bluuebunny/crossref_metadata_2025 huggingface dataset:
Monoclonal antibodies to human pancreatic trypsin 1 inhibit the activation of human trypsinogens 1 and 2
Authors: GUY‐CROTTE Odette , MISZCZUK‐JAMSKA Barbara , BRAYLE Alex , LAFONT Pannie , FIGARELLA Catherine
Published: March 2005

Two monoclonal antibodies (Mab) raised against human pancreatic trypsin 1, Mab G6 and A8, were previously isolated and characterized. The two Mab which recognize trypsinogen 1 are found to inhibit the activation of trypsinogen 1 by enterokinase. The inhibition of activation by the two Mab is concentration‐dependent, rapid and virtually complete with Mab G6. Activation of trypsinogen 2 is totally inhibited by Mab G6, while Mab A8 has no effect on the activation of trypsinogen 2. The two monoclonal antibodies have opposite effects on the proteolytic activity of trypsin 1; Mab G6 increases proteolytic activity while Mab A8 inhibits trypsin activity by as much as 40%. This inhibition is concentration dependent but cannot account for the complete inhibition of activation of trypsinogen 1. Neither monoclonal antibody significantly inhibits the esterolytic activity of either form of human trypsin. Western‐blot analysis of the reactivity of the two monoclonal antibodies with trypsinogens of various species shows that only Mab G6 cross‐reacts with dog trypsinogen.
Isolation and structural determination of three peptides from the insect Locusta migratoria
Authors: NAKAKURA Norihiko , HIETTER Hélène , VAN DORSSELAER Alain , LUU Bang
Published: March 2005

We have isolated three novel peptides from the aqueous extract of the pars intercerebralis of male and female adults of the insect Locusta migratoria. After extensive HPLC purification, the peptides were characterised by automated Edman degradation and electrospray mass spectrometry: one is a 35‐residue peptide (3752.3 ± 1.1 Da) containing six cysteines involved in three intramolecular disulfide bridges, the second is a 36‐residue peptide (3919.2 ± 0.9 Da), also cross‐linked by three intramolecular disulfide bridges. This second peptide is post‐translationally modified by a single fucose moiety, which was identified by gas chromatography coupled to mass spectrometry. These two peptides show a strong sequence similarity. Additionally, they were also found in larger amounts in the fat body of Locusta; this finding raises the question of their exact site of synthesis. The third peptide (5776.3 ± 0.9 Da), a 54‐residue peptide cross‐linked by six intramolecular disulfide bridges, is structurally related to the two other peptides, but to a lesser extent. Mass spectrometry has shown that all the cysteines within these three peptides are involved in intramolecular disulfide bridges; however, the location of these bridges is not yet established and is currently being investigated. A computer search of sequence data banks did not reveal any significant similarity of these three peptides with other known proteins.
Isolation and amino acid sequence of a glutamic acid specific endopeptidase from Bacillus licheniformis
Authors: SVENDSEN Ib , BREDDAM Klaus
Published: March 2005

An endopeptidase cleaving specifically at the carboxyl side of acidic amino acid residues, preferentially at glutamic acid, has been isolated from a commercial extract obtained by fermentation with Bacillus licheniformis. Using ion‐exchange chromatography and affinity chromatography on bacitracin‐Sepharose, it was possible, from 100 ml commercial extract, to isolate 100 mg homogeneous enzyme in a yield of 50%. It is the first description of a large‐scale isolation of a Glu/Asp‐specific enzyme. The preparation was essentially free of contaminating activities. The isolated enzyme consists of one peptide chain of 222 amino acid residues and has a calculated molecular mass of 23 589 Da. The determined amino acid sequence shows similarity to the Glu/Asp‐specific enzymes previously isolated from Staphylococcus aureus V8, Actinomyces sp. and Streptomyces thermovulgaris. The substrate preference of the enzyme has been investigated. Although non‐specific cleavages were observed after prolonged hydrolysis at high enzyme concentrations the enzyme appears to be essentially specific for Glu‐Xaa and Asp‐Xaa, with strong preference for the former. The isolated enzyme exhibits a bell‐shaped pH/activity profile with an optimum at pH 7.5–8.0. The activity is adversely affected by high ionic strength and beneficially affected by the inclusion of calcium ions in the assay medium. The enzyme is completely inhibited by diisopropylfluorophosphate, suggesting that it is a serine endopeptidase. It is partially inhibited by EDTA.
31P‐NMR assignment and conformational study of NADPH bound to Lactobacillus casei dihydrofolate reductase based on two‐dimensional 1H‐31P‐heteronuclear and 1H‐detected 1H‐31P‐shift‐correlation experiments
Authors: GEROTHANASSIS Ioannis P. , BIRDSALL Berry , BAUER Christopher J. , FEENEY James
Published: March 2005

For any detailed NMR conformational study of a protein – ligand complex it is essential to have specific resonance assignments. We have now assigned the pyrophosphate 31P resonances in spectra of NADPH bound to Lactobacillus casei dihydrofolate reductase (DHFR) by using a combination of 1H‐31P‐heteronuclear shift‐correlation (HETCOR), 1H‐31P‐heteronuclear multiple‐quantum‐coherence correlation spectroscopy (HMQC‐COSY), 1H‐1H COSY, homonuclear Hartmann‐Hahn (HOHAHA) and NOE spectroscopy (NOESY) experiments.

The nicotinamide pyrophosphate phosphorus, P(n), has been unequivocally assigned to a signal (−14.07 ppm) which shows a large 3JP‐O‐C‐H coupling constant. Such a coupling constant when combined with the appropriate Karplus relationship provides conformational information about the P‐O‐C‐H torsion angle. The torsion angle changes by 65°± 10° for the binary complex compared with the value in free NADPH. The observed coupling constants for the binary (DHFR – NADPH) and ternary (DHFR – NADPH – methotrexate) complexes (12.3 and 10.5 ± 0.6 Hz, respectively) indicate that the pyrophosphate group has a similar conformation in the two complexes.
Effects of ions on the intrinsic activities of c‐H‐ras protein p21
Authors: MISTOU Michel‐Yves , COOL Robbert H. , PARMEGGIANI Andrea
Published: March 2005

The influence of the ionic environment on the intrinsic GTPase activity and the guanine‐nucleotide interaction of Ha‐ras protein p21 were studied in various experimental conditions and compared with the behaviour of elongation factor (EF) Tu. To this purpose, nucleotide‐free p21 was prepared, which is much more stable than by any other reported method. Specific differences between p21 and EF‐Tu were found in the action of divalent anions which strongly enhance the dissociation rate of p21 · GDP without affecting that of EF‐Tu. Unlike EF‐Tu, the GTPase activity of p21 is only slightly dependent on the presence and concentration of monovalent cations. The concentrations of Mg2+ influencing the dissociation rate of the p21 · GDP complex are much higher than for the intrinsic GTPase activity, an effect also observed for EF‐Tu. These results point to two distinct roles of Mg2+: as a conformational regulator of the interaction with the substrate and as a key element for the hydrolysis of GTP. The GTPase activity of p21 is not affected by changes in pH over the range 6–9.2, different from that of EF‐Tu. However, stabilization by kirromycin confers a pH independence to the GTPase of EF‐Tu in the pH range 6.5–10, suggesting that the bell‐shaped behaviour of this activity in the absence of the antibiotic is due to denaturation. This implies similar properties in the catalytic mechanism of these two guanine‐nucleotide‐binding proteins.
Evidence against a role for phosphorylation/dephosphorylation in the regulation of acyl‐CoA: cholesterol acyl transferase
Authors: CORTON Julia M. , HARDIE D. Grahame
Published: March 2005

1. As detailed below, we have been able to reproduce observations of time‐dependent changes in the activity of acyl‐CoA: cholesterol acyl transferase (ACAT) in rat liver microsomes, that were suggested to represent evidence of a role for reversible phosphorylation in the regulation of cholesterol ester formation.

2. ACAT in washed rat liver microsomes was inactivated in a time‐dependent manner in the presence of Mg2+. However, this effect of Mg2+ appears to be caused by aggregation of microsomal vesicles rather than dephosphorylation, since it could be abolished by rehomogenization, and was mimicked by Ca2+, another agent which causes aggregation. Fluoride did not prevent this effect of Mg2+, but masked it by causing a rapid activation that appeared to be a non‐specific effect of increased ionic strength.

3. Under conditions where other proteins were rapidly dephosphorylated, microsomal ACAT activity from rat liver was not affected by incubation with the purified catalytic subunits of protein phosphatases 1, 2A or 2C. Similar results were obtained using protein phosphatases 1 or 2A on microsomes from a macrophage cell line (J774.2 cells). Incubation of cultured J774.2 cells with a cell‐ permeable inhibitor of these two protein phosphatases, okadaic acid, also had no effect on cholesterol ester formation.

4. A high‐speed‐centrifugation supernatant fraction (S303) from rat liver activated ACAT in the presence of MgATP. This effect was not abolished by prior heat‐treatment of the fraction, and the supernatant fraction could not be replaced by purified AMP‐activated protein kinase or a variety of other protein kinases.

5. The results above were obtained using assays involving endogenous cholesterol as the substrate. The MgATP‐dependent activation by S303 was reduced or abolished when the assays were carried out in the presence of the detergent Triton WR‐1339 plus cholesterol, or detergent alone.

6. These results do not support the idea that ACAT is regulated by reversible phosphorylation. The most likely explanation for the effect of S303 is that it is an artefact caused by changes in the availability of endogenous cholesterol to the enzyme.
Redox control of catalysis in ATP‐citrate lyase from rat liver
Authors: WELLS Timothy N. C. , SAXTY Barbara A.
Published: March 2005

In thiol redox buffers at pH 8.0, rat liver ATP‐citrate lyase is in equilibrium between an oxidised inactive form and a reduced active form. The reduced enzyme is inactivated by oxidised glutathione (GSSG) at a rate of 45 min−1· M−1 and the oxidised enzyme is activated by reduced glutathione (GSH) at a rate of 3.2 min−1· M−1. At redox equilibrium, the enzyme activity depends on the ratio [GSH]2/[GSSG]. The inactivation involves formation of a protein‐protein disulphide rather than a protein‐glutathione complex. This reaction has Keq= 78 ± 7 mM for the oxidative reaction. Activity can therefore be controlled by the redox state of the cell, being more active in the fed state than in the oxidatively stressed state.

This redox process is also important in the in vitro enzyme assay, where ATP‐citrate lyase is in redox equilibrium with oxygen and either dithiothreitol or 2‐mercaptoethanol. Reduction is a two‐step process, requiring high concentrations of reductant for full activation (30 mM dithiothreitol or 200 mM 2‐mercaptoethanol). The enzyme inhibitor, Medica‐16 raises the redox equilibrium constant to greater than 400 mM. It binds more tightly to the oxidised form of the enzyme, with Ki < 40 μM compared to 180 μM for the reduced form.
Repair of transcriptionally active and inactive genes during S and G2 phases of the cell cycle
Authors: RUSSEV George , BOULIKAS Teni
Published: March 2005

To study the effect of ultraviolet irradiation on S and G2 phases of the cell cycle, BB88 mouse cells synchronized by a double thymidine block were exposed to ultraviolet light, and rates of DNA synthesis and mitotic indexes were determined at regular intervals. It was found that with increasing ultraviolet dose, semiconservative DNA synthesis decreased and the sharp mitotic wave observed in the unirradiated cells gradually degenerated. To study repair, semiconservative DNA replication was inhibited with hydroxyurea at different time intervals after releasing cells from the block and the DNA synthesized as a result of repair of the ultraviolet damage was labeled with 5′‐bromodeoxyuridine (BrdU). The newly repaired DNA was separated from bulk DNA by immunoprecipitation with monoclonal anti‐BrdU antibody, labeled with 32P and hybridized to nine different gene and oncogene probes dot‐blotted in excess on nylon membranes to determine their abundance in the repaired DNA. The results showed that: (a) the most actively repaired segment was a 211‐bp sequence adjacent to the promotor region of the β‐actin gene; (b) all transcriptionally active genes were repaired at similar and constant rates throughout S and G2 phases; (c) the nontranscribed genes were repaired at much lower rates in early S phase, but later in S phase and especially in G2 phase, their repair rates increased and approached those of the transcribed genes.
Pharmacological and biochemical characterization of cholecystokinin/gastrin receptors in developing rat pancreas
Authors: HADJIIVANOVA Christina , DUFRESNE Marlène , POIROT Sandrine , SOZZANI Patricia , VAYSSE Nicole , MORODER Luis , FOURMY Daniel
Published: March 2005

Cholecystokinin/gastrin receptors in the pancreas of newborn (3‐day‐old) rats are of type A, as in control mature rats, revealed by pharmacological analysis of specific 125I‐Bolton‐Hunter‐reagent‐labelled [Thr34,Ahx37]cholecystokinin(31–39) (Ahx, aminohexanoic acid) binding. Also, by 1 day post‐partum, pancreatic cholecystokinin receptors were shown to be coupled to guanine‐nucleo‐tide‐binding regulatory (G) proteins. Scatchard analysis of 125I‐Bolton‐Hunter‐reagent‐labelled [Thr34,Ahx37]cholecystokinin(31–39) binding to pancreatic membranes from rats at different times after birth showed a slight increase in the binding capacity of cholecystokinin receptors between days 3 and 14 and a sixfold increase in 21‐day‐old rats, with no change in receptor affinity during development. SDS/PAGE analysis of pancreatic membranes affinity labelled with the photoactivable ligand 125I‐[2‐(p‐azidosalicylamido)‐1,3′‐dithiopropionate]‐labelled [Thr34,Ahx37]cholecystokinin‐(31–39) identified cholecystokinin receptors of 100–135 kDa in 3‐day‐old rats, 96–130 kDa in 7‐day‐old rats, 90–125 kDa in 10‐day‐old rats and 85–100 kDa in 14‐day‐old and 21‐day‐old rats, as found in control adult rats. Endo‐β‐N‐acetylglucosaminidase F treatment yielded a core protein of 42 kDa in all developmental stages. These findings are consistent with an age‐related postnatal expression of distinct glycoforms of pancreatic cholecystokinin receptors. Furthermore, it was observed that the period 2–3 weeks after birth, characterized by stabilization of the mass of the cholecystokinin receptor, precedes the dramatic increase in the receptor number.
Purification and characterization of maize seedling casein kinase IIB, a monomeric enzyme immunologically related to the α subunit of animal casein kinase‐2
Authors: DOBROWOLSKA Grazyna , MEGGIO Flavio , SZCZEGIELNIAK Jadwiga , MUSZYNSKA Grazyna , PINNA Lorenzo A.
Published: March 2005

Casein kinase IIB (CKIIB), a protein kinase related to animal casein kinase‐2 (CK2), has been purified to homogeneity. It appears to be a monomeric enzyme, composed by an individual 39 kDa subunit, homologous to the α/α′ subunits of animal CK2 and devoid of the autophosphorylatable 25‐kDa α subunit of animal CK2, which display an heterotetrameric α2β2/αα′β2 structure. Such a conclusion is supported by the following lines of evidence: (1) CKIIB displays an apparent 39000 Mr by gel filtration on Ultrogel AcA 34 and it gives rise to a single prominent protein band of similar Mr (38 000) upon SDS/PAGE; (2) upon incubation of the enzyme with [32P]ATP, no radiolabeled bands are detectable which might be attributable to either canonical or atypical β subunits; (3) the 39‐kDa band immunoreacts with antisera that recognize the α subunit of rat and chicken CK2; (4) conversely, no component immunologically related with the β subunit could be detected in CKIIB by Western‐blot analyses with antisera that recognize animal β subunits; (5) the recombinant β subunit of human CK2 is readily phosphorylated by CKIIB, the reaction being prevented, rather than stimulated, by polylysine, a behaviour typical of animal CK2 autophosphorylation.

While the responsiveness of CKIIB to either heparin inhibition or polylysine stimulation are reminiscent of those of animal CK2, its peptide substrate specificity is significantly different and its thermolability is increased. Altogether these data would indicate that maize seedling CKIIB represents a naturally occurring monomeric form of CK2 devoid of non‐catalytic subunits. Its properties, compared to those of animal CK2, suggest that the β subunits of animal CK2 may be responsible for structural modifications conferring an altered specificity and an increased stability to the catalytic subunit.
ADP‐ribosylation of actins by arginine‐specific ADP‐ribosyltransferase purified from chicken heterophils
Authors: TERASHIMA Masaharu , MISHIMA Koichi , YAMADA Kazuo , TSUCHIYA Mikako , WAKUTANI Tadao , SHIMOYAMA Makoto
Published: March 2005

We reported the purification and characterization of an arginine‐specific ADP‐ribosyltransferase and acceptor protein p33 in granules of chicken peripheral polymorphonuclear leukocytes (heterophils) [Mishima, K., Terashima, M., Obara, S., Yamada, K., Imai, K. & Shimoyama, M. (1991) J. Biochem. (Tokyo) 110, 388–394]. In the present study, we obtained evidence that chicken non‐muscle β/γ‐actin, skeletal muscle α‐actin and smooth‐muscle γ‐actin were ADP ribosylated by the heterophil ADP‐ribosyltransferase. The stoichiometry of ADP–ribose incorporation into these actins was 1.2 mol, 1.0 mol and 2.0 mol ADP–ribose/mol of β/γ‐actin, α‐actin and γ‐actin, respectively. The optimal pH for the ADP ribosylation was at pH 8.5, with the respective actin. Km values for NAD were calculated to be 30 μM with β/γ‐actin, 35 μM with α‐actin and 20 μM with γ‐actin. The Km values for the actin isoforms were 15 μM for β/γ‐actin, 2.5 μM for α‐actin and 10 μM for γ‐actin. ADP ribosylation of actin inhibited its capacity to polymerize, as determined by the increase in fluoresence intensity with N‐(1‐pyrenyl)iodoacetamide‐labelled actin. Filamentous actin (F‐actin) polymerized with the respective actin isoform was also ADP ribosylated, although the extent of the modification of F‐actin was lower than that of globular actin (G‐actin). In situ ADP ribosylation of β/γ‐actin was evidenced with chicken peripheral heterophils permeabilized with saponin. Thus, the endogenous ADP ribosylation of actin in the heterophils may be involved in the cellular processes such as phagocytosis, secretion and migration.
Evidence for a selective destabilization of an integral membrane protein, the cytochromeb6/fcomplex, during gametogenesis inChlamydomonas reinhardtii
Authors: BULTÉ Laurence , WOLLMAN Francis‐André
Published: March 2005

We studied the process of photosynthetic inactivation during gametogenesis of the unicellular green algaChlamydomonas reinhardtii. We show that it is caused by the selective destabilization of a single transmembrane protein complex, the cytochromeb6/fcomplex, which is initially accumulated in the thylakoid membranes of vegetative cells. This protein destabilization is controlled by the intracellular energy sources available in the gametes, i. e. the coupled electron flow in the mitochondria and the amount of starch accumulated in the chloroplast. It nevertheless requires the expression of gamete‐specific proteins. The loss of cytochromeb6/fcomplexes during gametogenesis is prevented by the addition of cycloheximide, but is chloramphenicol insensitive. Therefore, it is likely to involve some translation product of nuclear origin, specifically expressed during gametogenesis. Among the new polypeptides specifically found in the gametes, we detected a soluble polypeptide Mα (approximate molecular mass of 63 kDa), which shared common epitopes with cytochromef.Its synthesis displays an antibiotic sensitivity typical of a nuclear‐encoded polypeptide and is controlled by the same intracellular signals which control the destabilization of the cytochromeb6/fcomplexes in the thylakoid membranes.
Characterisation of a cysteine protease from bloodstream forms of Trypanosoma congolense
Authors: MBAWA Zeres R. , GUMM Ian D. , SHAW Elliott , LONSDALE‐ECCLES John D.
Published: March 2005

A cysteine protease (trypanopain‐Tc) with cathepsin‐L‐like properties has been purified from Trypanosoma congolense. The enzyme has an apparent molecular mass of 31–32 kDa by SDS/ PAGE and 66 kDa by gel chromatography. It has a pI 7.4 and a high affinity for concanavalin A. Trypanopain‐Tc catalyses the limited proteolysis of a variety of protein substrates such as fibrinogen, serum albumin and trypanosome variant‐surface glycoprotein. It has minimal or no activity against casein or elastin.

A variety of peptidyl amidomethylcoumarins and peptidyl diazomethanes were used to test the specificity of trypanopain‐Tc. The better substrates had Arg or Lys in P1 and hydrophobic amino acids in P2 and P3. The best substrate found for trypanopain‐Tc was Z‐Phe‐Arg‐NHMec (Z, benzyl‐oxycarbonyl; NHMec, 7‐amido‐4‐methylcoumarin). The kinetic constants for the hydrolysis of Z‐Phe‐Arg‐NHMec were kcat= 17.4 s−1, Km= 4.4 μM, kcat/Km= 4.0 μM−1· s−1, which are very similar to those of cathepsin L with this substrate. The specific substrates for cathepsin B (Z‐Arg‐Arg‐NHMec) and cathepsin H (Arg‐NHMec) were not hydrolysed by trypanopain‐Tc under the conditions tested. The pH optimum of trypanopain‐Tc against Z‐Phe‐Arg‐NHMec was pH 6.0 but it showed a broad peak of activity extending well into the alkaline region. The enzyme was activated by low‐molecular‐mass thiol compounds and inhibited by cystatin, L‐trans‐epoxysuccinyl‐4‐guanidinobutane (E‐64) and a variety of peptidyl diazomethanes. The most effective diazomethane inhibitors (Z‐Leu‐Leu‐Met‐CHN2, Z‐Leu‐Met‐CHN2 and Z‐Leu‐Lys‐CHN2, were inhibitory at nanomolar concentrations and were trypanocidal in vitro after 24–48 h incubation in ≥ 20 μM [inhibitor]. However, it is not clear whether the trypanocidal activity of these inhibitors is a consequence of the inhibition of trypanopains or of some other essential proteolytic activities within the parasites.
CecC, a cecropin gene expressed during metamorphosis in Drosophila pupae
Authors: TRYSELIUS Ylva , SAMAKOVLIS Christos , KIMBRELL Deborah A. , HULTMARK Dan
Published: March 2005

Cecropins are antibacterial peptides, induced in insects in response to bacterial infections. In Drosophila, three cecropin genes have previously been characterized, CecA1, CecA2, and CecB, in a dense cluster at 99E on the third chromosome. From the same locus, we now describe a fourth member of the cecropin gene family, CecC, which is mainly expressed at the early pupal stage. In situ hybridization to immunized pupae show that CecC is induced in the anterior end of the larval hindgut and in other larval tissues that are undergoing histolysis. Within these other tissues it is often expressed in distinct foci that may correspond to hemocytes. A similar pattern of expression in the metamorphosing pupa is also observed for the CecA and CecB genes. Comparing the DNA sequences of the cecropin genes, a conserved region is observed about 30 bp upstream of the TATA box. It consists of three shorter motifs, two of which are reminiscent of a putative promoter element in immune protein genes from the cecropia moth.
The axonally secreted cell adhesion molecule, axonin‐1
Authors: ZUELLIG Richard A. , RADER Christoph , SCHROEDER Annelies , KALOUSEK Markus B. , VON BOHLEN UND HALBACH Friedrich , OSTERWALDER Thomas , INAN Canan , STOECKLI Esther T. , AFFOLTER H.‐Urs , FRITZ Andreas , HAFEN Ernst , SONDEREGGER Peter
Published: March 2005

Axonin‐1 is an axon‐associated cell adhesion molecule (AxCAM) of the chicken, which promotes neurite outgrowth by interaction with the AxCAM L1(G4) of the neuritic membrane. Here we report the cloning and sequence determination of a cDNA encoding axonin‐1. Peptides generated by enzymatic cleavage showed similarity to the AxCAM F11. Degenerated polymerase chain reaction (PCR) primers were designed and an axonin‐1 fragment was amplified from mRNA of embryonic retina. Screening of a cDNA library from embryonic brain resulted in the isolation of a 4.0‐kb cDNA insert with an open reading frame of 3108 nucleotides. The deduced polypeptide of 1036 amino acids includes a putative hydrophobic N‐terminal signal sequence of 23 or 25 amino acids and a C‐terminal hydrophobic sequence of 29 amino acids which is suggestive of sequences serving as signal for the attachment of a glycosyl‐phosphatidylinositol (glycosyl‐PtdIns) anchor. The putative mature form of axonin‐1 comprises six immunoglobulin‐like repeats, followed by four fibronectin‐type III repeats. Axonin‐1 exhibits 75% amino acid identity with the AxCAM TAG‐1 of the rat, suggesting that it is the chicken homologue of TAG‐1. Like TAG‐1, axonin‐1 is glycosyl‐PtdIns‐anchored to the neuronal membrane; in contrast to TAG‐1, it does not exhibit an Arg‐Gly‐Asp sequence.
Cloning and expression of a human ATP‐citrate lyase cDNA
Authors: ELSHOURBAGY Nabil A. , NEAR Joseph C. , KMETZ Peter J. , WELLS Timothy N. C. , GROOT Pieter H. E. , SAXTY Barbara A. , HUGHES Stephen A. , FRANKLIN Michelle , GLOGER Israel S.
Published: March 2005

A full‐length cDNA clone of 4.3 kb encoding the human ATP‐citrate lyase enzyme has been isolated by screening a human cDNA library with the recently isolated rat ATP‐citrate lyase cDNA clone [Elshourbagy et al. (1990) J. Biol. Chem. 265, 1430]. Nucleic‐acid sequence data indicate that the cDNA contains the complete coding region for the enzyme, which is 1105 amino acids in length with a calculated molecular mass of 121419 Da. Comparison of the human and rat ATP‐citrate lyase cDNA sequences reveals 96.3% amino acid identity throughout the entire sequence. Further sequence analysis identified the His765 catalytic phosphorylation site, the ATP‐binding site, as well as the CoA binding site. The human ATP‐citrate lyase cDNA clone was subcloned into a mammalian expression vector for expression in African green monkey kidney cells (COS) and Chinese hamster ovary cells (CHO) cells. Transfected COS cells expressed detectable levels of an enzymatically active recombinant ATP‐citrate lyase enzyme. Stable, amplified expression of ATP‐citrate lyase in CHO cells was achieved by using coamplification with dihydrofolate reductase. Resistant cells expressed high levels of enzymatically active ATP‐citrate lyase (3 pg/cell/d). Site‐specific mutagenesis of His765→Ala diminishes the catalytic activity of the expressed ATP‐citrate lyase protein. Since catalysis of ATP‐citrate lyase is postulated to involve the formation of phosphohistidine, these results are consistent with the pattern of earlier observations of the significance of the histidine residue in catalysis of the human ATP‐citrate lyase.
Evolution of ruminant hemoglobins
Authors: GIARDINA Bruno , AREVALO Felicidad , CLEMENTI Maria E. , FERRARA Lucio , Di LUCCIA Aldo , LENDARO Eugenio , BELLELLI Andrea , CONDÒ Saverio G.
Published: March 2005

The ligand‐binding properties of hemoglobins from two homozygote phenotypes (AA and BB) of water buffalo (Bubalus bubalis) have been characterized by equilibrium and kinetic techniques. In the case of the BB phenotype, the two constituent hemoglobins have been purified and separately analysed.

Buffalo hemoglobins display the reduced sensitivity to organic phosphates characteristic of ruminant hemoglobins, their physiological effector probably being the chloride ion.

In contrast to the other known hemoglobins from ruminants, all the hemoglobins from the water buffalo display a significant temperature sensitivity, the ΔH for oxygen binding in the presence of physiological effectors approaching that of human hemoglobin (ΔH=−30.5 kJ/mol O2).

This discrepancy with the other ruminant hemoglobins (e.g. ox, ΔH=−10.4 kJ/mol O2), whose primary structure is very similar to that of buffalo, hemoglobins might be correlated to the different habitat and phylogenetic history of the two subfamilies (Bos and Bubalus) of Bovidae.
Isolation, characterization and properties of the oversulphated chondroitin sulphate proteoglycan from squid skin with peculiar glycosaminoglycan sulphation pattern
Authors: KARAMANOS Nikos K. , ALETRAS Alexis J. , TSEGENIDIS Theodore , TSIGANOS Constantine P. , ANTONOPOULOS Christos A.
Published: March 2005

Oversulphated chondroitin sulphate proteoglycan from squid skin was isolated from 4 M guanidine hydrochloride extract by ion‐exchange chromatography, gel chromatography and density gradient centrifugation. The proteoglycan had Mr 3.5 × 105, contained on average six oversulphated chondroitin sulphate chains (Mr4 × 104) bound on a polypeptide of Mr 2.8 × 104, and oligosaccharides consisting of both hexosamines, glucuronic acid, sulphates and fucose as the only neutral monosaccharide. The major amino acids of the proteoglycan protein core are glycine (corresponding to about one third of the total amino acids), aspartic acid/asparagine and serine, together amounting to 50% of the total. The proteoglycan was resistant to the proteolytic enzymes V8 protease, trypsin (treated with diphenylcarbamoyl chloride), α‐chymotrypsin and pronase, while it was completely degraded by papain and to a large extent by collagenase. Pretreated proteoglycan with chondroitinase AC was degraded by pronase to a large extent and slightly by V8 protease and trypsin. The proteoglycan did not interact with hyaluronic acid and did not form self‐aggregates. Oversulphated chondroitin sulphate chains were composed of unusual sulphated disaccharide units which were isolated and characterized by HPLC. In particular, it contained 2‐acetamido‐2‐deoxy‐3‐O‐(α‐L‐threo‐4‐enopyranosyluronic acid)‐D‐galactose 4‐sulphate (Δdi‐4S) and disulphated disaccharides (Δdi‐diS) [90% 2‐acetamido‐2‐deoxy‐3‐O‐(α‐L‐threo‐4‐enopyranosyluronic acid 2/3‐sulphate)‐D‐galactose 6‐sulphate (ΔdiS‐diSD) and 10% 2‐acetamido‐2‐deoxy‐3‐O‐(α‐L‐threo‐4‐enopyranosyluronic acid 2/3‐ sulphate)‐D‐galactose 4‐sulphate (Δdi‐diSK)] as the major disaccharides, significant amounts of trisulphated disaccharides (Δdi‐triS) and small amounts of 2‐acetamido‐2‐deoxy‐3‐O‐(α‐L‐threo‐4‐enopyranosyluronic acid)‐D‐galactose 6‐sulphate (Δdi‐6S) and 2‐acetamido‐2‐deoxy‐3‐O‐(α‐L‐threo‐4‐enopyranosyluronic acid)‐D‐galactose (Δdi‐OS). Trisulphated disaccharides contained sulphate groups at C‐4 and C‐6 of the galactosamine and at C‐2 or C‐3 of the glucuronic acid. By HPLC analysis of a pure preparation of oversulphated chondroitin sulphate, it was found that it contains glucose, galactose, mannose and fucose most likely as branches.
Bimodal distribution of phosphatidic acid phosphohydrolase in NG108–15 cells
Authors: ARIDOR‐PITERMAN Orly , LAVIE Yaakov , LISCOVITCH Mordechai
Published: March 2005

The properties and bimodal distribution of phosphatidic acid phosphohydrolase (PAP) were investigated in neuroblastoma X glioma hybrid NG108–15 cells. Two PAP activities distinguished by their differential sensitivity to Mg2+ and Triton X‐100 were identified in the cytosolic and microsomal fractions. A digitonin permeabilization method was employed to study the basal distribution of the cytosolic PAP and its redistribution upon cell exposure to amphiphilic lipids. Under conditions which release 100% of the cytosolic marker enzyme lactate dehydrogenase, only 60% of total cellular PAP activity was released into the medium through the digitonin‐induced membrane pores, suggesting that about 40% of the total are membrane associated. Elevated plasma‐membrane levels of phosphatidic acid, accomplished by incubating cells with Streptomyces chromofuscus phospholipase D, did not affect the distribution of cytosolic PAP. In contrast, oleic acid induced a marked concentration‐dependent redistribution of the cytosolic enzyme to the particulate fraction. PAP redistribution was completely abolished in the presence of the sphingoid base sphingosine, previously shown to inhibit PAP activity in vitro (Lavie, Y., Piterman, O. & Liscovitch, M. (1990) FEBS Lett. 277, 7–10). Thus, the distribution of cytosolic PAP is reciprocally regulated by a long‐chain (fatty) acid and a long‐chain (sphingoid) base which are breakdown products of phospholipids and sphingolipids, respectively. These effects might influence PAP function in glycerolipid metabolism and signal transduction under physiological and pathophysiological conditions.
Different conformational forms of Escherichia coli and rat liver 5S rRNA revealed by Pb(II)‐induced hydrolysis
Authors: CIESIOLKA Jerzy , LORENZ Siegfried , ERDMANN Volker A.
Published: March 2005

Different stable forms of Escherichia coli and rat liver 5S rRNA have been probed by Pb(II)‐induced hydrolysis. In the native A forms of 5S rRNA, Pb2+ reveal single‐stranded RNA stretches and regions of increased conformational flexibility or distorted by the presence of bulged nucleotides. Hydrolysis of urea/EDTA‐treated E. coli 5S rRNA (B form) shows the presence of two strong helical domains; helix A retained from the A form and a helix composed of RNA regions G33–C42 and G79–C88. Other RNA regions resistant to hydrolysis may be involved in alternative base pairing, causing conformational heterogeneity of that form. Pb(II)‐induced hydrolysis distinguishes two different forms of rat liver 5S rRNA; the native A form and the form obtained by renaturation of 5S rRNA in the presence of EDTA. Pb(II)‐hydrolysis data suggest that both forms are highly structured. In the latter form, the orientation of the bulged C66 is changed with respect to helix B. At the same time, a new helical segment is possibly formed, composed of nucleotides from helix C and loop c on one side and from helix E and loop d' on the other.
cDNA clones for mouse parotid proline‐rich proteins
Authors: LAYFIELD Roy , BANNISTER Andrew J. , PIERCE Eric J. , MCDONALD Charles J.
Published: March 2005

cDNA clones for mRNA sequences regulated by isoprenaline in mouse parotid glands were identified by differential colony hybridisation and all hybridised to a diagnostic proline‐rich protein (PRP) oligonucleotide. They were divided into two cross‐hybridisation groups, A and B, which were shown by hybrid‐selected translations to encode acidic PRP and basic PRP, respectively.

The A‐type subgroup consisted of sequences homologous to the previously identified mouse PRP genes MP2 and MP3. The B‐type subgroup comprised clones for the previously identified cDNA pUMP125 (MP4) as well as other PRP sequences. Six of the B‐type clones contained a novel PRP cDNA (MP5) and these were sequenced. The composite MP5 cDNA was 897 nucleotides long and contained an open reading frame capable of encoding a 260‐residue‐long salivary PRP precursor (30% Pro, 19% Gln and 18% Gly), containing nine variant repeat units of consensus PGNQQGPPPQGGPQQ(GPP)R(PPQ). MP5 was 80% identical to the sequence of MP4 and had a high degree of similarity (60%) at its 3′‐untranslated region to rat salivary glutamate/glutamine‐rich protein (GRP) cDNA. Two MP5 clones contained a 273‐bp intron‐like insertion in the 3′ untranslated region, being derived, therefore, from incompletely spliced MP5 transcripts.

Northern blotting showed that, although PRP mRNA species were induced by isoprenaline, a B‐type PRP mRNA was present in normal parotid glands. RNA dot‐blots probed with PRP‐genespecific oligonucleotides established that MP3, MP4 and MP5 PRP mRNA were all induced by isoprenaline.
The complete amino acid sequence of the Clostridium botulinum type‐E neurotoxin, derived by nucleotide‐sequence analysis of the encoding gene
Authors: WHELAN Sarah M. , ELMORE Michael J. , BODSWORTH Nicola J. , ATKINSON Tony , MINTON Nigel P.
Published: March 2005

The entire structural gene of the Clostridium botulinum NCTC 11219 type‐E neurotoxin (BoNT/E) has been cloned as five overlapping DNA fragments, generated by polymerase chain reaction (PCR). Analysis of triplicate clones of each fragment, derived from three independent PCR, has allowed the derivation of the entire nucleotide sequence of the BoNT/E gene. Translation of the sequence has shown BoNT/E to consist of 1252 amino acids and, as such, represents the smallest BoNT characterised to date. The light chain of the toxin exhibits the highest level of sequence similarity to tetanus toxin (TeTx, 40%). The light chains of BoNT/A and BoNT/D share 33% similarity with BoNT/E, while BoNT/C exhibits 32% similarity. In contrast, the TeTx heavy chain exhibits the lowest degree of similarity (35%) with BoNT/E, with the BoNT heavy chains sharing 46%, 36% and 37%, for neurotoxin types A, C and D, respectively. Comparisons with partial amino acid sequences of the light chain of BoNT/E from C. botulinum strain Beluga and that from the strains Mashike, Iwanai and Otaru, indicate single amino acid differences in each case. Alignment of all characterised neurotoxin sequences (BoNT/A, BoNT/C, BoNT/D, BoNT/E and TeTx) shows them to be composed of highly conserved amino acid domains interspersed with amino acid tracts exhibiting little overall similarity. The most divergent region corresponds to the extreme COOH‐terminus of each toxin, which may reflect differences in specificity of binding to neurone acceptor sites.
Structural diversity among sulfated α‐L‐galactans from ascidians (tunicates)
Authors: SANTOS Janayna A. , MULLOY Barbara , MOURÃO Paulo A. S.
Published: March 2005

Sulfated polysaccharides occurring in the tunic of different species of ascidians differ markedly in electrophoretic pattern and chemical composition. A purified sulfated α‐L‐galactan from Herdmania monus was studied using methylation analysis and NMR spectroscopy; it is composed mainly of 3‐sulfated 4‐linked α‐L‐galactopyranoyl units. This is the first description of a homo‐polymer of sulfated α‐L‐galactose. In contrast, the sulfated L‐galactan from Ciona intestinalis shows marked structural heterogeneity and a low sulfate content. These data indicate unusual structural diversity among sulfated L‐galactans from different species of ascidians.
Kinetic parameters for the uptake of anthracycline by drug‐resistant and drug‐sensitive K562 cells
Authors: TARASIUK Jolanta , GARNIER‐SUILLEROT Arlette
Published: March 2005

Fluorescence‐emission spectra from anthracycline‐treated cells suspended in buffer have been used to measure the uptake of three anthracycline derivatives: adriamycin, 4′‐O‐tetra‐hydropyranyladriamycin and aclacinomycin in drug‐sensitive and drug‐resistant K562 cells. The initial rate of uptake and the kinetics of active efflux under the effect of an integral membrane glycoprotein, P‐glycoprotein, have been measured as a function of temperature. The activation energies for the passage of the drugs through the plasma membrane have been calculated. In the case of 4′‐O‐tetrahydropyranyladriamycin, the activation energies for the passive diffusion of the drug equal 45 kJ · mol−1 and 37 kJ · mol−1 for sensitive and resistant cells, respectively. The activation energy for the active efflux of 4′‐O‐tetrahydropyranyladriamycin equal 25 kJ · mol−1.
The archaebacterial hypusine‐containing protein
Authors: BARTIG Dirk , LEMKEMEIER Kerstin , FRANK Juliane , LOTTSPEICH Friedrich , KLINK Friedrich
Published: March 2005

The amino acid hypusine is formed by post‐translational modification of a lysine residue in eukaryotes and archaebacteria but up to now only the eukaryotic translation initiation factor eIF‐5A has been known to contain this unique component. We isolated and purified a hypusine‐containing protein from the thermophilic archaebacterium Sulfolobus acidocaldarius. The mainly cytosolic protein comprised about 0.03% of the post‐ribosomal supernatant protein. No other hypusine‐containing protein could be detected in S. acidocaldarius. The molar ratio of hypusine/hypusine‐containing protein was 1:1. SDS/PAGE showed a molecular mass of 16.8 kDa; a pI of 7.8 for the native protein resulted from IEF. The N‐terminus was blocked. Four cyanogen bromide fragments were partially sequenced and used to derive two 17‐base oligonucleotide probes. A 3‐kb HindIII fragment of genomic DNA hybridizing with both probes was cloned. By sequencing of exonuclease III deletion clones an open reading frame of 405 nucleotides was found coding for a protein of 135 amino acids with a molecular mass of 15 kDa. It contained all cyanogen bromide sequences analysed. Sequence alignment revealed that seven of eight residues around Lys40 in the Sulfolobus hypusine‐containing protein were identical to the nonapeptides centered by hypusine in the three eIF‐5A proteins sequenced so far. The Edman procedure gave no phenylthiohydantoin derivative for this position. For a central region of 44 residues a sequence similarity of 54% between the archaebacterial and eukaryotic proteins was calculated; for the total sequence about 33% similarity resulted. In addition, there were a number of conservative changes. The unique lysine modification surrounded by a conserved sequence strongly suggests a common ancestry of archaebacterial hypusine‐containing protein and eIF‐5A. Together with similarities in molecular mass and intracellular localization, it may point to an analogous biochemical function.
An additional exon in the human vinculin gene specifically encodes meta‐vinculin‐specific difference peptide
Authors: KOTELIANSKY Victor E. , OGRYZKO Elena P. , ZHIDKOVA Natalia I. , WELLER Polly A. , CRITCHLEY David R. , VANCOMPERNOLLE Katia , VANDEKERCKHOVE Joel , STRASSER Peter , WAY Michael , GIMONA Mario , SMALL J. Victor
Published: March 2005

We have analyzed the structure, origin and expression of the high‐molecular‐mass muscle‐specific variant of vinculin, called meta‐vinculin. The meta‐vinculin‐specific inserts from the human and avian molecules have been isolated and sequenced and the sequences confirmed via cloning of the corresponding cDNA. Comparison of the human, avian and determined porcine sequences revealed cross‐species identity in the C‐terminal half of the insert. Human and porcine meta‐vinculin were highly similar in the insert region, showing only five amino acid exchanges; avian meta‐vinculin showed 22 exchanges in the same region compared to human meta‐vinculin and exhibited, in addition, one extra amino acid, making 69 in all. Each insert was flanked by characteristic KWSSK motifs.

Evidence for two vinculin mRNA species in human uterus smooth muscle was provided by reverse transcription combined with the polymerase chain reaction, as well as by ribonuclease‐mapping analysis of cDNA/mRNA hybrids. One of the mRNA species contained an additional 204‐nucleotide insert that precisely encoded the meta‐vinculin‐specific peptide.

Sequence analysis of the appropriate portion of the human vinculin gene showed that the section coding for the meta‐vinculin‐specific insert is present as a discrete exon. Thus, meta‐vinculin and vinculin mRNA are generated by alternative splicing.
Mössbauer characterization of the tetraheme cytochrome c3 from Desulfovibrio baculatus (DSM 1743)
Authors: RAVI Natarajan , MOURA Isabel , COSTA Cristina , TEIXEIRA Miguel , LeGALL Jean , MOURA José J. G. , HUYNH Boi Hanh
Published: March 2005

Mössbauer spectroscopy was used to study the tetraheme cytochrome c3 from Desulfovibrio baculatus (DSM 1743). Samples with different degrees of reduction were prepared using a redoxtitration technique. In the reduced cytochrome c3, all four hemes are reduced and exhibit diamagnetic Mössbauer spectra typical for low‐spin ferrous hemes (S= 0). In the oxidized protein, the hemes are low‐spin ferric (S= 1/2) and exhibit overlapping magnetic Mössbauer spectra. A method of differential spectroscopy was applied to deconvolute the four overlapping heme spectra and a crystal‐field model was used for data analysis. Characteristic Mössbauer spectral components for each heme group are obtained. Hyperfine and crystal‐field parameters for all four hemes are determined from these deconvoluted spectra.
Excimer fluorescence of pyrene‐maleimide‐labeled tubulin
Authors: PANDA Dulal , BHATTACHARYYA Bhabatarak
Published: March 2005

Excimer‐forming cysteines in tubulin are detected by the presence of excimer fluorescence in N‐(1‐pyrenyl)maleimide‐labeled tubulin. The ratio of excimer/monomer fluorescence of labeled protein remained unchanged upon its dilution. These results indicating that both partner of each pair(s) of cysteine are located in the same subunit. The excimer fluorescence is insensitive to prior treatment of tubulin with either colchicine or GTP, indicating that pairs of cysteines protected by those drugs are not involved in excimer formation. This excimer fluorescence of N‐(1‐pyrenyl)maleimide‐labeled tubulin disappeared upon treatment with SDS, guanidinium chloride (GdmCl) and urea. Studies with GdmCl induced unfolding of N‐(1‐pyrenyl)maleimide‐labeled tubulin showed that the loss of excimer fluorescence preceeds subunit dissociation. The loss of both colchicine‐binding activity and the excimer fluorescence with increasing temperature indicates a major conformational change of the tubulin molecule at elevated temperatures.
Site‐directed mutagenesis of the Pseudomonas aeruginosa cytotoxin for probing toxic activity
Authors: XIONG Guangming , LUTZ Frieder
Published: March 2005

The location of amino acids with direct influence on toxic activity in the pore‐forming cytotoxin from Pseudomonas aeruginosa was tested by site‐directed mutagenesis. Mutant fragments obtained by the polymerase‐chain reaction were subcloned into a cytotoxin gene‐bearing plasmid to minimize the possibility of amplification error. Our data suggest an important role of cysteines for toxic activity of cytotoxin. Furthermore, domain‐faciliting‐binding to plasma membranes is located on the C‐terminal side of Cys215.
Characterization of hepatocyte‐growth‐factor receptors on Meth A cells
Authors: KOMADA Masayuki , MIYAZAWA Keiji , ISHII Takehisa , KITAMURA Naomi
Published: March 2005

Hepatocyte growth factor (HGF) is a heparin‐binding polypeptide mitogen for a variety of cell types including hepatocytes. HGF also has cytotoxic activity on some tumor cell lines as well as scattering activity on epithelial cells. In this study, recombinant human HGF was used to identify HGF‐binding cell surface receptors on Meth A cells, whose growth is inhibited by HGF. Scatchard analysis of binding data indicated that there were two classes of binding sites with high affinity (Kd= 17 pM) and low affinity (Kd= 6.7 nM) and the average numbers were 6600 and 2600000 per cell, respectively. Affinity cross‐linking of 125I‐HGF to Meth A cells resulted in a major and a minor specifically labeled complex. Competition analysis followed by cross‐linking indicated that the HGF‐binding proteins were involved in the formation of the high‐affinity binding. The existence of the two HGF‐binding surface proteins was confirmed by HGF‐dependent immunoprecipitation of the binding proteins with an anti‐HGF polyclonal antibody. The molecular masses of the major and the minor surface proteins were 160 kDa and 130 kDa, respectively. The 160‐kDa protein was autophosphorylated in vitro on tyrosine residue and was immunoprecipitated with an antiserum against the c‐met proto‐oncogene product. These results indicate that the 160‐kDa HGF‐binding surface protein on Meth A cells is the c‐met protein. Furthermore, tyrosine phosphorylation of the c‐met protein was stimulated by HGF treatment of Meth A cells, suggesting that it may be involved in the signal transduction of the growth inhibition of Meth A cells by HGF.