hepatoma cells and in hepatocytes in culture, insulin, like glucose, also decreased PPrvck mRNA. While the effect of glucose and insulin was additive in FAO hepatoma cells, in primary hepatocytes in culture an effect of glucose by itself on PPrvck mRNA could only be demonstrated in the absence of insulin. Correspondingly also in vivo, the effect of glucose was demonstrated in the absence of insulin (provoked by streptozotocin diabetes); glucose application reduced the amount of hepatic PPrvck mRNA.

To summarize, glucose is capable of suppressing the effect of glucocorticoids and Bt2cAMP on increasing the PPrvck mRNA level. The carbohydrate reduces the rate of PPrvck geen transcription and accelerates the rate of PPrvck mRNA degradation. While in FAO hepatoma cells the effect is evident in the presence of insulin, in hepatocytes in culture the effect of glucose cannot be demonstrated in the presence of insulin, questioning its role under physiological conditions.