bluuebunny/crossref_metadata_2025 huggingface dataset:
Molecular cloning and amino acid sequence of the porcine 17β‐estradiol dehydrogenase
Authors: LEENDERS F. , ADAMSKI J. , HUSEN B. , THOLE H. H. , JUNGBLUT P. W.
Published: March 2005

We describe the cloning and sequencing of porcine 17β‐estradiol dehydrogenase. The enzyme performs oxidation 360‐fold more efficiently than reduction, both measured under optimal conditions. It is localized in specialized vesicles of epithelial cells. The cDNA clones were isolated from a λUNI ZAP XR library of porcine kidney and polymerase‐chain‐reaction‐amplified from templates of uterus epithelium. In both tissues, the same enzyme is coded by a transcript of 2.9 kb. It contains a 69‐b 5′‐noncoding region, an open reading frame of 2211 b and a 3′‐noncoding region of 624 b. The open reading frame of 737 amino acids with a predicted molecular mass 79973 Da was confirmed by amino acid sequencing of peptides. The 80‐kDa translation product is processed to the N‐terminal 32‐kDa enzyme, part of which is then covalently linked to actin. The estradiol dehydrogenase/actin complex and the 80‐kDa translation product comigrate in SDS/PAGE.
The purification, characterization, cloning and sequencing of the gene for a halostable and thermostable leucine dehydrogenase from Thermoactinomyces intermedius
Authors: OHSHIMA Toshihisa , NISHIDA Norikazu , BAKTHAVATSALAM Sundararaju , KATAOKA Kunishige , TAKADA Harumi , YOSHIMURA Tohru , ESAKI Nobuyoshi , SODA Kenji
Published: March 2005

Leucine dehydrogenase has been purified to homogeneity from a moderate thermophilic actinomycete, Thermoactinomyces intermedius IFO 14230. The enzyme can be stored without loss of its activity at a low temperature (e.g., 4°C) for over two years. The enzyme was more thermostable at higher concentrations of salts such as NaCl and KCl. It retained about 90% of activity on incubation at 70°C for at least 40 min in the presence of 3 M NaCl. The Michaelis constants for NAD, l‐leucine, NADH, 2‐oxoisocaproate and ammonia were determined to be 0.36, 2.0, 0.042, 0.63 and 118 mM, respectively, from initial‐velocity analyses. The enzyme showed pro‐S stereospecificity for hydrogen transfer of NADH in the reductive amination. The enzyme gene was cloned into Escherichia coli and its complete DNA sequence was determined. The leucine dehydrogenase gene (leudh) consists of a 1098‐bp open reading frame and encodes 366 amino acid residues corresponding to a subunit (Mr 40586) of the octameric enzyme. The amino acid sequence of the enzyme showed 80.7% similarity with that of the Bacillus stearothermophilus enzyme. The enzyme was overproduced in E. coli JM 109 having a recombinant plasmid, pULDH2, which was constructed from pUC18 and the leudh gene. The enzyme was purified from the cell extract to homogeneity in one day, with 78% recovery.
Differential nuclear localization of protein kinase C isoforms in neuroblastoma × glioma hybrid cells
Authors: BECKMANN Roland , LINDSCHAU Carsten , HALLER Hermann , HUCHO Ferdinand , BUCHNER Klaus
Published: March 2005

The protein kinase C (PKC) α, β and ε isoforms have distinct nuclear localizations in neuroblastoma × glioma hybrid cells NG 108–15. We found by immunoblotting that PKC α, βII, δ and ε are the predominant isoforms in these cells. In contrast to other neuronal cell lines, none of these isoforms is down‐regulated during differentiation.

Confocal immunofluorescence microscopy revealed that in undifferentiated cells PKC α is located in the cytoplasm and in the nucleus excluding nucleoli. In differentiated cells PKC α was almost exclusively located in the cytoplasm. Stimulation of the cells with phorbol ester resulted in translocation to the plasma membrane. PKC βII was not detectable in the nuclei. PKC δ was found in the nucleoli and in the cytoplasm, in differentiated cells particularly in the neurites. Phorbol ester failed to induce a translocation to other compartments. PKC δ was localized with the nuclear‐pore complexes at the nuclear envelope. In differentiated cells after stimulation with phorbol ester, partial translocation to the plasma membrane was observed.
cDNA cloning and deduced amino acid sequence of two ferritins: soma ferritin and yolk ferritin, from the snail Lymnaea stagnalis L.
Authors: VON DARL Matthias , HARRISON Pauline M. , BOTTKE Werner
Published: March 2005

Pulmonate freshwater snails contain two different ferritin types, soma ferritin and yolk ferritin. A cDNA library was constructed from midgut gland poly(A)‐rich RNA of the snail Lymnaea stagnalis L. and recombinant clones encoding both ferritin types were obtained by immunoscreening. The longest cDNA inserts had a length of 859 bp (soma ferritin) and 1548 bp (yolk ferritin) and the specificity of these inserts was confirmed by immunoprecipitation of both ferritin types translated in vitro from hybrid‐selected mRNAs. The 5′ untranslated region (UTR) of the soma ferritin mRNA contains a 28‐bp element which shows 64% sequence identity with the iron‐responsive element (IRE) of vertebrate ferritin mRNAs. The soma ferritin mRNA is strongly translated in the wheat germ system but poorly translated in rabbit reticulocyte lysate. The yolk ferritin mRNA, which contains no IRE, is equally well translated in both in vitro translation systems. The deduced amino acid sequence of the soma ferritin subunit (174 amino acid residues, Mr 20140) shows 50–70% sequence identity with subunits of vertebrate ferritins. After removal of an 18‐amino‐acid‐residue signal sequence the deduced protein sequence of yolk ferritin contains 221 amino acids (Mr 25438). Sequence identity of this chain with other eukaryotic ferritin chains is only 31–42%. Both snail ferritin sequences are more similar to the H‐subunit type of vertebrate ferritins than to the l‐type and both have the H‐specific amino acid residues of the ferroxidase centre. The yolk ferritin sequence has a 42‐amino‐acid‐residue insertion predicted to reside in the L loop of the subunit.
Heat‐induced synthesis and tunicamycin‐sensitive secretion of the putative storage glycoprotein conglutin γ from mature lupin seeds
Authors: DURANTI Marcello , SCARAFONI Alessio , GIUS Cristina , NEGRI Armando , FAORO Franco
Published: March 2005

SDS/PAGE, immune blotting with specific antibodies and amino acid sequence analyses revealed that 90% of the protein released from Lupinus albus seeds incubated in water at 60°C for about 3 h was conglutin γ, a putative storage glycoprotein already present in the protein bodies of mature seeds. Incorporation of [14C]leucine into the protein demonstrated that conglutin γ was newly synthesized during the treatment and the use of protein synthesis inhibitors ruled out the secretion of constitutive conglutin γ. Synthesis and secretion took place only over a narrow temperature range, 57.5–62.5°C, and in a short time interval, 135–180 min, of incubation of the seed. The amount of secreted conglutin γ, i.e. 1 mg/seed, was about three times that present inside the treated or untreated seed.

Secreted conglutin γ contained covalently linked carbohydrate as well as the constitutive protein. Inhibition of the glycosylation by tunicamycin did not affect conglutin γ synthesis, but prevented its secretion from the seed, as indicated by quantifying conglutin γ remaining in the seed. An accumulation of the protein outside the protein bodies and at the cotyledonary cell periphery was shown in these samples by immunocytochemistry.

Peptide mapping of the fragments obtained by incubation of constitutive and secreted conglutin γ with trypsin and pepsin revealed no difference between the two proteins.

Lupin seeds were still viable after the treatment. However no similarities between conglutin γ and heat‐shock proteins were observed either in the amino acid sequence or other molecular features.
Interactions of lipoprotein lipase with the active‐site inhibitor tetrahydrolipstatin (Orlistat)R
Authors: LOOKENE Aivar , SKOTTOVA Nina , OLIVECRONA Gunilla
Published: March 2005

Lipoprotein lipase (LPL) was rapidly inactivated by low concentrations of the active‐site inhibitor tetrahydrolipstatin (THL). The presence of amphiphils (e.g. long‐chain fatty acids) or of lipid/water interfaces (lipid emulsions) was required for inhibition to occur. Apolipoprotein CII increased the maximal inactivation rate constant by 1.8‐fold in the presence of an emulsion of long‐chain triacylglycerols, but had no effect in the presence of an emulsion of tributyrylglycerol. The fully inhibited enzyme had a ratio of THL/LPL of nearly 2, indicating that both subunits of the LPL homo‐dimer bound THL. The THL‐LPL complex was stable below pH 7.5. At higher pH reactivation occurred indicating that THL was slowly turned over by the enzyme. The apparent reactivation rate constant was increased about threefold by the presence of lipid/water interfaces.

Sucrose density gradient centrifugation revealed that THL induces tetramerisation of LPL. This aggregation was reversible on reactivation of the inhibited enzyme. Binding to heparin was not affected by THL. In contrast, binding to lipid droplets and to lipoproteins was increased, indicating exposure of hydrophobic regions in the inhibited LPL. It is suggested that THL induces local conformational changes in LPL, which may involve opening of the putative surface lid structure which covers the active‐site.
Identification of functional sites on bovine leukemia virus envelope glycoproteins using structural and immunological data
Authors: CALLEBAUT Isabelle , PORTETELLE Daniel , BURNY Arsène , MORNON Jean‐Paul
Published: March 2005

Sequence analysis using the sensitive hydrophobic cluster analysis method shows that the bovine leukemia virus envelope glycoproteins conserve the general organization of the influenza hemagglutinin into a ‘stem’, containing the external part of the transmembrane glycoprotein and the N‐terminal and C‐terminal parts of the external glycoprotein, and a ‘head’, containing only external glycoprotein residues. However, our analysis suggests, for the first time, that the bovine leukemia virus envelope head will not adopt the typical ‘jelly‐roll’ fold of the influenza A hemagglutinin head, but most likely folds into another type of ‘Greek‐key’ structure corresponding to the overall topology of constant immunoglobulin domains. We constructed a three‐dimensional model for the bovine leukemia virus envelope head by homology modeling using the crystal structure of the human histocompatibility antigen HLA‐A2 α3 domain. Furthermore, we propose a general model for the oligomeric organization of this head, based on the hemagglutinin trimer. The proposed structural organization of bovine leukemia virus external glycoprotein is further supported by antipeptide and monoclonal antibody reactivities. Our modeling study suggests that the loops of the two neutralizing peptides located in the head are adjacent at the top of the domain and define a potential interaction site of the external glycoprotein with its cellular receptor. This site is topologically similar to the binding site of hemagglutinin with its cellular receptor, sialic acid. The other neutralizing peptides are located within a small domain linking the head to the stem.

These data are of interest for defining other oncoviral glycoproteins heads and receptor‐binding sites.
O‐glycan biosynthesis in human colorectal adenoma cells during progression to cancer
Authors: VAVASSEUR Fabienne , DOLE Kiran , YANG Jimmy , MATTA Khushi L. , MYERSCOUGH Neil , CORFIELD Anthony , PARASKEVA Christos , BROCKHAUSEN Inka
Published: March 2005

A human colonic adenoma cell line PC/AA derived from a familial polyposis coli patient was passaged in culture to form an intermediate premalignant clonogenic variant AA/C1 and, upon treatment with differentiating and carcinogenic agents, a cell line AA/C1/SB10 which is tumourigenic in nude mice. These three mucin‐secreting cell lines have been used as a model to study the changes in O‐glycan biosynthesis during the progression to cancer. Several glycosyltransferases involved in the synthesis, elongation and termination of the common O‐glycan core structures were found to decrease in the progression sequence towards adenocarcinoma. Higher activity of a number of enzymes was seen in the intermediate cell line. O‐glycan biosynthesis in the original PC/AA cell line was closest to the normal human colonic phenotype, since all four common mucin O‐glycan cores and their extended structures could be synthesized; core 3 β3‐GlcNAc‐transferase and α6‐sialyltransferase acting on GalNAc‐mucin were still detectable and core 2 β6‐GlcNAc‐transferase activity was accompanied by core 4 and I β6‐GlcNAc‐transferase activities. During progression towards adenocarcinoma, the expression of α6‐sialyltransferase, core 3 β3‐GlcNAc‐transferase, core 4 and I β6‐GlcNAc‐transferases were turned off. Using monoclonal antibodies, Tn antigen, sialyl‐Tn antigen, O‐acetyl‐sialomucin and sialyl‐Lea determinants were not detected in secreted or cellular mucin isolated from any of the cell lines. The exposure of MUC1 epitopes was seen in the malignant line, whereas sialyl‐Lex determinants were found only in the premalignant PC/AA line. Sulfotransferase activities using core 1 substrate, Galβ1–3GalNAcα‐benzyl, were high in PC/AA cells and progressively decreased upon development to adenocarcinoma, and this decrease correlated with mucin sulfation. In summary, the synthesis of less abundant, sialylated, fucosylated and extended, unbranched core 1 structures should be facilitated in the malignant cells. This is the first report of glycosyltransferase changes in human premalignant cells developing to tumourigenic cells. The data demonstrate that these cell lines are an excellent model to study the changes and regulation of mucin oligosaccharide biosynthesis during progression to cancer.
Effect of low pH and heparin on the structure of acidic fibroblast growth factor
Authors: PINEDA‐LUCENA Antonio , NÚÑEZ DE CASTRO Ignacio , LOZANO Rosa M. , MUÑOZ‐WILLERY Isabel , ZAZO Mercedes , GIMÉNEZ‐GALLEGO Guillermo
Published: March 2005

Changes in the fluorescence of the single tryptophan of acidic fibroblast growth factor have been used to monitor the effect of low pH on the conformation of the molecule, and the consequences of heparin binding and high ionic strength under such conditions. These studies demonstrate that the conformation of the protein changes reversibly below pH 5, and that heparin, depending on the conditions, may either prevent that change or induce a new irreversible modification of the structure, which runs parallel to the partial inactivation of the protein. It is also demonstrated that secondary heparin‐binding sites appear at low pH, which favor the formation of precipitates at some protein/heparin ratios. Precipitation and inactivation of fibroblast growth factor at low pH may hinder its wound‐healing activity, since acidification seems frequent in wounds.
Stability and structural analysis of α‐amylase from the antarctic psychrophile Alteromonas haloplanctis A23
Authors: FELLER Georges , PAYAN Françoise , THEYS Fabienne , QIAN Minxie , HASER Richard , GERDAY Charles
Published: March 2005

The α‐amylase secreted by the antarctic bacterium Alteromonas haloplanctis displays 66% amino acid sequence similarity with porcine pancreatic α‐amylase. The psychrophilic α‐amylase is however characterized by a sevenfold higher kcat and kcat/Km values at 4°C and a lower conformational stability estimated as 10 kJ · mol−1 with respect to the porcine enzyme. It is proposed that both properties arise from an increase in molecular flexibility required to compensate for the reduction of reaction rates at low temperatures. This is supported by the fast denaturation rates induced by temperature, urea or guanidinium chloride and by the shift towards low temperatures of the apparent optimal temperature of activity.

When compared with the known three‐dimensional structure of porcine pancreatic α‐amylase, homology modelling of the psychrophilic α‐amylase reveals several features which may be assumed to be responsible for a more flexible, heat‐labile conformation: the lack of several surface salt bridges in the (β/α)8 domain, the reduction of the number of weakly polar interactions involving an aromatic side chain, a lower hydrophobicity associated with the increased flexibility index of amino acids forming the hydrophobic clusters and by substitutions of proline for alanine residues in loops connecting secondary structures. The weaker affinity of the enzyme for Ca2+ (Kd= 44 nM) and for Cl− (Kd= 1.2 mM at 4°C) can result from single amino acid substitutions in the Ca2+‐binding and Cl−‐binding sites and can also affect the compactness of α‐amylase.
Kinetics study of the oxidation of 4‐tert‐butylphenol by tyrosinase
Authors: ROS José Ramón , RODRÍGUEZ‐LÓPEZ José Neptuno , VARÓN Ramón , GARCÍA‐CÁNOVAS Francisco
Published: March 2005

The reaction between 4‐tert‐butylphenol (BuPhOH) and mushroom tyrosinase was investigated by following 4‐tert‐butyl‐ortho‐benzoquinone, whose high stability permits the reaction to be used as a model for the study of the monophenolase acfivity of tyrosinase. The system evolves to a pseudo‐steady state through an induction period (τ), the pseudo‐steady‐state rate (Vss) decreasing when the (BuPhOH) concentration increases. Increases in enzyme concentration result in a parabolic pattern with Vss, while τ is shortened. The addition of increasing catalytic amounts of 4‐tert‐butylcatechol at the start of the reaction reduces τ until it is totally abolished, an initial burst being observed at high 4‐t‐butylatechol concentrations. Initial bursts are also obtained at pH 4.5 or lower, indicating a lower affinity of the met‐tyrosinase or oxidized form for the monophenol at low pH. These experimental results can be explained by the reaction mechanism of tyrosinase.
Relationship between water activity and catalytic activity of lipases in organic media
Authors: VALIVETY Rao H. , HALLING Peter J. , PEILOW Alan D. , MACRAE Alasdair R.
Published: March 2005

The rates of synthesis of dodecyl decanoate in hexane have been measured as a function of water activity (aw), for various immobilised preparations of the lipases from Rhizomucor miehei and Candida rugosa. Only very large changes in the amount of enzyme adsorbed to the support affect the shape of the rate/aw profile; at the highest loadings the profiles tend to become somewhat flatter. A similar levelling can be obtained by pre‐adsorbing an inert protein. The effect is probably due to adjacent protein molecules effectively replacing water; it does not simply reflect mass transfer or interfacial area limitation. The activity/aw profile was essentially the same with most supports tested: polypropylene, anion‐exchange resin, celite, anion‐exchange modified silica. A hydrophobic porous glass support reduced the rate somewhat at intermediate aw values with both enzymes; a polyamide material had this effect only with the lipase from Rh. miehei. The shape of the activity/aw profile was not affected by large differences in purity of the lipase preparation, but did differ between forms that probably differ in glycosylation. Overall, relatively few manipulations of the system can significantly affect the shape of the rate/aw profiles, which seem to be mainly an intrinsic property of the enzyme molecules used.
Deletion analysis of the m4 muscarinic acetylcholine receptor
Authors: VAN KOPPEN Chris J. , SELL Alexandra , LENZ Wolfgang , JAKOBS Karl H.
Published: March 2005

In order to investigate whether coupling to and/or activation of guanine‐nucleotide‐binding proteins (G proteins) is involved in agonist‐induced internalization of m4 muscarinic acetylcholine receptors (mAChRs), a deletion mutant [des‐(264–394)mAChR] was constructed that lacks a substantial portion of the putative third intracellular loop. The wild‐type receptor and des‐(264–394)mAChR stably expressed in Chinese hamster ovary cells in essentially comparable amounts, exhibited identical antagonist‐binding affinities. Consistent with the reported importance of the third cytoplasmic loop for Gi protein activation, the des‐(264–394)mAChR showed a drastically reduced potential to mediate agonist‐induced inhibition of adenylyl cyclase. In contrast, the ability of the mutant receptor to couple to Gi proteins was not impaired, as demonstrated by a similar guanine‐nucleotide‐sensitive and pertussis‐toxin‐sensitive high‐affinity agonist‐receptor binding for both mAChRs. In contrast, des‐(264–394)mAChR was hardly able to stimulate the GTPase activity of G proteins, suggesting impaired activation of Gi proteins rather than ineffective coupling to Gi proteins. Internalization of wild‐type receptor and des‐(264–394)mAChR was observed with similar agonist concentrations and showed similar maximal values. However, des‐(264–394)mAChR displayed a significantly reduced rate of receptor internalization. A similar attenuation of wild‐type mAChR internalization was obtained upon pertussis toxin treatment. In conclusion, our data provide evidence that the molecular determinants of the m4 mAChR involved in Gi‐protein coupling and activation are not identical and that activation of, but not coupling to, Gi proteins regulates m4 mAChR internalization.
Comparison of the solution structures of the chimeric peptides galanin(1–12)‐Ala‐neuropeptide Y(25–36)amide and galanin(1–12)‐Pro‐neuropeptide Y(25–36)amide
Authors: ARVIDSSON Klas , LANGEL Ülo , EHRENBERG Anders
Published: March 2005

In a previous study [Arvdsson, K., Land, T., Langel, Ü., Bartfai, T. & Ehrenberg, A. (1993) Biochemistry 32, 7787–7798], the 25‐amino‐acid‐residue chimeric peptide M32, galanin(1–12)‐Pro‐neuropeptide Y(25–36)amide, was found to bind very strongly to galanin receptors and also to have considerable affinity to neuropeptide Y receptors. The solution structure of M32 in 30% (by vol.) 1,1,1,3,3,3‐hexafluoro‐2‐propanol, was determined from structural restraints obtained by two‐dimensional 1H‐NMR. Another peptide, M88, with Ala instead of Pro in position 13, was shown to bind with tenfold lower affinity to galanin receptors, i.e. with nearly the same affinity as galanin itself. The binding to neuropeptide Y receptors was altered in the opposite sense. The peptide M88 has now been examined by two‐dimensional 1H‐NMR under the same conditions as applied for M32. Using NMR‐derived restraints, we have calculated structures of M88 by means of the progarms CALIBA, HABAS and DIANA, and refined them by restrained energy minimisation and restrained molecular dynamics. The use of CALIBA and HABAS meant that the restraints were less restrictively formulated, than in the previous work. Hence, structures of M32 were recalculated in the same way as for M88, also including some additional restraints, possible to identify by comparison of the NMR spectra of the two peptides. The new structures of M32 confirm an α‐helix starting at Pro13, but now continuing until Gln23, instead of ending at IIe20. Conversely, no secondary structure is now expressed in the N‐terminal section. It is concluded that in the case of peptides the details of the calculated structures depend on how the restraints are calibrated. In M88, an α‐helix is found over a long section, approximately Ser6‐Gln23. Possible correlations between the binding affinities to receptors and the solution structures of the peptides M32 and M88 are discussed. This is done with particular reference to the length and stability of the α‐helix and the flexibility of the N‐terminal section and its bending direction versus the helix.
The introduction of a negative charge into the hydrophobic patch of Pseudomonas aeruginosa azurin affects the electron self‐exchange rate and the electrochemistry
Authors: VAN POUDEROYEN Gertie , MAZUMDAR Shyamalava , HUNT Nick I. , HILL H. Allen O. , CANTERS Gerard W.
Published: March 2005

By changing Met64 into a glutamate by means of site‐directed mutagenesis a negative charge was introduced into the hydrophobic patch of azurin from Pseudomonas aeruginosa. The three‐dimensional structure of the protein and the structure of the metal site in particular, appear unaffected by the mutation. The observed change of the midpoint potential of the mutant of 28 mV is ascribed to the deprotonation of Glu64. The electron‐self‐exchange rate constant equals that of the wild‐type protein at pH 4.5 but decreases by almost two orders of magnitude at high pH. Electron transfer is inhibited only when both of the reacting azurin molecules have an ionized glutamate at position 64 in their hydrophobic patch. Electron transfer at a graphite electrode is slowed down by the presence of the negative charge in the hydrophobic patch. The results demonstrate once again that the Cu‐ligand His117 in the hydrophobic patch is the likely entry and exit point for electrons. This observation holds both for the (homogeneous) electron‐self‐exchange reaction in solution as well as for the (heterogenous) reaction at an electrode.
Bovine retinal rod guanyl cyclase represents a new N‐glycosylated subtype of membrane‐bound guanyl cyclases
Authors: KOCH Karl‐Wilhelm , STECHER Patricia , KELLNER Roland
Published: March 2005

The molecular properties of retinal rod guanyl cyclase were investigated. Peptides were derived from a 112‐kDa protein previously identified as the particulate bovine retinal rod guanyl cyclase. The peptides showed 100% identity to the deduced amino acid sequence of the cloned human retina‐specific membrane guanyl cyclase, whereas identity to the members of the natriuretic peptide receptor guanyl cyclases was 14–59%. The 112‐kDa protein was further purified by a new approach using wheat‐germ agglutinin chromatography. This indicated N‐linked glycosylation in retinal rod guanyl cyclase. N‐glycosylation was unexpected from the sequence of the human retina‐specific membrane guanyl cyclase, although it is a common property of natriuretic peptide receptors. Therefore, we further analyzed the carbohydrate composition of bovine retinal rod guanyl cyclase by lectin binding using the lectins Galanthus nivalis agglutinin, Sambucus nigra agglutinin, Maackia amurensis agglutinin, Ricinus communis agglutinin, Datura stramonium agglutinin, peanut agglutinin and by chromatography of the purified enzyme using concanavalin‐A‐Sepharose. The oligosaccharide side chains were of the high‐mannose type or hybrid type, probably with mannose, N‐acetylglucosamine and sialic acid as terminal sugars. Enzymic deglycosylation by N‐glycosidase F was achieved after proteolytic digestion with endoproteinase Glu‐C. Lectins neither influenced the basal nor the stimulated guanyl‐cyclase activity at low calcium concentrations. Our results indicate that the particulate rod guanyl cyclase represents an unusual new subtype of membrane‐bound guanyl cyclases.
Different mechanisms of regulation of the human stromelysin and collagenase genes
Authors: YANG Maozhou , KURKINEN Markku
Published: March 2005

Tissue‐remodeling processes are largely controlled by matrix metalloproteinases that degrade the extracellular components of connective tissues. In this study, gene regulation of two human matrix metalloproteinases, stromelysin and collagenase, was investigated by a reverse‐transcription‐coupled (RT)‐PCR assay. Here, signals from both the heterogenous nuclear RNA (hnRNA) and mRNA are amplified, allowing the regulation of gene expression to be divided between transcriptional and/or post‐transcriptional control. In confluent human lung fibroblast cultures, tumor‐necrosis factor‐α and 12‐O‐tetradecanoyl‐phorbol 13‐acetate induce stromelysin and collagenase genes transcriptionally. Interleukin‐1β (IL‐1β) induces stromelysin gene transcription but has little, if any, effect on the collagenase gene transcription in cells cultured in the presence of 10% serum. By a competitive RT‐PCR assay, the IL‐1β‐reated cultures contain an average of 60 molecules of stromelysin mRNA/cell and the untreated cultures about 1.9 molecules/cell. In serum‐starved cells, both IL‐1β and serum induce transcription of the collagenase gene. Also, in serum‐starved cells type II collagen can induce collagenase mRNA but not stromelysin mRNA. Inhibition of protein synthesis with cycloheximide induces stromelysin gene transcription but has no effect on the collagenase gene. These data indicate different mechanisms of regulation of regulation of the Human stromelysin and collagenase genes in cultured cells.
Structural and spectral characterisation of the antenna complexes of Rhodocyclus gelatinosus
Authors: BRUNISHOLZ René A. , SUTER Franz , ZUBER Herbert
Published: March 2005

The core antenna/reaction‐centre complex RC‐B875 and the peripheral antenna complex B800–850 of the two strains DSM 149 and DSM 151 of the purple non‐sulphur bacterium Rhodocyclus gelatinosus have been isolated from photosynthetic membranes by means of lauryl‐N,N‐dimethyl‐amineoxide as a detergent and subsequent sucrose‐gradient centrifugation. The two complexes were characterised spectroscopically by absorption and circular dichroism (CD) spectroscopy at room temperature. CD measurements revealed very weak signals for the core antenna B875 whereas for the peripheral antenna B800–850, a strong biphasic CD signal was observed, attributable to the B850 pigments. There is apparently no CD signal present for the B800 pigments. The core and the peripheral antenna complex are built up by a distinct α/β‐polypeptide pair. The pigment/protein ratio in the peripheral antenna complex is 3 bacteriochlorophyll/(α/β)‐polypeptide pair. The amino acid sequences of the α and β polypeptides of both complexes from the two strains of Rc. gelatinosus were established by automated Edman degradation, chemical and enzymic digestion, amino acid composition analyses and carboxypeptidase digestion. In the case of the β polypeptides, the amino acid sequence determination was confirmed by ion‐spray MS of the isolated antenna apoproteins. The inter‐strain (DSM 149 and 151) positional identity between the equivalent apoproteins is extremely large and varies in the range 90–100%. The B875‐β polypeptide from Rc. gelatinosus exhibits shortened C‐termini, as detected for the analogous antenna apoproteins of Rhodobacter sphaeroides and Rhodobacter capsulatus, which can be correlated with weak core antenna near‐infrared CD signals. However, the B800–850‐α polypeptide of Rc. gelatinosus, with 71 amino acids, exhibits an extended C‐terminal portion indicative of the formation of a second transmembrane domain, which so far has not been observed for bacterial antenna apoproteins. This part of the molecule is extremely rich in alanine and proline residues. All the sequenced antenna apoproteins of Rc. gelatinosus exhibit a characteristic membrane‐buried histidine which is thought to ligate the B875 or the B850 pigments. In the B800–850‐β apoprotein, a second, so far β‐antenna‐apoprotein‐specific histidine, is replaced by a glutamine residue. A careful inspection of the determined antenna structures of Rc. gelatinosus revealed some remarkable structural similarities within presumed cofactor‐binding sites of Fe‐S‐type‐reaction‐centre apoproteins, indicating possible basic structural motifs for complexing bacteriochlorophyll molecules.
A liquid‐phase binding analysis for L‐selectin
Authors: YOSHIDA Tomoaki , FENNIE Christopher , LASKY Laurence A. , LEE Yuan C.
Published: March 2005

L‐selectin, together with E‐ and P‐selectins, forms a newer group of cell adhesion molecules which are believed to interact with carbohydrate ligands [Lasky, L. A., Singer, M. S., Yednoch, T. A., Dowbenko, D., Fennie, C., Rodriguez, H., Nguyen, T., Stachel, S. & Rosen, S. D. (1989) Cell 56, 1045–1055]. Using radiolabeled fucoidan as a reference ligand, we have developed a new liquid‐phase microcentrifugation assay where fine differences in binding affinity can be compared accurately. We found that glucan sulfates strongly inhibited the binding of fucoidan by murine L‐selectin‐IgG chimera. The efficacy of inhibition is extremely dependent on the size (up to 12 kDa) and the sulfate density (up to two sulfate groups/glucose molecule) of the glucan sulfates. The nature of the inter‐glucose linkages is also important. These data suggest that the binding by L‐selectin prefers certain clustering and proper spatial arrangement of the anionic groups.
Processing of the precursors of the human thyroid‐stimulating hormone receptor in various eukaryotic cells (human thyrocytes, transfected L cells and baculovirus‐infected insect cells)
Authors: MISRAHI Micheline , GHINEA Nicolae , SAR Sokhavuth , SAUNIER Bertrand , JOLIVET André , LOOSFELT Hugues , CERUTTI Martine , DEVAUCHELLE Gérard , MILGROM Edwin
Published: March 2005

The complementary DNA for human thyroid‐stimulating hormone (TSH) receptor encodes a single protein with a deduced molecular mass of 84.5 kDa. This protein is cleaved during its maturation in the human thyroid since the receptor protein has been shown to be composed of two subunits (α subunit of ∼ 53 kDa and β subunit of ∼ 38 kDa) held together by disulfide bridges [Loosfelt, H., Pichon, C., Jolivet, A., Misrahi, M., Caillou, B., Jamous, M., Vannier, B. & Milgrom, E. (1992) Proc. Natl Acad. Sci. USA 89, 3765–3769]. A similar processing occurs in an L cell line permanently expressing the human TSH receptor. The processing is however incomplete, resulting in a permanent accumulation of a 95‐kDa high‐mannose precursor which is present only in trace amounts in the thyroid. Pulse‐chase experiments show the successive appearance in the L cells of two precursors: initially the ∼ 95‐kDa high‐mannose glycoprotein followed by a ∼ 120‐kDa species containing mature oligosaccharides. This latter precursor is then processed into the α and β subunits. In primary cultures of human thyrocytes precursors of similar size are detected.

Spodoptera frugiperda insect cells (Sf9 and Sf21) infected with a recombinant baculovirus encoding the human TSH receptor synthesize a monomeric protein of about 90 kDa soluble only in denaturing conditions. Comparison with the product of in vitro transcription‐translation experiments (∼ 80 kDa), suggests that it may be incompletely or improperly glycosylated. The TSH receptor expressed in these cells is unable to bind the hormone.

Immunoelectron microscopy studies show that in human thyrocytes most of the receptor is present on the cell surface; in L cells the receptor is detected on the cell surface, as well as in the endoplasmic reticulum and in the Golgi apparatus (this intracellular pool of receptor molecules probably corresponding to the high‐mannose precursor); in insect cells nearly all the receptor molecules are trapped in the endoplasmic reticulum. These differences in receptor distribution are concordant with the differences observed for receptor processing.
Properties of cobalt‐substituted bovine serum amine oxidase
Authors: AGOSTINELLI Enzo , MORPURGO Laura , WANG Changqing , GIARTOSIO Anna , MONDOVÌ Bruno
Published: March 2005

Half‐copper‐depleted and fully copper‐depleted amine oxidase from bovine serum were reconstituted with either copper or cobalt. All samples were studied by high‐sensitivity scanning calorimetry, by enzyme activity analysis, and by reactivity with phenylhydrazine. The calorimetric profile of the protein was strongly modified by the removal of a single Cu ion approximately to the same extent as by complete copper removal, in agreement with the loss of over 80% enzymic activity. The thermograms of metal‐reconstituted species showed a marked similarity with that of the native enzyme, irrespective of whether copper or cobalt was present. Reactivity with phenylhydrazine and enzymic activity measurements showed that in cobalt‐substituted amine oxidase the organic cofactor was reactive and the enzyme was catalytically competent, although kinetically less efficient. These observations agree both with previous findings on the protein half‐site reactivity and with previous suggestions for a copper conformational role in bovine serum amine oxidase, namely of maintaining a functional conformation at the active site.
Cross‐reconstitution studies with polypeptides of Escherichia coli and bovine heart mitochondrial FoF1 ATP synthase
Authors: ZANOTTI Franco , GUERRIERI Ferruccio , DECKERS‐HEBESTREIT Gabriele , FIERMONTE Maria , ALTENDORF Karlheinz , PAPA Sergio
Published: March 2005

To characterize the role of supernumerary subunits of the mammalian FoF1 ATP synthase, crossreconstitution of mitochondrial and bacterial FoF1 complexes has been carried out. Escherichia coli F1 (EcF1) can be reconstituted with F1‐stripped everted membranes of E. coli (UPEc) and of bovine heart mitochondria (USMP). Bovine heart mitochondrial F1 (BHF1) can also be reconstituted with both membranes. Both EcF1 and BHF1, when reconstituted with UPEc, exhibited oligomycin‐insensitive ATP‐hydrolase activity. Subunits of the mammalian Fo, in particular FoI‐PVP protein, F6 and oligomycin‐sensitivity‐conferring protein (OSCP) conferred oligomycin sensitivity to the catalytic activity of EcF1 or BHF1 reconstituted with UPEc. Reaction of N, N′‐dicyclohexylcarbodiimide and development of inhibition of passive H+ conduction was, in UPEc, considerably slower and exhibited a lower apparent affinity than in USMP. The ATP hydrolase activity of UPEc+EcF1 or UPEc+BHF1 was, also, less sensitive to inhibition by N, N′‐dicyclohexylcarbodiimide than USMP+EcF1 or USMP+BHF1. Addition of mitochondrial FoI‐PVP to UPEc enhanced the sensitivity of H+ conduction to oligomycin. FoI‐PVP and OSCP added to UPEc, promoted inhibition by N, N′‐dicyclohexylcarbodiimide of passive H+ conduction and increased its binding affinity to subunit c of E. coli Fo. The presence of FoI‐PVP and OSCP also promoted inhibition by N, N′‐dicyclohexylcarbodiimide of the ATP‐hydrolase activity of EcF1 or BHF1 reconstituted with UPEc.
Two human antibodies reacting with different epitopes on integrin β3 of platelets and endothelial cells
Authors: JALLU Vincent , DIAZ‐RICART Maribel , ORDINAS Antonio , PICO Marta , VEZON Gérard , NURDEN Alan T.
Published: March 2005

Glanzmann's thrombasthenia is an inherited bleeding disorder that results from a deficit of glycoprotein (GP) IIb‐IIIa complexes in platelets. Patient (EBV) is an adult male with GP IIb‐IIIa levels <5% of normal values and a history of blood transfusions. Western‐blot analysis revealed a strong IgG antibody to GP IIIa in his plasma. The determinants were localized to the minimumsized fragment of GP IIIa (50 kDa) retained on chymotrypsin‐treated platelets and were lost on reduction of disulphides. A female patient (AF), previously described by us [Jallu, V., Pico, M., Chevaleyre, J., Vezon, G., Kunicki, T. J. & Nurden, A. T. (1992) Hum. Antibod. Hybridomas 3, 93–106] developed her anti‐GP‐IIIa antibody during pregnancy. This antibody was poorly reactive with the 50‐kDa proteolytic fragment, yet bound to 115‐kDa and 60‐kDa hydrolytic products of GP IIIa. Antibodies from both patients recognized the GP‐IIIa‐like protein of endothelial cells, thus confirming that they were directed against the integrin β3‐subunit. The (EBV) antibody reacted strongly with GP IIb‐IIIa in an antigen capture assay performed with each of a panel of four murine monoclonal antibodies (mAbs) recognizing different epitopes on GP IIb‐IIIa. In contrast, that from (AF) was specifically inhibited by AP‐3, a murine mAb whose epitope is thought to be localized between amino acids 324–422 of GP IIIa. The residual GP IIb and GP IIIa contents of platelets from each patient were assessed in Western blotting using chemiluminescence detection. SZ‐22, a murine mAb to the GP IIb heavy chain (140 kDa), located small amounts of a 130‐kDa protein in (EBV) platelets. The anti‐GP IIIa mAbs XII F9, P 37 and P 97 revealed trace amounts of protein with a relative mobility identical to that of GP IIIa in both (AF) and (EBV) platelets. This residual GP IIIa represented less than 0.5% of the amount in normal platelets. When, for each patient, plasma was tested in Western blotting against their own platelets, autoantibody activity to the residual GP IIIa was detected in both cases. Thus, patients (AF) and (EBV) have developed anti‐GP‐IIIa antibodies with restricted and distinct epitopes but recognizing self antigens.
DNA replication in vitro by recombinant DNA‐polymerase‐α‐primase
Authors: STADLBAUER Franz , BRUECKNER Andrea , REHFUESS Christoph , ECKERSKORN Christoph , LOTTSPEICH Friedrich , FÖRSTER Verena , TSENG Ben Y. , NASHEUER Heinz‐Peter
Published: March 2005

DNA‐polymerase‐α‐primase complex contains four subunits, p180, p68, p58, and p48, and comprises a minimum of two enzymic functions. We have cloned cDNAs encoding subunits of DNA‐polymerase‐α‐primase from human and mouse. Sequence comparisons showed high amino acid conservation among the mammalian proteins. We have over‐expressed the single polypeptides and co‐expressed various subunit complexes using baculovirus vectors, purified the proteins and investigated their biochemical properties. The purified mouse p48 subunit (Mp48) alone had primase activity. Purification of co‐expressed Mp48 and Mp58 subunits yielded stable DNA primase of high specific activity. Co‐expression of all four subunits yielded large quantities of tetrameric DNA‐polymerase‐α‐primase. The p180, p58 and p48 polypeptides were also co‐expressed and immunoaffinity purified as a trimeric enzyme complex. The tetrameric and trimeric DNA‐polymerase‐α‐primase complexes showed both DNA primase and DNA polymerase activities. The tetrameric recombinant DNA‐polymerase‐α‐primase synthesized double‐stranded M13 DNA and replicated polyoma viral DNA in vitro efficiently.
Large‐scale purification and further characterization of rat pristanoyl‐CoA oxidase
Authors: VAN VELDHOVEN Paul P. , VAN ROMPUY Patricia , FRANSEN Marc , DE BÉTHUNE Bernadette , MANNAERTS Guy P.
Published: March 2005

The elution of pristanoyl‐CoA oxidase from butyl‐Sepharose required unusually high concentrations of ethylene glycol, enabling the large‐scale purification of this oxidase in a single chromatographic step. The enzyme, the native molecular mass of which was estimated previously at 415 kDa by gel filtration (Van Veldhoven, P. P., Vanhove, G., Vanhoutte, F., Dacremont, G., Eyssen, H. J. & Mannaerts, G. P. (1991) J. Biol. Chem. 266, 24676–24683), migrated as a 513‐kDa protein during native gel electrophoresis. It showed a typical flavoprotein spectrum and probably binds 4 mol FAD/mol enzyme. Its amino acid composition was different from those of other known acyl‐CoA oxidases. Screening of different rat tissues, either for enzyme activity or by immunoblotting, revealed the highest level of pristanoyl‐CoA oxidase in liver, followed by kidney, intestinal mucosa, spleen and lung. The oxidase activities, measured with 2‐methylpalmitoyl‐CoA as the substrate, in livers from other vertebrates including man were low compared to rat. This was also confirmed by immunoblotting which provided a clear signal only in rat liver, possibly indicating that pristanoyl‐CoA oxidase might be a rat‐specific oxidase.
ATP synthase of yeast mitochondria
Authors: GIRAUD Marie‐France , VELOURS Jean
Published: March 2005

The δ‐subunit was isolated from the purified yeast F1. Partial protein sequences were determined by direct methods. From this information, degenerated primers were constructed. A part of the ATPδ gene was amplified by polymerase chain reaction from yeast genomic DNA. From the amplified DNA sequence, a nondegenerated oligonucleotide probe was constructed to isolate a 2.6‐kbp BamHI‐EcoRI DNA fragment bearing the whole gene. A 1036‐bp DraI fragment was sequenced. A 480‐bp open reading frame encoding a 160‐amino‐acid polypeptide is described. The deduced amino acid sequence is 22 amino acids longer than the mature protein, which is 138 amino acids long with a mass of 14555 Da. The δ‐subunit of Saccharomyces cerevisiae is 21%, 35%, 52% identical and 66%, 61% and 92% similar to the ɛ‐subunit of Escherichia coli and the δ‐subunits of beef heart and Neurospora crassa, respectively. A null mutant was constructed. The mutation was recessive and dramatically affected mitochondrial DNA stability since the transformed cells were 100% cytoplasmic petite. The double mutant (rho−, ATPδ::URA3) displayed low or no ATPase activity with an unstable catalytic sector, since a polyclonal antibody directed against the β subunit did not coprecipitate the α subunit.
An investigation on the role of vacuolar‐type proton pumps and luminal acidity in calcium sequestration by nonmitochondrial and inositol‐1,4,5‐trisphosphate‐sensitive intracellular calcium stores in clonal insulin‐secreting cells
Authors: BODE Hans‐Peter , EDER Bernadette , TRAUTMANN Michael
Published: March 2005

To test whether in RINm5F rat insulinoma cells luminal acidity and the activity of a vacuolar‐type proton pump are involved in calcium sequestration by intracellular calcium stores sensitive to inositol 1,4,5‐trisphosphate (InsP3) we examined the effects of various proton‐conducting ionophores and ammonium chloride, and of bafilomycin, a specific inhibitor of vacuolar proton pumps, on this parameter. Bafilomycin in concentrations up to 1 μM did not affect calcium sequestration by nonmitochondrial, InsP3‐sensitive stores at all; 50 μM carbonylcyanide m‐chlorophenylhydrazone, 50 μM monensin and 30 mM NH4Cl, which are diverse ways to dissipate transmembrane pH gradients, did not inhibit calcium sequestration. This argues against significant involvement of internal acidity and vacuolar proton pumps in calcium sequestration by InsP3‐sensitive stores in RINm5F cells. The proton—potassium‐exchanging ionophore nigericin (20–100 μM), however, inhibited calcium sequestration by nonmitochondrial and InsP3‐sensitive stores. This effect was dependent on the presence of potassium and could be reversed by inclusion of carbonylcyanide m‐chlorophenylhy‐drazone or acetate in the incubation medium. Thus, the inhibitory effect of nigericin appears to be based on proton extrusion coupled to potassium influx across the membrane of calcium stores in RINm5F cells, creating an internal alkalinization of these stores. The effect of nigericin implies the continuous maintenance of an outside‐to‐inside potassium concentration gradient by nonmitochondrial calcium stores in RINm5F cells. This feature will be of potential interest in the identification of InsP3‐sensitive calcium‐storing organelles.
Catabolite inactivation of heterologous fructose‐1,6‐bisphosphatases and fructose‐1,6‐bisphosphatase‐β‐galactosidase fusion proteins inSaccharomyces cerevisiae
Authors: GAMO Francisco‐Javier , NAVAS M. Angeles , BLAZQUEZ Miguel A. , GANCEDO Carlos , GANCEDO Juana M.
Published: March 2005

Fructose‐1,6‐bisphosphatase (FruP2ase) fromSaccharomyces cerevisiaeis rapidly inactivated upon addition of glucose to a culture growing on non‐sugar carbon sources. Under the same conditions the FruP2ases fromSchizosaccharomyces pombeorEscherichia coliexpressed inS. cerevisiaewere not affected. A chimaeric protein confaining the first 178 amino acids from the N‐terminal half ofS. cerevisiaeFruP2ase fused toE. coliβ‐galactosidase was susceptible to catabolite inactivation. Elimination of a putative destruction box, RAELVNLVG…KK….K., beginning at amino acid 60 did not prevent catabolite inactivation. Similarly a change of the vacuole‐targeting sequence QKKLD, amino acids 80–84, to QKNSD did not affect significantly the course of inactivation of β‐galactosidase. A fusion protein carrying only the first 138 amino acids from FruP2ase was inactivated at a higher rate than the one carrying the first 178, suggesting the existence of a protective region between amino acids 138 and 178. A fusion protein carrying the first 81 amino acids from FruP2ase was inactivated by glucose at a similar rate to the one carrying the 178 amino acids, but one with only the first 18 amino acids was resistant to catabolite inactivation.

Inactivation of FruP2ase in mutantsubr1that lack a protein required for ubiquitin‐dependent proteolysis, orpralthat lack vacuolar protease A, proceeded as in a wild type. Our results suggest that at least two domains of FruP2ase may mark β‐galactosidase for catabolite inactivation and that FruP2ase can be inactivated by a mechanism independent of transfer to the vacuole.