bluuebunny/crossref_metadata_2025 huggingface dataset:
Elevated levels and mitogenic activity of lysophosphatidylinositol in k‐ras‐transformed epithelial cells
Authors: FALASCA Marco , CORDA Daniela
Published: March 2005

In cell lines stably (KiKi) or reversibly (Ts) transformed by the k‐ras oncogene originated from a differentiated rat throid line (FRTL5 cells), k‐ras‐induced transformation has been associated with an increased phospholipase A2 activity. Here we provide evidence that this enzymic activity is phosphoinositide specific and leads to the formation of lysophosphatidylinositol. The levels of this lysolipid increased by 2–3‐fold in ras‐transformed cells (KiKi cells and Ts cells at the permissive temperature of 33°C) as compared to differentiated cells (FRTL5) or to Ts cells maintained at 39°C, i.e. at the temperature where ras‐p21, the product of the ras oncogene, is inactive. Since another lysoderivative, lysophosphatidic acid, has been shown to be a mitogen, we have tested whether lysophosphatidylinositol could have a similar activity on thyroid cells. Lysophosphatidylinositol (10–100 μM) induced a 5–10‐fold increase in [3H]thymidine incorporation in both FRTL5 and KiKi cells, whereas lysophosphatidic acid was active only in differentiated cells. Lysophosphatidylinositol (∼25 μM) and lysophosphatidic acid (50–100 μM) acted synergistically with insulin in increasing [3H]thymidine incorporation. Moreover, lysophosphatidylinositol at concentrations three‐fold higher than those found to be mitogenic, inhibited the activity of the GTPase‐activating protein. We conclude that lysophosphatidylinositol is a mitogen that might play a role in the modulation of k‐ras transformed cell proliferation.
Two mRNA species encoding the differentiation‐associated histone H1° are produced by alternative polyadenylation in mouse
Authors: BROCARD Marie‐Paule , ROUSSEAU Denis , LAWRENCE Jean‐Jacques , KHOCHBIN Saadi
Published: March 2005

Histone H1° is a differentiation‐specific member of the histone H1 family. The accumulation of the protein is associated with the terminal stage of cell differentiation and is regulated at various levels. In mouse, the analysis of the expression of the single copy gene encoding H1° has shown that another H1°‐related mRNA species (0.9 kb) is present in addition to the usual 2.1‐kb mRNA. In this study, we have cloned and sequenced the smaller H1°‐related mRNA. This mRNA seems to be produced by the use of an additional polyadenylation signal present in the 3′ untranslated region (UTR) of the initial transcript. This smaller H1°‐encoding mRNA is expressed only in mouse and is transferred into polysomes as efficiently as the larger version upon the induction of cell differentiation. The use of the described polyadenylation site removes over 1 kb of the 3′ UTR of H1° mRNA and seems to be involved in the regulation of H1° mRNA stability.
Expression of the gene encoding an insulin‐related peptide inLocusta (Insecta, Orthoptera)
Authors: KROMER‐METZGER Elisabeth , LAGUEUX Marie
Published: March 2005

A peptide with significant sequence similarity to mammalian insulins,Locustainsulin‐related peptide (LIRP) has recently been isolated from neurohaemal lobes ofLocusta migratoriaand the corresponding cDNA has been cloned. We report here the cloning of the LIRP gene and we show that this gene is present as a single copy/haploid genome. The organization of this gene is similar to that of mammalian insulin genes. There are at least two LIRP transcripts, LIRP T1 and LIRP T2, differing in their 5′ untranslated regions and in the 5′ end of the coding region resulting in an additional ATG present in LIRP T2. The two transcripts are differentially expressed inLocusta; whereas LIRP T1 is only expressed in neurosecretory cells of the brain, LIRP T2 is present at low levels in all tissues. Our data indicate that the LIRP gene has at least two promoters, the alternative usage of which could account for a differential regulation of expression of LIRP in the neurosecretory cells and in various peripheral tissues.
Asp650 is crucial for catalytic activity of neutral endopeptidase 24‐11
Authors: LE MOUAL Hervé , DION Natalie , ROQUES Bernard P. , CRINE Philippe , BOILEAU Guy
Published: March 2005

Neutral endopeptidase (NEP) is a membrane‐bound mammalian ectopeptidase that contains a catalytic zinc ion in its active site. Previous studies showed that the active site, and especially the zinc‐binding site of NEP have features in common with the prototypical bacterial zinc protease, thermolysin. Sequence comparison reveals that both enzymes have a conserved Asp residue (Asp650 in NEP and Asp170 in thermolysin) located four positions on the C‐side of the third zinc ligand. In thermolysin, this residue is involved in a carboxylate‐histidine‐zinc interaction whose functional role has never been established [Christianson, D. W. & Alexander, R. S. (1990) Nature 346, 225]. To test the hypothesis that, in NEP, this residue is important for catalysis, we have changed Asp650 of NEP by site‐directed mutagenesis and expressed the mutant enzymes in COS‐1 cells. Substitution of Glu, Asn or Ala for Asp650 resulted in mutant enzymes exhibiting drastic decreases in specific activity. Binding experiments using the zinc‐chelating inhibitor [3H]‐N‐[(2RS)‐4‐(hydroxyamino)‐1,4‐dioxo‐2‐(phenylmethyl)butyl]glycine suggested that the zinc ion is present in the active site of these mutant enzymes. These results strongly support the conclusion that Asp650 in NEP is crucial for hydrolytic activity.
Efficient but aberrant cleavage of mitochondrial precursor proteins by the chloroplast stromal processing peptidase
Authors: BASSHAM Diane C. , CREIGHTON Alison M. , ARRETZ Michael , BRUNNER Michael , ROBINSON Colin
Published: March 2005

Cytosol‐synthesised chloroplast and mitochondrial precursor proteins are proteolytically processed after import by highly specific, metal‐dependent soluble enzymes: the stromal processing peptidase (SPP) and the matrix processing peptidase (MPP), respectively. We have used in vitro processing assays to compare the reaction specificities of highly purified preparations of pea SPP and Neurospora crassa MPP, both of which are unable to cleave a variety of ‘foreign’ proteins. We show that SPP can cleave all five mitochondrial precursor proteins tested, namely cyclophilin, the β subunit of the F1‐ATPase complex, the Rieske FeS protein, the α‐MPP subunit and cytochrome b2. In contrast, MPP is unable to cleave any chloroplast precursor proteins tested. Several of the mitochondrial precursor proteins are cleaved more efficiently by SPP than are many authentic chloroplast precursor proteins but, in each case, cleavage takes place at a site or sites which are N‐terminal to the authentic MPP site; pre‐cyclophilin is cleaved 5 residues upstream of the MPP site and the precursor of the β subunit of the F1‐ATPase complex is cleaved at sites 5 and 12 residues upstream. We discuss the implications of these data for the SPP reaction mechanism.
Characterization and primary structure of amphioxus troponin C
Authors: TAKAGI Takashi , PETROVA Tatiana , COMTE Michelle , KUSTER Thomas , HEIZMANN Claus W. , COX Jos A.
Published: March 2005

Troponin C (TnC) from amphioxus (Protochordate) was purified and its primary structure determined. Unlike the case of vertebrates and other invertebrates, amphioxus TnC is found in the soluble fraction after extractions at physiological ionic strength in the presence of Ca2+. Edman sequencing combined with mass spectroscopy indicate that the protein contains 163 amino acid residues. It possesses an acetylated N‐terminus (although a small percentage has a free Ser N‐terminus) and either ɛ‐N‐methyllysine or ɛ‐N‐dimethyllysine in position 20. It displays about 50% sequence identity with vertebrate skeletal‐muscle and cardiac‐muscle TnC, 44% with TnC of sea squirt, also a Protochordate, and 30% with other invertebrate TnC. Like vertebrate TnC, amphioxus TnC contains a N‐terminal α‐helix plus the usual four ancestral Ca2+‐binding regions, but analysis of the sequence suggests that the fourth site is not functional. Flow dialysis shows that amphioxus TnC binds three Ca2+ with the mean apparent affinity constant K′ of 3.4 ± 1.5 105 M−1. No cooperativity exists between the sites, and the presence of up to 10 mM Mg2+ does not influence the Ca2+‐binding isotherm, indicating that the metal‐binding sites are Ca2+‐specific at physiological Mg2+ concentrations. It forms a Ca2+‐dependent, 1:1 complex with melittin and rabbit or crayfish troponin I (TnI). Amphioxus TnC possesses one Trp residue in position 151 and one at the C‐terminus. Trp fluorescence suggests that one or both residues are solvent‐exposed in the metal‐free form and efficiently shielded in the Ca2+ form. Although Mg2+ has no effect on the Ca2+ binding, the Trp fluorescence is influenced by millimolar Mg2+, suggesting the presence of one or more independent Mg2+‐binding site(s). A phylogenetic analysis clearly shows that amphioxus TnC is positioned on the branch of the Chordates, but at a distance from the vertebrate TnC. Its place on the phylogenetic tree is in accordance with the consensus evolutionary phylogeny.
Catabolic ornithine carbamoyltransferase of Pseudomonas aeruginosa
Authors: TRICOT Catherine , SCHMID Sergio , BAUR Heinz , VILLERET Vincent , DIDEBERG Otto , HAAS Dieter , STALON Victor
Published: March 2005

Ornithine carbamoyltransferases (OTCases) catalyse the formation of citrulline and phosphate from ornithine and carbamoylphosphate by a thermodynamically favoured reaction. In vivo, catabolic OTCase of Pseudomonas aeruginosa promotes the reverse reaction, the phosphorolysis of citrulline. Although the enzyme is assayed in vitro in the direction of citrulline synthesis, the enzyme cannot perform this reaction in vivo due to poor affinity for carbamoylphosphate and high cooperativity towards this substrate. Furthermore, the dodecameric catabolic OTCase is an allosteric enzyme; the enzyme is stimulated by nucleoside monophosphates and inhibited by polyamines (e.g. spermidine). A previous study showed that a modification of the C‐terminus of the catabolic OTCase alters the homotropic cooperativity of the enzyme. We have now investigated the importance of the C‐terminus for homotropic and heterotropic cooperativity by site‐directed mutagenesis. Deletion of the C‐terminal Ile335 residue strongly reduced cooperativity for carbamoylphosphate and sensitivity to spermidine. These properties were essentially restored when the two C‐terminal amino acids (Asp334 and Ile335) were removed by deletion. However, in this variant enzyme, AMP failed to abolish carbamoylphosphate cooperativity completely, whereas the wild‐type enzyme was rendered virtually non‐cooperative by AMP. An extension of catabolic ornithine carbamoyltransferase by 15 amino acid residues interfered with both homotropic and heterotropic interactions and lowered the maximal velocity. All variant enzymes had the same dodecameric structure as the wild type and differed only slightly in affinity for the second substrate ornithine. A structural model of the dodecamer, at 0.3‐nm resolution, suggests that the C‐terminus could be involved in trimer/trimer interaction. We propose that modifications at the C‐terminus alter the trimer/trimer interface and, in addition, removes the salt bridge His5‐Ile335 within a monomer. These changes profoundly and indirectly modify the allosteric transition and consequently the interactions of the dodecamer with carbamoylphosphate and effectors.
Residues essential for catalytic activity of soybean β‐amylase
Authors: TOTSUKA Atsushi , Van NONG Hai , KADOKAWA Hiroya , KIM Chan‐Shick , ITOH Yoshifumi , FUKAZAWA Chikafusa
Published: March 2005

To determine which amino acid residues are essential for the catalytic activity of soybean β‐amylase, deoxyoligonucleotide site‐directed mutagenesis was employed against aspartyl, glutamyl, and cysteinyl residues located in highly conserved regions found in β‐amylase family to date. Both substitution of aspartic acid at position 101 and that of glutamic acid at position 186 of the enzyme by neutral and acidic amino acids, respectively, led to the complete elimination of activity, but did not induce any significant changes in circular dichroic spectra or the binding affinity for cyclomaltohexaose, a substrate analogue.

Taking account of the results obtained here, the above two amino acid residues are involved in the catalytic site of soybean β‐amylase. The replacement of glutamic acid at position 345 decreased activity to below 6% of the non‐mutant level, implying that this residue may also play a crucial role in β‐amylase activity, although it may not be involved at the catalytic site itself. In contrast, substitution of cysteinyl residue at position 95 by a serinyl residue led to a drastic reducing of the optimal temperature (from 50°C to 30°C), suggesting that this cysteinyl residue is responsible for the thermal stability of the enzyme.
Purification and characterisation of a brain‐derived neurotrophic factor/ neurotrophin‐3 (BDNF/NT‐3) heterodimer
Authors: JUNGBLUTH Stefan , BAILEY Karen , BARDE Yves‐Alain
Published: March 2005

The neurotrophins nerve growth factor (NGF), brain‐derived neurotrophic factor (BDNF), and neurotrophin‐3 (NT‐3) are known to exist in solution as non‐covalently linked homodimeric proteins. The recent elucidation of the crystal structure of the NGF homodimer, as well as the conservation of structural motifs in the neurotrophins, raised the possibility that neurotrophin heterodimers might also occur. The formation of BDNF/NT‐3 heterodimers was explored using a vaccinia virus expression system. Upon co‐infection of cells with viruses expressing BDNF and NT‐3, we could identify a BDNF/NT‐3 heterodimer as a biosynthetic product and separate it from the BDNF and NT‐3 homodimers. We could also show that the BDNF/NT‐3 heterodimers can be formed irrespective of wild‐type or exchanged prosequences, indicating that prosequence specificity does not influence dimer formation. In all neuronal survival assays that were used, the BDNF/NT‐3 heterodimer was shown to be 10‐fold less active compared with a mixture of BDNF and NT‐3 homodimers. This lower specific activity was also measured in a neuronal population co‐expressing receptors for BDNF and NT‐3. The low biological activity of the heterodimer observed with neurons was not paralleled by a reduced ability of the heterodimer to interact with trkB or trkC receptors, as assessed by the induction of tyrosine phosphorylation of these receptors expressed by fibroblast cell lines.
The local and global unfolding of coiled‐coil tropomyosin
Authors: ISHII Yoshiharu
Published: March 2005

The thermal unfolding of a two‐stranded α‐helical coiled coil of tropomyosin was studied using circular dichroism and excimer fluorescence of N‐(1‐pyrenyl)iodoacetamide‐labeled tropomyosin. Tropomyosin unfolds with two transitions, namely local and global unfolding at high salt (greater than 0.1 M NaCl) and pH 7.5. The local unfolding was masked by the global unfolding at low salt (less than 0.1 M NaCl), at high pH (greater than pH 9.0), and in the presence of methanol, where the global unfolding temperature was similar to or lower than the local unfolding temperature. The local and global unfolding are different in nature. A comparison of the helix thermal unfolding of N‐(1‐pyrenyl)iodoacetamide–tropomyosin with unlabeled tropomyosin showed that tropomyosin had an inherent less‐stable region, when Cys190. was N‐(l‐pyrenyl)iodoacetamide‐labeled, disulfide cross‐linked, or reduced. Instead, the chemical state of Cys190, determined the stability of the local unfolding region, because a strain created by the disulfide cross‐link or the pyrene/pyrene interaction decreases the stability of the local unfolding region. Thus, these data show that the excimer fluorescence of N‐(1‐pyrenyl)iodoacetamide‐labeled tropomyosin is useful for studying the local and global unfolding of tropomyosin.
Molecular cloning and characterisation of a neutrophil chemotactic protein from porcine platelets
Authors: POWER Christine A. , PROUDFOOT Amanda E. I. , MAGNENAT Edith , BACON Kevin B. , WELLS Timothy N. C.
Published: March 2005

In our search for novel chemoattractant factors, we have purified a heparin‐binding protein from porcine platelets which is a potent chemoattractant for human neutrophils. The protein has 80 amino acids and a molecular mass of 8597.5Da as measured by electrospray mass spectrometry. It has been characterised by amino acid sequencing and shown to have highest identity to members of the human platelet basic‐protein‐family. Its N‐terminal sequence is intermediate in length between the human connective‐tissue‐activating polypeptide III (CTAP‐III) and neutrophil‐activating polypeptide‐2 (NAP‐2). The porcine NAP‐2/CTAP‐III shows the classic CXC cysteine spacing found towards the N‐terminus in the chemokine α family and contains the ELR motif which has been shown to be essential for neutrophil chemotaxis. We have isolated mRNA from porcine platelets and constructed a cDNA library containing 1.0×106 independent clones. Using probes based on the protein sequence we have isolated a full length‐clone for this gene, with an open reading frame containing 119 amino acids. Despite overall similarity between the human and porcine proteins, the N‐terminal region is almost completely different between the two species, with only two identical amino acids. The proteolytic cleavage sites required for processing of human platelet basic protein are completely missing in the porcine homologue, implying a different processing pathway or mechanism. The porcine protein is capable of agonizing certain effects of both NAP‐2 and CTAP‐III when incubated with human cells indicating that the same porcine protein may be involved in both processes.
Characterization of the essential yeast gene encoding N‐acetylglucosamine‐phosphate mutase
Authors: HOFMANN Michael , BOLES Eckhard , ZIMMERMANN Friedrich K.
Published: March 2005

A previously cloned gene of Saccharomyces cerevisiae, which complements the growth defect of a phosphoglucomutase (pgm1Δ/pgm2Δ) double deletion mutant on a pure galactose medium [Boles, E., Liebetrau, W., Hofmann, M. & Zimmermann, F. K. (1994) Eur. J. Biochem. 220, 83–96], was identified as the structural gene encoding N‐acetylglucosamine‐phosphate mutase. The complete nucleotide sequence of the gene, AGM1, and surrounding regions was determined. AGM1 codes for a predicted 62‐kDa protein with 557 amino acids and is located on chromosome V adjacent to the known gene PRB1 encoding protease B. No extended nucleotide or amino acid sequence similarities could be found in the databases, except for a small region of amino acids with high similarity to the active‐site consensus sequence of hexosephosphate mutases. Three putative pheromone‐responsive elements have been identified in the upstream region of the AGM1 gene. The gene is essential for cell viability. An agm1 deletion mutant progresses through only approximately five cell cycles to form a ‘string' of undivided cells with an abnormal cell morphology resembling glucosamine auxotrophic mutants. Expression of the AGM1 gene on a multi‐copy plasmid led to a significantly increased N‐acetylglucosamine‐phosphate mutase activity. Unlike over‐expression of the AGM1 gene in a pgm1/pgm2 double deletion mutant which could restore phosphoglucomutase activity, over‐expression of the PGM2 gene encoding the major isoenzyme of phosphoglucomutase did not increase N‐acetylglucosamine‐phosphate‐mutase activity and did not restore growth of agm1 deletion mutant cells. Our observations indicate that the different hexosephosphate mutases of S. cerevisiae have partially overlapping substrate specifities but, nevertheless, distinct physiological functions.
Conformational analysis of 3′‐fluorinated A(2′‐5′)A(2′‐5′)A fragments
Authors: VAN DEN BOOGAART Jacquelien E. , KALINICHENKO Elena N. , PODKOPAEVA Tatjana L. , MIKHAILOPULO Igor A. , ALTONA Cornelis
Published: March 2005

A one‐ and two‐dimensional NMR study has been performed on seven A(2′–5′)A(2′–5′)A fragments containing 9‐(3′‐fluoro‐3′‐deoxy‐β‐D‐xylofuranosyl)‐adenine (AF) or 3′‐fluoro‐3′‐deoxyadenosine (AF) residues at different positions, and on the corresponding monomers. A(2′–5′)A(2′–5′)A served as a reference compound. The fluoro substituent governs the conformation of the sugar ring: an AF residue displays mainly N‐type sugar and the ring is considerably flattened (øN∼ 30°) compared to AF residues (øS∼ 40°), which exhibit almost pure S‐type conformation. Moreover, in AF moieties the rotamer distribution around torsion angle γ (O5′‐C5′‐C4′‐C3′) and the base orientation are influenced to a large extent by the preseñce of the fluorine substituent. The sugar rings of nonfluorinated residues in the trimers appear rather flexible.

A possible correlation between the conformational characteristics of the fluorinated fragments and their biological activity has been found: the fragments that meet the prerequisites for binding to RNase L indeed show enhanced binding to this endonuclease. Furthermore, substitution of the 3′‐OH group of the second residue by hydrogen or of the 3′‐OH group of the 2′‐terminal residue by fluorine or hydrogen results in increased resistance towards 2′–5′‐phosphodiesterase.
Comparative analysis of the protein components from 5S rRNA · protein complexes of halophilic archaebacteria
Authors: McDOUGALL John , WITTMANN‐LIEBOLD Brigitte
Published: March 2005

The 5S RNA · protein complexes have been isolated from the 50S subunit of the halophilic archaebacteria Halobacterium cutirubrum, Halobacterium halobium, Halobacterium salinarium, Haloferax valcanii and Haloarcula marismortui. The 50S subunits from most of the halophiles released a multiprotein ribonucleoprotein particle similar to that previously observed with the H. cutirubrum 5S RNA · protein complex, which contained proteins from the L5 and L18 ribosomal protein families. Ribosomes from H. marismortui, however, released an RNA · protein complex containing a single protein (L18) that is homologous to the single protein found in the eukaryotic 5S ribonucleoprotein complexes. N‐terminal sequence analyses of the halophilic 5S RNA‐binding proteins suggest that the L18 protein primary structure is highly conserved, with only the H. marismortui protein having a sequence difference in at least the first twenty amino acids. Although the L5 group of ribosomal proteins also shows a high conservation, it appears that the proteins may have had more freedom to diverge throughout evolution.
Cytochrome c553 from Desulfovibrio vulgaris (Hildenborough)
Authors: VERHAGEN Marc F. J. M. , WOLBERT Ronnie B. G. , HAGEN Wilfred R.
Published: March 2005

An electrochemical study of the periplasmic cytochrome c553 of Desulfovibrio vulgaris (Hildenborough) is presented. The dependence of the midpoint potential on temperature and pH was studied with cyclic voltammetry. The voltammograms obtained were reversible and revealed that this cytochrome showed fast electron transfer on a bare glassy carbon electrode. The midpoint potential at pH 7.0 and 25°C was found to be 62 mV versus the normal hydrogen electrode. It was observed that the temperature dependence was discontinuous with a transition temperature at 32°C. The standard reaction entropy at the growth temperature of the organism (37°C) was calculated to be ΔS°′=–234 J mol−1 K−1. The pH dependence of the midpoint potential could be described with one pK of the oxidized form with a value of 10.6. The second‐order rate constant for electron transfer between cytochrome c553 and the Fe‐hydrogenase from D. vulgaris (H) was also determined with cyclic voltammetry. The equivalent rate constant for cytochrome c3 and hydrogenase was measured for comparison. The second‐order rate constants are 2×107M−1 s−1 for cytochrome c553 and 2×108M−1 s−1 for cytochrome c3. The kinetic parameters of the hydrogenase for both cytochromes were determined using the spectrophotometric hydrogen consumption assay. With cytochrome c553 this resulted in a Km of 46 μM and a maximum turnover number of 7.1×102s−1 in the H2 consumption assay. The values with cytochrome c3 were 17 μM and 6.4×102s−1, respectively. The importance of the different kinetic parameters for contrasting models proposed to describe the function of the Fe‐hydrogenase are discussed.
Intrinsic tryptophan fluorescence of bovine liver adenosine kinase, characterization of ligand binding sites and conformational changes
Authors: ELALAOUI Abdelaziz , DIVITA Gilles , MAURY Georges , IMBACH Jean‐Louis , GOODY Roger S.
Published: March 2005

Bovine liver adenosine kinase is a 45‐kDa monomeric protein which exhibits a characteristic intrinsic tryptophan fluorescence with a maximal excitation at 284 nm and an emission peak centered at 335 nm. A total of three tryptophan residues/molecule has been estimated by using a fluorescence titration method. Low values of Stern‐Volmer quenching constants in the presence of either acrylamide or iodide (4.2 M−1 or 1.5 M−1, respectively) indicated that the tryptophan residues are relatively buried in the native molecule. Tryptophan residues also showed a high heterogeneity, with a fractional accessible fluorescence value for iodide of 0.65. The enzyme fluorescence was very sensitive to substrate binding, which induced a marked fluorescence quenching, a lower tryptophan accessibility to acrylamide and iodide, and an increase in the tryptophan heterogeneity. ADP or ATP showed a monophasic saturation curve consistent with the existence of one binding site. In contrast, adenosine and AMP gave biphasic saturation curves, suggesting the existence of at least two binding sites, with a high and a low affinity. The presence of MgCl2 increased the affinity of ATP or ADP, whereas the binding of adenosine or AMP was not affected.
Structural organization of the genes for rat von Ebner's gland proteins 1 and 2 reveals their close relationship to lipocalins
Authors: KOCK Kai , AHLERS Christiane , SCHMALE Hartwig
Published: March 2005

The rat von Ebner's gland protein 1 (VEGP 1) is a secretory protein, which is abundantly expressed in the small acinar von Ebner's salivary glands of the tongue. Based on the primary structure of this protein we have previously suggested that it is a member of the lipocalin superfamily of lipophilic‐ligand carrier proteins. Although the physiological role of VEGP 1 is not clear, it might be involved in sensory or protective functions in the taste epithelium.

Here, we report the purification of VEGP 1 and of a closely related secretory polypeptide, VEGP 2, the isolation of a cDNA clone encoding VEGP 2, and the isolation and structural characterization of the genes for both proteins. Protein purification by gel‐filtration and anion‐exchange chromatography using Mono Q revealed the presence of two different immunoreactive VEGP species. N‐terminal sequence determination of peptide fragments isolated after protease Asp‐N digestion allowed the identification of a new VEGP, named VEGP 2, in addition to the previously characterized VEGP 1. The complete VEGP 2 sequence was deduced from a cDNA clone isolated from a von Ebner's gland cDNA library. The VEGP 2 cDNA encodes a protein of 177 amino acids and is 94% identical to VEGP 1.

DNA sequence analysis of the rat VEGP 1 and 2 genes isolated from rat genomic libraries revealed that both span about 4.5 kb and contain seven exons. The VEGP 1 and 2 genes are non‐allelic distinct genes in the rat genome and probably arose by gene duplication. The high degree of nucleotide sequence identity in introns A–C (94–100%) points to a recent gene conversion event that included the 5′ part of the genes.

The genomic organization of the rat VEGP genes closely resembles that found in other lipocalins such as β‐lactoglobulin, mouse urinary proteins (MUPs) and prostaglandin D synthase, and therefore provides clear evidence that VEGPs belong to this superfamily of proteins.
1H‐NMR assignments and secondary structure of human plasminogen kringle 1
Authors: REJANTE Marita R. , LLINÁS Miguel
Published: March 2005

The 1H‐NMR spectrum of the kringle 1 domain of human plasminogen complexed with 6‐aminohexanoic acid, an antifibrinolytic drug, has been assigned. Elements of secondary structure have been identified on the basis of sequential, medium and long‐range dipolar interactions, backbone amide spin‐spin couplings (3JHN‐Hα) and 1H‐2H exchange rates. The kringle contains scarcely any repetitive secondary structure: eight reverse turns and two short β‐sheets. These comprise 40% and 12% of the domain, respectively. No α‐helix was found. An aromatic cluster formed by His31, Phe36, Trp62, Phe64, Tyr72 and Tyr74 is indicated by several inter‐residue Overhauser connectivities. Contacts between the methyl groups of Leu46 and the side chains of Phe36, Trp62 and Trp25 are observed. A second hydrophobic cluster formed by Tyr9, Ile77 and Leu78 is also indicated. A comparison of secondary structure elements among plasminogen kringles 1 and 4 and tissue‐type plasminogen activator kringle 2 suggests that there is variability in the position and number of reverse turns on going from one kringle to another; however, the β‐sheets are conserved among the homologs.
The nutrient factor queuine protects HeLa cells from hypoxic stress and improves metabolic adaptation to oxygen availability
Authors: REISSER Thomas , LANGGUT Werner , KERSTEN Helga
Published: March 2005

Queuine (q), a cyclopentendiol derivative of 7‐aminomethyl‐7‐deazaguanine, is a nutrient factor for lower and higher eukaryotes, except yeast; it is synthesized in eubacteria partly at the level of tRNA. In eukaryotes q is preferentially inserted into the wobble position of specific tRNAs in differentiated and adult tissues, but occurs mainly free in embryonic and fast proliferating cells. HeLa cells grow to a higher cell density under aerobic than under hypoxic conditions only when supplemented with q. Here we show that in hypoxically grown HeLa cells, sufficiently supplied with q, free q accumulated when serum factors become limiting while the respective tRNAs remained completely q deficient. In these cells the levels of lactate dehydrogenase A (LDH A) mRNA and of LDH A protein were at least twofold higher than in aerobically grown cells, independent of the absence or presence of q. In response to q the LDH A4 isoenzyme was further activated by a post‐translational mechanism. In q‐deficient HeLa cells the activity of the major anoxic stress protein, LDHk, increased as a result of hypoxia; this increase was suppressed by q. In aerobically grown, q‐deficient cells significant activities of LDH A4 and LDHk were present; both activities were markedly lowered by q, while the mitochondrial electron flow was imporved. The results show that free q is essential for relieving hypoxic stress in HeLa cells that results from oxygen limitation.
Insertion in barnase of a loop sequence from ribonuclease T1
Authors: VUILLEUMIER Stéphane , FERSHT Alan R.
Published: March 2005

Barnase was mutated by inserting into its active site loop sequences found in the related enzyme ribonuclease T1 (RNase T1), according to either structural or sequential similarity alignments. The barnase/RNase T1 hybrid corresponding to the structural alignment of the two proteins, endo‐[RNaseT1‐(93–99)]102abarnase, contains RNase T1 residues at positions 93–99 inserted between residues at positions 102 and 103 of barnase. The other constructed mutant, endo‐[RNaseT1‐(95–98)]104abarnase, has RNase T1 residues at positions 95–98 inserted between residues at positions 104 and 105 in barnase, corresponding to published sequence alignments of the two proteins in this region. The mutants were characterized by absorbance, fluorescence and CD spectroscopy; the stability, folding and unfolding kinetics, and catalytic activity were measured and compared with the wild‐type enzyme. Endo‐[RNaseT1‐(93–99)]102abarnase, the mutant protein corresponding to the structural alignment of barnase with ribonuclease T1, shows a slightly higher stability (approximaely 5 kJ/mol) towards urea and heat denaturation than the mutant endo‐[RNaseT1‐(95–98)]104abarnase, designed according to a sequence alignment between the two enzymes. Both mutants have very low catalytic activity, although the effect of mutation is almost entirely limited to kcat in the case of the mutant corresponding to the structural alignment between barnase and ribonuclease T1, while both kcat and Km are affected in the mutant corresponding to the sequence alignment between the two enzymes. Thus, the superiority of structural over sequential alignments cannot be supported conclusively by direct experiment in the present case.
Cysteine‐specific ADP‐ribosylation of actin
Authors: JUST Ingo , WOLLENBERG Peter , MOSS Joel , AKTORIES Klaus
Published: March 2005

Incubation of lysate from human polymorphonucleated neutrophils and human platelets with [32P]NAD resulted in the labeling of a 42‐kDa protein. Phosphodiesterase (Crotalus durissus) released 5′‐AMP from the radiolabeled protein. The 42‐kDa protein was identified as actin by binding to DNAse‐I, two‐dimensional gel electrophoresis and partial proteolysis. The rate of ADP‐ribosylation was greater with [32P]ADP‐ribose than with [32P]NAD, indicating a non‐enzymic modification. ADP‐ribose also modified actin in the actin–DNAse‐I complex, but denatured actin was not modified by ADP‐ribose. Only cytoplasmic β/γ‐actin isoforms were non‐enzymically ADP‐ribosylated but not muscle α‐actin. The acceptor amino acid was identified as a cysteine residue whereas the bacterial ADP‐ribosyltransferase C. perfringens iota toxin catalyzes incorporation of ADP‐ribose to Arg177 of actin. Alkylation of cysteine residues of actin with N‐ethylmaleimide prevented subsequent non‐enzymic ADP‐ribosylation but not the toxin catalyzed modification. Non‐enzymically ADP‐ribosylated actin was further modified by C. perfringens iota toxin. The F‐actin stabilizing mycotoxin phalloidin blocked the non‐enzymic ADP‐ribosylation and, conversely, ADP‐ribosylation inhibited the phalloidin‐induced polymerization of ADP‐ribosylated actin. The data indicate that cytoplasmic actin is non‐enzymically ADP‐ribosylated by ADP‐ribose at a cysteine residue to inhibit actin polymerization.
Chirality of the interaction of mithramycin with phospatidylcholine‐bilayer membranes in the presence of Mg2+ or Zn2+
Authors: DEMICHELI Cynthia , GARNIER‐SUILLEROT Arlette
Published: March 2005

Binding of the antitumor compound mithramycin to dipalmitoyl‐sn‐glycerophosphocholine and dimyristoyl‐sn‐glycerophosphocholine (Myr2GroPCho) model membrane (small unilamellar vesicles, SUV) bilayers was shown to be dependent on the molecular packing of the lipids and on the presence of Zn2+ or Mg2+. The CD properties of mithramycin have been used to follow its complexation to Zn2+ and to Mg2+. In the absence of SUV, Mg2+ and Zn2+ are each able to form with mithramycin two types of complexes of different chirality: at low molar ratios of M2+ to mithramycin, a complex in the left‐handed conformation is obtained whereas at high molar ratios the complex formed has the right‐handed conformation. In the presence of Zn2+ or Mg2+, mithramycin binds to SUV in the gel‐phase state as a dimer in the left‐handed conformation. No binding is observed when SUV are in liquid‐crystal state.
Deactivation of F0F1 ATPase in intact plant mitochondria
Authors: VALERIO Marie , DIOLEZ Philippe , HARAUX Francis
Published: March 2005

By using a method especially adapted to intact (pea leaf) mitochondria, we studied the regulation of the F0F1 ATPase by the electrochemical proton gradient (Δμ˜H+) and by the matricial pH. The kinetics of decay of the ATP hydrolase activity was studied immediately after the collapse of the electrochemical proton gradient by an uncoupler. At pH 7.5, three inhibitors of the ATPase (venturicidin, tri‐n‐butyl tin and aurovertin), used at non‐saturating concentrations, inhibited ATP hydrolysis to the same extent throughout the decay. This showed that the activity was totally controlled by the ATPase during all the decay and rules out any involvement of the phosphate or nucleotide carriers. This interpretation was confirmed by the fact that carboxyatractyloside, an inhibitor of the ATP/ADP antiporter, had a strong effect only on the initial rate of ATP hydrolysis, but not on the rate measured after some tens of seconds of decay. Oligomycin, at variance with the other ATPase inhibitors, interfered with the deactivation process, suggesting that its effect depends on the conformational state of the enzyme. Between pH 6.5 and 7.5, the hydrolase activity rose continuously and was still kinetically controlled by the ATPase. At higher pH value, the activity slightly decreased and appeared limited by at least one of the carriers. The activity of the ATPase itself, free of any transport process, seemed to increase monotonously with pH from 6.5 to 8.

The electrochemical proton gradient is required to maintain the ATPase active, whereas no effect can be observed on transport processes. Matricial pH, while modulating the apparent catalytic turnover, has no marked effect on the rate of deactivation. These results, obtained with intact mitochondria, extend previous observations on the isolated enzyme and question the binding of IF1 as a rate‐limiting step for ATPase deactivation.
Calcium and magnesium binding to rat parvalbumin
Authors: EBERHARD Marc , ERNE Paul
Published: March 2005

Ca2+ and Mg2+ binding to rat parvalbumin was measured by means of the fluorescent Ca2+ indicator fluo‐3 using a method developed earlier [Eberhard, M. & Erne, P. (1991) Eur. J. Biochem. 202, 1333–1338]. We demonstrate that rat parvalbumin contains two equivalent Ca2+/Mg2+ binding sites and that Ca2+ and Mg2+ compete for the same sites. Dissociation constants (Kd) for Ca2+ and Mg2+ in Hepes buffer containing 150 mM K+ at 35°C and pH 7.2 are 11.0 ± 1.8 nM and 41 ± 8 μM, respectively. At an ionic strength below 0.2 M, Kd values of Ca2+ binding to rat parvalbumin are approximately proportional to the ion concentration. Kd values of Ca2+ binding were found to be about fourfold larger in the presence of Na+ as compared with K+, indicating that Na+ distinctly influences Ca2+ binding to rat parvalbumin. Both Ca2+ and Mg2+ binding to parvalbumin are exothermic whereas Ca2+ and Mg2+ binding to fluo‐3 are endothermic entropy‐driven processes.
Purification of Golgi adaptor protein 1 from bovine adrenal gland and characterization of its β1 (β′) subunit by microsequencing
Authors: UNGEWICKELL Ernst , PLESSMANN Uwe , WEBER Klaus
Published: March 2005

A method for the purification of the Golgi adaptor protein 1 from bovine adrenal gland tissue was devised to investigate the relationship of its β1 (formerly referred to as β′) subunit to known β‐type sequences. Adrenal gland tissue was chosen for this study because it yielded 2–3 times more adaptor protein 1 than a comparable preparation from bovine brain. Like its neuronal isoform, the β1 subunit from adrenal gland adaptor protein 1 is readily cleaved by trypsin into a 63‐kDa N‐terminal fragment and a 40‐kDa C‐terminal fragment, while the γ subunit is largely refractory to digestion. Based on microsequencing of 167 residues from the 63‐kDa fragment, we noted 11 differences to the corresponding region of the β2 (formerly β) subunit of the plasma membrane adaptor protein 2, but only one difference to the corresponding region of a β‐type protein encoded by the rat cDNA clone AP105a which is supposed to be a variant of the β2 subunit of the plasma membrane adaptor protein 2 [Kirchhausen, T., Nathanson, K. L., Matsui, W., Vaisberg, A., Chow, E. P., Burne, C., Keen, J. H. & Davis, A. E. (1989) Proc. Natl Acad. Sci. USA 84, 8805–8809]. Alignment of 187 residues from the 40‐kDa β1 C‐terminal fragment revealed differences in 77 positions to the corresponding region of the β2 subunit and differences in 23 positions compared to the supposed β2‐like protein. These findings suggest that the protein encoded by the rat cDNA clone AP105a is more closely related to the β1 subunit of the bovine adrenal Golgi adaptor protein 1 than to the β2 subunit of the rat plasma membrane adaptor protein 2.
Purification of rat liver plasma membrane glutathione transferase
Authors: HORBACH Martina E. , SIES Helmut , AKERBOOM Theodorus P. M.
Published: March 2005

Glutathione transferases purified from plasma membrane and microsomal fractions from rat liver share similar enzymic properties. The activity of both proteins with 1‐chloro‐2,4‐dinitrobenzene can be stimulated about 10–15‐fold by N‐ethylmaleimide. No activation is observed using p‐nitrobenzylchloride as a substrate.

The enzymes are immunologically related as indicated by Western‐blot analysis using antibodies against the microsomal glutathione transferase or against a synthetic peptide corresponding to the amino acid positions 55–64 of microsomal glutathione transferase. Isolated plasma membrane and microsomal glutathione transferases possess the same amino‐terminal amino acid sequence and digestion with different proteases results in identical fragment patterns as displayed by SDS/PAGE. These data suggest that plasma membrane and microsomal glutathione transferase are identical proteins.
Cell‐specific expression and function of adenylyl cyclases in rat pituitary tumour cell lines
Authors: PAULSSEN Ruth H. , JOHANSEN Per W. , GORDELADZE Jan O. , NYMOEN Øystein , PAULSSEN Eyvind J. , GAUTVIK Kaare M.
Published: March 2005

The present study demonstrates cell‐specific distribution and describes distinct functional regulation of different adenylyl cyclases (AC, types I–VI) in rat pituitary cell tumor cell lines (GH12C1, GH3 and GH4C1 cells) and pituitary tissue.

Northern‐blot analysis revealed a distinct pattern of cell‐specific expression of the different AC types; Ca2+/calmodulin (CaM)‐insensitive AC type II was found in all cell lines tested except GH12C1 cells. The Ca2+‐inhibitable AC type VI was found in all cell types tested. We observed a lack of the Ca2+/CaM‐sensitive AC type I in GH3 and GH4C1 cells. GH12C1 cells exclusively contained both Ca2+/CaM‐sensitive AC types I and III, the latter previously believed to be specific for olfactory tissue. An additional transcript of AC type III was found in rat brain and rat liver tissue. AC type IV, which is Ca2+/CaM insensitive, could be detected in the prolactin‐producing GH3 and GH4C1 cells and pituitary tissue but not in growth‐hormone‐producing GH12C1 cells.

Basal and vasoactive‐intestinal‐peptide‐(VIP)‐releasing‐hormone, somatostatin (SRIF) and thyrothropin‐releasing‐hormone (TRH)‐modulation of AC activity was measured in the presence of 100 μM EGTA, anti‐CaM serum (dilution 1:2000) or 10 μM trifluoroperazine. Antisera against guanine‐nucleotide‐binding protein (G‐protein) α subunits (Gi‐2α, Gsα) and β subunits (Gβ35/36) and CaM were added for functional studies of the SRIF and VIP‐modulated AC in GH12C1 and GH3 cells. These experiments indicate that the VIP and the SRIF receptors are coupled to a Ca2+/CaM‐sensitive AC in GH12C1 cells, different from the AC involved in the regulation of cAMP levels in GH3 and GH4C1 cells. In addition, the βγ‐complex is possibly able to modulate SRIF‐inhibited AC activity by potentiating the inhibitory effect. The TRH receptor in GH3 and GH4C1 cells is coupled to a Ca2+/CaM‐sensitive AC which is different from the already cloned forms of AC types I and III.

We, therefore, conclude that hormone regulation of pituitary tumour cell functions differs between the GH cell lines, due to specific utilisation of AC types.
Site‐directed mutagenesis of cysteine residues of hepatitis B surface antigen Analysis of two single mutants and the double mutant
Authors: ANTONI Beth A. , RODRÍGUEZ‐CRESPO Ignacio , GÓMEZ‐GUTIÉRREZ Julián , NIETO Marta , PETERSON Darrell , GAVILANES Francisco
Published: March 2005

The structure of hepatitis B surface antigen (HBsAg) is mainly maintained by an intricate disulfide network responsible for most of its structural and antigenic properties. Characterization of three cysteine‐replacement mutants of HBsAg has been performed by both structural and immunological methods. Replacement of Cys121 or Cys124 with serine results in mutant proteins that show diminished binding titres to both monoclonal antibodies and to a polyclonal serum, indicating that a structural change has taken place. Circular dichroism analysis shows that the substitution of either of these two residues also diminishes the helical content of the protein. However, the double mutant, in which both cysteine residues have been simultaneously changed, reverts the properties of the single mutations, and shows similar behaviour to the wild‐type protein. Both the single and double cysteine mutants are efficiently glycosylated and secreted from Chinese hamster ovary cells and, in all cases, the mutant proteins assemble into spherical particles of similar buoyant density to both the wild‐type and serum‐derived HBsAg.
Purification and characterization of an extracellular enzyme from Streptomyces antibioticus that converts inactive glycosylated oleandomycin into the active antibiotic
Authors: QUIRÓS Luis M. , HERNÁNDEZ César , SALAS José A.
Published: March 2005

Cell‐free extracts from the oleandomycin producer, Streptomyces antibioticus, possess an intracellular glycosyltransferase capable of inactivating oleandomycin by glysosylation of the 2′‐hydroxyl group in the desosamine moiety of the molecule [Vilches, C., Hernández, C., Méndez, C. & Salas, J. A. (1992) J. Bacteriol. 174, 161–165]. Using a four‐step purification procedure, we have purified an enzyme activity from the culture supernatants from this organism which is able to release glucose from the inactive glycosylated molecule thus reactivating the antibiotic activity. This enzyme activity appeared in the culture supernatants immediately before oleandomycin is detected. The enzyme (molecular mass 87 kDa) showed a high degree of substrate specificity, not acting on other glycosylated macrolides such as methymycin, lankamycin and rosaramicin which are substrates for the glycosyltransferase. A second activity was detected corresponding to a 34‐kDa polypeptide which probably originates from proteolytic cleavage of the larger polypeptide. The 87‐kDa polypeptide possibly catalyses the last biosynthetic step in oleandomycin biosynthesis by S. antibioticus.