bluuebunny/crossref_metadata_2025 huggingface dataset:
Ca2+ and Mg2+ as modulators of mitochondrial l‐glycerol‐3‐phosphate dehydrogenase
Authors: BELEZNAI Zsuzsa , SZALAY Lucia , JANCSIK Veronika
Published: March 2005

Physiological concentrations of either Ca2+ or Mg2+stimulated l‐glycerol 3‐phosphate oxidation by intact mitochondria isolated from various mammalian tissues (hamster brown adipose tissue, rat brain, liver of normal and hyperthyroid rats). A higher cation concentration was required for stimulation by Mg2+ than by Ca2+. l‐glycerol‐3‐phosphate dehydrogenase was the target of the stimulation by both cations as revealed by measurements with intact mitochondria as well as with the solubilized enzyme. With different electron acceptors Ca2+ and Mg2+ stimulation occurred at significantly different cation concentrations.

Substrate activation of mitochondrial l‐glycerol‐3‐phosphate dehydrogenase was observed in intact mitochondria and with the solubilized enzyme isolated from hyperthyroid rats in the absence of Ca2+ and Mg2+. According to kinetic analysis two independent binding sites, functioning with different turnovers and with different affinities for the substrate, could account for the phenomenon. In the presence of Ca2+ or Mg2+ substrate activation could not be detected; the kinetic parameters apparently correspond to the tight substrate‐binding site functioning with high turnover.

Thiol group(s), which in the absence of Ca2+ and Mg2+ did not participate in the functioning of the enzyme, played an essential role in the binding of these cations to the enzyme, as shown by chemical modification studies.

From the solubilized mitochondrial proteins l‐glycerol‐3‐phosphate dehydrogenase was bound selectively to the hydrophobic phenyl‐Sepharose 4B matrix in the presence Ca2+, and the bound enzyme could be eluted with EDTA. This suggests that Ca2+ caused an alteration in the conformation of the enzyme.
Subunit structure of a yeast site‐specific endodeoxyribonuclease, endoSceI
Authors: NAKAGAWA Kei‐ichi , HASHIKAWA Jun‐ichi , MAKINO Osamu , ANDO Tadahiko , SHIBATA Takehiko
Published: March 2005

EndoSceI is a eucaryotic site‐specific endoDNase of 120 kDa that causes double‐stranded scission at well‐defined sites, but is distinguishable from procaryotic restriction endonucleases by its mode of sequence recognition and lack of related specific DNA modification. In purified preparations of endoSceI, only two polypeptide species of 75 kDa (75‐kDa peptide) and 50 kDa (50‐kDa peptide) are detected in apparently equal amounts. We prepared mouse monoclonal IgGs that bound specifically to the 75‐kDa peptide (but not the 50‐kDa peptide) without inhibiting the endoSceI activity. Immunoprecipitation experiments with these IgGs revealed that the 75‐kDa peptide and the 50‐kDa peptide are physically associated with each other and with the endonucleolytic activity. Full endoSceI activity was recovered by mixing the purified 75‐kDa peptide and the partially purified 50‐kDa peptide, each of which exhibited little or no endonuclease activity alone. These observations indicate that endoSceI consists of two non‐identical subunits of 75 kDa and 50 kDa, and that both subunits are required for full enzyme activity.
A myosin site involved in energy transduction during muscle contraction
Authors: MIYANISHI Takayuki , HORIUTI Keisuke , ENDO Makoto , MATSUDA Genji
Published: March 2005

An antibody was developed against a 23‐kDa fragment of myosin which contains a part of the ATP‐binding site, and applied to skinned muscle fibers. The antibody abolished active tension generation of the fibers, but did not block their assumption of a rigor state nor their release from this state by ATP. The primary amino acid sequence of the antigenic site on the fragment was found to be the region containing residues 77–80. This sequence region is predicted to have interesting secondary structures, and is distinct from the proposed ATP‐binding site. We discuss the possibility that the region is responsible for the energy‐transducing step in muscle contraction.
The human von Willebrand factor gene
Authors: BONTHRON David , ORKIN Stuart H.
Published: March 2005

Von Willebrand factor (vWF) is a multimeric glycoprotein product of endothelial cells and platelets, that is important for normal hemostasis. Recently the complete primary structure of the vWF precursor was deduced from cloned complementary DNAs (cDNAs). To approach the analysis of DNA elements that mediate tissue‐specific expression, we have characterized the region of the human vWF gene surrounding the transcriptional initiation site, and including the first five exons. The putative vWF promoter region includes an (A + T)‐rich ‘TATA’‐like element approximately 30 bp upstream of the transcription start site, but no CCAAT or ‘GC’ box elements (typical motifs of other promoters). A (GT)n, repeat element of uncertain significance is located about 650 bp farther upstream. The DNA sequence of the 5′ flanking region suggests the presence of potentially novel elements involved in regulation of the restricted expression of vWF.
Purification and characterization of a bacterial dehalogenase with activity toward halogenated alkanes, alcohols and ethers
Authors: JANSSEN Dick B. , GERRITSE Jan , BRACKMAN Josephine , KALK Christiaan , JAGER Dick , WITHOLT Bernard
Published: March 2005

An enzyme that is capable of hydrolytic conversion of halogenated aliphatic hydrocarbons to their corresponding alcohols was purified from a 1,6‐dichlorohexane‐degrading bacterium. The dehalogenase was found to be a monomeric protein of relative molecular mass 28000. The affinity for its substrates was relatively low with Km values for short‐chain haloalkanes in the range 0.1–0.9 mM. The aliphatic dehalogenase showed a much broader substrate range than has been reported for halidohydrolases so far. Novel classes of substrates include dihalomethanes, C5–C9 1 ‐halo‐n‐alkanes, secondary alkylhalides, halogenated alcohols and chlorinated ethers. Several of these compounds are important environmental pollutants, e.g. methylbromide, dibromomethane, 1,2‐dibromoethane, 1,3‐dichloropropene, and bis(2‐chloroethyl)ether. The degradation of chiral 2‐bromoalkanes appeared to proceed without stereochemical preference. Optically active 2‐bromobutane was converted with inversion of configuration at the chiral carbon atom, suggesting that the dehalogenase reaction proceeds by a nucleophilic substitution involving a carboxyl group or base catalysis.
cDNA clones for human platelet GPIIb corresponding to mRNA from megakaryocytes and HEL cells
Authors: UZAN Georges , FRACHET Philippe , LAJMANOVICH Alicia , PRANDINI Marie‐Hélène , DENARIER Eric , DUPERRAY Alain , LOFTUS Joseph , GINSBERG Mark , PLOW Edward , MARGUERIE Gérard
Published: March 2005

Platelet glycoprotein (GP) IIb is one of the two subunits of the common platelet adhesion receptor, GPIIb‐IIIa. The isolation, characterization and sequencing of cDNA clones encoding for the two polypepetide chains of GPIIb are described. A number of clones were isolated from λgt11 libraries constructed with mRNA from an erythroleukemic cell line, HEL, and human megakaryocytes. Two of these clones, λIIb1, from HEL cells, and λIIb2, from megakaryocytes, cross‐hybridized and were selected for detailed analysis. ythe identification of these as authentic GPIIb clones was based on immunological criteria and confirmed by the presence of nucleotide sequences in each insert encoding for known protein sequences of platelet GPIIb. These clones contained inserts of 1.54 kb and 1.39 kb, respectively, with an overlapping sequence of 801 bp. The nucleotide sequence of the overlapping region was identical indicating that HEL cells produce a protein closely related, if not identical, to platelet GPIIb. The determined nucleotide sequence of two inserts included a coding sequence for 648 amino acid residues, a TAG stop codon and 185 nucleotides of 3′ non‐coding sequence followed by a poly(A) tail. The coding sequence contained a portion of the heavy chain, the junction between the heavy and light chains and the entire light chain including a potential transmembrane‐spanning domain and a short cytoplasmic tail. When these cDNA were used to probe for GPIIb mRNA, a single mRNA species of 3.9 kb was identified in both HEL cells and human megakaryocytes. A comparison of the deduced amino acid sequence for GPIIb with those of the α Subunit of the vitronectin and the fibronectin receptors revealed extensive homologies. These homologies further establish that GPIIb‐IIIa from platelets, together with the vitronectin and the fibronectin receptors, are members of a supergene family of adhesion receptors with a recognition specificity for Arg‐Gly‐Asp amino acid sequences.
Reduction of myosin‐light‐chain phosphorylation and of parvalbumin content in myotonic mouse muscle and its reversal by tocainide
Authors: JOCKUSCH Harald , REININGHAUS Jutta , STUHLFAUTH Ingo , ZIPPEL Monika
Published: March 2005

In muscle of the myotonic mouse mutant, ‘arrested development of righting response’, ADR, a reduced level of fast‐myosin‐light‐chain‐2 (LC2f) phosphorylation was observed in addition to a lowered parvalbumin content. In fast muscles, average phosphorylation levels of LC2f (LC2‐P/LC2 total) were 0.76 mol/mol for wild type and 0.59 mol/mol for the myotonic mutant. The difference was not due to short‐term activity prior to freezing because it was also found in curare‐paralyzed muscles. Long‐term treatment of genetically myotonic animals with the membrane‐stabilizing drug, tocainide, led to an increase of parvalbumin content and LC2‐P level. In wild‐type mice, tocainide had a similar effect, leading to supranormal parvalbumin concentrations. It is concluded that both the basal level of LC2‐P and parvalbumin concentration are regulated by a common factor, related to long‐term muscle activity.
Hydrogen peroxide (H2O2) induces actin and some heat‐shock proteins in Drosophila cells
Authors: COURGEON Anne‐Marie , ROLLET Emmanuelle , BECKER Jacqueline , MAISONHAUTE Claude , BEST‐BELPOMME Martin
Published: March 2005

Drosophila cells of a clone derived from line Kc were treated with various concentrations of hydrogen peroxide (H2O2). The concentration of 10 mM was lethal, whereas concentrations of 1–100 μM did not affect cell viability, rate of multiplication or protein synthesis. The intermediate concentration of 1 mM H2O2 was used to study the response of the cells to an oxidative stress.

We observed a transitory decrease of the global protein synthesis, which was accompanied by changes in the polypeptide pattern. There was a 2.5‐fold increase of the synthesis of the heat‐shock proteins 70–68 and 23. The most prominent response was a 6.5‐fold increase of actin synthesis 3 h after a 1 mM H2O2 treatment. When aminotriazole (an inhibitor of catalase) was added in association with H2O2, the increase of actin synthesis became 8.5‐fold. Experiments in which catalase was added at various times after H2O2 showed that a 10‐min treatment with H2O2 was sufficient to induce actin and heat‐shock protein synthesis 3 h later. H2O2 was shown to induce the transcriptional activation of an actin gene and of the heat‐shock protein genes 70 and 23 within minutes.

These results are coherent with the hypothesis that the byproducts of O2 reduction (the superoxide ion and hydrogen peroxide) could be inducers of the heat‐shock response. Whether the increase of actin synthesis is a stress‐related response, and the mode of action of H2O2 are discussed
Control of enzyme synthesis in the lysine biosynthetic pathway of Saccharomyces cerevisiae
Authors: RAMOS Fernando , DUBOIS Evelyne , PIÉRARD André
Published: March 2005

Several enzymes of the lysine pathway of Saccharomyces cerevisiae were found to respond to an induction mechanism mediated by the product of gene LYS14 in the presence of 2‐aminoadipate semialdehyde, an intermediate of this pathway. This novel regulatory mechanism appears independent of the specific repression by lysine and of the general control of amino acid biosynthesis. Genes LYS1, LYS9 and LYS14 have been cloned and their DNAs used to assay the corresponding messenger RNAs. The results suggest that the induction mechanism, as well as the specific and general regulations, operate at the transcriptional level.

The synthesis of saccharopine dehydrogenase (glutamate‐forming), previously shown to require the unlinked genes LYS9 and LYS14, is also affected by the induction mechanism. The leaky auxotrophic behaviour of lys14 mutants is explained by the low basal level of expression of LYS9, the structural gene of this enzyme, in the absence of induction by 2‐aminoadipate semialdehyde.
Adipose tissue protein phosphatase inhibitor‐2
Authors: STRÅLFORS Peter
Published: March 2005

Rat fat cells contain three species of spontaneously active inhibitor proteins of protein phosphatase 1, as resolved by SDS‐PAGE, with apparent molecular masses of 40 kDa, 33 kDa and 28 kDa respectively. The 33‐kDa, thermostable inhibitor was highly purified from bovine adipose tissue and shown to be very similar to inhibitor‐2 of skeletal muscle. It was phosphorylated, on threonine only, by glycogen synthase kinase 3. It formed an inactivated complex with protein phosphatase 1, that was reactivated by incubation with ATP‐Mg and glycogen synthase kinase 3. By gel filtration it had a Stokes radius of 3.4 nm. Peptide and phosphopeptide maps, generated by Staphylococcus aureus V8 proteinase, trypsin or thermolysin, of the inhibitor and of the skeletal muscle inhibitor‐2 were similar. The 40‐kDa inhibitor, which was denatured by boiling, represents a novel protein phosphatase inhibitor protein or an undegraded precursor of inhibitor‐2. The total activity of inhibitor‐2‐like material (thermostable and macromolecular) in an adipocyte cytosol extract corresponded to an intracellular concentration of 0.3 μM inhibitor‐2.
Purification and characterization of a liver microsomal cytochrome P‐450 isoenzyme with a high affinity and metabolic capacity for coumarin from pyrazole‐treated D2 mice
Authors: JUVONEN Risto O. , SHKUMATOV Vladimir M. , LANG Matti A.
Published: March 2005

Microsomal coumarin 7‐hydroxylase activity is regulated differently from several other monooxygenase enzymes, at least in mice [Wood, A. W. and Conney, A. H. (1974) Science (Wash. DC) 612–614]. Recently we found that in D2 mice this activity is strongly and selectively induced by pyrazole [Juvonen, R. O., Kaipainen, P. K. and Lang, M. A. (1985) Eur. J. Biochem. 152, 3–8]. This paper describes the purification of the pyrazole‐inducible cytochrome P‐450 isoenzyme. Because of the lability of the protein, a special procedure for the purification was developed. The procedure is based on a combination of hydrophobic and ion‐exchange chromatography and the presence of 100 μM coumarin in the preparations throughout the whole purification.

Coumarin effectively protected the P‐450 from degradation and also converted the pyrazole‐inducible P‐450 to its high‐spin state. This enabled us to choose only those fractions for further purification where the P‐450(s) was in its high‐spin state (rather than measuring the content of the total P‐450). As a result the purified protein had an apparent molecular mass of 49.7 kDa, a specific content of 19.9 nmol/mg protein and a very high affinity and metabolic capacity for coumarin.
Metabolism of 2‐oxoaldehyde in mold
Authors: INOUE Yoshiharu , RHEE Hae‐ik , WATANABE Kunihiko , MURATA Kousaku , KIMURA Akira
Published: March 2005

Two kinds of methylglyoxal reductases were purified to apparent homogeneity from Aspergillus niger and designated MGR I and MGR II. Both enzymes consisted of a single polypeptide chain with a relative molecular mass of 36000 (MGR I) and 38000 (MGR II). NADPH was specifically required for the activities of both enzymes and Km values for NADPH were 54 μM (MGR I) and 6.8 μM (MGR II). MGR I was specific to 2‐oxoaldehydes [glyoxal, methylglyoxal (Km=15.4 mM) and phenylglyoxal], whereas MGR II was active on both 2‐oxoaldehydes [glyoxal (Km= 10mM), methylglyoxal (Km= 1.43 mM), phenylglyoxal (Km=4.35 mM) and 4,5‐dioxovalerate] and some aldehydes (propionaldehyde and acetaldehyde). Optimal pH values for MGR I and MGR II activities were 9.0 and 6.5 respectively. Both enzymes were inactivated by a brief incubation with 2‐oxoaldehydes (glyoxal, methylglyoxal and phenylglyoxal) in the absence of NADPH. MGR I activity was competitively inhibited by NADP+ and the Ki value for NADP+ was calculated to be 0.49 mM. On the other hand, the inhibition of MGR II activity by NADP+ was of mixed type, the Ki value for NADP+ being 45 μM. MGR I was different from MGR II in amino acid composition.
Type B nucleoside‐diphosphatase of rat brain
Authors: SANO Shin‐ichiro , MATSUDA Yoshihiro , NAKAGAWA Hachiro
Published: March 2005

Type B nucleoside‐diphosphatase was purified from membranes of rat brain by solubilization with a non‐ionic detergent and successive column chromatographies on DEAE‐cellulose DE‐52, concanavalin‐A–Sepharose, Bio‐Gel HT, blue‐Sepharose CL‐6B, chelating Sepharose 6B, Ultrogel AcA44 and TSK gel G3000 SW. The purified enzyme gave a single protein band on SDS/polyacrylamide gel electrophoresis and its molecular mass was estimated to be 75 kDa. It hydrolyzed thiamin diphosphate as well as GDP, IDP and UDP. Thiamin diphosphate (TPP) was hydrolyzed twice as efficiently as nucleoside diphosphates in the presence of Mn2+ at pH 7.4. The Km values for TPP, GDP, IDP and UDP were 0.66, 0.40, 0.54 and 1.06 mM respectively. ATP, ADP and pyridoxal 5′‐phosphate inhibited thiamin‐pyrophosphatase activity competitively and their Ki values were 2.3 mM, 1.0 mM and 0.59 mM respectively. The optimum pH of thiamin‐pyrophosphatase activity was 7.4 in the presence of Mn2+ and that of GDP‐hydrolytic activity was 6.5 in the presence of Mg2+.
Distributions of retinoids, retinoid‐binding proteins and related parameters in different types of liver cells isolated from young and old rats
Authors: HENDRIKS Henk F. J. , BLANER William S. , WENNEKERS Herm M. , PIANTEDOSI Roseann , BROUWER Adriaan , de LEEUW A. Margreet , GOODMAN DeWitt S. , KNOOK Dick L.
Published: March 2005

The levels of retinoids, retinol‐binding protein, cellular retinol‐binding protein, cellular retinoic‐acid‐binding protein, transthyretin and the activities of retinyl palmitate hydrolase and cholesteryl oleate hydrolase were determined in purified parenchymal, fat‐storing, endothelial and Kupffer cell preparations, and in liver homogenates from young adult (6‐month‐old) and old (36‐month‐old) rats. Retinoid levels were also determined in the plasma from young and old rats. Retinoid contents were determined by HPLC. The binding proteins and transthyretin were measured by specific radioimmunoassays; retinyl palmitate and cholesterol oleate hydrolases were measured by sensitive microassays. The retinoid content of both the liver homogenates and of the fat‐storing, and parenchymal cell preparations increased between 6 months and 36 months of age. The cellular distribution of retinoids was similar for the two age groups analyzed with the fat‐storing cells being the main retinoid storage sites in the rat liver.

Concentrations of retinol‐binding protein and transthyretin were high in parenchymal cell preparations. Cellular retinol‐binding protein was enriched both in parenchymal and in fat‐storing cell preparations; the highest concentrations of cellular retinoic‐acid‐binding protein were present in fat‐storing cell preparations. No major differences were observed between the two age groups in the cellular concentrations and distributions of any of these binding proteins. High activity of cholesterol oleate hydrolase was measured in parenchymal and in Kupffer cell preparations; endothelial cell preparations also contained considerable activities. The distribution of this activity over the various cell types reflects their role in lipoprotein metabolism. Retinyl palmitate hydrolase activity was specifically enriched in parenchymal and in fat‐storing cell preparations, consistent with the roles of these cells in retinoid metabolism. No major differences were observed between the two age groups in the cellular distributions of the two hydrolase activities. This study indicates that no major changes occur in the retinoid‐related parameters analyzed with age, suggesting that rat liver retinoid metabolism does not change dramatically with age and that retinoid homeostasis is maintained.
The hopanoids of the purple non‐sulfur bacteria Rhodopseudomonas palustris and Rhodopseudomonas acidophila and the absolute configuration of bacteriohopanetetrol
Authors: NEUNLIST Serge , BISSERET Philippe , ROHMER Michel
Published: March 2005

Five complex hopanoids have been detected in the purple non‐sulfur bacterium Rhodopseudomonas acidophila. Next to the polyfunctionalized methylcyclopentane bacteriohopanetetrol ether already isolated from Methylobacterium organophilum, 35‐carbamoylbacteriohopane‐32,33,34‐triol, 34,35‐dicarbamoylbacteriohopane‐32,33‐diol and two nucleoside analogues, (22R)‐30‐(5′‐adenosyl)hopane and (22S)‐30‐(5′‐adenosyl)hopane were isolated and identified by spectroscopic and chemical methods. In Rhodopseudomonas palustris, however, only 35‐amino‐bacteriohopane‐32,33,34‐triol was detected.

Chemical correlation between adenosylhopane and bacteriohopanetetrol, as well as comparison of derivatives obtained from bacterial and synthetic hopanoids, permitted the determination of the configurations of all asymmetric centres of the side‐chain of bacteriohopanetetrol as 22R, 32R, 33R and 34S. According to the stereochemistry, this side‐chain could be a d‐ribose derivative linked through its C‐5 carbon atom to the hopane skeleton.
Luteal progesterone synthesis and high‐density lipoprotein
Authors: AITKEN John W. , BOOTH Roger , STANSFIELD David A.
Published: March 2005

We have shown previously that corpus luteum cells isolated from the superovulated ovaries of rats treated with 4‐amino‐pyrazolo[3,4‐d]pyrimidine constitute a suitable experimental system by which to investigate the mechanism in which plasma high‐density lipoprotein (HDL) plays a role in luteal cellular progesterone synthesis. In the present study, the rate of luteal cellular progesterone synthesis was shown to be stimulated by 125I‐labelled HDL up to about 70% of the rate achieved in the presence of native HDL. The concentration of HDL needed for half‐maximal stimulation of progesterone synthesis in the presence of lutropin was not significantly different irrespective of whether radioiodinated HDL or unlabelled HDL was used. Experimental conditions for studying the binding of 125I‐labelled HDL to isolated luteal cells have been defined and cellular binding affinity and binding capacity have been measured. Exposure of the luteal cells to pronase virtually abolished their capacity to bind 125I‐HDL and made them unable to respond to added HDL by increasing their rate of progesterone synthesis in the presence of lutropin. Control experiments showed this effect of pronase on cellular progesterone synthesis not to be due to destruction of cellular lutropin receptors, nor to general cellular damage. This evidence supports the view that luteal cellular binding of HDL is part of the mechanism by which HDL acts in luteal progesterone synthesis. Cellular binding capacity and affinity for 125I‐labelled HDL were the same irrespective of whether or not lutropin was present during incubation. Furthermore, the binding capacity and affinity of cells from the ovaries of rats not treated with 4‐amino‐pyrazolo[3,4‐d]pyrimidine were the same as in luteal cells isolated from rats that had been treated.
Elicitor‐induced biosynthesis of psoralens in Ammi majus L. suspension cultures
Authors: HAMERSKI Daria , MATERN Ulrich
Published: March 2005

Suspension cultures of Ammi majus L. cells produce various linear furanocoumarins in response to treatment with elicitor preparations from either Alternaria carthami Chowdhury or Phytophthora megasperma f.sp. glycinea. Microsomes which were isolated from these cells 14 h after addition of the elicitor efficiently catalyzed the conversion of demethyl [3 –14C]suberosin into labelled (+)marmesin in the presence of NADPH and oxygen. In contrast to the chemical cyclization of demethylsuberosin by m‐chloroperoxybenzoic acid, the reaction catalyzed by the marmesin synthase proceeded rapidly and no intermediate demethylsuberosin epoxide could be recovered. Significant blue‐light‐reversible inhibition by carbon monoxide and inhibition by various chemicals known to inhibit reactions dependent on cytochrome P450 suggested that the marmesin synthase is a cytochrome‐P450‐dependent monooxygenase.

Upon prolonged incubation, a subsequent major labelled product originated from (+)marmesin, which was identified as psoralen. The psoralen synthase was also characterized as a cytochrome‐P450‐dependent monooxygenase. Both the marmesin synthase and the psoralen synthase, as well as enzymes catalyzing the formation of demethylsuberosin and O‐prenylumbelliferone from umbelliferone and dimethylallyl diphosphate, were associated with the endoplasmic reticulum in Ammi majus cells and their activities were concomitantly induced by elicitor treatment of the cells. We propose that in vivo these enzymes are active in the lumen of the endoplasmic reticulum from where the furanocoumarin phytoalexins are excreted into the cell culture fluid.
A human embryonic lung fibroblast with a high density of muscarinic acetylcholine receptors
Authors: ANDRÉ Claudine , MARULLO Stefano , CONVENTS André , LÜ Bao Zhang , GUILLET Jean Gérard , HOEBEKE Johan , STROSBERG A. Donny
Published: March 2005

Binding studies with the radiolabeled muscarinic antagonists dexetimide, quinuclidinyl benzilate and N‐methylscopolamine showed that the human embryonic lung fibroblast CCL137 possesses approximately 2 × 105 muscarinic receptors/cell, i.e. 2.1 pmol/mg membrane protein. These receptors showed a marked stereoselectivity towards dexetimide and levetimide and only low affinity for another antagonist, pirenzepine. The muscarinic agonist carbamylcholine inhibited forskolin‐stimulated adenylate cyclase and induced phosphatidylinositide turnover in the intact cells. Both effects were inhibited by the muscarinic antagonist atropine. Affinity labeling with tritiated propylbenzylcholine mustard revealed a protein of 72 kDa. Finally, down‐regulation of the membrane receptors following prolonged treatment with the agonist carbamylcholine was assessed by means of the hydrophilic antagonist N‐methylscopolamine.
The Na+/K+/Cl− cotransport in C6 glioma cells
Authors: CHASSANDE Olivier , FRELIN Christian , FARAHIFAR Dariush , JEAN Thierry , LAZDUNSKI Michel
Published: March 2005

The role of the Na+/K+/Cl− cotransporter in the regulation of the volume of C6 astrocytoma cells was analyzed using isotopic fluxes and cell cytometry measurements of the cell volume. The system was inhibited by ‘loop diuretics’ with the following order of potency: benzmetanide > bumetanide > piretanide > furosemide. Under physiological conditions of osmolarity of the incubation media, equal rates of bumetanide‐sensitive inward and outward K+ fluxes were observed. Blockade of the Na+/K+/Cl− cotransporter with bumetanide did not lead to a modification in the mean cell volume. When C6 cells were incubated in an hyperosmotic solution, a cell shrinkage was observed. It was accompanied by a twofold increase in the activity of the Na+/K+/Cl− cotransport, which then catalyzed the net influx of K+. In spite of this increased activity, no cell swelling could be measured. Incubation of the cells in an iso‐osmotic medium deprived of either Na+, K+ or Cl− also produced cell shrinkage. Large activations (up to tenfold) of the Na+/K+/Cl− cotransport together with a cell swelling back to the normal volume were observed upon returning ion‐deprived C6 cells to a physiological solution. This cell swelling was completely prevented in the presence of bumetanide. It is concluded that the Na+/K+/Cl− cotransport system is one of the transport systems involved in volume regulation of glial cells. The system can either be physiologically quiescent or active depending on the conditions used. A distinct volume regulating mechanism is the Na+/H+ exchange system.
Determination of the backbone conformation of secretin by restrained molecular dynamics on the basis of interproton distance data
Authors: CLORE G. Marius , NILGES Michael , BRÜNGER Axel , GRONENBORN Angela M.
Published: March 2005

The backbone conformation of the 27‐residue polypeptide hormone secretin has been investigated using nuclear magnetic resonance spectroscopy and restrained molecular dynamics calculations under conditions where it adopts a fully ordered structure (40% v/v trifluoroethanol). The basis for the restrained molecular dynamics calculations consists of 52 nuclear‐Overhauser‐enhancement‐derived interproton distance restraints involving the NH, CαH and CβH protons. It is shown that convergence to similar extended structures is achieved starting from four different initial structures, namely an α helix, a mixed α/β structure, a β strand and a polyproline helix. The converged structures are made up of short N‐ and C‐terminal strand‐like regions and a central region comprising two irregular helices connected by a ‘half‐turn’.
Human erythrocyte glucose‐6‐phosphate dehydrogenase
Authors: CAMARDELLA Laura , CARUSO Carla , RUTIGLIANO Bruno , ROMANO Mario , DI PRISCO Guido , DESCALZI‐CANCEDDA Fiorella
Published: March 2005

Human erythrocyte glucose‐6‐phosphate dehydrogenase contains a reactive lysyl residue, which can be labelled with pyridoxal 5′‐phosphate. The binding of one mole of pyridoxal 5′‐phosphate per mole of enzyme subunit produces substantial inactivation. The substrate glucose‐6‐phosphate prevents the loss of activity, suggesting that the reaction site is close to the substrate‐binding site. A tryptic peptide containing the pyridoxal‐5′‐phosphate‐binding lysyl residue has been isolated and characterised. The reactive lysyl residue has been identified in the glucose‐6‐phosphate dehydrogenase amino acid sequence. Comparison with glucose‐6‐phosphate dehydrogenase from other sources shows a high homology with a peptide containing a reactive lysyl residue, isolated from the enzyme from Saccharomyces cerevisiae; glucose‐6‐phosphate dehydrogenase from Leuconostoc mesenteroides also contains a region highly homologous with the sequence around the reactive lysyl residue in the human enzyme.

The results of this communication provide the first direct evidence for the association of an essential catalytic function with a specific region of the molecule of human erythrocyte glucose‐6‐phosphate dehydrogenase.
Preferential positioning of nucleosomes on pBR322 as evaluated via Fourier transform of data from electron microscopy
Authors: CAFFARELLI Elisa , DE SANTIS Pasquale , LEONI Luisa , PALLESCHI Antonio , SAVINO Maria
Published: March 2005

Nucleosome positioning on pBR322 DNA has been evaluated by electron microscopy visualization, after psoralen cross‐linking. The distribution function of nucleosomes on pBR322 DNA has been calculated analyzing the data of the electron microscopy via Fourier transform. This function shows definite maxima, which indicate differential interactions of the histone octamer to different DNA sequences.
Albumin in rabbit skeletal muscle
Authors: HEILIG Annegard , PETTE Dirk
Published: March 2005

A 68‐kDa protein is present in increasing amounts in the soluble fractions of fast, slow and cardiac muscles of the rabbit. Induced contractile activity by chronic 10‐Hz stimulation caused in rabbit fast‐twitch muscle a rapid increase of this protein, reaching fivefold to sixfold higher levels than normal after one week of stimulation. The 68‐kDa protein was identified as albumin by its mobility in one‐dimensional and two‐dimensional electrophoreses and by its reactivity with a polyclonal antibody directed against rabbit serum albumin. Immunohistochemistry revealed a localisation pattern corresponding to a distribution mainly in the interstitium. The assumption that albumin is not an intrinsic protein of the muscle fiber was strongly supported by the failure to show its synthesis by in vivo labeling. Injection of [35S]methionine into normal and chronically stimulated muscles did not lead to a detectable incorporation of the precursor into muscle albumin. In addition, albumin could not be detected as a product formed by in vitro translation of RNA preparations from normal and chronically stimulated muscles.
Production in Escherichia coli and one‐step purification of bifunctional hybrid proteins which bind maltose
Authors: BEDOUELLE Hugues , DUPLAY Pascale
Published: March 2005

Two enzymes, the secreted Staphylococcus aureus nuclease A and the Klenow fragment of the cytoplasmic Escherichia coli DNA polymerase I, were fused, at the genetic level, to MalE, the periplasmic maltose‐binding protein of E. coli, or to a signal‐sequence mutant. The hybrid proteins were synthesized in large amounts by E. coli under control of promoter malEp. The synthesis was repressed with glucose and could be totally switched off in a malT mutant strain. The hybrid between MalE and the nuclease was exported into the periplasmic space. Several criteria demonstrated that a fraction of the hybrid chains with the Klenow polymerase was exported to the periplasm in a signal‐sequence‐specific manner and ruled out the possibility of a membrane leakage. The hybrid with the Klenow polymerase was not exported and remained in the cytoplasm when carrying a tight signal‐sequence mutation in its MalE portion. The hybrid proteins were purified in one step by affinity chromatography on cross‐linked amylose. Most of the hybrid chains in the periplasm but only a fraction of those in the other cell compartments had their MalE portion correctly folded. The nuclease and the Klenow polymerase had their full specific activities in the purified hybrids. The potential of MalE as a vector for the production, export and purification of desirable proteins in E. coli is discussed.
Depression in gene expression for poly(ADP‐ribose) synthetase during the interferon‐γ‐induced activation process of murine macrophage tumor cells
Authors: TANIGUCH Taketoshi , YAMAUCHI Kiyoshi , YAMAMOTO Tadashi , TOYOSHIMA Kumao , HARADA Nobuhiro , TANAKA Hideaki , TAKAHASHI Seiichi , YAMAMOTO Hiroshi , FUJIMOTO Shigeyoshi
Published: March 2005

A 2.7‐kb cDNA clone coding for bovine poly(ADP‐ribose) synthetase was isolated from a λgt11 expression library by direct immunological screening with an antiserum to the enzyme. The cDNA hybridizes to an approximately 3.8‐kb bovine thymus polyadenylated RNA, which translates an immunoprecipitable 120‐kDa protein with the antibody to the enzyme. The partial DNA sequence of the cDNA was determined and portions of the predicted amino acid sequence matched the sequence of 26 amino acids at the N terminal of the 41‐kDa α‐chymotryptic fragment and two cyanogen‐bromide‐cleaved peptides of the enzyme.

A subcloned fragment from the coding region of the cDNA was used as a probe to estimate the level of mRNA for the enzyme during the interferon‐γ‐induced activation process of the murine macrophage tumor P388D1 cell line. The amount of mRNA for the enzyme decreased nearly completely within 24 h after incubation in a medium containing interferon‐γ, while mRNA of the Ia antigen, one of the major histocompatibility gene products, was increased in the macrophage tumor cells by interferon‐γ as confirmed by the I‐Aβ cDNA as a probe. These results suggest that the gene expression for poly(ADP‐ribose) synthetase is depressed during the interferon‐γ‐induced activation process of macrophage tumor cells.
Mapping of catalytic domains and phosphorylation sites in the multifunctional pyrimidine‐biosynthetic protein CAD
Authors: CARREY Elizabeth A. , HARDIE D. Grahame
Published: March 2005

We have examined the domain organization, and the locations of the sites phosphorylated by the cyclic‐AMP‐dependent protein kinase, in the multifunctional polypeptide of the pyrimidine‐biosynthetic protein, CAD. Fragments produced after limited proteolysis by elastase or trypsin were separated by SDS/polyacrylamide gel electrophoresis and transferred onto nitrocellulose. The blots were probed with antibodies raised against the core aspartate carbamoyltransferase (ACTase) and dihydroorotase (DHOase) fragments to locate fragments containing these domains, and we also examined the locations of the phosphorylation sites by complete tryptic digestion of blotted, 32P‐labelled fragments, followed by analytical isoelectric focussing.

Our results are consistent with the domain order glutaminase(GLNase)‐carbamoyl‐phosphate synthetase‐(CPSase)‐DHOase‐ACTase, as suggested by recently reported homologies between the predicted amino acid sequence for the Drosophila rudimentary gene product, and monofunctional CPSases/ACTases/DHOases. In particular, the finding of a 95‐kDa elastase fragment which cross‐reacted with both anti‐DHOase and anti‐ACTase antibodies rules out the previously suggested domain order: DHOase‐GLNase‐CPSase‐ACTase.

Phosphorylation by cyclic‐AMP‐dependent protein kinase accelerates cleavage of native CAD by both elastase and trypsin, and abolishes the protective effect of UTP. Site 1 is located close to the C‐terminal end of the 160‐kDa GLNase/CPSase region. Comparison with the predicted amino acid sequence of the Drosophila rudimentary gene revealed a strong homology between the tryptic peptide containing site 1 from hamster CAD, and a region at the extreme C‐terminal end of the CPSase II domain of the Drosophila enzyme. Alignment of the Drosophila sequence and that of rat liver CPSase I, which is not phosphorylated by cyclic‐AMP‐dependent protein kinase, revealed that this putative site 1 region is missing in CPSase I.

Site 2 could not be located with certainty, either from the limited proteolysis data, or from comparison of the sequence around this site and the sequence of the rudimentary gene. There were also one or more previously undetected minor phosphorylation site(s) located in the protease‐sensitive hinge region between the DHOase and ACTase domains.
Characterization of phosphofructokinase 2 and of enzymes involved in the degradation of fructose 2,6‐bisphosphate in yeast
Authors: FRANÇOIS Jean , VAN SCHAFTINGEN Emile , HERS Henri‐Géry
Published: March 2005

Phosphofructokinase 2 from Saccharomyces cerevisiae was purified 8500‐fold by chromatography on blue Trisacryl, gel filtration on Superose 6B and chromatography on ATP‐agarose. Its apparent molecular mass was close to 600 kDa. The purified enzyme could be activated fivefold upon incubation in the presence of [γ‐32P]ATP‐Mg and the catalytic subunit of cyclic‐AMP‐dependent protein kinase from beef heart; there was a parallel incorporation of 32P into a 105‐kDa peptide and also, but only faintly, into a 162‐kDa subunit.

A low‐Km, (0.1 μM) fructose‐2,6‐bisphosphatase could be identified both by its ability to hydrolyze fructose 2,6‐[2‐32P]bisphosphate and to form in its presence an intermediary radioactive phosphoprotein. This enzyme was purified 300‐fold, had an apparent molecular mass of 110 kDa and was made of two 56‐kDa subunits. It was inhibited by fructose 6‐phosphate (Ki= 5 μM) and stimulated 2–3‐fold by 50 mM benzoate or 20 mM salicylate. Remarkably, and in deep contrast to what is known of mammalian and plant enzymes, phosphofructokinase 2 and the low‐Km fructose‐2,6‐bisphosphatase clearly separated from each other in all purification procedures used. A high‐Km (∼ 100 μM), apparently specific, fructose 2,6‐bisphosphatase was separated by anion‐exchange chromatography. This enzyme could play a major role in the physiological degradation of fructose 2,6‐bisphosphate, which it converts to fructose 6‐phosphate and Pi, because it is not inhibited by fructose 6‐phosphate, glucose 6‐phosphate or Pi.

Several other phosphatases able to hydrolyze fructose 2,6‐bisphosphate into a mixture of fructose 2‐phosphate, fructose 6‐phosphate and eventually fructose were identified. They have a low affinity for fructose 2,6‐bisphosphate (Km, > 50 μM), are most active at pH 6 and are deeply inhibited by inorganic phosphate and various phosphate esters.
Purification, properties and cation activation of galactosyltransferase from lactating‐rat mammary Golgi membranes
Authors: NAVARATNAM Naveenan , WARD Simon , FISHER Cynthia , KUHN Nicholas J. , KEEN Jeffery N. , FINDLAY John B. C.
Published: March 2005

Galactosyltransferase was purified from Golgi membranes of lactating‐rat mammary gland and studied with respect to its physical and enzymic (lactose synthetase) properties.

The enzyme occurred in both monomeric (43–46 kDa) and apparently dimeric (90 kDa) forms. It was very unstable except in the presence of phospholipid, detergent, or cations binding to site 2. The amino acid composition and the N‐terminal sequence closely resembled that of the human and bovine milk enzymes, particularly in respect to a Pro‐Pro‐Pro‐Pro sequence.

Kinetic studies demonstrated a high‐affinity Mn2+‐binding site (1) essential for activity, and a low‐affinity Mn2+‐binding site (2) that could also bind spermidine or clupeine. Mn2+ binding at site 2 raised Vmax fivefold. Spermidine binding at site 2 enhanced Mn2+ binding at site 1, and influenced binding of glucose. At physiological glucose concentration, clupeine or spermidine activated nearly as well as 15 mM MnCl2 and are regarded as models of a natural cation activator that remains to be isolated. Evidence is given for an essential histidine residue in the galactosyltransferase.

It is proposed that site 1 Mn2+ participates directly in the reaction mechanism, whereas site 2 is a regulator site allosterically activated by a basic protein.
Primary structure of neutral oligosaccharides derived from respiratory‐mucus glycoproteins of a patient suffering from bronchiectasis, determined by combination of 500‐MHz 1H‐NMR spectroscopy and quantitative sugar analysis
Authors: KLEIN André , LAMBLIN Geneviève , LHERMITTE Michel , ROUSSEL Philippe , BREG Jan , VAN HALBEEK Herman , VLIEGENTHART Johannes F. G.
Published: March 2005

Carbohydrate chains of respiratory‐mucus glycopeptides from a patient (blood group O) suffering from bronchiectasis with a Kartagener's syndrome have been released by alkaline borohydride treatment. Application of high‐performance liquid chromatography using subsequently two silica columns, one bonded with aminopropyl groups and the other with octadecyl groups, afforded 39 neutral fractions; 35 oligosaccharide‐alditol structures have been characterized by employing 500‐MHz 1H‐NMR spectroscopy in conjunction with sugar analysis.

Here, 16 oligosaccharide structures, possessing a core consisting of Galβ(1→3)GalNAc‐ol with or without branching through a GlcNAc residue linked β(1→6) to the GalNAc residue (core type 2 or core type 1, respectively), are described. Ten oligosaccharide‐alditols with these types of cores (2, 3, 10a, 14, 7, 11a, 15a, 16a, 12 and 16c) have been identified previously in human bronchial mucins of patients suffering from cystic fibrosis [Lamblin, G., Boersma, A., Lhermitte, M., Roussel, P., Mutsaers, J. H. G. M., Van Halbeek, H. and Vliegenthart, J. F. G. (1984) Eur. J. Biochem. 143, 227–236].

Of the remaining six compounds, one is a partial structure of oligosaccharides previously described:
image

The structures 17a and 20 contain the Y determinant, i.e., Fucα(1→2)Galβ(1→4)[Fucα(1→3)]GlcNAcβ(1→). High‐resolution 1H‐NMR spectroscopy is able to distinguish whether the Y determinant is β(1→3) or β(1→6) linked in such oligosaccharide‐alditols.
The interaction of α‐tocopherol and homologues with shorter hydrocarbon chains with phospholipid bilayer dispersions
Authors: KAGAN Valerian E. , QUINN Peter J.
Published: March 2005

The intrinsic fluorescence of tocopherol homologues with hydrocarbon chains ranging from 1 carbon (C1) to 16 carbons (α‐tocopherol, C16) and their ability to quench the fluorescence of 9‐anthroyloxy derivatives of fatty acids with the fluorophore located at different positions in the hydrophobic domain of phospholipid bilayers has been used to model the interaction of tocopherol with lipid bilayer membranes. All the tocopherol homologues used, C1 C6, Cll and α‐tocopherol, showed a similar fluorescence emission intensity at 325 nm in cyclohexane but were almost completely self‐quenched by aggregation in water. Fluorescence was restored when dispersions of dimyristoylglycerophosphocholine were added but the maximum intensity was lower with the longer‐chain homologues. Full intensity was observed in all homologues on addition of the detergent Triton X‐100. Studies using 9‐anthracenecarboxylic acid and 9‐anthracenecarboxymethyl ester, 6‐(9‐anthroyloxy)stearic acid and 16‐(9‐anthroyloxy)palmitelaidic acid showed that the tocopherol homologues partitioned into the hydrophobic domain of phospholipid dispersions composed of dimyristoylglycerophosphocholine at 40°C and dioleoylglycerophosphocholine at 40°C. The 9‐anthroyloxy fatty acids and 1,6‐diphenyl‐1,3,5‐hexatriene were quenched by all the homologues and Stern‐Volmer plots of the concentration dependence of the quenching indicated that this was predominantly via dynamic processes. No fluorescence energy transfer was observed between diphenylhexatriene and tocopherols but efficient transfer was recorded to the 9‐anthroyloxy Fatty acid probes. The results are consistent with a model in which the chromanol nucleus of tocopherol is oriented towards the lipid‐water interface of the phospholipid bilayer. As the phytol chain length increases there is an increasing tendency for the chromanol nucleus to reside in the hydrophobic interior of the structure. α‐Tocopherol appears to form clusters within the phospholipid dispersion which are not fluorescent and do not quench the fluorescence of the different fluorescent probes nor transfer fluorescence energy to them. It is suggested that the monomeric form is responsible for the vitamin effects of tocopherol and the aggregated form acts as a reservoir that does not markedly perturb bilayer stability.
Comparative study of the lateral motion of extrinsic probes and anthracene‐labelled constitutive phospholipids in the plasma membrane of Chinese hamster ovary cells
Authors: DUPOU Laurence , LOPEZ André , TOCANNE Jean‐François
Published: March 2005

9‐(2‐Anthryl)‐nonanoic acid is a new photoactivatable fluorescent probe which has been designed for the study of the lateral diffusion and distribution of lipids in biological membranes by means of the anthracene photodimerization reaction. This anthracene fatty acid can be incorporated metabolically into the glycerophospholipids (phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, phosphatidylinositol) of Chinese hamster ovary (CHO) cells in culture. The diffusion coefficient of intrinsic lipids in the plasma membrane of these eukaryotic cells can thus be measured using the fluorescence recovery after a photobleaching technique, since illumination of the fluorescent anthracene groups yields non‐fluorescent photodimers. For the sake of comparison, the extrinsic lipophilic probes 5‐(N‐hexadecanoyl)‐aminofluorescein, 12‐(9‐anthroyloxy)‐stearic acid, 9‐(2‐anthryl)‐nonanoic acid and a synthetic anthracene‐phosphatidylcholine were also used to label the plasma membrane of CHO cells. The diffusion coefficients for the extrinsic and intrinsic probes ranged over 1 – 2 × 10‐9 cm2/s. Small but significant differences were observed between the various probes reflecting differences they exhibit in size and polarity. All the extrinsic probes were free to diffuse, with a mobile fraction close to 100%. In contrast, a fractional recovery of only 75% was observed for the intrinsic anthracene‐labelled phospholipids, suggesting that the anthracene fatty acid was metabolically incorporated into membrane lipid regions which were inaccessible to the extrinsic probes.