bluuebunny/crossref_metadata_2025 huggingface dataset:
Aortic smooth muscle contains guanylate‐cyclase‐coupled 130‐kDa atrial natriuretic factor receptor as predominant receptor form
Authors: ABE Tetsuaki , NISHIYAMA Kozo , SNAJDAR Rudolf , HE Xiaolan , MISONO Kunio S.
Published: March 2005

Photoaffinity labeling of atrial natriuretic factor (ANF) receptor in the plasma membranes from bovine aortic smooth muscle tissue using Nα5‐(4‐azidobenzoyl)‐ANF‐(5‐28)‐peptide labeled with 125I yielded a 130‐kDa band. However, when smooth muscle cells from the same bovine aorta were placed in culture, the 130‐kDa receptor quickly disappeared and a 60‐kDa band began to appear at high density. After three passages, essentially no 130‐kDa band was found and only the 60‐kDa band was strongly labeled. The primary structures of the two receptor forms were compared by radiochemical peptide mapping after endoproteinase Glu‐C digestion of photoaffinity‐labeled and detergent‐solubilized 130‐kDa receptor from the aorta or the 60‐kDa receptor from the cultured cells. The peptide mapping showed courses of digestion that were significantly different from each other, suggesting difference in their primary structures. The basal guanylate cyclase activity in the aortic membranes was 1.0 pmol cGMP produced · min−1· mg protein−1 at 37°C using Mn2+‐GTP as substrate. The corresponding activity in the membranes from the cultured cells was 20 fmol cGMP · min−1· mg protein−1· Binding studies gave a density of binding sites (Bmax) of 82 fmol/mg protein for the aortic membranes and 850 fmol/mg protein for the cultured cell membranes. These data suggest that the major form of ANF receptor in the cultured cells, namely the 60‐kDa receptor, lacked guanylate cyclase activity. Northern blot analysis of poly(A)‐RNA extracted form bovine thoracic aorta or adrenal cortex gave a single 3.6‐kb band when 32P‐labeled human A‐type ANF receptor cDNA was used as a hybridization probe. However, no band was detected when C‐receptor cDNA was used as a probe. In addition to the major 130‐kDa band, extended SDS/PAGE revealed two additional faint bands with estimated molecular masses of 126 kDa and 135 kDa. Treatment with endoglycosidase H resulted in disappearance of the 126‐kDa band and appearance of a 100‐kDa band. The 130‐kDa and 135‐kDa bands were unchanged. Treatment by endoglycosidase F or glycopeptidase F reduced all three bands to a single 100‐kDa band. These results suggest that the slight difference in mobility is due to different states of glycosylation. Competitive protection experiments showed binding specificity in the order of ANF > BNP ≫ CNP, AP‐I for all three bands, indicating that the state of glycosylation had no effect on the ligand specificity. Photoaffinity labeling of bovine adrenal cortex membranes gave a single 130‐kDa band. The same experiment with lung membranes yielded a 130‐kDa band and a 60‐kDa band at about 1: 1 ratio. The data presented in this report indicate that the vascular smooth muscle contains predominantly the 130‐kDa guanylate‐cyclase‐coupled ANF receptor in vivo, and that C‐receptors found in cultured vascular smooth muscle cells have been induced artificially from cell culturing.
Inhibition by the protein kinase C activator 4β‐phorbol 12‐myristate 13‐acetate of the prostaglandin F2α‐mediated and noradrenaline‐mediated but not glucagon‐mediated activation of glycogenolysis in rat liver
Authors: PÜSCHEL Gerhard P. , MIURA Hisayuki , NEUSCHÄFER‐RUBE Frank , JUNGERMANN Kurt
Published: March 2005

In perfused rat livers, infusion of prostaglandin F2α (PGF2α) or noradrenaline increased glucose and lactate output and reduced flow. Glucagon increased glucose output and decreased lactate output without influence on flow. Infusion of phorbol 13‐myristate 14‐acetate (PMA) for 20 min prior to these stimuli strongly inhibited the metabolic and hemodynamic effects of noradrenaline, reduced the metabolic actions of PGF2α but did not alter the effects of glucagon.

In isolated rat hepatocytes PGF2α noradrenaline and glucagon activated glycogen phosphorylase but only PGF2α and noradrenaline increased intracellular inositol 1,4,5‐trisphosphate (InsP3). The noradrenaline‐ or PGF2α‐elicited activation of glycogen phosphorylase and increase in InsP3 were largely reduced after preincubation of the cells for 10 min with PMA, whereas the glucagon‐mediated enzyme activation was not affected. In contrast to PMA, the phorbol ester 4α‐phorbol 13,14‐didecanoate, which does not activate protein kinase C, did not attenuate the PGF2α‐ and noradrena‐line‐elicited stimulation of glucose output, glycogen phosphorylase and InsP3 formation. Stimulation of InsP3 formation by AlF4−, which activates phospholipase C independently of the receptor, was not attenuated by prior incubation with PMA.

Plasma membranes purified from isolated hepatocytes had both a high‐capacity, low‐affinity and a low‐capacity, high‐affinity binding site for PGF2α. The Kd of the high‐capacity, low‐affinity binding site was close to the concentration of PGF2α that increased glycogen phosphorylase activity half‐maximally. Binding to the high‐capacity, low‐affinity binding site was enhanced by guanosine 5′‐O‐(3‐thio)triphosphate (GTP[S]). This high‐capacity, low‐affinity site might thus represent the receptor. The Bmax and Kd of the high‐capacity site, as well as the enhancement by GTP[S] of PGF2α binding to this site, remained unaffected by PMA pretreatment.

It is concluded that, in hepatocytes, activation of protein kinase C by PMA interrupted the InsP3‐mediated signal pathway from PGF2α via a PGF2α receptor and phospholipase C to glycogen phosphorylase at a point distal of the receptor prior to phospholipase C.
Immunoelectron microscopic localization of ribosomal proteins BS8, BS9, BS20, BL3 and BL21 on the surface of 30S and 50S subunits from Bacillus stearothermophilus
Authors: SCHWEDLER Gerda , ALBRECHT‐EHRLICH Renate , RAK Karl‐Heinz
Published: March 2005

The locations of ribosomal proteins BS8, BS9 and BS20 on the 30S subunit of Bacillus stearothermophilus ribosomes, and of BL3 and BL21 on the 50S subunit, were determined by immunoelectron microscopy. BL3 was found to lie half‐way down the body of the 50S subunit on the interface side, below the L7/L12 stalk, in agreement with the placement of the corresponding protein in Escherichia coli, by neutron‐scattering; BL21 was located at a similar position on the solvent side of the subunit, as predicted by cross‐linking experiments with E. coli ribosomes. Similarly, BS8 was found in the upper region of the body of the 30S subunit on the solvent side, and BS9 on the top of the head of the subunit, also on the solvent side, both positions being in good agreement with neutron‐scattering data and other immunoelectron microscopy results. In contrast, BS20 was found to lie at the extreme base of the body of the 30S subunit; this placement is not compatible with the location of E. coli S20 by neutron‐scattering but fits very plausibly with other biochemical data, such as sites of RNA–protein footprinting on 16S RNA, relating to the location of S20 in E. coli.
Identification of a 100‐kDa protein associated with nuclear ribonuclease P activity in Schizosaccharomyces pombe
Authors: ZIMMERLY Steven , DRAINAS Denis , SYLVERS Lee A. , SÖLL Dieter
Published: March 2005

Ribonuclease P from the fission yeast Schizosaccharomyces pombe has been purified to apparent homogeneity. A purification of 23000‐fold was achieved by four fractionation steps with DEAE‐cellulose chromatography, phosphocellulose chromatography, glycerol‐gradient fractionation and finally tRNA‐affinity chromatography. A 100‐kDa protein was present in the most pure preparations in amounts approximately stoichiometric with the previously identified RNA components of the enzyme, K1‐RNA and K2‐RNA [Krupp, G., Cherayil, B., Frendeway, D., Nishikawa, S. & Söll, D. (1986) EMBO J. 5, 1697–1703]. A cross‐linking experiment utilizing a 4‐thiouridine‐substituted precursor tRNA demonstrated that the 100‐kDa protein interacts with the ribonuclease P substrate in a specific fashion. We therefore conclude that the protein component of S. pombe ribonuclease P is a 100‐kDa protein.
Primary structure of a porcine leukocyte serpin
Authors: TESCHAUER Winfried F. , MENTELE Reinhart , SOMMERHOFF Christian P.
Published: March 2005

Inhibitors of neutral serine proteinases (serpins) have been shown to be colocalized with their target enzymes in leukocytes of several mammalian species. Here we report the purification and complete primary structure of a cytosolic inhibitor from porcine granulocytes which is directed against neutrophil elastase. Two molecular mass forms of the leukocyte neutral proteinase inhibitor (LNPI) were isolated by affinity and ion‐exchange chromatography followed by gel filtration, and identified as the inhibitorily active monomer and homodimer of the inhibitor protein. According to the amino acid sequence the molecular mass of the non‐glycosylated inhibitor was calculated to 42 597 Da (378 amino acid residues). A sequence idendity of 81% was found between LNPI and the homologous elastase inhibitors from both human and equine leukocytes, whereas only 50% of the positions are identical in LNPI and human plasminogen activator inhibitor 2. These data suggest that LNPI is a member of a new group of cytosolic serpins closely related to the ovalbumin branch of the superfamily.
Purification, nucleotide sequence and some properties of a bifunctional isomerase/decarboxylase from the homoprotocatechuate degradative pathway of Escherichia coli C
Authors: ROPER David I. , COOPER Ronald A.
Published: March 2005

A 1.8‐kbp region of DNA that appeared from deletion subcloning to code for 2‐hydroxyhepta‐2,4‐diene‐1,7‐dioate isomerase and 5‐oxopent‐3‐ene‐1,2,5‐tricarboxylate decarboxylase was investigated further. By nucleotide sequencing, a single open reading frame was found encoding a polypeptide of Mr 44514. One of the deletion subclones expressed the decarboxylase and isomerase activities at elevated levels and was used to facilitate purification of the enzyme(s). Both activities copurified, indicating that they were distinct activities of the same protein. Some kinetic properties of the purified isomerase/decarboxylase protein were investigated and it was shown that there is a 49000‐fold preference for 2‐hydroxyhepta‐2,4‐diene‐1,7‐dioate over the structurally related compound 5‐carboxymethyl‐2‐hydroxymuconate, the substrate of a second isomerase in the same catabolic pathway. Comparison of the amino acid sequences of the two isomerases showed only a low level of similarity, suggesting that these two enzymes are not evolutionarily related. However, comparison of the N‐terminal half of the isomerase/decarboxylase sequence (residues 1–202) with the second half (residues 203–406) showed significant similarity, suggesting that a duplication may have occurred to produce the bifunctional gene.
[1‐13C]Glucose metabolism in rat cerebellar granule cells and astrocytes in primary culture
Authors: MARTIN Magali , PORTAIS Jean‐Charles , LABOUESSE Julie , CANIONI Paul , MERLE Michel
Published: March 2005

The metabolism of [1‐13C]glucose in rat cerebellum astrocytes and granule cells was investigated using 13C‐ and 1H‐NMR spectroscopy. Near homogeneous primary cultures of each cell type were incubated with [1‐13C]glucose, under the same conditions. Analysing the relative 13C enrichments of metabolites in spectra of cell perchloric acid extracts, on the one hand, the 13C‐1H spin‐coupling patterns in 1H‐NMR spectra of cell medium lactate and the 13C‐13C spin‐coupling patterns in 13C‐NMR spectra of purified cell glutamate, on the other hand, showed significant differences, between the two cell types, in the activity of various metabolic ways. First, the carbon flux through the oxidative branch of the hexose monophosphate shunt, which leads to unenriched lactate, was found higher in granule cells than in astrocytes. Second, although the specific 13C enrichment of lactate was higher in astrocytes than in granule cells, the fraction of 13C‐enriched acetyl‐CoA entering the citric acid cycle was more than twice as high in granule cells as in astrocytes. Lactate C3 and acetyl‐CoA C2 enrichments were very similar in granule cells, whereas acetyl‐CoA C2 enrichment was 60% lower than that of lactate C3 in astrocytes. These results can be explained by the fact that granule cells used almost exclusively the exogenous glucose to fuel the citric acid cycle, whereas astrocytes used concomitantly glucose and other carbon sources. Last, in the case of granule cells, glutamate C2 and C3 enrichments were equivalent; the carbon flux through the pyruvate carboxylase route was evaluted to be around 15% of the carbon flux through the citrate synthetase route. In astrocytes, glutamate C2 enrichment was higher than that of C3, which could be explained by a pyruvate carboxylase activity much more active in these cells than in granule cells.
3‐Hydroxy‐3‐methylglutaryl‐coenzyme‐A synthase from Blattella germanica
Authors: MARTÍNEZ‐GONZÁLEZ José , BUESA Carlos , PIULACHS María‐Dolos , BELLÉS Xavier , HEGARDT Fausto G.
Published: March 2005

Insects do not synthezise cholesterol; the 3‐hydroxy‐3‐methylglutaryl‐coenzyme‐A (HMG‐CoA) produced by HMG‐CoA synthase is transformed to mevalonate by HMG‐CoA reductase for the production of non‐sterol isoprenoids, which are essential for growth and differentiation. To understand the regulation and developmental role of HMG‐CoA synthase, we have cloned a 1658 bp cDNA that encompasses the entire transcription unit of the HMG‐CoA synthase gene from the cockroach Blattella germanica. This cDNA clone was isolated using as a probe a partial cDNA of B. germanica HMG‐CoA synthase, amplified using the polymerase chain reaction. Analysis of the nucleotide sequence reveals that the cDNA encodes a polypeptide of 453 amino acids (Mr 50338) that is similar to vertebrate HMG‐CoA synthase (74–76% conserved residues). The B. germanica cDNA has been expressed as a fusion protein in Escherichia coli and exhibits HMG‐CoA synthase activity. The HMG‐CoA synthase transcript was differentially expressed throughout B. germanica development. Analysis of RNA samples from different adult female tissues shows high HMG‐CoA synthase mRNA levels in the ovary and lower levels in brain and muscle.
The phospholipase‐A2 reaction leads to increased monocyte adhesion of endothelial cells via the expression of adhesion molecules
Authors: YOKOTE Koutaro , MORISAKI Nobuhiro , ZENIBAYASHI Mika , UEDA Shiro , KANZAKI Tetsuto , SAITO Yasushi , YOSHIDA Sho
Published: March 2005

Mononuclear cell invasion into the vascular‐vessel wall is a very important initial step in the development of atherosclerotic lesions. Hypercholesterolemia leads to a marked adhesion of circulating blood monocytes to arterial endothelial cells in vivo, and minimally oxidized low‐density lipo‐protein enhances monocyte adhesion to endothelial cells in vitro. The activation of phospholipase A2 (PLA2) is also important in the oxidation of low‐density lipoprotein by endothelial cells. In this study, we investigated the role of PLA2 activation in the adhesion of a leukemic monocyte cell line (THP‐1 cells) to endothelial cells in vitro using an adhesion assay and a cell‐ELISA technique. The treatment of human umbilical‐cord‐vein endothelial cells with PLA2 stimulators such as interleukin‐1β, tumor necrosis factor and lipopolysaccharide all increased the adhesion of THP‐1 cells to endothelial cells. Exogeneous PLA2 also increased the adhesion of these cell types. The increased adhesion induced by these PLA2 stimulators, as well as PLA2 itself, was reversed by various inhibitors of the PLA2 reaction. A product of the PLA2 reaction, lysophosphatidylcholine, also increased cell adhesion. A cell‐ELISA technique showed the enhanced expression of vascular‐cell‐adhesion‐molecule 1 and intercellular‐adhesion‐molecule 1 to endothelial cells after treatment with PLA2 stimulators, PLA2 or lysophosphatidylcholine. These results suggest that the PLA2 reaction enhances monocyte adhesion to endothelial cells through the expression of cellular adhesion molecules.
Characterization and purification of the solubilized pituitary adenylate‐cyclase‐activating polypeptide‐1 receptor from porcine brain using a biotinylated ligand
Authors: SCHÄFER Heiner , SCHMIDT Wolfgang E.
Published: March 2005

A specific receptor for the brain‐gut neuropeptide pituitary adenylate‐cyclase‐activating polypeptide (PACAP‐1 receptor) was solubilized with Chapso from porcine brain plasma membranes and purified. Binding of 125I‐PACAP(1–27) to the solubilized material was reversed equipotently by unlabeled PACAP(1–27) and PACAP(1–38). Soluble receptors retained the binding affinities and specificities of the plasma membrane fraction. Scatchard analysis of equilibrium‐binding data indicated the existence of a single high‐affinity binding site (Kd= 0.23 nM, Bmax = 1.2 pmol/mg protein). Binding of 125I‐PACAP(1–27) to solubilized receptors was not affected by guanosine nucleotides, suggesting that solubilization dissociates the PACAP‐1 receptor/guanosine‐nucleotide‐binding protein complex. Affinity cross‐linking of 125I‐PACAP(1–27) to soluble PACAP‐1 receptors identified a specifically labeled 60‐kDa protein. Enzymic deglycosylation of soluble affinity‐labeled receptors reduced the apparent molecular mass by 10 kDa. The solubilized receptor glyco‐protein was purified 4–5‐fold by lectin‐adsorption chromatography on wheatgerm agglutinin immobilized on agarose. S‐Biotinyl[Ala28–34, Cys35]PACAP(1–35) was synthesized, immobilized on streptavidin‐coated magnetic Sepharose beads and used to further affinity‐purify wheatgerm‐agglutinin‐eluted receptor material. This more than 6000‐fold enriched PACAP‐1‐receptor‐preparation retained single‐class high‐affinity binding and consisted of an almost homogenous 55–60‐kDa protein identified by silver staining. In conclusion, we established a rapid method for purification of PACAP‐1 receptors, allowing further studies to be performed by protein chemistry.
Molecular cloning, expression and functional characterization of rabbit anticoagulant vitamin‐K‐dependent protein S
Authors: HE Xuhua , DAHLBÄCK Björn
Published: March 2005

Vitamin‐K‐dependent protein S is an anticoagulant plasma protein which functions as cofactor to activated protein C (APC) in the degradation of coagulation factors Va and VIIIa. In addition, it interacts with C4b‐binding protein (C4BP), a regulator of the complement system. Using a human protein S cDNA clone as probe, cDNA clones for rabbit protein S were isolated from a rabbit liver cDNA library. The cDNA sequence encoded the mature protein S and 12 residues of the leader sequence. The amino acid sequence of the single‐chain 634‐amino‐acid‐residue‐long rabbit protein S molecule was 82% and 81% identical to those of human and bovine protein S, respectively. Northern blotting demonstrated protein S mRNA not only in liver but also in reproductive organs (testis, ovary and uterus), in lung and brain. Recombinant rabbit protein S was expressed in eucaryotic cells and found to be post‐translationally modified, i.e. it had the correct amino terminus, contained N‐linked carbohydrate side chains, γ‐carboxyglutamic acid residues and β‐hydroxylated aspartic acid and asparagine residues. Recombinant rabbit protein S bound calcium like its human counterpart, as judged by its migration in the presence of calcium on agarose‐gel electrophoresis. Rabbit protein S has been reported to be species specific with respect to its interaction with APC and not to function with bovine APC. However, we found it to act as cofactor to both human and bovine APC, albeit it was somewhat more efficient with human than with bovine APC. Rabbit protein S, like its human and bovine counterparts, bound human C4BP in a reaction which was associated with the loss of its APC‐cofactor activity. However, unlike human plasma, rabbit plasma appeared to contain only the free form of protein S as a radiolabeled rabbit protein S tracer added to rabbit plasma migrated as free protein S on agarose‐gel electrophoresis. Addition of human C4BP to rabbit plasma resulted in the formation of a C4BP–protein‐S complex, suggesting an explanation for the absence of complexed protein S in rabbit plasma to be sought for in the structure of rabbit C4BP.
Four 9‐kDa proteins excreted by somatic embryos of grapevine are isoforms of lipid‐transfer proteins
Authors: COUTOS‐THEVENOT Pierre , JOUENNE Thierry , MAES Olivier , GUERBETTE Françoise , GROSBOIS Michèle , LE CAER Jean Pierre , BOULAY Michel , DELOIRE Alain , KADER Jean Claude , GUERN Jean
Published: March 2005

Four 9‐kDa small extracellular proteins produced by embryogenic cultures in the absence of auxin have been purified from the extracellular medium of grapevine somatic embryo cultures through cation‐exchange chromatography and hydrophobic‐interaction chromatography. The partial amino‐acid sequences reflect high similarities between the four proteins as well as with the sequences established for carrot, spinach, millet and maize nonspecific lipid‐transfer proteins. All these sequences show conservation of three cysteines at positions 4, 14 and 30–32, as well as glycine, valine, tyrosine and lysine residues at positions 5, 7, 17 and 37, respectively. In‐vitro lipid‐transfer assays reveal that the four proteins catalyze the transfer of phosphatidylcholine from liposomes towards motochondria with an efficiency similar or higher than that of a purified maize lipid‐transfer protein.
Disulfide interchange reactions in 11S globulin subunits of Cruciferae seeds
Authors: INQUELLO Valérie , RAYMOND Jacques , AZANZA Jean Louis
Published: March 2005

Cruciferin, the main storage protein in rapeseed (Brassica napus L.), is a legumin‐like 11S globulin. Using SDS/PAGE cruciferin was shown to be composed of different subunits consisting of αS and βS polypeptides, which were disulfide linked, and also closely related free αf and βf polypeptides which were not covalently linked. The origin of these latter free chains was found to be the result of disulfide interchange reactions occurring during SDS‐induced dissociation of the hexameric 11S complex. These reactions involved the rupture of the αS‐βS interchain disulfide link and, as shown by specific thiol fluorescent labeling, the subsequent formation of an intra‐β‐chain disulfide bond. N‐terminal amino acid sequence determinations revealed that these reactions were related to particular cruciferin subunit β chains having an additional cysteine residue (position 11) near the residue (position 7) implicated in the inter‐chain disulfide bridges. Such a sequence could explain the origin of the free and linked polypeptides as also shown in SDS/PAGE analysis of radish (Raphanus sativum L.) and Arabidopsis thaliana H. cruciferins. These two polypeptide classes may be considered to represent phenotypic biochemical markers of two different Cruciferae globulin gene families.
Structural requirements for processing of pro‐adipokinetic hormone I
Authors: RAYNE Richard C. , O'SHEA Michael
Published: March 2005

We found that a seven‐residue sequence in pro‐adipokinetic hormone I (proAKH I) which precedes the endopeptidase cleavage site is predicted to form an Ω loop. Molecular modelling experiments indicated that a stable Ω loop may form at this site, and suggested that loop stability may depend on the C‐terminal loop residue, Lys12. The importance of this residue in proAKH I processing was confirmed by the observation that replacement of Lys12 by thialysine, a Lys analog with an altered side chain, prevented processing in vivo. In addition we showed by molecular modelling that this side‐chain alteration may prevent formation of an Ω loop. Together, these approaches lead us to propose that an Ω loop may serve as a recognition motif in proAKH I processing.
Expression of human interferon ω1 in Sf9 cells
Authors: VOSS Tilman , ERGÜLEN Erkan , AHORN Horst , KUBELKA Viktoria , SUGIYAMA Kenji , MAURER‐FOGY Ingrid , GLÖSSL Josef
Published: March 2005

Human interferon ω1 (IFN‐ω1) was expressed in Spodoptera frugiperda Sf9 insect cells using the baculovirus expression system. Half of the protein purified by immunoaffinity chromatography was shown to be N‐glycosylated at the same site as the natural IFN‐ω1. The degree of glycosylation was independent of the expression rate. While natural IFN‐ω1 was shown to carry complex‐type oligosaccharides [Adolf, G. R., Maurer‐Fogy, I., Kalsner, I. & Cantell, K. (1990) J. Biol. Chem. 265, 9290–9295], the insect cell produced protein which was demonstrated by lectin blot, mass spectroscopy and HPLC analysis to contain only the core oligosaccharide. Two different structures, (Man)2(GlcNAc)2[Fuc] and (Man)3(GlcNAc)2[Fuc] were identified. The fucosylation was identified to be (α1–6)‐linked to the core saccharide. Sialic acid residues were clearly absent. IFN‐ω1 expressed in S. frugiperda cells was shown to be partially truncated at the C‐terminus by nine residues; its antiviral activity when glycosylated was significantly lower than the activity of IFN‐ω1 produced by Sendai‐virus‐stimulated leukocytes. Circular dichroism and fluorescence spectroscopy did not reveal any structural differences between glycosylated and nonglycosylated IFN‐ω1. This implies the importance of a complex‐type glycosylation for the maximal biological activity of human IFN‐ω1.
Purification of the large mobilization protein of plasmid RSF1010 and characterization of its site‐specific DNA‐cleaving/DNA‐joining activity
Authors: SCHERZINGER Eberhard , KRUFT Volker , OTTO Sabine
Published: March 2005

A site‐specific and strand‐specific nick, introduced into the RSF1010 plasmid origin of transfer (oriT), initiates unidirectional DNA transfer during bacterial conjugation. We have previously reproduced this nicking at the duplex oriT in vitro using purified preparations of the three known RSF1010‐mobilization proteins: MobA (78‐kDa form of RSF1010 primase), MobB and MobC [Scherzinger, E., Lurz, R., Otto, S. & Dobrinski, B. (1992) Nucleic Acids Res. 20, 41–48]. In this study we report the purification of MobA to apparent homogeneity and demonstrate that this 78‐kDa protein by itself is capable of creating the oriT‐specific nick if the DNA is present in the single‐stranded form. By studying the cleavage of sets of oligodeoxyribonucleotides varying successively by single nucleotides at the 5′ or 3′ end, the minimal substrate for cleavage has been defined. The results identify the MobA recognition sequence within the 11‐residue oligonucleotide AAGTGCGCCCT which is cleaved at the 3′ side of the G at position 7. During the cleavage reaction, MobA becomes covalently linked to the 5′‐phosphate end of each broken DNA molecule and retains its activity for the rejoining reaction. It can transfer the attached DNA to an incoming acceptor strand provided that the DNA molecule contains at its 3′ end at least the seven nucleotides upstream of the nick site. The covalent MobA‐DNA linkage has been determined by two‐dimensional thin‐layer electrophoresis to be a tyrosyl phosphate. Extensive digestion of the 32P‐labeled MobA‐oligonucleotide complex with lysine carboxypeptidase yielded a single DNA‐bound peptide which was purified and sequenced. The resulting peptide sequence consists of amino acid residues at positions 22–30 in the MobA sequence and identifies Tyr24 as the residue linked to DNA in the covalent complex.
Catalytic mechanism of 3‐deoxy‐d‐manno‐2‐octulosonate‐8‐phosphate synthase
Authors: BAASOV Timor , SHEFFER‐DEE‐NOOR Shani , KOHEN Amnon , JAKOB Abla , BELAKHOV Valery
Published: March 2005

The proposed mechanistic pathway for the reaction catalyzed by 3‐deoxy‐d‐manno‐2‐octuloson‐ate‐8‐phosphate (Kdo8P) synthase was examined in terms of the structure of the putative bisphosphate intermediate. Two 2‐deoxy analogues of the product Kdo8P, having been structurally prohibited from undergoing the ring‐opening and possessing the stereochemistry of either the α‐pyronase (compound 1) or the β‐pyranose form (compound 2) of the product, were synthesized and probed as inhibitors for the synthase. It was found that both analogues bind to the enzyme and are competitive inhibitors with respect to phosphoenolpyruvate binding, having Ki values of 470 μM and 303 μM, respectively. Comparison of this data to the Ki value of the tautomeric mixture of the product Kdo8P (Ki= 590 μM) suggests that both the α‐ and the β‐pyranose anomers (65.8% and 3.1%, respectively at neutral pH) bind to the enzyme with a slight (1.13 kJ/mol) preference for the β‐anomer, and that the C2 hydroxyl does not contribute to the binding. This uncertain stereochemical preference exhibited by the enzyme for the stereoisomers at the anomeric carbon suggests that the carboxylate binding site of the product is indistinct, while the hydroxyl and carboxylate binding sites may be interchangeable. More importantly, however, the isosteric phosphonate analogue 2,6‐anhydro‐3‐deoxy‐2β‐phosphonylmethyl‐8‐phosphate‐d‐glycero‐d‐talo‐octonate (3), which mimics the topological and electrostatic properties of the proposed cyclic intermediate, was found to be the most potent inhibitor of the enzyme with a Ki, value of 5 μM. Two hitherto unrecognized aspects of the mechanism of the synthase were identified. First, the results showing that the cyclic analogues 1, 2 and 3 are inhibitors of the enzyme whereas the previously reported acyclic analogue, which contains no carbonyl group at C2 and may thus resemble the open‐chain form of Kdo8P, is not an inhibitor, suggest that the pyranose form and not the open‐chain acyclic form of the putative bisphosphate intermediate is handled by the enzyme. Second, since the overall stereochemical course of the transformation mediated by the synthase has been shown to involve si face addition of phosphoenol‐pyruvate to the re face of the carbonyl of arabinose 5‐phosphate, the present observation involving analogue 3 suggests that the bisphosphate intermediate formed during the initial steps of synthesis may have the pyranose structure with the anomeric phosphate located in the β‐configuration.
Thermodynamics of the maleate and citraconate hydration reactions catalysed by malease from Pseudomonas pseudoalcaligenes
Authors: VAN DER WERF Mariët J. , VAN DEN TWEEL Will J. J. , HARTMANS Sybe
Published: March 2005

Malease from Pseudomonas pseudoalcaligenes catalyses the hydration of both maleate and citraconate to D‐malate and D‐citramalate, respectively. The Kapp for these hydration reactions were 2050 and 104, respectively, under standard biochemical conditions (25°C, pH 7.0, I= 0.1). The influence of the pH (6.0–8.5) on Kapp was determined. The Gibbs‐free‐energy changes under standard biochemical conditions for the hydration of the dianionic acids were calculated to be –19.28 kJ · mol−1 and –11.65 kJ · mol−1, respectively. From the obtained data together with data from the literature, the Gibbs free energy of formation of maleate2− and citraconate2− were calculated to be ‐588.91 kJ · mol−1 and –600.56 kJ · mol−1, respectively. The influence of the temperature (10–40°C) on Kapp was determined for both hydration reactions. The enthalpy change (ΔH°′) and entropy change (ΔS°′) under standard biochemical conditions for the maleate2− (ΔH°′=−18.07 kJ · mol−1, ΔS°′= 2.94 J · mol−1· K−1) and citraconate2− (ΔH°′=−22.55 kJ · mol−1, ΔS°′=−35.92 kJ · mol−1· K−1) hydration reactions were calculated. The reaction rate of malease from Ps. pseudoalcaligenes was studied for both hydration reactions as a function of temperature. From these studies, the Gibbs free energies of activation for the maleate and citraconate hydration reactions catalysed by malease from Ps. pseudoalcaligenes were calculated to be 62.21 kJ · mol−1 and 63.43 kJ · mol−1, respectively.
The putative zinc‐finger protein WZF1 interacts with a cis‐acting element of wheat histone genes
Authors: SAKAMOTO Ayako , MINAMI Maki , HUH Gyung Hye , IWABUCHI Masaki
Published: March 2005

A nonamer motif (CATCCAACG) that is one of the cis‐acting elements identified in the proximal promoter region of some wheat histone genes is included in the region that interacts with the wheat DNA‐binding protein, HBP (histone gene‐binding protein)‐2. To obtain structural and functional information about this DNA‐binding protein, we attempted to isolate a cDNA clone encoding HBP‐2 on the basis of its ability to bind to a nonamer‐containing 38‐bp DNA fragment. Southwestern screening of a wheat cDNA library with concatenated 38‐residue oligonucleotides as the probe produced one candidate clone. Nucleotide sequence analyses of this cDNA clone and the corresponding genomic clone showed that the protein deduced from the nucleotide sequence consisted of 261 amino acids and contained a set of zinc‐finger motifs similar to those found in many eukaryotic transcription factors. The protein, named WZF1 (wheat zinc‐finger protein 1), which was expressed from the cDNA in Escherichia coli cells, bound specifically and metal‐ion‐dependently to the nonamer‐containing oligonucleotide. The WZF1 mRNA was highly expressed in the root apexes of wheat seedlings, but less so in the proximal portion of young leaves; whereas, histone H3 mRNA was highly expressed in both tissues. The expression patterns of the WZF1 and histone H3 genes in the early stages of germination differed, expression of the WZF1 gene being almost constant but not that of the H3 gene. The relationship of WZF1 and HBP‐2 and the possible role of WZF1 in the histone gene expression were discussed.
Structural and functional properties of a multi‐enzyme complex from spinach chloroplasts
Authors: GONTERO Brigitte , MULLIERT Guillermo , RAULT Magali , GIUDICI‐ORTICONI Marie‐Thérèse , RICARD Jacques
Published: March 2005

The carboxylase activity of free ribulose 1,5‐bisphosphate carboxylase‐oxygenase has been compared to that of the five‐enzyme complex present in chloroplasts. Kinetic results have shown that the V/active site is lower for the free enzyme than for the complex. Conversely the Km is smaller for the complex than for the free enzyme. This implies that the catalytic activity of the enzyme is enhanced when it is embedded in the complex.

Under reducing conditions and in the presence of reduced thioredoxin, inactive oxidized phosphoribulokinase, free in solution or inserted in the multi‐enzyme complex, becomes active. The kinetics of this activation process has been studied and shown to be exponential. The time constant of this exponential decreases, for the free enzyme, as thioredoxin concentration is increased. Alternatively, for the enzyme embedded in the complex, this time constant increases with thioredoxin concentration almost in a linear fashion. This implies that the complex is much more rapidly activated by reduced thioredoxin than is the free phosphoribulokinase.

The variation of the amplitude of this activation process as a function of thioredoxin concentration is a hyperbola. The concentration of thioredoxin which results in half the asymptotic value of this hyperbola is smaller for the complex than for the free enzyme.

A kinetic model has been proposed and the dynamic equations resulting from this model have been derived. They fit the experimental results exactly. From the variation of the amplitude of the activation process one may derive the binding constants of thioredoxin on either the oxidized enzyme or on a partly dithiothreitol‐reduced enzyme (both of them free or inserted in the complex). In either case, the affinity of reduced thioredoxin is larger for the complex than for the free enzyme. The individual values of some of the rate constants have also been estimated from the variation of the time constants as a function of thioredoxin concentration.

Taken together, these results show that at least two enzymes, ribulose 1,5‐bisphosphate carboxylase‐oxygenase and phosphoribulokinase, have quite different kinetic properties depending on whether they are in free solution or embedded in the multi‐enzyme complex.
Mechanism of activation of liver acetyl‐CoA carboxylase by cell swelling
Authors: BAQUET Arnaud , GAUSSIN Vinciane , BOLLEN Mathieu , STALMANS Willy , HUE Louis
Published: March 2005

The activation of hepatic glycogen synthase by the amino‐acid‐induced cell swelling has been attributed to the stimulation of [glycogen‐synthase]‐phosphatase resulting from an increase in the intracellular content in glutamate and aspartate, and a decrease in intracellular Cl−, which is a compensatory response to cell swelling [Meijer, A. J., Baquet, A., Gustafson, L., van Woerkom, G. M. & Hue, L. (1992) J. Biol. Chem. 267, 5823–5828]. Here we studied whether the activation of acetyl‐CoA carboxylase by cell swelling could be explained by the same mechanism. The activation of endogenous or purified acetyl‐CoA carboxylase was measured in gel‐filtered liver extracts or cytosols. No activation could be observed under basal conditions but a fivefold stimulation was obtained with concentrations of glutamate (20–25 mM) found in hepatocytes incubated with glutamine. A similar stimulation was also observed with other dicarboxylic acids such as malonate and succinate, or with metal ions like Mg2+, Ca2+ and Mn2+ (10 mM). The addition of 50–100 mM Cl− was found to inhibit the activation of acetyl‐CoA carboxylase by some 20–30%. Mg2+ was also found to stimulate the activation of the endogenous glycogen synthase. The glutamate‐stimulated and Mg2+‐stimulated activation of glycogen synthase and acetyl‐CoA carboxylase was unaffected by 10 μM inhibitor‐2, a specific inhibitory protein of protein phosphatase‐1, but could be nearly completely blocked by the phosphatase inhibitor microcystin‐LR. Our data suggest that the amino‐acid‐induced activation of acetyl‐CoA carboxylase and glycogen synthase in the liver occurs by a common ionic mechanism.
Pseudomonas aeruginosa cytotoxin‐binding protein in rabbit erythrocyte membranes
Authors: LUTZ Frieder , MOHR Manuela , GRIMMIG Magdalene , LEIDOLF Regina , LINDER Dietmar
Published: March 2005

Rabbit erythrocyte membrane glycosylated 28‐kDa protein was investigated in the membranebound as well as in the soluble state on an example of a Pseudomonas aeruginosa cytotoxin‐binding component. When membranes were treated with trypsin/N‐glycosidase F, a 13.5‐kDa‐binding active peptide residue is obtained as revealed by a ligand‐blot technique after separation by SDS/PAGE under reducing conditions and electrophoretic transfer to nitrocellulose. Target‐size analysis of intact membranes by radiation inactivation using 2–450 kGy gave a value of 29, 40 and 60 kDa for the binding‐protein structure. This suggests that the native form of the binding peptide is associated as an oligomer. As seen in ligand‐blot technique, 125I‐cytotoxin binds with high affinity to waterchannel integral protein CHIP28 from human erythrocyte membranes. The 20 N‐terminal amino acids of the deglycosylated rabbit cytotoxin‐binding protein show high similarity to transmembrane channel‐like proteins.
Phenotype of recombinant Leishmania donovani and Trypanosoma cruzi which over‐express trypanothione reductase
Authors: KELLY John M. , TAYLOR Martin C. , SMITH Keith , HUNTER Karl J. , FAIRLAMB Alan H.
Published: March 2005

Trypanothione reductase is thought to be important in maintaining an intracellular reducing environment in trypanosomatids. To investigate the role of trypanothione reductase we transfected Leishmania donovani and Trypanosoma cruzi with an expression vector containing the L. donovani trypanothione reductase gene and achieved over‐expression of enzyme activity (10–14‐fold) in transformed cells. Following treatment of L. donovani cells with the thiol‐oxidizing agent diamide, the ability to regenerate dihydrotrypanothione from trypanothione disulphide was considerably enhanced in cells which over‐expressed trypanothione reductase. However, the growth of transformed and control cells was equally sensitive to inhibition by nifurtimox, nitrofurazone and gentian violet, drugs that are thought to act by inducing oxidative damage. Likewise, growth of transformed and control cells were equally susceptible to inhibition by hydrogen peroxide, and control and transformed L. donovani promastigotes metabolized hydrogen peroxide at comparable rates. Thus, these experiments suggest that the ability to regenerate dihydrotrypanothione from trypanothione disulphide is not a rate‐limiting step in the metabolism of hydrogen peroxide.
Increased phosphorylation of eukaryotic initiation factor 4α during early activation of T lymphocytes correlates with increased initiation factor 4F complex formation
Authors: Morley Simon J. , RAU Michael , KAY John E. , PAIN Virginia M.
Published: March 2005

Mature porcine peripheral blood mononuclear cells (PPBMCs) exist in a resting state both in vivo and when maintained in culture, with low translation rates consistent with their non‐proliferative state. When cultured in the presence of the appropriate mitogen, there is a 2–4‐fold increase in the rate of protein synthesis per ribosome within 4 h of stimulation [Kay, J. E., Ahern, T. and Atkins, M. (1971) Biochim. Biophys. Acta 247, 322–334]. Studies on extracts prepared from unstimulated cells have suggested lesions in initiation factor activity, primarily affecting the binding of mRNA to ribosomes [Ahern, T., Sampson, J. and Kay, J. E. (1974) Nature 248, 519–521].

In these studies, we have demonstrated that activation of quiescent PPBMCs with the phorbol ester phorbol 12‐myristate 13‐acetate or concanavalin A leads to a rapid 2–4‐fold increase in the rate of protein synthesis within 1 h or 4 h, respectively, which is insensitive to the transcriptional inhibitor, 5,6‐dichlorobenzimidazole riboside. Relative to control cells, both phorbol ester and concanavalin A induce a 2–4‐fold increase in labelling of the eukaryotic initiation factor eIF‐4α with phosphate in vivo, which primarily reflects a small net increase in phosphorylation rather than phosphate turnover on eIF‐4α. Similarly, with the human leukaemic T cell line JURKAT, stimulation of the T cell receptor with the monoclonal antibody, OKT‐3, or treatment with phorbol ester induces a 2–3‐fold increase in eIF‐4α phosphorylation within 30 min. Analysis of phosphorylation by two‐dimensional gel electrophoresis and measurement of kinase activity towards synthetic peptides, indicate that this increased labelling also reflects increased eIF‐4α kinase activity rather than phosphate turnover on eIF‐4α.

Of central importance is the finding that, concomitant with increased rates of protein synthesis following stimulation of PPBMCs with either phorbol ester or concanavalin A, there is a significant increase in the level of eIF‐4α recovered in high‐molecular‐mass complexes. These data suggest that, in quiescent PPBMCs, eIF‐4F may be limiting and that the association of eIF‐4α and eIF‐4γ into high‐molecular‐mass complexes is regulated by phosphorylation and may play a pivotal role in translational control.
Partial purification and characterization of a circulating hypertensive factor in spontaneously hypertensive rats
Authors: SCHLÜTER Hartmut , KLUTH Brigitte , BÖRJESSON‐STOLL Regina , NORDHOFF Eckhard , ZIDEK Walter
Published: March 2005

Parabiosis and cross‐circulation experiments with spontaneously hypertensive and normotensive rats gave indications for a previously unidentified circulating hypertensive agent. In this study, plasma from normotensive and hypertensive rats was fractionated and the vasopressor action of the corresponding fractions was measured in the isolated perfused rat kidney. One of three vasoactive fractions obtained by gel filtration (Biol‐Gel P2) from hypertensive rats showed a significantly higher activity (increase in perfusion pressure by 1502.9 ± 438.9 Pa) than that from normotensive rats (increase in perfusion pressure by 505.4 ± 186.2 Pa, P<0.01). Further chromatographic separations of this fraction revealed that the hypertensive factor is hydrophilic and has no ionic groups or vicinal diol groups. The molecular mass was estimated by dialysis and the matrix‐assisted laser desorption/ionization mass spectrometry to be in the range of 1 kDa. The vasopressor is heat resistant and not degradable with trypsin or carboxypeptidase Y. The vasopressor action was not inhibited with the angiotensin‐II‐receptor antagonist saralasin, the α‐receptor antagonist phentolamine, the thromboxane‐receptor antagonist carbocyclic thromboxane A2 or the serotonin antagonist ketanserine.

The results confirm the existence of a vasopressor factor in the plasma of hypertensive rats and, in a lower concentration, of normotensive rats, which is possibly related to the pathogenesis of essential hypertension. The chromatographic behaviour suggests that this factor is different from the parathyroid hypertensive factor described recently.
Aminoacyl chloromethanes as tools to study the requirements of NADPH oxidase activation in human neutrophils
Authors: CHOLLET‐PRZEDNOWED Emmanuelle , LEDERER Florence
Published: March 2005

Previous studies from this laboratory described the kinetic characteristics of the inhibition by tosylphenylalanine chloromethane (TosPheCH2Cl) on superoxide anion production by human neutrophils (PMN) stimulated with a phorbol ester (PMA). In this study we present further evidence concerning the potential role of the chloromethane target in the normal cellular activation of NADPH oxidase. When PMN are treated with TosPheCH2Cl and subsequently PMA, or with the two reagents in the reverse order, the inhibition of superoxide production by the intact cells is still present in a particulate NADPH oxidase fraction prepared from these cells. Nevertheless, when cells incubated only with the chloromethane and not with PMA are disrupted, both their cytosolic and membrane fractions are fully competent in the cell‐free activation assay. Thus, the chloromethane target has a role in NADPH oxidase activation exclusively at the cellular level. This observation constitutes additional evidence in favour of the idea that activation in the cell‐free system reflects only partially the events which occur in the cells.

When cells are activated with PMA, their cytosol displays a loss of activating capacity in the cell‐free activation assay in the presence of arachidonate, as was shown before with SDS as activator [Ambruso, D. R., Bolsher, B. G. J. M., Stockman, P. M., Verhoeven, A. J. & Roos, D. (1990) J. Biol. Chem. 265, 924–930]. This phenomenon was shown to arise most probably from the translocation of cytosolic factors to the membrane, resulting in a depleted cytosol. When superoxide production was inhibited by cell treatment with TosPheCH2Cl, either before or after activation with PMA, the cytosol from inhibited cells showed a recovery of activation capacity in the cell‐free system. This effect probably results from TosPheCH2Cl inhibiting the translocation of the cytosolic factors when added before PMA. This results in an insufficient activation at the membrane level, which was previously considered as an inhibition. The effect of TosPheCH2Cl, when added after PMA, can best be explained again as an inhibition of translocation in the frame of the continuous replenishment‐deactivation hypothesis proposed by Akard et al. [Akard, L. P., English, D. & Gabig, T. G. (1988) Blood 72, 322–327]. Thus, TosPheCH2Cl is apparently a promising new tool for studying the activation of NADPH oxidase at the cellular level.

In this study, we have also found that [3H]TosPheCH2Cl‐labeled cells show a prominent radiola‐beled band of Mr 15000 after SDS/PAGE analysis of the cytosolic proteins from inhibited cells; this species has the same migration as the labeled membrane protein detected previously, but appears much more abundant on a protein/cell basis. The potential role of the TosPheCH2Cl protein target is discussed.