bluuebunny/crossref_metadata_2025 huggingface dataset:
Identification of a functionally important carboxyl group in cellobiohydrolase I from Trichoderma reesei
Authors: Tomme Peter , Claeyssens Marc
Published: July 2002

Several aspects of the specific modification of cellobiohydrolase I (CBH I) from Trichoderma reesei with Woodward's reagent K (N‐ethyl‐5‐phenylisoxazolium‐3′‐sulfonate) are presented. The pH dependence of the resulting inactivation points to the implication of an ionising group with a pK
a of approx. 5.5. The rapid inactivation kinetics, the specific protection and the stoichiometry of modification (3 versus 2 residues), together with the isolation and amino acid sequencing of the putative active site peptide, provide a large body of evidence for the presence of a catalytically important carboxyl residue in the 125–135 region of the CBH I amino acid sequence. From the striking homology between this peptide sequence and those of the active site regions of different lysozymes, glutamic acid 126 is retained as the most plausible catalytic residue (proton donor) in CBH I, equivalent to glutamic acid 35 in hen egg white lysozyme. Glutamic acid 127 is proposed as a potential active site residue to the homologous endoglucanase I (EG I) isolated from the same Trichoderma species.
Stimulation of adenosine receptor enhances α1 ‐adrenergic receptor‐mediated activation of phospholipase C and Ca2+ mobilization in a pertussis toxin‐sensitive manner in FRTL‐5 thyroid cells
Authors: Okajima Fumikazu , Sato Koichi , Sho Kimie , Kondo Yoichi
Published: July 2002

Norepinephrine (NE) stimulated FRTL‐5 thyroid cells via an α1‐adrenergic receptor, resulting in cytosolic Ca2+ ([Ca2+]i) mobilization and activation of phospholipase C. Adenosine and its receptor agonist, phenylisopropyladenosine (PIA), although not exerting a direct effect, markedly enhanced the NE‐induced changes. Basal NE action was not totally abolished whereas the permissive action of adenosine and PIA was completely abolished by pretreatment of the cells with islet‐activating protein (IAP), pertussis toxin. The decrease in cAMP level induced by adenosine or PIA is not the cause of their permissive effect, since this effect was not reversed by the addition of cAMP‐increasing agents. We conclude that an IAP substrate GTP‐binding protein(s) plays a novel role in forming a stimulatory coupling between an adenosine receptor and an α1‐adrenergic receptor‐coupled phospholipase C system.
Human recombinant interleukin‐ 1α increases biosynthesis of collagenase and hyaluronic acid in cultured human chorionic cells
Authors: Katsura Morimasa , Ito Akira , Hirakawa Shun , Mori Yo
Published: July 2002

The influence of human recombinant interleukin‐1α (hrIL‐1) on biosynthesis of collagenase and glycosaminoglycans was investigated with fibroblast‐like cells of human chorionic membrane. hrIL‐1 stimulated cells to produce procollagenase in a dose‐dependent manner. Furthermore, it similarly accelerated both biosynthesis and secretion of hyaluronic acid in chorionic cells, but did not modulate the biosynthesis of sulfated glycosaminoglycans. Therefore, the relative concentration of hyaluronic acid vs total glycosaminoglycans increased significantly. These results are connected with the decrease in tensile strength observed in ruptured fetal membranes. Thus, it is proposed that IL‐1 from effused leukocytes in fetal membranes plays an important role in connective tissue metabolism, especially in premature rupture of membranes with chorioamnionitis.
Cytochrome aa3 from the thermoacidophilic archaebacterium Sulfolobus acidocaldarius
Authors: Anemüller S. , Schäfer G.
Published: July 2002

Cytochrome aa
3 serves as a terminal oxidase in the thermoacidophilic archaebacterium Sulfolobus acidocaldarius. A procedure for its isolation is described. The purified preparation consists of only one major polypeptide of 38 kDa apparent molecular mass. The enzyme contains two heme α molecules with midpoint potentials of + 220 and + 370 mV, respectively. The copper content is at least 2 Cu/aa
3. It has only negligible capacity to oxidize cytochrome c, but rather serves as an oxidase for reduced caldariella quinone as present in the membrane of Sulfolobus.
Two isozymes of PKC found in HL‐60 cells show a difference in activation by the phorbol ester TPA
Authors: Beh Ingrid , Schmidt Rainer , Hecker Erich
Published: July 2002

Cytosol from untreated cells and a detergent extract of the particulate fraction from TPA‐treated HL‐60 cells were analyzed for protein kinase C activity by consecutive column chromatography on Mono Q and hydroxyapatite. From both preparations two separate peaks of enzyme activity were obtained. The first peak, eluting at lower salt concentrations, is activated at lower TPA concentrations (3 × 10−9 M) than the other (10−7 M), which was eluted at higher salt concentrations.
Transforming growth factors β1 and β2 as well as milk growth factor decrease anti‐CD3‐induced proliferation of human lymphocytes without inhibiting the anti‐CD3‐mediated increase of [Ca2+]i and the activation of protein kinase C
Authors: Stoeck Michael , Sommermeyer Henning , Miescher Sylvia , Cox David , Alkan Sefik , Szamel Marta
Published: July 2002

Porcine transforming growth factor 1 and 2 (pTGF‐β1 and ‐β2) and milk growth factor (MGF) at 1 ng/ml significantly inhibited the proliferation of human lymphocytes induced by anti‐CD3 antibodies. In contrast, the anti‐CD3‐mediated increase of intracellular Ca2+ and the activation and translocation of protein kinase C were not affected by the transforming growth factors.
Structural analyses of the polymorphic area in type II collagen gene
Authors: Vikkula Miikka , Peltonen Leena
Published: July 2002

The structure of type II collagen gene is extremely well conserved but contains a cluster of high frequency polymorphisms in a 2.2 kb area. Here we report the nucleotide sequence of this DNA area, essential for the PCR‐facilitated RFLP‐analyses of this gene. In the structural analyses we found four differences in the deduced human amino acid sequence when compared to the published bovine amino acid sequence. The donor and acceptor signals and branch point signals required for the splicing events were in agreement with mammalian consensus sequences. The frequency of inverted repeats which could provoke the DNA strand to loop formation and consequently to deletion mutations did not differ from that found in other sequenced genes coding for fibrillar collagens.
Modulation of cytochrome oxidase kinetics by indirect antibody action
Authors: Nicholls P. , Cooper C.E.
Published: July 2002

Polyclonal antibodies raised against isolated subunit V from beef heart cytochrome oxidase or against the intact enzyme increase its apparent affinity for the substrate cytochrome c at the high‐affinity site while diminishing the turnover at that site. At the low‐affinity site the major action of both types of antibody is to reduce the apparent affinity for cytochrome c. At high ionic strengths the kinetic effect of anti‐subunit V is very small although it still binds to the enzyme. The results are interpreted in terms of a model for the enzyme in which antibodies can modulate cytochrome oxidase kinetics by affecting the binding of cytochrome c, even if the antibody‐binding site is on a subunit not directly involved in substrate binding.
Cleavage of the O antigen 4,5,12 of Salmonella typhimurium by hydrofluoric acid
Authors: Helander Ilkka M. , Kitunen Veikko
Published: July 2002

The effect of hydrofluoric acid (aqueous 48% HF) upon different lipopolysaccharides (LPS) was studied, employing conditions (48 h at + 4°C) that are commonly used to dephosphorylate LPS. From the LPS of Salmonella typhimurium having the O antigen 4,5,12 almost all of the O‐antigenic sugars (Abe, Gal, Glc, Man, Rha) were liberated in dialysable form, whereas the saccharide chains of Salmonella LPS with O antigen 6,7 (Man, Glc, GlcNAc) were resistant to HF. The lability towards HF was shown to be due to the presence of the deoxysugar L‐rhamnose in the saccharide backbone of the O antigen 4,5,12, since only Rha was found as the terminal sugar in the corresponding dialysable material. Hydrofluoric acid can thus be used to specifically cleave Rha‐containing polysaccharides.
Identity of GD1C, GT1a and GQ1b synthase in Golgi vesicles from rat liver
Authors: Iber Heinrich , Sandhoff Konrad
Published: July 2002

Competition experiments using GM1b, GD1a and GT1b as substrates, and as mutual inhibitors for ganglioside sialyltransferase activity in preparations of Golgi vesicles derived form rat liver, suggested that sialyl transfer to these three respective compounds, leading to gangliosides GD1C , GT1a and GQ1b, respectively, is catalyzed by one enzyme. These results are incorporated into a model for ganglioside biosynthesis and its regulation.
Crystallisation and preliminary analysis of glucose isomerase from Streptomyces albus
Authors: Dauter Zbigniew , Dauter Miroslawa , Hemker Jörg , Witzel Herbert , Wilson Keith S.
Published: July 2002

The glucose isomerase of Streptomyces albus has been crystallised from a dilute solution of magnesium chloride buffered at a pH of 6.8–7.0. The crystals are in the space group I222 with cell dimensions a=93.9 Å, b=99.5 Å and c=102.9 Å. There is one monomer of the tetrameric molecule per asymmetric unit of the crystal and the packing density is 2.93 Å3·Da−1. The tetramer sits on the 222 symmetry point of the crystal. Native data have been recorded to a resolution of 1.9 Å and the crystals diffract to about 1.5 Å. The α‐carbon coordinates of the Arthrobacter glucose isomerase and the backbone coordinates of the S.olivochromogenes enzyme determined by other groups have been oriented in the present cell. The structure is currently being refined. The binding of several metal ions to the two metal sites has been analysed.
Conformational properties of deltorphin: New features of the δ‐opioid receptor
Authors: Temussi P.A. , Picone D. , Tancredi T. , Tomatis R. , Salvadori S. , Marastoni M. , Balboni G.
Published: July 2002

Deltorphin is an opioid peptide with the sequence H‐Tyr‐D‐Met‐Phe‐His‐Leu‐Met‐Asp‐NH2, recently isolated from the skin of Phyllomedusa sauvagei. Its enormous selectivity towards the δ‐opioid receptor and the similarity of the N‐terminal part of the sequence with that of dermorphin (H‐Tyr‐D‐Ala‐Phe‐Gly‐Tyr‐Pro‐Ser‐NH2), a μ selective peptide isolated from the same natural source, prompted a comparative conformational study. A 1H‐NMR study in two different solvent systems showed that the conformational preferences of the N‐terminal sequences of the two peptides are similar. The different selectivities towards opioid receptors have been interpreted in terms of charge effects. Besides a general trend consistent with the role of the membrane in the preselection of the peptides, the present study demonstrates the crucial role played by charged residues in the interaction inside the receptors.
Effect of phenobarbital on the expression of glutathione S‐transferase isoenzymes in cultured rat hepatocytes
Authors: Vandenberghe Yves , Morel Fabrice , Foriers André , Ketterer Brian , Vercruysse Antoine , Guillouzo André , Rogiers Vera
Published: July 2002

Cultured adult rat hepatocytes were treated daily with 3.2 mM phenobarbital (PB) in order to study its effect on the expression of cytosolic glutathione S‐transferase isoenzymes. Glutathione S‐transferase (GST) activities, using 1‐chloro‐2,4‐dinitrobenzene and 1,2‐dichloro‐4‐nitrobenzene as substrates, were increased when PB was present in the culture medium. After purification and separation of GST on glutathione Sepharose 6 B and reversed‐phase HPLC, respectively, it was observed in vitro that PB caused an increase in the relative amounts of subunits 1, 3 and 7 compared to subunits 2 and 4. Using Northern blot technique, elevated levels of GST subunit 1/2 and 7 mRNA were measured, after addition of PB to the cultures.
Scapharca hemoglobins, type cases of a novel mode of chain assembly and heme‐heme communication
Authors: Petruzzelli Raffaele , Boffi Alberto , Barra Donatella , Bossa Francesco , Ascoli Franca , Chiancone Emilia
Published: July 2002

The sequences of the A and B chains of the Scapharca inaequivalvis tetrameric hemoglobin (HbII) are reported. They are homologous to the corresponding chains of other Arcid hemoglobins. Moreover, a comparison of the present data with the sequence of the S. inaequivalvis dimeric hemoglobin (HbI), for which high‐resolution X‐ray data are available, allows the identification of the residues that direct the assembly of the two components and of those that may be involved in the interdimeric contacts.
Import and processing of proteins by castor bean leucoplasts
Authors: Halpin Claire , Musgrove Janet E. , Lord J.Michael , Robinson Colin
Published: July 2002

The precursor of plastocyanin, a thylakoid lumen protein, is imported by castor bean leucoplasts and processed to an intermediate form by a stromal processing peptidase of similar properties and specificity to the corresponding chloroplast enzyme. No further maturation is observed, indicating that the internal protein transport and maturation system present in the chloroplast is absent in leucoplasts.
Molecular cloning and characterization of the complementary DNA coding for the B‐chain of murine Clq
Authors: Petry Franz , Reid Kenneth B.M. , Loos Michael
Published: July 2002

cDNA clones coding for the B‐chain of murine Clq were isolated from a mouse macrophage library. The characterized clones include the total coding region plus a leader sequence. High homology was found with human Clq B‐chain in the coding region (81%). Northern blot analysis of total RNA from different tissues of Balb/c mice showed one band of approximately 1.2 kb. The highest signal was found in RNA preparations of thioglycolate‐activated peritoneal macrophages. The probe also hybridized with mRNA from spleen, thymus and heart. Extremely weak signals were found in liver, kidney, lung and intestine tissues.
Guanine nucleotides modify calcium entry induced by insulin‐like growth factor‐I
Authors: Kojima Itaru , Kitaoka Masafumi , Ogata Etsuro
Published: July 2002

When calcium influx rate was measured in the cell suspension system instead of confluent monolayer culture, IGF‐I (1 nM) augmented calcium entry independent of the cell cycle. In a cell suspension system, NaF increased calcium entry in a dose‐dependent manner, which was sensitive to tetramethrin. When GTP‐γS was introduced into the cells by employing ATP4−, calcium influx rate was elevated whereas ATP‐γS was without effect. In cells loaded with GDP‐βS, IGF‐I did not stimulate calcium entry. These results suggest that a G‐protein is involved in the signal transduction of IGF‐I action.
Feedback regulation of ornithine decarboxylase expression
Authors: Autelli Riccardo , Holm Ingvar , Heby Olle , Persson Lo
Published: July 2002

The rate‐controlling enzyme in polyamine synthesis, ornithine decarboxylase (ODC), is subject to feedback regulation by the polyamines at the level of translation. In the present study we used a cell‐free translation system to further investigate the mechanism by which this regulation occurs. Lysates of ODC‐overproducing cells were capable of synthesizing large amounts of ODC. The degree of initiation was poor in the lysates and the synthesis of ODC was mainly a result of continued elongation of peptide chains on pre‐initiated ribosomes. By determining the amount of ODC produced in the lysate, we obtained an estimate of the number of ribosomes that were actively translating ODC mRNA at the moment of lysis. Using this polysomal run‐off assay we demonstrated that the polyamine‐mediated regulation of ODC synthesis occurs without any change in the number of ribosomes associated with the message. This finding indicates that the polyamines exert a coordinate effect on initiation and elongation.
A chymosin‐like extracellular acidic endoprotease from Myxococcus xanthus DK101
Authors: Carias Jean-Robert , Raingeaud Joël , Mazaud Catherine , Vachon Gilles , Lucas Nathalie , Cenatiempo Yves , Julien Raymond
Published: July 2002

SDS‐PAGE and N‐tenninal sequence analysis of hydrolysis products from 3 substrates containing a unique sensitive bond usually recognized by chymosin (k‐casein, a synthetic hexapeptide and a recombinant tripartite protein) revealed that a 45 kDa endoprotease of Myxococcus xanthus DK101 cleaved the same characteristic Phe‐Met bond with high specificity. Such an enzyme, easy to obtain from culture supernatant and to use in acidic conditions, could be a new tool for protein engineering.
A circular dichroism study of the pH‐dependent activation/inactivation equilibrium in the glutamate dehydrogenase of Clostridium symbiosum
Authors: Syed Shabih E.-H. , Engel Paul C. , Martin Stephen R.
Published: July 2002

Glutamate dehydrogenase from Clostridium symbiosum has been studied by circular dichroism spectroscopy. The far UV spectrum shows negative bands at 205 nm and 220 nm typical of α‐helical structure. Change in the pH from 7 to 8.8 has little effect on this spectrum, suggesting no major change in secondary structure. The near UV spectrum is strongly negative with a maximum at 208 mn, shoulders at 293, 287, 274 and 266 nm and a trough at 258 nm. This spectrum is intensified by about 50% on‐going from pH 7 to 8.8 indicating a perturbation of the aromatic chromophores in keeping with the conformational change suggested by the concomitant time‐dependent inactivation of the enzyme. The dichroic absorption at 280 nm was used to follow the time course of change from pH 8.8 (inactive) to pH 7 (active). This transition at 30°C was virtually complete in 15 min. The pH dependence of the CD changes suggests a pK of 7.8 for the conformational change.
Modification methylase M.Sau3239I from Streptomyces aureofaciens 3239
Authors: Zelinková E. , Pauliček M. , Zelinka J.
Published: July 2002

By chromatography on phosphocellulose and Heparin‐Sepharose the modification methylase M.Sau3239I was detected and partly purified from cells of Streptomyces aureofaciens 3239. Methylation by this enzyme protects DNA from cleavage by the restriction endonuclease R.Sau3239I. The enzyme catalyzes methylation of adenine to N‐6‐methyladenine in the 5'‐CTCGmAG‐3' recognition sequence.
A factor that specifically binds to the 5'‐untranslated region of encephalomyocarditis virus RNA
Authors: Borovjagin A.V. , Evstafieva A.G. , Ugarova T.Yu. , Shatsky I.N.
Published: July 2002

A protein factor that specifically binds to the 5'‐untranslated region of encephalomyocarditis virus (EMCV) RNA has been found in extracts of ascites carcinoma Krebs‐2 cells. This was done using UV‐irradiation on extracts supplemented with in vitro synthesized 32P‐labelled transcripts followed by analysis of crosslinked proteins by SDS‐polyacrylamide gel electrophoresis. The transcripts represented the viral RNA sequence from nt 315 to 1155, its derivatives with internal deletions or truncated forms. This set of transcripts has allowed us to find out that the factor (p58) binds to EMCV RNA within the sequence 315–485.
The redox‐cycling assay is not suited for the detection of pyrroquinoline quinone in biological samples
Authors: van der Meer Robert A. , Groen Barene W. , Jongejan Jaap A. , Duine Johannis A.
Published: July 2002

Based on the results of the so‐called redox‐cycling assay it has been claimed that various common foods and beverages as well as mammalian body fluids and tissues contain substantial quantities (μM) of free PQQ [M. Paz et al. (1989) in: PQQ and Quinoproteins (J.A. Jongejan and J.A. Duine, eds.) Kluwer Academic Publishers, Dordrecht, pp. 131–143 and J. Killgore et al. (1989) Science 245, 850–852]. However, by investigating samples from such sources with a biological assay of nM sensitivity, we could not confirm these claims. Analysis of the samples with procedures that proved adequate for the detection of PQQ adducts and conjugates gave equally negative results. To account for the positive response in the redox‐cycling assay, as opposed to the negative results obtained by other methods, a search was made for those substances in these samples that caused the false‐positive reactions. It was found that a number of commonly occuring biochemicals like ascorbic and dehydroascorbic acid, riboflavin and to a lesser extent pyridoxal phosphate, gave a positive response in the redox‐cycling assay. The amounts of these interfering substances that were determined in the samples by independent methods could well explain the response. In separate experiments it was found that the effect of PQQ added to biological samples was obscured over an appreciable range of concentrations. For these reasons it must be concluded that the redox‐cycling assay is not suited for the detection of PQQ in these samples. Any claims that are based on the results of this method should be disregarded.
CD4 molecules are restricted to the basolateral membrane domain of in vitro differentiated human colon cancer cells (HT29‐D4)
Authors: Rabenandrasana Chantal , Baghdiguian Stephen , Marvaldi Jacques , Fantini Jacques
Published: July 2002

The CD4 glycoprotein serves as a receptor for the human immunodeficiency virus HIV, the etiologic agent of acquired immunodeficiency syndrome (AIDS). We have examined the expression of CD4 molecules in a clone (HT29‐D4) derived from a human colon adenocarcinoma cell line. HT29‐D4 cells synthesized a 60 kDa polypeptide immunoprecipitated with two anti‐CD4 monoclonal antibodies after metabolic or cell surface labeling. This 60 kDa polypeptide was also immunodetected using the same antibodies in human acute lymphoblastic leukemia cells CEM which are known to express CD4. HT29‐D4 cells can be induced to differentiate into enterocyte‐like cells by removing glucose from the culture medium. Under these conditions, HT29‐D4 cells form a polarized epithelial monolayer in which tight junctions separate the plasma membrane in an apical and a basolateral domain. The localization of CD4 molecules in differentiated HT29‐D4 cells was exclusively restricted to the basolateral membrane domain as demonstrated by radioimmunoassay and indirect immunofluorescence studies. Therefore the HT29‐D4 clonal cell line represents a unique model for polarized HIV infection of colonic epithelial cells and may be useful to understand some of the gastrointestinal disorders occurring in AIDS patients.
Fixation of a highly reactive form of α‐chymotrypsin by micellar matrix
Authors: Levashov A.V. , Klyachko N.L. , Bogdanova N.G. , Martinek K.
Published: July 2002

Using reversed micelles of surfactants solvated by water‐organic cosolvent mixtures as a matrix for enzyme entrapping, it is possible to fix the highly reactive α‐chymotrypsin form. The reactivity of α‐chymotrypsin towards nonspecific substrates increases to the extent comparable with that observed in reactions involving specific substrates.
A cellular 57 kDa protein binds to two regions of the internal translation initiation site of foot‐and‐mouth disease virus
Authors: Luz N. , Beck E.
Published: July 2002

A ribosome‐associated 57 kDa protein from rabbit reticulocytes was linked to the internal translation initiation site of foot‐and‐mouth disease virus by mild UV‐irradiation. Binding studies with different RNA fragments revealed that this protein interacts with two distinct sites within the translational control region. One site is located approximately 400 nucleotides upstream from the translational start codon and the second binding site could be confined to 60 nucleotides preceding this codon. Both sequences coincide with hairpin structures at the two opposite ends of a secondary structure model of the internal ribosomal entry site proposed by Pilipenko et al. [(1989) Nucleic Acids Res. 17, 5701‐5711].
Application of 13C(ω1)‐half‐filtered [1H,1H]‐NOESY for studies of a complex formed between DNA and a 13C‐labeled minor‐groove‐binding drug
Authors: Leupin W. , Otting G. , Amacker H. , Wüthrich K.
Published: July 2002

The complex formed between the anticancer drug 4‐[p‐[p‐(4‐quinolylamino)benzamido]anilino]pyridine (SN 6999) and the decadeoxyribonucleoside nonaphosphate d‐(GCATTAATGC)2 was investigated using two‐dimensional nuclear Overhauser enhancement spectroscopy (NOESY) with a 13C(ω1)‐half‐filter. The two quaternary methyl groups in SN 6999 had been labeled with 13C for these experiments. The simplified subspectra of [1H,1H]‐NOESY obtained with this procedure greatly facilitate the identification and assignment of intermolecular NOEs. Quite generally, the combined use of isotope labeling and heteronuclear filters in [1H,1H]‐NOESY provides an improved experimental basis for structural studies of drug/ DNA complexes.
Use of ion exchange chromatography for the study of RecA‐DNA interaction
Authors: Takahashi Masayuki , Hagmar Per
Published: July 2002

In vitro binding or RecA protein to double‐stranded DNA (dsDNA) was studied using ion‐exchange liquid chromatography. The method allowed quantification of both free DNA and free protein. The results unambiguously showed a binding stoichiometry or 3 base pairs per RecA monomer. The binding exhibited cooperativity, and the stoichiometry suggested that RecA does not form complexes with two molecules or dsDNA. More than 90% of RecA molecules in the sample were active for DNA binding.
Glycine‐144 is required for efficient folding of outer membrane protein PhoE of Escherichia coli K12
Authors: de Cock Hans , Quaedvlieg Nicolette , Bosch Dirk , Scholten Monica , Tommassen Jan
Published: July 2002

Short stretches of similar sequences have been detected in unrelated bacterial outer membrane proteins (Nikaido and Wu (1984) Proc. Natl. Acad. Sci. USA 81, 1048‐1052). In the most pronounced similarity region, only a glycine residue is absolutely conserved. To investigate whether this glycine residue is essential for outer membrane incorporation, oligonucleotide‐directed mutagenesis was applied to replace this residue, i.e. Gly‐144, as well as two other Gly‐residues in pore protein PhoE. Substitution of Gly‐52 and Gly‐258 by Ala And Val, respectively, did not influence outer membrane incorporation. However, the substitution of Gly‐144 by Leu affected the efficiency of outer membrane incorporation. After in vitro synthesis this mutant protein was less efficiency precipitated with monoclonal antibodies that recognize coformational epitopes than wild‐type PhoE, showing that the mutation interferes with correct folding of the protein.
Correct intron splicing generates a new type of a putative zinc‐binding domain in a transcriptional activator of Aspergillus nidulans
Authors: Kulmburg Peter , Prangé Thierry , Mathieu Martine , Sequeval Daria , Scazzocchio Claudio , Felenbok Béatrice
Published: July 2002

alcR is the pathway‐specific transcriptional activator of the ethanol regulon in the filamentous fungus, Aspergillus nidulans. The deduced amino acid sequence of a cDNA clone, including the 5′ part of the alcR‐mRNA, shows that a putative Zn‐binding of the all‐cysteine class, exemplified by GAL4 is present. This structure presents some striking features. At variance with other structures of this class, the binding domain is strongly asymmetrical. Model building indicates that the zinc‐binding motif of alcR could adopt an helix‐turn‐helix structure. We propose that the DNA binding motif of alcR could participate in two types of DNA‐binding structures: the zinc‐cluster and the helix‐turn‐helix.
Eukaryotic DNA primase abortive synthesis of oligoadenylates
Authors: Podust V.N. , Vladimirova O.V. , Manakova E.N. , Lavrik O.I.
Published: July 2002

Calf thymus DNA polymerase α‐primase, human placenta DNA polymerase α‐primase and human placenta DNA primase synthesized oligoriboadenylates of a preferred length of 2–10 nucleotides and multimeric oligoribonucleotides of a modal length of about 10 monomers on a poly(dT) template. The dimer and trimer were the prevalent products of the polymerization reaction. However, only the oligonucleotides from heptamers to decamers were elongated efficiently by DNA polymerase α.
Di(1,N6‐ethenoadenosine)5′, 5‴‐P1,P4‐tetraphosphate, a fluorescent enzymatically active derivative of Ap4A
Authors: Rotilán P. , Ramos A. , Pintor J. , Torres M. , Miras-Portugal M.T.
Published: July 2002

Di(1,N6‐ethenoadenosine) 5′, 5‴‐P1, P4‐tetraphosphate, ϵ‐(Ap4A), a fluorescent analog of Ap4A has been synthesized by reaction of 2‐chloroacetaldehyde with Ap4A. At neutral pH this Ap4A analog presents characteristic maxima at 265 and 274 nm, shoulders at ca 260 and 310 nm and moderate fluorescence (λexc 307 nm, λem 410 nm). Enzymatic hydrolysis of the phosphate backbone produced a slight hyperchromic effect but a notorious increase of the fluorescence emission. Cytosolic extracts from adrenochromaffin tissue as well as cultured chromaffin cells were able to split ϵ(Ap4A) and catabolize the resulting ϵ‐nucleotide moieties up to ϵ‐Ado.
The transcriptional control proteins c‐Myb and v‐Myb contain a basic region DNA binding motif
Authors: Carr Mark D. , Mott Richard F.
Published: July 2002

A class of transcriptional control proteins, typified by c‐Myb, are characterised by a highly conserved N‐terminal DNA binding domain, which is composed of either 2 or 3 imperfect repeats of about 52 amino acids. A sequence homology search of the SWISSPROT protein sequence data bank with this region from mouse c‐Myb has allowed us to identify an area near the C‐terminus of repeat 2 that contains a short sequence motif, known as the basic region, which forms the DNA binding site in both leucine zipper and helix‐loop‐helix transcription factors. We therefore propose that this region of repeat 2 and the homologous part of repeat 3 will form the Myb DNA binding site.
Differential processing of hormone precursor Independent production of somatostatins 14 and 28 in transfected neuroblastoma 2A cells
Authors: Brakch Noureddine , Rholam Mohamed , Nault Christiane , Boileau Guy , Cohen Paul
Published: July 2002

Neuro 2A cells infected with a retroviral vector carrying human prosomatostatin cDNA expressed and processed correctly the precursor into somatostatins‐14 and ‐28 [(1989) EMBO J. 8, 2911–2916]. In order to study the mechanisms by which the active hormone sequences arise, site directed mutagenesis was performed on either the dibasic (ArgLys) or monobasic (Arg) cleavage sites involved in the production of somatostatins‐14 and ‐28, respectively. Radioimmunochemical analysis of the somatostatin‐related products indicated that replacement of either Arg−2‐Lys−1 by Asn−2‐Asn−1 or of Arg−15 by Asn−12 resulted in the exclusive production of either somatostatin‐28 or ‐14, respectively. Moreover only prosomatostatin[1–76] was detected and no somatostatin‐28[1–12] could be measured in cell extracts. Selective suppression of either somatostatin‐14 or somatostatin‐28 release by mutation did not affect the level of production of the other hormone but resulted in a correlative increase of unprocessed prosomatostatin. It is concluded that in this cell type (i) somatostatin‐14 is exclusively generated by dibasic cleavage at the Arg−2‐Lyst1̄ site of the intact precursor with concomitant product of prosomatostatin[1–76], and (ii) no direct interactions between the monobasic and dibasic processing domains occur.
Chemical and functional analysis of components of adenylyl cyclase from human platelets treated with phorbolesters
Authors: Simmoteit Robert , Schulzki Horst-Dieter , Palm Dieter , Mollner Stefan , Pfeuffer Thomas
Published: July 2002

Human platelets, prelabeled with [32P]phospate were treated with tetradecanoylphorbol acetate (TPA) for 5 min at 37°C. Phosphorylation of the components of adenylyl cyclase was determined in membranes using specific antibodies against G‐proteins and the catalytic moiety. Less than 0.01 mol of [32P]phosphate/mol could be detected in immunoprecipitates using antibodies against sequences within the α‐subunit of the GTP binding protein Gi. TPA, however, caused the incorporation of 0.67–1.1 mol of [32P]phosphate per mol of catalyst while 0.13‐0.2 mol were found in the absence or TPA. Lack of modification of the α‐subunit of Gi was also indicated by the results of reconstitution experiments with purified Giα from bovine brain: adenylyl cyclase in membranes from untreated platelets was significantly more inhibited by added Giα, than that from TPA treated cells. While β,γ‐subunits were like‐wise inhibitory no difference dependent on platelet‐pretreatment could be observed.
Identification of a 42 kDa protein as a substrate of protein phosphatase 1 in cilia from Paramecium
Authors: Klumpp Susanne , Schultz Joachim E.
Published: July 2002

Okadaic acid, a specific inhibitor of protein phosphatase 1 in Paramecium causes sustained backward swimming in response to depolarising stimuli (S. Klumpp et al. (1990) EMBO J. 9, 685). Here, we employ okadaic acid, tautomycin, microcystin LR and inhibitor 1 as phosphatase inhibitors to identify a 42 kDa protein in the excitable ciliary membrane that is dephosphorylated by protein phosphatase 1. Identification of the 42 kDa protein was facilitated by the finding that the protein kinase responsible for its phosphorylation uses Ca‐ATP as a substrate just as effectively as Mg‐ATP. Notably, dephosphorylation of the 42 kDa protein is specifically inhibited by cyclic AMP; cyclic GMP has no effect.
H+/e− stoichiometry of mitochondrial cytochrome complexes reconstituted in liposomes Rate‐dependent changes of the stoichiometry in the cytochrome c oxidase vesicles
Authors: Capitanio N. , Capitanio G. , De Nitto E. , Villani G. , Papa S.
Published: July 2002

The H+/e− stoichiometry of protonmotive cytochrome c oxidase, isolated from bovine heart mitochondria and reconstituted in liposomes, has been determined by making use of direct spectrophotometric measurements of the initial rates of e− flow and H+ translocation. It is shown that the ←H+/e− ratio for redox‐linked proton ejection by the oxidase varies from around 0 to a maximum of 1 as a function of the rate of overall electron flow in the complex.
Mycoplasma Gallisepticum strain S6 genome contains three regions hybridizing with 16 S rRNA and two regions hybridizing with 23 S and 5 S rRNA
Authors: Scamrov A. , Beabealashvilli R.
Published: July 2002

Southern hybridization and cloning experiments revealed existence of 3 regions hybridizing with 16 S rRNA and 2 regions hybridizing with 23 S and 5 S rRNA in Mycoplasma Gallisepticum strain S6 genome thus forming 4 separate contiguous regions. One set of a putative rRNA genes is organized classically for eubacteria order 16 S‐23 S‐5 S. The other two 16 S rRNA and the other one 23 S‐5 S rRNA hybridizing regions are separated from each other and from the complete rRNA operon for a distance of more than 6 kb.
Lysine residues on ferredoxin‐NADP+ reductase from Anabaena sp. PCC 7119 involved in substrate binding
Authors: Medina Milagros , Mendez Enrique , Gomez-Moreno Carlos
Published: July 2002

Ferredoxin‐NADP+ reductase from Anabaena sp. PCC 7119 is chemically modified by pyridoxal 5′‐phosphate. The incorporation of 2±0.3 mol pyridoxal 5′‐phosphate/mol ferredoxin‐NADP+ reductase inhibited NADPH‐cytochrome c reductase activity by up to 95% while 55% of diaphorase activity still remained. Considerable protection against inactivation was afforded by ferredoxin. Chymotryptic cleavage of the modified enzyme was performed, the peptides were separated by high performance liquid chromatography, and the peptides containing pyridoxamine 5′‐phosphate were identified by their fluorescence and by their absorbance at 325 nm. Three major labelled peptides were found. Their sequences were comprised of residues 46–54, 231–235 and 289–295. Lys‐53 and ‐294 were the residues which presented the highest degree of modification and seem to be involved in the ferredoxin binding site of ferredoxin‐NADP+ reductase from Anabaena sp. PCC 7119.
Sequence‐specific resonance assignment and conformational analysis of subtilin by 2D NMR
Authors: Chan Weng C. , Bycroft Barrie W. , Leyland Mark L. , Lian Lu-Yun , Yang J.C. , Roberts Gordon C.K.
Published: July 2002

Subtilin, a 32‐amino acid peptide with potent antimicrobial activity, has been isolated from Bacillus subtilis ATCC6633. The chemical structure has been confirmed by the unambiguous sequence‐specific assignment of its 1H NMR spectrum. Detailed NMR analysis revealed that subtilin is a rather flexible molecule; the only observed conformational contraints were those imposed by the cyclic structures created by the tanthionine and 3‐methyllanthionine residues. These results suggest that in aqueous solution subtilin and the homologous peptide nisin have similar conformation.
C‐CAM (Cell‐CAM 105) is a calmodulin binding protein
Authors: Blikstad Ingrid B. , Wikström Tarja , Aurivillius Magnus , Öbrink Björn
Published: July 2002

C‐CAM (Cell‐CAM 105) is a transmembrane cell adhesion molecule belonging to the immunoglobulin superfamily. It mediates intercellular adhesion of rat hepatocytes and occurs in various isoforms in several epithelia, vessel endothelia and leukocytes. We now report that purified liver C‐CAM interacts specifically with calmodulin. Binding was observed both when 125I‐labeled C‐CAM was used in a dot‐blot assay and when 125I‐labeled calmodulin was used in a gel overlay assay. Experiments with protease‐generated peptides indicated that calmodulin bound to the cytoplasmic domain of C‐CAM. Analyses of whole liver membranes demonstrated that C‐CAM is one of five major proteins that bind calimodulin in a calcium‐dependent manner.
Interchain and intrachain crosslinking of actin thiols by a bifunctional thiol reagent
Authors: Faulstich Heinz , Heintz Daniela , Drewes Gerard
Published: July 2002

From 1,9‐nonylenedithiol and Ellman's Reagent the bifunctional asymmetric disulfide n‐nonylene‐1,9‐bis‐[5‐dithio‐(2‐nitrobenzoic acid)] (NBDN) was prepared. By monovalent reaction with cysteine‐374 the crosslinker could be introduced into monomeric actin, with release of one equivalent of yellow 2‐nitro‐5‐thiobenzoate (NTB). From the monovalent actin derivative we prepared a crosslinked actin dimer (Cys‐374–Cys‐374′) as well as a monomer with a crosslink between Cys‐374 and Cys‐10. Neither crosslinked actin species was able to polymerize the crosslinked monomer even in the presence of phalloidin. The crosslinked monomer polymerized on the addition of dithiothreitol, thus providing the first unpolymerizable actin species whose polymerizability can be restored under mild conditions. We suggest the use of NBDN as a thiol‐specific crosslinker that reacts under spectrophotometric control and can be removed by the addition of thiols.
31P NMR investigation of the backbone conformation and dynamics of the hexamer duplex d(5′‐GCATGC)2 in its complex with the antibiotic nogalamycin
Authors: Searle Mark S. , Lane Andrew N.
Published: July 2002

Heteronuclear chemical shift correlation experiments confirm that the two down‐field shifted 31P resonances in the spectrum of the (nogalamycin)2‐d(GCATGC)2 complex correspond to the phosphodiesters CpA and TpG at the intercalation sites. 31P relaxation measurements (R1, R2 and {1H}‐31P NOE) at 4.7 and 9.4 T permit the correlation time of each phosphate to be determined together with their chemical shift anisotropies. Significant differences in deoxyribose H3′−31P coupling constants and chemical shift anisotropy contributions are observed, consistent with an asymmetric DNA backbone conformation for the phosphate groups at the intercalation sites. Large amplitude internal motions of the phosphates do not appear to contribute significantly to relaxation.
Baculovirus expression of mammalian G protein α subunits
Authors: Labrecque Jean , Caron Mireille , Torossian Krikor , Plamondon Josée , Dennis Michael
Published: July 2002

Complementary DNAs encoding three subtypes of the α subunit (αi−1, αo and αx) of rat guanyl nucleotide regulatory proteins were used to construct recombinant baculoviruses which direct high‐level expression of the corresponding proteins in cultured Sf9 insect cells. The expressed proteins were recognized by polyclonal antisera specific for the different α chains, and co‐migrated with the native proteins from rat brain membranes in immunoblotting analyses. Soluble and particulate forms of all three immunoreactive α chains were observed following ultracentrifugation of cell lysates. Biosynthetic radiolabelling of infected cells with [35S]methionine or [3H]myristate showed that both soluble and particulate forms of αi−1 and αo were myristoylated; in contrast, α, did not incorporate myristate. The soluble fractions from cells expressing α chains showed high levels of GTP‐binding activity over that observed in uninfected cells, or in cells infected with wild‐type virus. The peak expression levels observed at 72 h post‐infection were highest for αo at ca, 400 pmol of GTP‐γ‐35S/mg protein, or roughly 2% of the total soluble protein. The results of this work show that the baculovirus system can be employed for high‐level production of mammalian G protein α chains which retain GTP‐binding activity and are appropriately modified by myristoylation.
Validity of putative calcium binding loops of photoprotein aequorin
Authors: Oishi Osamu , Nagatomo Akinori , Kohzuma Takuji , Oda Naoko , Miyazima Tohru , Ohno Motonori , Sakaki Yoshiyuki
Published: July 2002

Three peptides containing the putative Ca2+ binding loops, I, II and III, respectively, of a photoprotein, aequorin, from jellyfish Aequorea victoria were synthesized by a solid‐phase procedure. The peptides bound Ca2+ with dissociation constants or 10−3 to 10−4 M, providing evidence for the assumption that Ca2+ binding loops are actually responsible for the binding of Ca2+. When the highly conserved 6th glycine residue in the 12‐residue loops was replaced by arginine, no large effect was observed on Ca2+ binding. Exposure to a hydrophobic environment and the binding or Ca2+ brought about conformational changes to the peptides.
A critical role of protein‐bound water in the catalytic cycle of cytochrome P‐450 camphor
Authors: Di Primo Carmelo , Sligar Stephen G. , Hoa Gaston Hui Bon , Douzou Pierre
Published: July 2002

The rates of NADH oxidation during the hydroxylation of camphor by cytochrome P‐450cam were followed in the presence of co‐solvents used to increase the osmotic pressure surrounding the protein‐bound water. As a result, the measured V
max decreases independently of the perturbant tested. Roughly 28 molecules of water, involved during the catalytic cycle, are deduced from the variation of V
max as a function of osmotic pressure. These molecules, in part, could be those present in the cytochrome P.450cam‐putidaredoxin interface.
p33, an endogenous target protein for arginine‐specific ADP‐ribosyltransferase in chicken polymorphonuclear leukocytes, is highly homologous to mim‐1 protein (myb‐induced myeloid protein‐1)
Authors: Yamada Kazuo , Tsuchiya Mikako , Mishima Koichi , Shimoyama Makoto
Published: July 2002

We have determined the partial amino acid sequence of p33, an endogenous substrate protein for arginine‐specific ADP‐ribosyltransferase in chicken polymorphonuclear leukocytes (heterophils), and found that the sequence was completely identical with the regions of amino acid sequences deduced from mim‐1 (named for myb‐induced myeloid protein‐1, which is expressed in chicken promyelocytes) cDNA [(1989) Cell, 59, 1115–1125], except for one amino acid difference (Tyr297→Ile). These results together with data on cellular and subcellular distributions of p33 in heterophils suggest that mim‐1 may encode the precursor protein of p33.
Distinct forms of the haem o‐Cu binuclear site of oxidised cytochrome bo from Escherichia coli
Authors: Watmough Nicholas J. , Cheesman Myles R. , Gennis Robert B. , Greenwood Colin , Thomson Andrew J.
Published: July 2002

Oxidised, formate‐bound and fluoride‐bound forms of E. coli cytochrome bo give rise to an electronic absorption band near 630 nm, diagnostic of high‐spin ferrric haem o, whose position is sensitive to the nature of the bound anion. In all three forms, haem o remains spin‐coupled to cuB(II), resulting in distinct broad X‐band EPR signals. Those of formate‐bound cytochrome bo are similar to the signals seen in slow cytochrome aa
3 but cannot be induced by incubation at acid pH suggesting that the endogenous earboxylate believed to be important in slow cytochrome aa
3 is not present in cytochrome bo. The oxidised form gives rise to novel EPR signals at g = 3.74 and g = 3.08 which have not been detected in cytochrome aa
3 and may arise from a weak magnetic coupling between high‐spin haem o, S = 5/2, and Cub(ii), S = ½.
Phosphorylated residues as specificity determinants for an acidophilic protein tyrosine kinase
Authors: Donella-Deana Arianna , Marin Oriano , Brunati Anna Maria , Cesaro Luca , Piutti Claudia , Pinna Lorenzo A.
Published: July 2002

Spleen TPK‐IIB is an acidophilic protein tyrosine kinase, devoid of autophosphorylation activity, whose phosphorylation of the src‐peptide NEYTA is crucially specified by Glu‐2 [(1991) J. Biol. Chem. 266,17798‐17803]. We show that phosphothreonine, phosphotyrosine and phosphoserine are, in this order, specificity determinants even more effective than glutamic acid if they are replacing Glu‐2, to give the phosphopeptides NTpYTA, NYpYTA, NSpYTA, respectively. Non‐phosphorylated threonine, tyrosine and serine are conversely ineffective. Consequently also the heptapeptide GEGTYGV reproducing the phosphoacceptor and inhibitory site of p34cdc2 is not appreciably affected by TPK‐IIB, unless its threonyl residue is previously phosphorylated, the phosphoderivative GEGTpYGV being readily phosphorylated at its tyrosyl residue. Such a behaviour is unique for TPK‐IIB among the protein tyrosine kinases tested (lyn‐TPK, fgr‐TPK and EGF‐receptor, besides TPK‐IIB). These data provide the first evidence that, in some instances, the targeting by protein tyrosine kinases can be specifically determined by the previous phosphorylation of the peptide substrate, thus extending the concept of ‘hierarchal phosphorylation’ [(1991) J. Biol. Chem. 266, 14139‐14142] to tyrosyl residues as well.
The primary structure of inhibitor of cysteine proteinases from potato
Authors: Kriz̆aj I. , Drobnic̆-Kos̆orok M. , Brzin J. , Jerala R. , Turk V.
Published: July 2002

The complete amino acid sequence of the cysteine proteinase inhibitor from potato tubers was determined. The inhibitor is a single‐chain protein having 180 amino acid residues. Its primary structure was elucidated by automatic degradation of the intact protein and sequence analysis of peptides generated by CNBr, trypsin and glycyl endopeptidase. A search through the protein sequence database showed homology to other plant proteinase inhibitors of different specificities and non‐inhibitory proteins of M
r around 20,000. On the basis of sequence homology, prediction of secondary structure and fold compatibility, based on a 3D‐1D score to the three‐dimensional profile of Erythrina caffra trypsin inhibitor, we suggest that the potato cysteine proteinase inhibitor belongs to the superfamily of proteins that have the same pattern of three‐dimensional structure as soybean trypsin inhibitor. This superfamily would therefore include proteins that inhibit three different classes of proteinases ‐ serine, cysteine and aspartic proteinases.
RPII15 codes for the Mr 15,000 subunit 9 of Drosophila melanogaster RNA polymerase II
Authors: Liu Zhihong , Kontermann Roland E. , Schulze Rüdiger A. , Petersen Gabriele , Bautz Ekkehard K.F.
Published: July 2002

The RPII15 gene product of Drosophila melanogaster, which has recently been identified by sequence comparison, possesses a high similarity to subunit 9 of yeast RNA polymerase II. Using the polymerase chain reaction the coding region of RPII15 was isolated from genomic DNA of adult flies. Sequence analysis shows four amino acid substitutions in comparison to the previously reported sequence. Antisera were generated against bacterially expressed RPII15 and were used for immunoblotting experiments with RNA polymerase II of Drosophila melanogaster. This analysis identified the M
r, 15,000 subunit 9 as gene product of RPII15.