bluuebunny/crossref_metadata_2025 huggingface dataset:
Lithium, an inhibitor of cAMP‐induced inositol 1,4,5‐trisphosphate accumulation in Dictyostelium discoideum, inhibits activation of guanine‐nucleotide‐binding regulatory proteins, reduces activation of adenylylcyclase, but potentiates activation of guanylyl cyclase by cAMP
Authors: PETERS Dorien J. M. , SNAAR‐JAGALSKA B. Ewa , VAN HAASTERT Peter J. M. , SCHAAP Pauline
Published: March 2005

Li+ drastically alters pattern formation in Dictyostelium by inhibiting cAMP‐induced presporegene expression and promoting cAMP‐induced prestalk‐gene expression. We reported previously that Li+ inhibits inositol monophosphatases in this organism and strongly reduces basal and cAMP‐stimulated inositol 1,4,5‐trisphosphate levels. We show here that Li+ also reduces cAMP‐induced accumulation of cAMP. but promotes cAMP‐induced accumulation of cGMP. This effect is not due to inhibition of cGMP hydrolysis or inhibition of adaptation and may therefore reflect stimulation of guanylyl‐cyclase activation. Li+ does not affect the binding of cAMP to surface receptors but interferes with the interaction between receptors and guanine‐nucleotide‐binding regulatory (G) proteins. These effects are complex; in the absence of Mg2+, Li+ increases guanosine 5′‐[γ‐thio]triphosphate(GTP[S])‐binding activity to similar levels as 1 mM Mg2+. However, while Mg2+ potentiates cAMP‐induced stimulation of GTP[S]‐binding activity, Li+ effectively inhibits stimulation. Li+ also inhibits cAMP‐stimulated, but not basal high‐affinity GTP‐ase activity, indicating an inhibitory effect on cAMP‐induced activation of G‐proteins.

Our data suggest that in addition to inositolphosphate metabolism, the activation of G‐proteins may be a second biochemical target for Li+ effects on pattern formation and signal transduction in Dictyostelium.
Tissue selectivity of pravastatin sodium, lovastatin and simvastatin
Authors: KOGA Teiichiro , FUKUDA Kuniaki , SHIMADA Yohko , FUKAMI Masaharu , KOIKE Hiroyuki , TSUJITA Yoshio
Published: March 2005

Tissue selectivity of pravastatin sodium (pravastatin), lovastatin and simvastatin, 3‐hydroxy‐3‐methylglutaryl‐CoA reductase inhibitors was examined by measuring inhibition of de novo sterol synthesis and active drug concentrations in the liver, spleen and testis in rats after a single oral administration (25 mg/kg) of these drugs. Regarding tissue drug concentrations, all three drugs were liver selective: concentrations of drugs in the liver were about ten‐times higher than those in the spleen and testis. On the other hand, pravastatin was far more liver selective in inhibiting sterol synthesis than two other inhibitors: pravastatin inhibited de novo sterol synthesis in the liver but minimally in the spleen and testis, whereas lovastatin and simvastatin inhibited in all three tissues. Microautoradiographic and in vitro cellular‐uptake studies demonstrated that pravastatin remained in the extracellular space in the spleen, whereas the other drugs entered the cell. We conclude that pravastatin exhibits a liver‐selective inhibition of sterol synthesis because the agent permeates the cell membrane in the liver, but not in non‐hepatic tissues.
Revision of the haem‐core architecture in the tetraheam cytochrome c from Desulfovibrio baculatus by two‐dimensional 1H NMR
Authors: COUTINHO Isabel B. , TURNER David L. , LEGALL Jean , XAVIER António V.
Published: March 2005

The haem‐core architecture in cytochrome c3 isolated from Desulfovibrio baculatus (Norway 4) was probed using two‐dimensional 1H NMR. Interhaem connectivities detected in NOE spectroscopy experiments performed at short mixing times are incompatible with the structure of the protein determined by X‐ray crystallography, but agree instead with the haem arrangement found in cytochrome c3 from Desulfovibrio vulgaris (Miyazaki). These experiments show unequivocally that the relative orientation of the four haems in the two proteins is the same and does not invofve the 180° rotation of haems I and IV indicated in the X‐ray structure determined for the cytochrome c3 from D. baculatus (Norway 4).
Purification and enzymic characterization of the cytoplasmic pyrophosphatase from the thermoacidophilic archaebacterium Thermoplasma acidophilum
Authors: RICHTER Oliver‐Matthias H. , SCHÄFER Günter
Published: March 2005

Cytoplasmic pyrophosphatase has been isolated from the thermoacidophilic archaebacterium Thermoplasma acidophilum. The enzyme was purified to electrophoretic homogeneity by combining ion‐exchange and affinity‐chromatographic separations. This soluble pyrophosphatase probably consists of six identical subunits, since SDS/PAGE gave an estimate of about 22 kDa for a single subunit and size‐exclusion chromatography under non‐denaturing conditions indicates a molecular mass of 110 ± 5 kDa.

The two most prominent catalytic features of this enzyme are the absolute requirement for divalent cations for catalytic action, Mg2+ conferring the highest activity, and the pronounced specificity for PPi. The catalytic behavior apparently follows simple Michaelis‐Menten kinetics with a Km of about 7 μM for PPi and a specific acitivity of about 1200 U/mg at 56°C. Surprisingly, maximum activity could be observed at 85°C which is more than 20°C above the temperature for optimal growth.

Several cytoplasmic extracts of eubacteria and archaebacteria have been probed with a polyclonal antiserum raised against the purified archaebacterial protein. The only noticeable cross‐reactivity could be detected with an extract from the methanogen Methanosarcina barkeri although this probably does not reflect the inferred phylogenetic relationship between methanogens and Thermoplasma acidophilum.
Cloning and sequence of the gene for the cytoplasmic inogranic pyrophosphatase from the thermoacidophilic archaebacterium Thermoplasma acidophilum
Authors: RICHTER Oliver‐Matthias H. , SCHÄFER Günter
Published: March 2005

The gene (ppa) from the thermoacidophilic archaebacterium Thermoplasma acidophilum, encoding the cytoplasmic pyrophosphatase, has been cloned. Two degenerate oligonucleotide probes, synthesized according to the N‐terminal amino acid sequence of the isolated protein, were used to screen subgenomic libraries.

The DNA‐derived amino acid sequence of the archaebacterial enzyme allows, for the first time, comparative studies of cytoplasmic pyrophosphatases to be extended to all three urkingdoms. The archaebacterial pyrophosphatase more closely resembles the eubacterial enzymes on the basis of sequence similarity and subunit size. The majority of amino acid residues considered to be essential for hydrolysis of pyrophosphate seem to have been conserved throughout evolution, as inferred from the results of an alignment of sequences from all three urkingdoms.
Characterization and molecular cloning of a flavoprotein catalyzing the synthesis of phytofluene and ζ‐carotene in Capsicum chromoplasts
Authors: HUGUENEY Philippe , RÖMER Susanne , KUNTZ Marcel , CAMARA Bilal
Published: March 2005

In plants, ζ‐carotene is the first visible carotenoid formed in the biosynthetic pathway through the following two‐step desaturation reaction: phytoene→phytofluene→ζ‐carotene. Using Capsicum annuum chromoplast membranes and the reconstitution system previously described [Camara, B., Bardat, F. & Monéger, R. (1982) Eur. J. Biochem. 127, 255–258], we have attempted to purify the desaturase(s) catalyzing these reactions. The two activities were coincidental during all the purification procedures. Only a single polypeptide with 56 ± 2 kDa was detected by SDS/PAGE of all active fractions. The enzyme contained protein‐bound FAD. Antibodies raised against the purified polypeptide selectively precipitated the phytoene and the phytofluene desaturase activities, thus demonstrating that the enzyme is a bifunctional flavoprotein. The antibodies were used to isolate a full‐length cDNA clone from which was deduced the primary structure of the desaturase which contains a characteristic dinucleotide‐binding site. Overexpression of the cDNA in Escherichia coli allowed the production of a recombinant desaturase which had all the properties of the chromoplast desaturase. The phytoene/phytofluene desaturase mRNA levels were extremely low in green fruits and increased slightly before detectable carotenoid synthesis and remained constant throughout ripening. However, the desaturase activity and protein levels were found to increase significantly during the chloroplast to chromoplast transition in C. annuum fruits.
Interactions of phospholipids with the mitochondrial cytochrome‐c reductase studied by spin‐label ESR and NMR spectroscopy
Authors: HAYER‐HARTL Manajit , SCHÄGGER Hermann , VON JAGOW Gebhard , BEYER Klaus
Published: March 2005

Protein/phospholipid interactions in the solubilized mitochondrial ubihydroquinone: cytochrome‐c oxidoreductase (bc1 complex) were studied by spin‐label electron‐spin resonance and by 31P‐NMR spectroscopy. Spin‐labelled phospholipids were employed to probe the relative binding affinities of a number of phospholipids with regard to the significance of phospholipids for the activity and stability of this multisubunit complex. The protein was titrated with spin‐labelled cardiolipin (1,3‐bisphosphatidyl‐sn‐glycerol) and with the spin‐labelled analogues of PtdCho and PtdEtn, both of which have been shown recently to elicit a substantial increase in electron‐transport activity [Schägger, H., Hagen, T., Roth, B., Brandt, U., Link, T. A. & von Jagow, G. (1990) Eur. J. Biochem. 190, 123–130]. A simplified distribution model showed that neutral phospholipids have much lower protein affinity than cardiolipin. In contrast to the transient weak lipid binding detected by spin‐label electron‐spin resonance, 31P NMR revealed a tightly bound cardiolipin portion, even after careful delipidation of the complex. Considerable line narrowing was observed after phospholipase A2 digestion of the bound cardiolipin, whereas addition of SDS resulted in complete release. Relative proportions and line widths of mobile and immobilized lipids were obtained by deconvoluting the partially overlapping signals. The current results are discussed with reference to similar findings with other mitochondrial membrane proteins. It is assumed that activation by neutral phospholipids reflects a generalized effect on the protein conformation. Cardiolipin binding is believed to be important for the structural integrity of the mitochondrial protein complexes.
Fast kinetics studies of Escherichia coli electrotransformation
Authors: EYNARD Nathalie , SIXOU Sophie , DURAN Nathalie , TEISSIE Justin
Published: March 2005

Direct gene transfer is achieved in Escherichia coli by use of square wave electric pulsing. As observed by video monitoring, the field pulse causes bacteria to orientate parallel to the field lines. Rapid kinetic turbidity changes indicate that this process happens quickly. In these circumstances, and in pulsing conditions prone to inducing transformation, only caps are affected by the field. Considerable cytoplasmic ion leakage occurs during the pulse, affecting the interfacial ionic concentration. The pulsing‐buffer osmolarity has to be close to that used with protoplasts. Contact between the plasmid and the bacteria can be very short before the pulse but must be present during the pulse. The plasmid remains accessible to externally added DNases up to 5 days after the pulse, suggesting that the transfer step is slow. Electric‐field‐mediated transfer can be described in two steps: the anchoring process during the pulse, followed by the crossing of the membrane.
Effect of anisotonic cell‐volume modulation on glutathione‐S‐conjugate release, t‐butylhydroperoxide metabolism and the pentose‐phosphate shunt in perfused rat liver
Authors: SAHA Nirmalendu , STOLL Barbara , LANG Florian , HÄUSSINGER Dieter
Published: March 2005

1. Addition of 1‐chloro‐2,4‐dinitrobenzene to isolated perfused rat liver results in the rapid formation of its glutathione‐S‐conjugate [S‐(2,4‐dinitrophenyl)glutathione], which is released into both, bile and effluent perfusate. Ahisotonic perfusion did not affect total S‐conjugate formation, but release of the S‐conjugate into the perfusate was increased (decreased) following hypertonic (hypotonic) exposure at the expense of excretion into bile. Stimulation of S‐conjugate release into the perfusate following hypertonic exposure paralleled the time course of volume‐regulatory net K+ uptake.

2. Basal steady‐state release of oxidized glutathione (GSSG) into bile was 1.30 ± 0.12 nmol · g−1· min−1 (n= 18) during normotonic (305 mOsmol/I) perfusion and was 3.8±0.3 nmol · g−1· min−1 in the presence of t‐butylhydroperoxide (50 μmol/I). Hypotonic exposure (225 mOsmol/1) lowered both, basal and t‐butylhydroperoxide(50 μmol/I)‐stimulated GSSG release into bile by 35% and 20%, respectively, whereas hypertonic exposure (385 mOsmol/I) increased. Anisotonic exposure was without effect on t‐butylhydroperoxide removal by the liver. GSSG release into bile also decreased by 33% upon liver‐cell swelling due to addition of glutamine plus glycine (2 mmol/I, each).

3. Hypotonic exposure led to a persistent stimulation 14CO2 production from [1‐14C]glucose by about 80%, whereas 14CO2 production from [6‐14C]glucose increased by only 10%. Conversely, hypertonic exposure inhibited 14CO2 production from [1‐14C]glucose by about 40%, whereas 14CO2 production from [6‐14C]glucose was unaffected. The effect of anisotonicity on 14CO2 production from [1‐14C]glucose was also observed in presence of t‐butylhydroperoxide (50 μmol/1), which increased 14CO2 production from [1‐14C]glucose by about 40%.

4. t‐Butylhydroperoxide (50 μmol/1) was without significant effect on volume‐regulatory K+ fluxes following exposure to hypotonic (225 mOsmol/1) or hypertonic (385 mOsmol/1) perfusate. Lactate dehydrogenase release from perfused rat liver under the influence of t‐butylhydroperoxide was increased by hypertonic exposure compared to hypotonic perfusions.

5. The data suggest that hypotonic cell swelling stimulates flux through the pentose‐phosphate pathway and diminishes loss of GSSG under conditions of mild oxidative stress. Hypotonically swollen cells are less prone to hydroperoxide‐induced lactate dehydrogenase release than hypertonically shrunken cells. Hypertonic cell shrinkage stimulates the excretion of glutathione‐S‐conjugates into the sinusoidal circulation at the expense of biliary secretion.
Effect of heat shock on expression of proteins not involved in the heat‐shock regulon
Authors: FABISIEWICZ Anna , JANION Celina
Published: March 2005

The effect of heat shock on the expression of some genes of Escherichia coli was tested. To avoid side effects, promoters of the genes were fused to lacZ and their expression measured by the level of β‐galactosidase. The results show that expression of umuC, recA and polB, after induction of the SOS response, was somewhat higher in the heat‐shocked than in the non‐shocked cells, whereas expression of ada, alkB and alkA genes, after induction of the adaptive response, was about the same. Unexpectedly, it was found that expression of lacZ from its own promoter was drastically lowered in the heat‐shocked cells. This effect, however, seems not to be dependent on the induction of heat‐shock proteins.
X‐ray crystallographic characterisation of type‐2‐depleted ascorbate oxidase from zucchini
Authors: MESSERSCHMIDT Albrecht , STEIGEMANN Wolfgang , HUBER Robert , LANG Gunter , KRONECK Peter M. H.
Published: March 2005

The type‐2 depleted form of ascorbate oxidase from zucchini has been prepared in crystals and characterised by X‐ray crystallography and EPR spectroscopy. The X‐ray structure analysis by difference‐Fourier techniques and refinement shows that, on average, about 1.3 Cu atoms/ascorbate oxidase monomer are removed. The copper is lost from the trinuclear site whereby the EPR‐active type‐2 copper is depleted most; type‐1 copper is not affected. This observation indicates preferential formation of a 1 Cu‐depleted form with the hole equally distributed over all three copper sites. Each of these 1 Cu‐depleted species may represent an anti‐ferromagnetically coupled copper pair which is EPR‐silent and could explain the disappearance of the type‐2 EPR signal.
Iron release from ferrisiderophores
Authors: HALLÉ Félix , MEYER Jean‐Marie
Published: March 2005

Release of iron from various ferrisiderophores (ferripyoverdines, ferrioxamines B and E, ferricrocin, ferrichrome A, ferrienterobactin and its analog ferric N,N′,N”‐tri(1,3,5‐Tris) 2,3‐dihydroxybenzoylaminomethylbenzene) was obtained through an enzymic reduction of iron, involving NADH, FMN and the ferripyoverdine reductase of Pseudomonas aeruginosa PAO1. The iron released from the same complexes was also obtained through chemical reduction of iron involving FMNH2. Evidence is given that the enzymic process acts through a FMNH2 reduction; the P. aeruginosa enzyme, purified according to its ferripyoverdine‐reductase activity [Hallé, F. & Meyer, J. M., Eur. J. Biochem. 209, 613–620], functions as a NADH:FMN oxidoreductase, the FMNH2 produced being able to chemically reduce the iron complexed by siderophores. The general occurence of such a multi‐step mechanism, which denies the existence of specific ferrisiderophore reductases, is discussed.
Characterization and molecular cloning of neurotoxic phospholipases A2 from Taiwan viper (Vipera russelli formosensis)
Authors: WANG Ying‐Ming , LU Pei‐Jung , HO Chewn‐Lang , TSAI Inn‐Ho
Published: March 2005

Two phospholipases A2 (PLA2s), designated as RV‐4 and RV‐7 were purified from venom of the Taiwan Russell's viper (Vipera russelli formosensis) by gel‐filtration and reverse‐phase HPLC. Their primary structures were solved by both protein sequencing and cDNA cloning and sequencing. The cDNA synthesized was amplified by the polymerase‐chain reaction using a pair of synthetic oligonucleotide primers corresponding to the N‐ and the C‐terminal flanking regions of the enzymes. The deduced amino acid sequences of RV‐4 and RV‐7 were 92% identical to those of the vipoxin and vipoxin inhibitor, respectively, from the Bulgarian Vipera a. ammodytes. RV‐4 itself was neurotoxic, whereas RV‐7 had much lower enzymatic activity and was not toxic. The low enzymatic activity of RV‐7 may be attributed to five acidic residues at positions 7, 17, 59, 114 and 119, which presumably impair its binding to aggregated lipid substrates. Based on the sequence comparison among all the known group II PLA2s, residues 6, 12, 76–81, and 119–125 were identified as important for the neurotoxicity. RV‐4 and RV‐7 exist in the crude venom as heterodimers, which were again formed by mixing together the HPLC‐purified RV‐4 and RV‐7. Moreover, RV‐7 inhibited the enzymatic activity of RV‐4 in vitro but potentiated its lethal potency and neurotoxicity. It is suggested that RV‐7 may facilitate the specific binding of RV‐4 to its presynaptic binding sites, probably by preventing its non‐specific adsorption.
1,4–Diamino‐2‐butyne as the mechanism‐based pea diamine oxidase inhibitor
Authors: PEČ Pavel , FRÉSORT Ivo
Published: March 2005

1,4‐Diamino‐2‐butyne is a mechanism‐based inhibitor of diamine oxidase (EC 1.4.3.6) from pea cotyledons. It shows saturation kinetics Km= 1 mM like a substrate, but its interaction leads to time‐dependent loss of enzyme activity which is not restored by gel filtration. The substrate 1,4‐diaminobutane and the competitive inhibitor 1,4‐diamino‐2‐butanone protect the enzyme against inactivation. Changes in the enzyme electronic spectra with 1,4‐diamino‐2‐butyne were found. The mechanism of the interaction involves an intermediate aminoallenic compound, which is formed with covalent bound pyrrole in the reaction of the nucleophile with the enzyme. The presence of a pyrrole in the inactivated enzyme was confirmed by reaction with Ehrlich's reagent. The kinetic data obtained in this study indicate that 1,4‐diamino‐2‐butyne is a mechanism‐based inactivator with number of turnovers, r= 17 and characteristic constants K′= 0.32 mM and km= 4.89 min−1.
The mechanism of stimulation of MgATPase activity of chloroplast F1‐ATPase by non‐catalytic adeninenucleotide binding
Authors: MURATALIEV Marat B. , BOYER Paul D.
Published: March 2005

The presence of ATP at non‐catalytic sites of the chloroplast F1‐ATPase (CF1) eliminates a considerable lag in onset of enzyme activity that otherwise occurs in the presence of bicarbonate [Milgrom, Y. M., Ehler, L. & Boyer, P. D. (1991) J. Biol. Chem. 266, 11551–11558]. Sulfite is known to be much more effective than bicarbonate in stimulating ATPase activity CF1. Results reported here show that when assayed in the presence of sulfite, CF1, with some non‐catalytic sites empty or filled with GT(D)P, is able to hydrolyze both ATP and GTP. Thus, the presence of adenine nucleotides at non‐catalytic sites is not necessary for catalytic turnover of CF1. However, even though CF1 with empty non‐catalytic sites shows a significant initial activity, the prior binding of adenine nucleotides at non‐catalytic site(s) results in further activation of MgATPase and MgGTPase activities, even at relatively high sulfite and substrate concentrations. Although extensive activation of CF1 results from the presence of sulfite, with or without nucleotide binding at non‐catalytic sites, theKm remains constant, at about 50 γM for MgATP and 400 γM for MgGTP.

The results obtained show that the ATPase activity of CF1 is determined by the fraction of the active enzyme. The inactive CF1· ADP · Mg2+ formed during MgATP hydrolysis can be rapidly trapped by azide to provide a measure of the fraction of inactive enzyme. Increasing the concentration of sulfite increases the fraction of active CF1 in the assay medium. Measurements with radioactively labeled nucleotides show that the presence of ATP at non‐catalytic sites promotes the ATP‐dependent release of inhibitory ADP from a catalytic site. The activating effect of ATP binding at non‐catalytic sites results from increasing the portion of CF1 in an active state during steady‐state ATP hydrolysis.
Mechanism of the inhibition of cell growth by N1, N12‐bis(ethyl)spermine
Authors: FUKUCHI Jun‐ichi , KASHIWAGI Keiko , KUSAMA‐EGUCHI Kuniko , TERAO Kiyoshi , SHIRAHATA Akira , IGARASHI Kazuei
Published: March 2005

The mechanism of the antiproliferation effect of N1, N12‐bis(eithyl)spermine(BESPM) was studied in detail using mouse FM3A cells, since this polyamine analogue mimics the functions of spermine in several aspects [Igarashi, K., Kashiwagi, K., Fukuchi, J., Isobe, Y., Otomo, S. & Shirahata, A. (1990) Biochem. Biophys. Res. Commun. 172, 715–720]. Our results indicate that not only the decrease in spcrimine and spermine caused by BESPM but also its accumulation play important roles on the inhibition of cell growth by BESPM, since BESPM accumulated in cells at a concentration fivefold that of spermidine in control cells. In comparison with the polaymine‐deficient cells caused by α‐difluoromethylormthine, an inhibitor of ornithine decarboxylase, and ethylglyoxal bis‐(guanylhydrazone), an inhibitor of S‐adenosylmethionine decarboxylase, the behavior of polyamine‐deficient cells caused by BESPM was different as follows: the inhibition of cell growth by BESPM was not abrogated by spermine or spermidine; polyamine uptake, which is stimulated during polyamine deficiency, was greatly inhibited, while spermidine/spermine N−1‐acetyltransferase activity, which is inhibited during polyamine deficiency, was enhanced in BESPM‐treated cells; thymidine kinase activity did not decrease in BESPM‐treated cells; inhibition of cell growth and macromolecule synthesis by BESPM correlated with the swelling of mitochondria and the decrease in ATP content; BESPM caused cell death when incubated together for several days. The role of BESPM accumulation on inhibition of cell growth is discussed.
Characterization of Saccharomyces cerevisiae mutants lacking the E1α subunit of the pyruvate dehydrogenase complex
Authors: WENZEL Thibaut J. , VAN DEN BERG Marco A. , VISSER Wiebe , VAN DEN BERG Johan A. , STEENSMA H. Yde
Published: March 2005

Pyruvate dehydrogenase mutants of Saccharomyces cerevisiae were isolated by disruption of the PDAl gene. To this end, the PDAl gene encoding the E1α subunit of the pyruvate dehydrogenase complex was replaced by the dominant Tn5ble marker. Disruption of the PDAl gene abolished production of the E1α subunit and pyruvate dehydrogenase activity. Two additional phenotypes were observed in the Pdh− mutants: (a) a reduced growth rate in glucose medium which was partially complemented by the amino acid leucine; (b) an increase in formation of petites which lack mitochondrial DNA [rho°], during growth on glucose. Both phenotypes were shown to be a result of inactivation of the PDAl gene. Explanations for these phenotypes are discussed.
Cloning, overexpression and mechanistic studies of carboxyphosphonoenolpyruvate mutase from Streptomyces hygroscopicus
Authors: POLLACK Scott J. , FREEMAN Sally , POMPLIANO David L. , KNOWLES Jeremy R.
Published: March 2005

The enzyme carboxyphosphonoenolpyruvate mutase catalyses the formation of one of the two C‐P bonds in bialaphos, a potent herbicide isolated from Streptomyces hygroscopicus. The gene encoding the enzyme has been cloned from a subgenomic library from S. hygroscopicus by colony hybridisation using an exact nucleotide probe. An open reading frame has been identified that encodes a protein of molecular mass 32700 Da, in good agreement with the subunit molecular mass of the carboxyphosphonoenolpyruvate mutase recently isolated from this source [Hidaka, T., Imai, S., Hara, O., Anzai, H., Murakami, T., Nagaoka, K. & Seto, H. (1990) J. Bacteriol. 172, 3066–3072]. The gene shares significant sequence similarity with that of phosphoenolpyruvate mutase, an enzyme that catalyses the related interconversion of phosphoenolpyruvate and phosphonopyruvate. When the carboxyphosphonoenolpyruvate‐mutase gene was subcloned into the vector pET11a, the mutase was expressed as about 20% of the total soluble cellular protein in Escherichia coli. The mutase has been purified to homogeneity in three steps in 40% yield. With malate dehydrogenase/NADH, (hydroxyphosphinyl)pyruvate gives (hydroxyphosphinyl)lactate (Kcat 164 s−1 and Km 680 μM) and this spectrophotometric assay for the product of the mutase reaction has been employed in the mechanistic studies. The kinetics for the mutase reaction have been evaluated for the substrate, carboxyphosphonoenolpyruvate, and for the putative reaction intermediate carboxyphosphinopyruvate, both of which have been prepared by chemical synthesis. Carboxyphosphonoenolpyruvate is converted to (hydroxyphosphinyl)pyruvate with a kcat of 0.020 s−1 and a Km of 270 μM, and carboxyphosphinopyruvate is converted to (hydroxyphosphinyl)pyruvate with a kcat of 7.6 × 10−4 s−1 and a Km of 2.2 μM. Although the exogenously added intermediate is not kinetically competent, these results suggest that the mechanism for the mutase reaction involves an initial rearrangement to the intermediate carboxyphosphinopyruvate, followed by decarboxylation to yield the product (hydroxyphosphinyl)pyruvate.
Characterization of tryptophan phosphorescence of aspartate aminotransferase from Escherichia coli
Authors: CIONI Patrizia , ONUFFER James Joseph , STRAMBINI Giovanni Battista
Published: March 2005

The Trp phosphorescence spectrum, intensity and decay kinetics of apo‐aspartate aminotransferase, pyridoxamine‐5P‐aspartate‐aminotransferase and pyridoxal‐5P‐aspartate aminotransferase were measured over a temperature range 160–273 K. The fine structure of the phosphorescence spectra in low‐temperature glasses, with 0–0 vibrational bands centered at 408, 415 and 417 nm, for both apoenzyme and pyridoxamine‐5P‐enzyme reveals a marked heterogeneity of the chromophore environments. Only for the pyridoxal‐5P form of the enzyme is the triplet emission strongly quenched and, in this case, the spectrum displays a unique 0–0 vibrational band centered at 415 nm. Concomitant to quenching, there is Trp‐sensitized delayed fluorescence of the Schiff base, an indication that quenching of the excited triplet state is due, at least in part, to a process of triplet singlet energy transfer to the ketoenamine tautomer.

All three forms of the enzyme are phosphorescent for temperatures up to 273 K. However, across the glass transition temperature the pyridoxal‐5P enzyme shows a decrease in lifetime‐normalized phosphorescence intensity, a thermal quenching that reduces even further the number of phosphorescing residues at ambient temperature. In fluid solution, the triplet decay is nonexponential and multiple lifetimes stress the heterogeneity in dynamical structure of the chromophores' sites. For the pyridoxal‐5P enzyme, where only one or at most two residues are phosphorescent at 273 K, the nonexponential nature of the decay implies the presence of different conformers of the protein not interconverting in the millisecond time scale.
Role of protein kinase C in induction of gene expression and inhibition of cell proliferation by interferon α
Authors: JAMES Robert I. , MENAYA Juan , HUDSON Kati , DEVALIA Vinod , RYVES Jonathan , EVANS Fred J. , THOMAS Shaun , CLEMENS Michael J.
Published: March 2005

Recent studies have suggested that protein kinase C (PKC) may be involved in the mechanism of signal transduction by which members of the interferon (IFN) family regulate gene expression and cell phenotype. We have investigated the role of PKC in the control of cell growth and gene expression by IFNα in Daudi cells. Treatment of these cells with two analogues of staurosporine, which are potent inhibitors of PKC, completely blocked the induction by IFNα of the mRNA for 2′,5′‐oligoadenylate synthetase and the 6–16 gene. These compounds also inhibited cell proliferation and thymidine incorporation in this system. In contrast, the protein kinase inhibitor 1‐(5‐isoquinolinylsulphonyl)‐2‐methylpiperazine (H7) did not significantly inhibit the induction of these genes by IFNα and had no effect on Daudi cell growth or thymidine incorporation in the presence or absence of IFNα. No effect of IFNα on total PKC activity could be observed, and there were no significant changes in the overall levels of individual PKC isoforms or their mRNA following IFNα treatment. In contrast, treatment of Daudi cells with the phorbol ester 12‐O‐tetradecanoylphorbol 13‐acetate, which also inhibits cell proliferation, strongly down‐regulated PKC. These data suggest that the activity of a PKC species, or a closely related enzyme, may be required both for continued cell proliferation and the response to IFNα in Daudi cells, but that IFN‐induced growth inhibition does not involve overall down‐regulation or change in activity of PKC.
Wheat Ec metallothionein genes
Authors: KAWASHIMA Ichiro , KENNEDY Theresa D. , CHINO Mitsuo , LANE Byron G.
Published: March 2005

A cDNA library was prepared from the bulk mRNA of mature wheat embryos and screened with mixed 32P‐labeled oligonucleotide probes that encoded parts of the partial amino‐acid sequence for the Zn‐containing Ec protein. Each DNA insert in 11 positives from a screen of 105 plaques encoded a 5′ untranslated and a 3′ untranslated region, in addition to an open reading frame (of 81 amino acids) which, in every case, corresponded to at least 56 of the 59 amino acids in the partial polypeptide sequence previously determined for the Ec protein. The three different mRNA sequences encoded in the cDNA probably correspond to single‐copy genes in the A, B and D genomes of hexaploid wheat. A wheat genomic library was screened with 32P‐labeled cDNA and gave a single positive in a screen of 5 × 105 plaques. A 3.1‐kb genomic fragment (gf‐3.1) was sequenced and a cap site for the encoded mRNA was determined by primer extension. The gf‐3.1 sequence encodes an intronless mRNA for the Ec protein and contains appreciable amounts of 5′ and 3′ flanking sequences. In addition to a putative TATA box, two inverted‐repeat sequences and one direct‐repeat sequence, the 5′ flank in gf‐3.1 contains a sequence similar to the abscisic‐acid‐responsive element in other higher‐plant genes but does not contain sequences similar to the metal‐responsive elements in animal metallothionein genes. Consistent with these findings, RNA blotting shows that accumulation of Ec mRNA is abundant in immature embryos, undetectable in germinated embryos and can be induced by adding abscisic acid, but not by adding Zn2+ to the medium in which mature wheat embryos are germinated. The findings suggest that the wheat Ec metallothionein genes, like mammalian liver metallothionein genes, are conspicuously expressed during embryogenesis.
A novel insect defensin mediates the inducible antibacterial activity in larvae of the dragonfly Aeschna cyanea (Paleoptera, Odonata)
Authors: BULET Philippe , COCIANCICH Stéphane , REULAND Michel , SAUBER Ferd , BISCHOFF Rainer , HEGY Gilbert , Van DORSSELAER Alain , HETRU Charles , HOFFMANN Jules A.
Published: March 2005

The injection of low doses of bacteria into the aquatic larvae of dragonflies (Aeschna cyanea, Odonata, Paleoptera) induces the appearance in their hemolymph of a potent antibacterial activity. We have isolated a 38‐residue peptide from this hemolymph which is strongly active against Gram‐positive bacteria and also shows activity against one of the Gram‐negative bacteria which was tested. The peptide is a novel member of the insect defensin family of inducible antibacterial peptides, which had so far only been reported from the higher insect orders believed to have evolved 100 million years after the Paleoptera. Aeschna defensin is more potent than defensin from the dipteran Phormia, from which its structure differs in several interesting aspects, which are discussed in the paper.
Unusual amino acid substitution in the anion‐binding site of Lactobacillus plantarum non‐allosteric L‐lactate dehydrogenase
Authors: TAGUCHI Hayao , OHTA Takahisa
Published: March 2005

In Lactobacillus plantarum non‐allosteric L‐lactate dehydrogenase (L‐LDH), the highly conserved His188 residue, which is involved in the binding of an allosteric effector, fructose 1,6‐bisphosphate [Fru(1,6)P2], in allosteric L‐LDH is uniquely substituted by an Asp. The mutant L. plantarum L‐LDH, in which Asp188 is replaced by a His, showed essentially the same Fru(1,6)P2‐independent catalytic activity as the wild‐type enzyme, except that the Km and Vmax values were slightly decreased. However, the addition of Fru(1,6)P2 induced significant thermostabilization of the mutant enzyme, as in the case of many allosteric L‐LDHs, while Fru(1,6)P2 showed no significant effect on the stability of the wild‐type enzyme, indicating that only the single‐point mutation, G°C, sufficiently induces the Fru(1,6)P2‐binding ability of L. plantarum L‐LDH. The mutant enzyme showed higher thermostability than the wild‐type enzyme in the presence of Fru(1,6)P2. In the absence of Fru(1,6)P2, on the other hand, the mutant enzyme was more labile below 65°C but more stable above 70°C.
Cloning and specific polymerised‐chain‐reaction amplification of a third charge‐separable human metallothionein isoform
Authors: SOUMILLION Ann , Van DAMME Jo , De LEY Marc
Published: March 2005

Metallothionein (MT) fractions isolated from human adult liver tissue are readily separated by anion‐exchange chromatography in two isoforms, MT‐1 and MT‐2, which differ from each other in the nature of the amino acid residue at position 11. In fetal liver tissue, the presence of a third charge‐separable MT isoform has been previously reported. We determined its partial amino acid sequence and the sequence of a cDNA clone encoding this MT form. This confirmed the existence of another human MT isoform, hereafter named MT‐0, which is characterized by the presence of a negatively charged amino acid at position 8, and by a Glu23 to Lys substitution in a strictly conserved region of the protein. Taking into account these substitutions, we are able to classify human MT isoforms into three instead of two charge‐separable groups, based on the nature of three amino acid residues.

The unique presence of Glu8 in MT‐0 enabled us to develop an MT‐0‐specific amplification by the polymerase chain reaction, which revealed the presence of MT‐0 mRNA in adult liver RNA samples, in spite of the total absence of this isoform at the protein level. This suggests the involvement of post‐transcriptional regulatory mechanisms in the expression of this fetal MT form.
Characterization of fly rhodopsin kinase
Authors: DOZA Yair N. , MINKE Baruch , CHOREV Michael , SELINGER Zvi
Published: March 2005

Rhodopsin kinase activity of Musca domestica was characterized in a reconstitution assay, using urea‐treated eye membranes as substrate and a purified fraction of eye cytosol as the enzyme. Analysis of kinase activity in fly eye, brain and abdomen extracts by reconstitution assays revealed that fly rhodopsin kinase is an eye‐specific enzyme. It preferentially phosphorylates the light‐activated form of rhodopsin (metarhodopsin) and has little activity with other protein substrates. Rhodopsin kinase binds to metarhodopsin and is released from rhodopsin‐containing membranes. Metarhodopsin is a poor substrate for kinases from tissues other than the eye, making it a unique substrate for rhodopsin kinase. Rhodopsin kinase is inhibited by heparin, but not by the protein inhibitor of cAMP‐dependent protein kinase. Its Km for ATP is 9 μM. Since fly rhodopsin is coupled to phospholipase C, studies of the interaction of rhodopsin with rhodopsin kinase can be useful in analysis of the reactions that lead to termination of the inositol‐phospholipid‐signaling pathway.
Tenascin: a modulator of cell growth
Authors: END Peter , PANAYOTOU George , ENTWISTLE Alan , WATERFIELD Michael D. , CHIQUET Matthias
Published: March 2005

The large, multidomain extracellular matrix protein tenascin displays a markedly restricted tissue distribution during embryogenesis and remains present only in a few adult tissues. The protein is re‐expressed, however, during wound healing and in the stroma of malignant tumours. While a variety of studies have dealt with the important role of tenascin in the development of neural and non‐neural tissues, there is growing evidence that tenascin expression may be associated with proliferation of cells lining these tissues. The presence of repeating domains in tenascin similar to those in epidermal growth factor prompted us to investigate the ability of tenascin to modulate the growth of different cell types. Tenascin was actually found to be mitogenic for several cell types. This mitogenic activity, however, appears to be associated with a region in the fibronectin type III domains. The mitogenic mechanism is clearly distinct from pathways used by peptide growth factors such as epidermal growth factor and platelet‐derived growth factor, which activate the intrinsic tyrosine kinase activity of their cell‐surface receptors. However, we show that this large extracellular matrix molecule is efficiently internalised and may be processed by responding cells.
Amino acid and DNA sequences of an extracellular basic protease of Dichelobacter nodosus show that it is a member of the subtilisin family of proteases
Authors: LILLEY Glenn G. , STEWART David J. , KORTT Alexander A.
Published: March 2005

A DNA fragment encoding an extracellular basic protease (pI ∼ 9.5) from Dichelobacter nodosus, a Gram‐negative obligate anaerobe and the causative agent of ovine footrot, has been cloned and expressed in Escherichia coli and sequenced. E. coli harbouring a plasmid with a 3‐kb DNA fragment containing the D. nodosus basic‐protease gene exhibited proteolytic activity when tested on skim‐milk plates. The sequence of the native basic protease isolated from D. nodosus was also determined by direct amino acid sequencing. Comparison of the deduced sequence of the primary translation product (603 residues) and that of the native protease (344 residues) indicates that the protease is synthesized as a precursor molecule, containing a signal peptide (21 residues), a 111 amino acid pro‐peptide and a 127 residue C‐terminal extension which is subsequently processed to the mature active form.

Comparison of the D. nodosus basic protease sequence with that of other serine proteases showed that it is related to the subtilisin family of proteases with strong conservation of sequence identity around the catalytic site residues. A remarkable similarity in structure was found to the serine protease of Xanthomonas campestris, a plant pathogen, with respect to the length of the precursor segments, conservation of disulfide bridges and approximately 50% sequence identity of the mature proteases.
The effect of metal binding on the structure of annexin V and implications for membrane binding
Authors: LEWIT‐BENTLEY Anita , MORERA Solange , HUBER Robert , BODO Gerhard
Published: March 2005

The structure of annexin V, crystallised in the presence of two calcium or barium ions for each protein molecule, was solved by molecular replacement to 0.24 nm resolution. Two metal ions are found in domains I and IV, i.e. on the same side of the channel that lies in the centre of the molecule. The structures of the barium and calcium form are extremely close, the only differences localised in the metal‐binding sites that lie on the surface of the molecule. The occupancies of the metal ions, however, are lower for barium than for calcium, expressing the lower affinity of the protein for the former. The packing of the annexin molecules in the crystal asymmetric unit may represent a model for the calcium driven association of membrane‐bound annexins that leads to membrane fusion.
Substrate‐analogue‐induced changes in the nickel‐EPR spectrum of active methyl‐coenzyme‐M reductase from Methanobacterium thermoautotrophicum
Authors: ROSPERT Sabine , VOGES Markus , BERKESSEL Albrecht , ALBRACHT Simon P. J. , THAUER Rudolf K.
Published: March 2005

Methyl‐coenzyme‐M reductase (MCR) catalyzes the formation of methane from methyl‐coenzyme M [2‐(methylthio)ethanesulfonate] and 7‐mercaptoheptanoylthreonine phosphate in methanogenic archaea. The enzyme contains the nickel porphinoid coenzyme F430 as a prosthetic group. In the active, reduced (red) state, the enzyme displays two characteristic EPR signals, MCR‐red1 and MCR‐red2, probably derived from Ni(I). In the presence of the substrate methyl‐coenzyme M, the rhombic MCR‐red2 signal is quantitatively converted to the axial MCR‐red1 signal. We report here on the effects of inhibitory substrate analogues on the EPR spectum of the enzyme. 3‐Bromopropanesulfonate (BrPrSO3), which is the most potent inhibitor of MCR known to date (apparent Ki= 0.05 μM), converted the EPR signals MCR‐red1 and MCR‐red2 to a novel axial Ni(I) signal designated MCR‐BrPrSO3.

3‐Fluoropropanesulfonate (apparent Ki < 50 μM) and 3‐iodopropanesulfonate (apparent Ki < 1 μM) induced a signal identical to that induced by BrPrSO3 without affecting the line shape, despite the fact that the fluorine, bromine and iodine isotopes employed have nuclear spins of I= 1/2, I= 3/2 and I= 5/2, respectively. This finding suggests that MCR‐BrPrSO3 is not the result of a close halogen‐Ni(I) interaction.

7‐Bromoheptanoylthreonine phosphate (BrHpo ThrP) (apparent Ki= 5 μM), which is an inhibitory substrate analogue of 7‐mercaptoheptanoylthreonine phosphate, converted the signals MCR‐red1 and MCR‐red2 to a novel axial Ni(I) signal, MCR‐BrHpoThrP, similar but not identical to MCR‐BrPrSO3. The results indicate that inhibition of MCR by the halogenated substrate analogues investigated above is not via oxidation of Ni(I)F430. The different MCR EPR signals are assigned to differnt enzyme/substrate and enzyme/inhibitor complexes.
The 70‐kilodalton heat‐shock proteins of the SSA subfamily negatively modulate heat‐shock‐induced accumulation of trehalose and promote recovery from heat stress in the yeast, Saccharomyces cerevisiae
Authors: HOTTIGER Thomas , De VIRGILIO Claudio , BELL Walter , BOLLER Thomas , WIEMKEN Andres
Published: March 2005

In the yeast, Saccharomyces cerevisiae, the disaccharide trehalose is a stress‐related metabolite that accumulates upon exposure of cells to heat shock or a variety of non‐heat inducers of the stress response. Here, we describe the influence of mutations in individual heat‐shock‐protein genes on trehalose metabolism. A strain mutated in three proteins of the SSA subfamily of 70‐kDa heat‐shock proteins (hsp70) overproduced trehalose during heat shock at 37°C or 40°C and showed abnormally slow degradation of trehalose upon temperature decrease from 40°C to 27°C. The mutant cells were unimpaired in the induction of thermotolerance; however, the decay of thermotolerance during recovery at 27°C was abnormally slow. Since both a high content of trehalose and induced thermotolerance are associated with the heat‐stressed state of cells, the abnormally slow decline of trehalose levels and thermotolerance in the mutant cells indicated a defect in recovery from the heatstressed state. A similar albeit minor defect, as judged from measurements of trehalose degradation during recovery, was detected in a hsp 104 mutant, but not in a strain deleted in the polyubiquitin gene, UB14. In all our experiments, trehalose levels were closely correlated with thermotolerance, suggesting a thermoprotective function of trehalose. In contrast, heat‐shock proteins, in particular hsp70, appear to be involved in recovery from the heat‐stressed state rather that in the acquisition of thermotolerance. Cells partially depleted of hsp70 displayed an abnormally low activity of neutral trehalase when shifted to 27°C after heat shock at 40°C. Trehalase activity is known to be under positive control by cAMP‐dependent protein kinases, suggesting that hsp70 directly or indirectly stimulate these protein‐kinase activities. Alternatively, hsp70 may physically interact with neutral trehalase, thereby protecting the enzyme from thermal denaturation.
Expression, purification and characterisation of a functional phosphatidylinositol‐specific phospholipase C‐δ1 protein in Escherichia coli
Authors: GINGER Rebecca S. , PARKER Peter J.
Published: March 2005

Inositol‐lipid‐specific phospholipase C‐δ1 (PtdIns‐PLCδ1) was expressed in Escherichia coli as a fusion protein containing a short 22‐amino‐acid lac‐Z‐derived amino terminus. Under appropriate conditions, the phospholipase constituted approximately 0.2% of the detergent‐soluble protein and could be purified to near homogeneity in a simple three step protocol. The catalytic properties of the purified enzyme closely resemble those of the eukaryote‐derived protein. The suitability of bacterial expression for the investigation of PtdIns‐PLCδ regulation is discussed.
α‐Crystallin exists in a non‐spherical form
Authors: van HAERINGEN Bart , EDEN Don , van den BOGAERDE Marco R. , van GRONDELLE Rienk , BLOEMENDAL Michael
Published: March 2005

Native α‐crystallin, obtained from the cortex of calf lenses with FPLC (Pharmacia) was characterized by means of transient‐electric‐birefringence measurements and ultraviolet linear‐dichroism spectroscopy. These techniques were also performed on 6‐M‐urea‐dissociated and reconstituted α‐crystallin. Transient‐electric‐birefringence measurements offer the possibility to characterize the often observed, but usually neglected, non‐spherical occurrences of α‐crystallin in more detail. Although not distinguishable with size‐exclusion chromatography, we could identify at least two different classes of both native and reconstituted α‐crystallin, from which at least one consists of non‐spherical molecules. The results are compared with those obtained with electron microscopy using different staining methods. From the three independent techniques used we find evidence that a fraction of the α‐crystallin exists in a more extended quaternary structure. The results are difficult to explain with a concentric three‐layer model for α‐crystallin as proposed by Tardieu et al. [Tardieu, A., Laporte, D., Licinio, P., Krop, B. & Delaye, M. (1986) J. Mol. Biol. 192, 711–724].