bluuebunny/crossref_metadata_2025 huggingface dataset:
Baculovirus‐expressed myogenic determination factors require E12 complex formation for binding to the myosin‐light‐chain enhancer
Authors: BRAUN Thomas , GEARING Katharine , WRIGHT Woodring E. , ARNOLD Hans H.
Published: March 2005

Two recombinant baculoviruses BcV‐myf4 and BcV‐myf5 have been constructed to synthesize the human myogenic determination factors myogenin (myf4) and myf5 in eucaryotic cells. Both recombinant proteins are localized to the nucleus of virus‐infected Spodoroptera frugiperda (sf) insect cells and can be recovered as soluble factors. The virus‐produced proteins exhibit high‐affinity binding to a muscle‐specific DNA sequence in the presence of the ubiquitous helix‐loop‐helix (HLH) protein E12, but only marginal binding in unsupplemented sf nuclear extracts. Both baculovirus‐encoded myogenic factors are able to heterooligomerize with E12 in the absence of DNA‐binding sites. We conclude from our results that these muscle‐specific HLH proteins produced in eucaryotic cells largely depend on dimerization with E12 or similar HLH proteins to recognize the myosin‐light‐chain‐enhancer‐MEF‐1‐binding site. We have no evidence for intracellular protein modifications exerting major effects on the interaction between these factors and DNA.
NMR study of Galeorhinus japonicus myoglobin
Authors: YAMAMOTO Yasuhiko , CHÛJÔ Riichirô , SUZUKI Tomohiko
Published: March 2005

The heme molecular structure of the met‐azido form of the myoglobin from the shark Galeorhinus japonicus has been investigated by 1H NMR. A nuclear Overhauser effect (NOE) was clearly observed among the heme peripheral side‐chain proton signals of this complex, which undergoes thermal spin equilibrium between high‐spin (S= 5/2) and low‐spin (S= 1/2) states, and the NOE connectivities provided the assignment of the resonances from the heme C131H2 and C171H2 protons. Chemical shift inequivalence of these proton resonances not only provided information about the orientation of these methylene protons with respect to the heme plane, but also allowed characterization of the time‐dependent build‐up of the NOE between them, which yields the correlation time for the internal motion of the inter‐proton vector. The relatively large mobility found for the C171H2 group suggests that the carboxyl oxygen of the heme C17 propionate is not anchored to the apo‐protein by a salt bridge. It has been shown that the ferric high‐spin form of G. japonicus Mb possesses a penta‐coordinated heme [Suzuki, T. (1987) Biochim. Biophys. Acta 914, 170–176; Yamamoto, Y., ōsawa, A., Inoue, Y., Chûjô, R. & Suzuki, T. (1990) Eur. J. Biochem. 192, 225–229] and that the conformation of both heme propionate groups is fixed with respect to the heme, as well as the apo‐protein, by a salt bridge [Yamamoto, Y., Inoue, Y., Chûjô, R. & Suzuki, T. (1990) Eur. J. Biochem. 189, 567–573]. Therefore the weakening or interruption of the interaction between the C17 propionate and His FG3 upon the changes of the coordination and spin state of the heme iron, during azide ion binding to ferric high‐spin G. japonicus Mb, is attributed to the displacement of the FG corner of the apoprotein away from the heme C17 propionate group. A similar structural alteration has been revealed by X‐ray structural analyses of unliganded and liganded forms of ferrous hemoproteins [Baldwin, J. & Chothia, C. (1979) J. Mol. Biol. 129, 175–220; Phillips, S. E. V. (1980) J. Mol. Biol. 142, 531–554].
Multisite contacts involved in coupling of the β‐adrenergic receptor with the stimulatory guanine‐nucleotide‐binding regulatory protein
Authors: MÜNCH Gerald , DEES Christian , HEKMAN Mirko , PALM Dieter
Published: March 2005

Synthetic peptides, 12–22 amino acid residues long, comprising the presumed coupling sites of the β‐adrenergic receptor with the stimulatory guanine‐nucleotide‐binding regulatory protein (Gs), were examined for their ability to modulate Gs activation in turkey erythrocyte membranes. Three peptides corresponding to the second cytoplasmic loop, the N‐terminal region of the third cytoplasmic loop, and the N‐terminal region of the putative fourth cytoplasmic loop, compete synergistically with the hormone‐stimulated receptor for Gs activation with median effector concentrations of 15–35 μM, or 3–4 μM for combinations of two peptides. One peptide, corresponding to the C‐terminal region of the third cytoplasmic loop, carries the unique ability to activate the Gs‐adenylate‐cyclase complex independent of the signalling state of the receptor. These observations are consistent with a dynamic model of receptor‐mediated G‐protein activation in membranes, where domains composed of the second, third and fourth intracellular loop of the receptor bind to and are interactive with the G‐protein heterotrimer, resulting in ligand‐induced conformational changes of the receptor. In response to hormone binding, the extent or the number of sites involved in interaction with Gs may be readjusted using a fourth site. Modulation of coupling sites may elicit congruent conformational changes within the Gs heterotrimer, with qualitatively different effects on GTP/GDP exchange in the α subunit of Gs and downstream effector regulation. This model corroborates and expands a similar model suggested for activated rhodopsin‐transducin interaction [König, B., Arendt, A., McDowell, J. H., Kahlert, M., Hargrave, P. A. & Hofmann, K. P. (1989) Proc. Natl Acad. Sci. USA 86, 6878–6882.
Acetone‐regulated synthesis and degradation of cytochrome P4502E2 and cytochrome P4502B1 in rat liver
Authors: RONIS Martin J. J. , JOHANSSON Inger , HULTENBY Kjell , LAGERCRANTZ Jacob , GLAUMANN Hans , INGELMAN‐SUNDBERG Magnus
Published: March 2005

The regulation of CYP2E1 and 2B1 was studied by following mRNA levels, catalytic activities and the subcellular distribution of the apoproteins in rat liver 0, 6, 12, 24, 48 and 96 h after a single intragastric dose of acetone. No changes were observed in hepatic CYP2E1 mRNA levels at any time after acetone treatment, whereas rapid rises were observed in the microsomal amount of CYP2E1 protein and CYP2E1‐catalyzed 4‐nitrophenol hydroxylase and carbon‐tetrachloride‐initiated lipid‐peroxidation activities. However, CYP2E1‐dependent catalytic activities declined much faster than the immunodetectable CYP2E1 protein, suggesting that this cytochrome P‐450 is inactivated prior to degradation. Similar results were seen in primary hepatocyte cultures. By contrast, concomitant changes in levels of CYP2B1 and CYP2B1‐dependent O‐depentylation of pentoxyresorufin were observed in the same microsomal preparations. Investigation of the degradative mechanism of both CYP2E1 and CYP2B1 by immunoquantitation of the proteins in lysosomes and by immunohistochemistry indicated their degradation via an autophagic‐lysosomal pathway. The data suggest that CYP2E1 is acutely inactivated in the endoplasmic reticulum and that degradation of this isozyme occurs, at least in part, by the lysosomal route. By contrast, CYP2B1 is principally controlled at the level of synthesis.
Purification and identification of 91‐kDa neutrophil gelatinase
Authors: MASURE Stefan , PROOST Paul , VAN DAMME Jo , OPDENAKKER Ghislain
Published: March 2005

Human neutrophils were found to release a 91‐kDa gelatinase that is serologically related to tumor‐derived gelatinolytic enzymes, as evidenced by immunoprecipitation. In order to identify the neutrophil gelatinase, the activity in conditioned medium from human neutrophil suspensions was purified by affinity chromatography on a gelatin substrate. The 91‐kDa active enzyme was further separated from other stainable protein bands by classical SDS/PAGE and blotting to a solid support. Amino‐terminal sequence analysis of blotted proteins showed that the 91‐kDa enzyme is a truncated form of tumor‐derived 92‐kDa gelatinase (type IV collagenase), lacking eight residues at the NH2‐terminus. Sequence analysis of enzymatically inactive cleavage products of this neutrophil gelatinase demonstrated that the gelatin‐binding part of the molecule is restricted to the amino‐terminal third.

Exocytosis of gelatinase‐containing granules from neutrophils occurred spontaneously within 6 h after neutrophil plating. When the cells were triggered with the phorbol ester phorbol 12‐myristate 13‐acetate, a strong secretagogue, rapid gelatinase release was observed. When granulocytes were stimulated with the neutrophil‐activating peptide interleukin‐8, maximal exocytosis occurred within 1 h. The almost immediate release of neutrophil gelatinase after stimulation of the cells with a chemotactic factor might play a key role in remodeling of the extracellular matrix during granulocyte movement in response to chemotactic stimuli.
A model of the lac repressor–operator complex based on physical and genetic data
Authors: KISTERS‐WOIKE Brigitte , LEHMING Norbert , SARTORIUS Jürgen , von WILCKEN‐BERGMANN Brigitte , MÜLLER‐HILL Benno
Published: March 2005

Computer graphics were used to build a molecular model of the complex of Lac repressor and lac operator. The model is based (a) on the NMR data of the Kaptein group [Boelens, R., Lamerichs, R. M. J. N., Rullmann, J. A. C., van Boom, J. H. & Kaptein, R. (1988) Protein Sequence Data Anal. 1, 487–498] and (b) on our genetic and biochemical data including specificity changes [Lehming, N., Sartorius, J., Kisters‐Woike, B., von Wilcken‐Bergmann, B. & Müller‐Hill, B. (1990) EMBO J. 9, 615–621]. Effects of amino acid exchanges in the recognition helix could be predicted by the model and were subsequently tested and confirmed by genetic experiments. Comparison of the modelled lac complex with the known crystallographic structures of several helix‐turn‐helix DNA complexes reveals striking similarities and suggests rules which govern the recognition between particular amino acid side chains and particular base pairs in these systems.
Functionally important regions of glucose‐6‐phosphate dehydrogenase defined by the Saccharomyces cerevisiae enzyme and its differences from the mammalian and insect forms
Authors: PERSSON Bengt , JÖRNVALL Hans , WOOD Irene , JEFFERY Jonathan
Published: March 2005

The primary structure of Saccharomyces cerevisiae glucose‐6‐phosphate dehydrogenase has been determined. It consists of 503 amino acid residues, with an acetyl‐blocked N‐terminus. The structure shows equally extensive differences from the corresponding mammalian and fruit fly enzymes (52% residues non‐identical). Residues conserved in all the forms constitute about 40% of the structures and include two histidines. One of these (His200 in the numbering of the rat enzyme) occurs in a 10‐residue conserved segment, including the reactive Lys204, probably related to substrate binding. Two segments with conserved Gly‐Xaa‐Xaa‐Gly‐Xaa‐Xaa‐Gly/Ala pattern constitute possibilities for the coenzyme‐binding site. One is N‐terminally located (positions 37–43) with two conserved arginine residues nearby (positions 56 and 71), of interest for phosphate binding. The other (positions 241–247) is in a middle region, with many residue identities, containing the conserved residues Arg256 and His264.
The H+‐motive and Na+‐motive respiratory chains in Bacillus FTU subcellular vesicles
Authors: KOSTYRKO Vitaly A. , SEMEYKINA Anna L. , SKULACHEV Vladimir P. , SMIRNOVA Irina A. , VAGHINA Margarita L. , VERKHOVSKAYA Marina L.
Published: March 2005

Respiration‐dependent pumping of Na+ and H+ into the inside‐out subcellular vesicles of alkalotolerant and halotolerant Bacillus FTU grown at alkaline pH was studied.

The vesicles were shown to be competent in Na+ and H+ transport coupled to ascorbate oxidation via N,N,N′,N′‐tetramethyl‐p‐phenylenediamine or diaminodurene. The uphill Na+ uptake is strongly stimulated by either protonophores or valinomycin, whereas H+ uptake is stimulated by valinomycin and completely inhibited by protonophores. The salt of a penetrating weak base and of the penetrating weak acid, diethylammonium acetate, potentiates the stimulating effect of protonophores on Na+ uptake and abolishes H+ uptake. Na+ transport, supported by ascorbate oxidation, is resistant to 2‐heptyl‐4‐hydroxyquinoline N‐oxide, but sensitive to Ag+ and Na+ ionophore, N,N′‐dibenzyl‐N,N′‐diphenyl‐1,2‐phenylenediacetamide. Micromolar concentrations of cyanide specifically inhibit the H+ uptake but does not affect Na+ uptake. These cyanide concentrations are shown to cause 70% inhibition of respiration, complete reduction of a‐type cytochromes and partial reduction of c/b‐type cytochromes. To inhibit the remaining respiratory activity and Na+ uptake, approximately 100‐fold higher cyanide concentrations are necessary. High cyanide concentrations cause some additional increase in absorbance in the region of cytochromes c and/or b. In the presence of a high cyanide concentration, Na+ uptake can be supported by NADH oxidation by fumarate. This Na+ transport is stimulated by protonophores and diethylammonium acetate, being sensitive to very low concentrations of 2‐heptyl‐4‐hydroxyquinoline N‐oxide and Ag+. The NADH‐fumarate reductase reaction is also found to be competent in H+ uptake, which is inhibited by protonophores and by much higher 2‐heptyl‐4‐hydroxyquinoline N‐oxide concentrations, and is resistant to Ag+.

It is inferred that Bacillus FTU possesses two respiratory chains: the H+‐motive and the Na+‐motive, which strongly differ in their inhibitor sensitivities. Each chain comprises at least two energy‐coupling sites which are localized in their initial and terminal segments. It has been indicated that common redox carrier(s) are present in the two chains.
Different glycoforms of human thrombomodulin
Authors: KOYAMA Takatoshi , PARKINSON John F. , SIÉ Pierre , BANG Nils U. , MÜLLER‐BERGHAUS Gert , PREISSNER Klaus T.
Published: March 2005

The relationship between thrombomodulin‐associated O‐linked glycosaminoglycans (GAGs) and the exogenous GAGs heparin or dermatan sulfate was studied in the inhibition of thrombin by antithrombin III (AT III) or heparin cofactor II (HC II). Both rabbit thrombomodulin (TM) and two glycoforms (a high‐Mr form containing GAGs and a low‐Mr form lacking the majority of O‐linked GAGs) of a recombinant human TM deletion mutant (rec‐TM) were used. The rapid inactivation of thrombin by HC II in the presence of dermatan sulfate was prevented by both the high‐Mr rec‐TM and the rabbit TM. In contrast, both rabbit TM treated with chondroitin ABC lyase to remove O‐linked GAGs and the low‐Mr form of rec‐TM had only weak protecting effects. In the absence of exogeneous dermatan sulfate, thrombin inhibition by a high concentration of HC II was slightly accelerated by the high‐Mr form of rec‐TM but protected by rabbit TM. When thrombin inhibition by AT III in the presence of heparin was studied, both high‐Mr rec‐TM and rabbit TM again invoked a similar reduction of inactivation rates, whereas in the absence of exogenous heparin, both high‐Mr forms accelerated thrombin inhibition by AT III. The diverse reactivities of various forms of TM towards HC II and AT III were also observed during protein C activation by the thrombin‐TM complex. These results suggest that thrombin activity at the vessel wall or in fluid phase may undergo major kinetic modulations depending on the type of protease inhibitor, the presence or absence of exogenous GAGs and the glycosylation phenotype of TM. The dependence of TM anticoagulant function on the presence of an intrinsic GAG moiety suggests that variant glycoforms of this endothelial cell cofactor may be expressed differently in a species‐, organ‐, or tissue‐specific manner as a means to regulate TM function in diverse vasculatures.
Evidence that the decarboxylation reaction occurs before the first methylation in ubiquinone biosynthesis in rat liver mitochondria
Authors: KANG Dongchon , TAKESHIGE Koichiro , ISOBE Ryoichi , MINAKAMI Shigeki
Published: March 2005

Biosynthesis of ubiquinone‐9 was studied by incubating rat liver mitochondria with p‐hydroxy[U‐14C]benzoate, solanesyl diphosphate and S‐adenosyl‐l‐methionine. When methylation reactions were inhibited by replacing S‐adenosyl‐l‐methionine with S‐adenosyl‐l‐homocysteine, nonaprenyl p‐hydroxybenzoate and three other labeled peaks, designated as P1, P2 and P3 according to their retention times on HPLC, were observed. No carboxyl group was present in P1, P2 or P3 because the radioactivities disappeared when p‐hydroxy[U‐14C]benzoate was replaced by p‐hydroxy[carboxyl‐14C]benzoate. Compound P2 seemed to be hydroxylated but not methylated because its radioactivity markedly diminished under anaerobic conditions and the radioactivity was not incorporated into the compound from S‐adenosyl‐l‐[methyl‐3H]methionine, suggesting that P2 is 6‐hydroxynonaprenylphenol. The complete correspondence of the retention times of P2 and chemically synthesized 6‐hydroxynonaprenylphenol on HPLC further confirmed this possibility. P2 was a precursor of ubiquinone‐9 because the radioactivity of the compound was incorporated into ubiquinone when incubated with mitochondria. The results suggest that the decarboxylation may occur prior to the first methylation in the ubiquinone biosynthesis in rat liver mitochondria, though it has been generally considered that in eukaryotes the first methylation precedes the decarboxylation.
Mechanism of the permissive action of dexamethasone on the glucagon‐dependent activation of the phosphoenolpyruvate carboxykinase gene in cultured rat hepatocytes
Authors: RUNGE Dieter , SCHMIDT Helga , CHRIST Bruno , JUNGERMANN Kurt
Published: March 2005

Rat hepatocytes were cultured for 24 h in the presence or absence of 100 nM dexamethasone (DX). After a medium change, phosphoenolpyruvate carboxykinase (PCK) was induced by addition of glucagon at different concentrations, from physiological 0.1 nM to hyperphysiological 10 nM, again in the presence or absence of 100 nM dexamethasone.

1. With dexamethasone addition during the culture and induction phase (DX+/+), 10 nM glucagon increased PCK mRNA abundance (Northern blot analysis) and activity (in vitro translation) synchronously to the same extent with maxima after 2 h and PCK enzyme activity after a time lag with a maximum after 6 h. The total detectable PCK mRNA amount was apparently also translationally active. 10 μM N6,2′‐O‐dibutyryladenosine 3′,5′‐(cyclic)phosphate (Bt2cAMP) as the second messenger had essentially the same effect as 10 nM glucagon.

2. In the absence of dexamethasone during the preculture and the induction phase (DX‐/‐), 10 nM glucagon and 10 μM Bt2cAMP could enhance PCK mRNA only about half‐maximally. Glucagon or dexamethasone added alone in physiological concentrations of 0.1 nM and 100 nM, respectively, were unable to increase PCK mRNA. However, treatment of the cells with dexamethasone also enabled 0.1 nM glucagon to enhance PCK mRNA to a maximum after 2 h, independent of the presence of dexamethasone during the induction period (DX+/+ and DX+/‐ cells). Thus, dexamethasone was a permissive agent in that it shifted the sensitivity of the cells towards glucagon into the physiological concentration range.

3. In the presence of dexamethasone during the culture and induction phase (DX+/+) 0.1 nM glucagon maximally enhanced the transcription of the PCK gene (nuclear run on) fourfold after 30 min; in the absence of dexamethasone during both phases (DX‐/‐) glucagon was without any effect. The overall transcriptional rate was not significantly different in cells with and without dexamethasone during the culture and induction phase (DX+/+ vs. DX‐/‐). Thus, dexamethasone acted permissively mainly on the transcription of the PCK gene.

4. With culture in the presence of dexamethasone over decreasing periods of time, 1 nM glucagon could induce submaximal PCK mRNA amounts already after 1–3 h steroid culture. This restitution by dexamethasone of the PCK mRNA inducibility by glucagon was inhibited by cycloheximide. This suggested that ongoing protein synthesis was required for the permissive action of dexamethasone on the expression of the PCK gene.

The results allow the following conclusions. In the presence of dexamethasone a trans‐acting protein factor was formed which allowed glucagon via cAMP to enhance the expression of the PCK gene in physiological concentrations. The dexamethasone‐induced trans‐acting factor exerted its permissive action mainly at a transcriptional level.
Structural and functional analyses of glycosylation on the distinct molecules of human GM‐CSF receptors
Authors: SHIBUYA Kyoichi , CHIBA Shigeru , MIYAGAWA Kiyoshi , KITAMURA Toshio , MIYAZONO Kohei , TAKAKU Fumimaro
Published: March 2005

We have previously demonstrated that granulocyte‐macrophage colony‐stimulating factor (GM‐CSF) receptors are composed of at least two molecules of 80 and 135 kDa, which were denoted α‐ and β‐chains, respectively [Chiba, S., Shibuya, K., Piao, Y.‐F., Tojo, A., Sasaki, N., Matsuki, S., Miyagawa, K., Miyazono, K. & Takaku, F. (1990) Cell Regul. 1, 327–335]. In this paper, we describe an investigation of the biochemical disparity noted between the α‐ and β‐chains of GM‐CSF receptors using proteolytic and deglycosidic enzymes, and further demonstrate the potential importance of carbohydrate structures of the GM‐CSF receptors using different lectins and glycoprotein synthesis inhibitors. Cross‐linked α‐ and β‐chains with 125I‐GM‐CSF were digested by Staphylococcus aureus V8 protease and gave a different pattern. Furthermore, the size of the α‐chain was reduced by 25 kDa by the removal of the N‐linked oligosaccharides with peptidase: N‐glycosidase F treatment, whereas that of the β‐chain remained unmodified by the enzyme. These results suggest that the α‐chain of GM‐CSF receptors agrees with the recently cloned low‐affinity GM‐CSF receptor [Gearing, D. P., King, J. A., Gough, N. M. & Nicola, N. A. (1989) EMBO J. 8, 3667–3676] having approximately 30% N‐linked oligosaccharides and is biochemically different from the αβ‐chain. By analyses using lectins, some of the oligosaccharides in the α‐chain seem to be the complex‐type and/or hybrid‐type, because wheat germ agglutinin and leukoagglutinating phytohemagglutinin inhibited both GM‐CSF‐induced proliferation and GM‐CSF binding to its receptors. Further analyses using glycoprotein synthesis inhibitors showed that N‐linked processing of the α‐chain, especially glucose removal by glucosidase I and II (whose activities are inhibited by deoxynojirimycin), appeared to be required for the expression onto the cell surface although the β‐chain expression was little affected by their inhibitors. Thus the β‐chain, probably located near the α‐chain on the cell surface, was associated with a high‐affinity class of GM‐CSF receptors.
EPR and 1H‐NMR spectroscopic studies on the paramagnetic iron at the active site of phenylalanine hydroxylase and its interaction with substrates and inhibitors
Authors: MARTÍNEZ Aurora , ANDERSSON Kristoffer K. , HAAVIK Jan , FLATMARK Torgeir
Published: March 2005

The paramagnetic iron at the active site of highly purified, catalytically active phenylalanine hydroxylase was studied by EPR at 3.6 K and one‐dimensional 1H‐NMR spectroscopy at 293 K. The EPR‐detectable iron of the bovine enzyme was found to be present as a high‐spin form (S= 5/2) in different ligand field symmetries depending on medium conditions (buffer ions) and the presence of ligands known to bind at the active site. At 3.6 K and in phosphate buffer, the paramagnetic iron is coordinated in an environment of rhombic symmetry (g= 4.3), whereas Tris buffer favours an environment of axial ligand field symmetry (g= 6.7, 5.3 and 2.0). The latter axial type of signals resembles those observed at g= 7.0, 5.2 and 1.9 for the enzyme in phosphate buffer when L‐noradrenaline is added as an active‐site ligand (inhibitor). The same proportion of iron that coordinates to L‐noradrenaline seems to be reduced by the pterin cofactor and participate in catalysis. Experimental evidence is presented that Tris inhibits the enzyme by interacting with the enzyme‐bound ferric iron and decreases its rate of reduction by the tetrahydropterin cofactor. Preincubation with dithiothreitol also inhibits the enzyme activity and prevents the reduction of its catalytically active ferric iron by pterin cofactors as well as binding of catecholamines to the enzyme.

1H‐NMR spectroscopy revealed that the substrate (l‐phenylalanine) and l‐noradrenaline bind close to the paramagnetic iron, and that the catecholamine displaces the substrate from its binding at the active site. The results support our recently proposed model for the cooperative binding of inhibitor and substrate at the active site [Martínez, A. et al. (1990) Eur. J. Biochem. 193, 211–219].
Purification and characterization of a novel 5′ exodeoxyribonuclease from the yeast Saccharomyces cerevisiae
Authors: DÖLBERG Martin , BAUR Claus‐Peter , KNIPPERS Rolf
Published: March 2005

We have isolated from yeast cells an exonuclease which preferentially attacks double‐stranded DNA from the 5′ ends producing 5′‐mononucleotides as reaction products. A second typical product is a full‐length single‐stranded DNA complement, suggesting that the enzyme hydrolyzes one DNA strand in a processive manner before it associates with another DNA substrate to initiate a new reaction cycle. Its biochemical properties suggest that the enzyme is unlike the yeast exonucleases which have been reported so far. However, the new exonuclease is strikingly similar to the well characterized 5′ exonuclease of bacteriophage λ
Differential degradation of a recombinant albumin‐binding receptor in Escherichia coli
Authors: MURBY Maria , NYGREN Per‐Åke , RONDAHL Helena , HELLMAN Ulf , ENFORS Sven‐Olof , UHLÉN Mathias
Published: March 2005

The degradation in Escherichia coli of the recombinant serum‐albumin‐binding receptor derived from streptococcal protein G was investigated using a dual‐affinity fusion approach. The proteolytic degradation of the receptor was characterized when fused to human proinsulin and human secretin. Several cleavages occurred at sequences not normally regarded as proteolytically sensitive, such as the dipeptide sequences Ile‐Gly, Val‐Ser and Ser‐Ala. Depending on the fusion partner, large differences in the degradation of the albumin‐binding domain were observed. Thus, susceptibility to proteolysis of a recombinant protein can be affected by a neighbouring domain.
An embryo‐specific protein of barley (Hordeum vulgare)
Authors: CLARK Anthony J. , HIGGINS Patricia , MARTIN Helen , BOWLES Dianna J.
Published: March 2005

An immunological approach has been used to identify embryo‐specific products that can be used as molecular markers of embryogenesis. Immunoadsorption of antisera to remove antigens common to embryos, meristematic cells and callus, revealed one major embryo‐specific antigen, a polypeptide of 17 kDa. The antigen appeared at mid‐stages of zygotic embryo formation and remained at similar levels up to six days post‐germination of the seedling. The polypeptide could not be detected by protein staining, suggesting it is a non‐abundant product. Appearance of the antigen could be induced by culture of zygotic embryos in vitro on abscisic acid (1 μM) or mannitol (9% mass/vol.). Cross‐reactive products of near‐identical molecular mass were observed in embryos of wheat, rye and oats but not of distantly related cereals, nor embryos from dicotyledonous species. The timing of the appearance of the antigen was different in embryos formed from microspores during anther culture in vitro. In the cultured material, the 17‐kDa polypeptide preceded the appearance of morphologically distinct embryonic structures.
Analysis and nucleotide sequence of the genes encoding the surface‐layer glycoproteins of the hyperthermophilic methanogens Methanothermus fervidus and Methanothermus sociabilis
Authors: BRÖCKL Günther , BEHR Michael , FABRY Stefan , HENSEL Reinhard , KAUDEWITZ Hubert , BIENDL Elisabeth , KÖNIG Helmut
Published: March 2005

The genes (slgA) encoding the surface‐layer glycoproteins of the hyperthermophilic methanogens Methanothermus fervidus and Methanothermus sociabilis were cloned and sequenced. The nucleotide sequences of these genes differ at only nine positions, resulting in three amino acid differences.

In both organisms, the transcription start site was localized by primer extension analyses. The DNA sequence at this site conforms to the promotor box B motif for promotors of archaea. 24 nucleotides upstream of the transcription start is an A + T‐rich region, which closely resembles the consensus box A motif of promoters of methanogens. Ribosome binding sites are exactly complementary to the 3′ end of the 16S rRNA of these methanogens.

Both slgA genes encode for a precursor of the mature surface‐layer protein containing 593 amino acid residues with a putative N‐terminal signal sequence of 22 amino acid residues. The deduced protein sequences contain 20 sequon structures representing possible carbohydrate‐binding sites.

In comparison with other surface‐layer proteins, these obtained from the two hyperthermophilic methanogens contain unusually high amounts of isoleucine, asparagine and cysteine residues. Predicted secondary structures have a high content of β‐sheet structure (44%) and only 7%α‐helix structures.
The interaction of paramagnetic ions chelated to ATP with pyridoxal analogues
Authors: KWOK Francis , CHURCHICH Jorge E.
Published: March 2005

Fluorescence spectroscopy, static and dynamic, was applied to deduce proximity relationships between paramagnetic metal ions chelated to ATP and the inhibitor pyridoxal oxime bound to the site of the substrate pyridoxal. The fluorescence yield of free pyridoxal oxime is considerably reduced in the presence of CoCl2 (1 mM) due to the formation of a non‐fluorescent complex.

The fluorescence properties of pyridoxal oxime bound to pyridoxal kinase (50 μM, Kd= 1 μM) remain invariant in the presence of CoCl2 (1 mM) and ATP (1 mM). The results are interpreted to mean that the distances are too great for direct or first‐coordination‐sphere interaction between pyridoxaloxime and Co(II) centers.

The bidentate complex Cr(III) · ATP is a competitive inhibitor of ATP (Ki= 83 μM) and its binding to pyridoxal kinase (50 μM) complexed to pyridoxal oxime (50 μM) was monitored by static and dynamic fluorescence spectroscopy. The fluorescence yield of bound pyridoxal oxime is reduced by 25% in the presence of Cr(III) · ATP (0.8 mM) leading to a decrease in fluorescence lifetime from 〈τ〉= 6.60 ns to 〈τ〉= 4.20 ns. The decrease in fluorescence yield together with a diminution in fluorescence lifetime can be explained in terms of radiationless energy transfer from excited pyridoxal oxime to Cr(III) chelated to ATP.

Using Forster's equation and an efficiency of energy transfer of 0.25, it was found that a distance of 1.3 nm separates pyridoxal oxime from the Cr(III) center.
The C‐terminal domain of plant catalases Implications for a glyoxysomal targeting sequence
Authors: GONZÁLEZ Elma
Published: March 2005

A castor bean (Ricinus communis cv. Hale) cDNA encoding catalase was cloned and sequenced. The cDNA encoding the carboxy‐terminal domain of catalase was compared to the corresponding sequences of six other plant catalases. The deduced amino acid sequences were compared according to the chemical attributes of each amino acid within each carboxy‐terminal domain. A tripeptide sequence having the chemical attributes of the peroxisomal targeting sequence [Gould, S. J., Keller, G.‐A., Hosken, N., Wilkinson, J. & Subramani, S. (1989) J. Cell Biol. 108, 1657–1664] was common to all the glyoxysomal/peroxisomal plant catalases. This sequence motif was located six amino acids from the carboxy terminus of each of the plant catalases. An identical motif was also found within the carboxy‐terminal domain of three mammalian catalases previously sequenced. We hypothesize that these motifs are at least part of the targeting mechanism for catalase entry into plant glyoxysomes/peroxisomes.
Cell‐cycle‐regulated expression of thymosin β4 in thymocytes
Authors: SCHÖBITZ Bernd , NETZKER Roland , HANNAPPEL Ewald , BRAND Karl
Published: March 2005

Thymosin β4 belongs to a family of ubiquitous peptides present at a high cellular content but still with an unknown intracellular function. The expression of this peptide was studied in concanavalin‐A‐stimulated, proliferating rat thymocytes during cell cycle progression. An early, transient 10‐fold increase of the peptide occurred 1 h after stimulation without elevation of the corresponding mRNA level. This increase coincided with that of thymosin β4 biosynthesis. The sharp decline of the thymosin β4 content was not due to a secretion of the peptide into the medium. During S phase and mitosis, the biosynthetic rates as well as mRNA content, but not the cellular thymosin β4 concentration, increased again. After 96 h of culture the values returned to those of quiescent cells.
Two‐step mobilization of arachidonic acid in platelet activation induced by low concentrations of TP 82, a monoclonal antibody against CD9 antigen
Authors: OZAKI Yukio , MATSUMOTO Yuki , YATOMI Yutaka , HIGASHIHARA Masaaki , KUME Shoji
Published: March 2005

Arachidonic acid mobilization in platelets activated by low concentrations (≤ 1.6 μg/ml) of TP 82, a monoclonal antibody against CD9, appears to consist of two distinct phases. In the first phase, limited arachidonic acid release occurs concomitantly with a shape change induced by TP 82. This appears to be dependent upon phospholipase A2 activation, since it is well preserved in the presence of aspirin, which completely blocked both intracellular Ca2+ elevation and phosphatidic acid formation which would indicate phospholipase C activation. The Na+/H7plus; exchange was also found to participate in the first phase of arachidonic acid mobilization, since extracellular Na+ depletion and ethylisopropylamiloride, a specific inhibitor of the Na+/H+ exchanger, effectively blocked this limited mobilization of arachidonic acid. The second, much larger, phase of arachidonic acid mobilization occurs with the beginning of platelet aggregation. A limited amount of thromboxane A2 formed during the first phase of arachidonic acid release plays an important role in induction of the massive arachidonic mobilization in the second phase. Factors, as yet unidentified, also appear to work synergistically with thromboxane A2 to induce the full picture of arachidonic acid mobilization.
Identification of the disulphide bonds in human platelet glycocalicin
Authors: HESS Daniel , SCHALLER Johann , RICKLI Egon E. , CLEMETSON Kenneth J.
Published: March 2005

The glycoprotein Ib/IX complex on platelets is responsible for the first stage of haemostasis as an essential component in the primary adhesion of platelets to damaged vessel walls. Glycocalicin is the extracellular part of platelet glycoprotein Ibα and contains the von Willebrand factor and thrombin binding sites. Disulphide bonds are implicated in the von Willebrand binding site and studies with peptides point towards a region of glycocalicin with four cysteines as containing the binding sites for both von Willebrand factor and thrombin. The position and linkage of these two disulphide bonds are now determined to be 209–248 and 211–264 and the relevance of this double‐loop structure for glycoprotein Ib/IX function is discussed.
On the mechanism of transcellular lipoxin formation in human platelets and granulocytes
Authors: EDENIUS Charlotte , STENKE Leif , LINDGREN Jan Åke
Published: March 2005

Endogenous arachidonic acid was converted to lipoxins A4, B4 and (6S)‐lipoxin A4, in ionophore‐A23187‐stimulated mixtures of human platelets and granulocytes, while no lipoxins were formed when these cells were incubated separately. However, pure platelet suspensions transformed exogenous leukotriene A4 to lipoxins, including lipoxin A4 and (6S)‐lipoxin A4, but not lipoxin B4. This compound was produced exclusively in the presence of granulocytes.

A common unstable tetraene intermediate in lipoxin formation, 15‐hydroxy‐leukotriene A4 [5(6)‐epoxy‐15‐hydroxy‐7,9,13‐trans‐11‐cis‐eicosatetraenoic acid], was indicated by trapping experiments with methanol. Thus, identical profiles of less polar tetraene‐containing derivatives were formed from leukotriene A4 in platelet suspensions, from exogenous 15‐hydroxyeicosatetraenoic acid in granlocyte suspensions and from endogenous substrate in mixed platelet/granulocyte suspensions.

Evidence for the involvement of 12‐lipoxygenase in platelet‐dependent lipoxin formation was obtained. Thus, lipoxin synthesis from leukotriene A4 and 12‐hydroxyeicosatetraenoic acid production from arachidonic acid by human platelets was equally inhibited by 15‐hydroxyeicosatetraenoic acid with 50% inhibition obtained at 7.0 μM and 8.2 μM, respectively. In experiments with subcellular preparations from platelets, lipoxin synthesis was observed in both the particulate and soluble fraction and was paralleled by the 12‐lipoxygenase activity. Furthermore, lipoxin formation from leukotriene A4 in platelet sonicates was dose‐dependently inhibited by exogenous arachidonic acid. Finally, 12‐lipoxygenase‐deficient platelets from a patient with chronic myelogenous leukemia were totally unable to produce lipoxins from exogenous or granulocyte‐derived leukotriene A4.

It is concluded that the transcellular lipoxin synthesis is dependent on the platelet 12‐lipoxygenase and proceeds via the unstable intermediate, 15‐hydroxy‐leukotriene A4. This tetraene epoxide is transformed to lipoxin B4 by a granulocyte epoxide hydrolase activity or to lipoxin A4 and lipoxins A4/B4 isomers by enzymatic or non‐enzymatic hydrolysis.
Hydroperoxides of α‐ketols
Authors: GRECHKIN Alexander N. , KURAMSHIN Rustem A. , LATYPOV Shamil K. , SAFONOVA Yelena Yu. , GAFAROVA Tatiana E. , ILYASOV Akhat V.
Published: March 2005

Metabolism of [1‐14C]linolenic acid, 13‐hydroperoxy‐9(Z),11(E),15(Z)‐[1‐14C]octadecatrienoic acid (13‐HPOT) and 9‐hydroperoxy‐10(E),12(Z),15(Z)‐[1‐14C]octadecatrienoic acid (9‐HPOT) was studied by enzyme preparations from flax, wheat and corn seeds, containing two enzymes of fatty acid metabolism, namely, lipoxygenase and hydroperoxide dehydrase. Along with the previously known products of the hydroperoxide dehydrase pathway, the radiolabel was incorporated into some more polar metabolites. These polar products 1 and 4, formed from 13‐HPOT and 9‐HPOT, respectively, were purified by reversed‐phase and normal‐phase HPLC, and investigated by ultraviolet spectroscopy, chemical‐ionization and electron‐impact mass spectrometry, and 1H‐NMR. The data obtained suggest that metabolites 1 and 4 are 9‐hydroperoxy‐12‐oxo‐13‐hydroxy‐10(E),15(Z)‐octadecadienoic acid and 9‐hydroxy‐10‐oxo‐13‐hydroperoxy‐11(E),15(Z)‐octadecadienoic acid, respectively. 12‐oxo‐13‐hydroxy‐9(Z),15(Z)‐[1‐14C]octadecadienoic acid (12,13‐α‐ketol) and 9‐hydroxy‐10‐oxo‐12(Z),15(Z)‐[1‐14C]octadecadienoic acid (9,10‐α‐ketol) are the direct precursors of metabolites 1 and 4, respectively. Metabolites 1 and 4 are formed from the corresponding HPOT precursors in two stages; (a) conversion of hydroperoxide into the α‐ketol by hydroperoxide dehydrase and (b) the lipoxygenase oxidation of the α‐ketol. Different lipoxygenases were found to oxidize α‐ketols. Oxidation of the 3(Z)‐buten‐1‐onyl moiety of α‐ketols presents an unusual and previously unknown type of lipoxygenase reaction.
Purification of tobacco nuclear proteins binding to a CACGTG motif of the chalcone synthase promoter by DNA affinity chromatography
Authors: STAIGER Dorothee , BECKER Frank , SCHELL Jeff , KONCZ Csaba , PALME Klaus
Published: March 2005

The activity of various light‐regulated and developmentally regulated plant gene promoters critically depends upon the presence of a conserved sequence with a central CACGTG motif. Using band‐shift assays, we have identified nuclear factor(s) from Nicotiana tabacum, termed CG‐1, that specifically recognize(s) this transcriptional element in the ultraviolet‐light‐regulated Antirrhinum majus chalcone synthase promoter. CG‐1 activity is constitutively expressed in tobacco seedlings grown in the absence of ultraviolet light as well as in seedlings induced for chalcone synthase gene expression by ultraviolet light irradiation. CG‐1 activity has also been detected in flower tissue. DNA‐protein cross‐linking studies identified three polypeptides with apparent molecular masses of 20, 32 and 42 kDa binding to the CACGTG motif. Proteins interacting with the CACGTG motif were purified from N. tabacum seedlings using differential sequence‐specific DNA affinity chromatography employing wild‐type and mutated CG‐1‐binding sites. Denaturing polyacrylamide gel electrophoresis revealed major polypeptides of approximately 20, 30 and 40 kDa which are highly enriched in the affinity‐purified fractions binding specifically to the CACGTG motif.
Action of trypsin on glycinin
Authors: SHUTOV Andrei D. , PINEDA Julio , SENYUK Vitalyi I. , REVA Vyacheslav A. , VAINTRAUB Iosif A.
Published: March 2005

The formation of a relatively stable high‐molecular‐mass product on trypsin hydrolysis of glycinin (glycinin‐T) is interpreted to be a result of ‘zipper’ proteolysis. Evidence of parallel one‐by‐one degradation of glycinin occurring after the formation of glycinin‐T is presented. At a relatively low concentration of the substrate, the one‐by‐one proteolysis proceeds as a first‐order reaction. A method of determination of the changes in the molecular mass of a protein during the mixed‐type proteolysis and some other parameters of this process is developed on the basis of the analysis of the proteolysis kinetics. The value of the molecular mass of glycinin‐T calculated by means of this method makes up 70% of the initial molecular mass and coincides with the result of direct determination by gradient gel electrophoresis.
Characterisation of the electron self‐exchange rates in hexametaphosphate‐cytochrome‐c aggregates measured using high‐resolution 1H‐NMR spectroscopy
Authors: CONCAR David W. , WHITFORD David , WILLIAMS Robert J. P.
Published: March 2005

1H‐NMR spectroscopy has been used to measure the rate of unimolecular electron exchange between cytochrome c molecules in protein aggregates stabilised by the addition of sodium hexametaphosphate. The average intracomplex electron exchange rate is measured from line broadening of hyperfine‐shifted resonances of ferricytochrome c in an equimolar mixture of reduced and oxidised protein. The line‐broadening due to electron exchange is significantly greater than that due to protein aggregation and reaches a maximum value between 1 – 2 mol hexametaphosphate/mol protein. Significantly the exchange‐induced broadening is a first‐order process and is directly proportional to the size of the cytochrome c oligomer. From the temperature dependence of exchange broadening the activation enthalpy was estimated to be 75.8 kJ mol−1 whereas the activation entropy was 295 J mol−1 K−1 for a dimer of cytochrome c at a hexametaphosphate/protein molar ratio of 1. Both activation parameters decrease in magnitude as the order of the cytochrome c oligomer increases. The rates of intracomplex electron exchange in Saccharomyces cerevisiae iso‐2 and Candida krusei cytochromes c are lower than that of the horse protein, implying that primary sequence plays a fundamental part in determining the rate of exchange. The relevance of these observations is discussed in terms of the function of cytochrome c.
Characterization and purification of the fusicoccin‐binding complex from plasma membranes of Commelina communis
Authors: OECKING Claudia , WEILER Elmar W.
Published: March 2005

The fungal phytotoxin fusicoccin binds with high affinity to plasma membranes of the monocotyledonous plant, Commelina communis L. The sites bind the toxin with an apparent Kd of 5.2 nM and a pH optimum of 6.0. They occur at a level of approximately 6–8 pmol/mg plasma membrane protein. Photoaffinity labeling with the biologically active fusicoccin derivative 9′‐nor‐8′‐(4‐azido[3,5‐3H]benzoyl) diaminoethylfusicoccin identified a polypeptide of 31.5 kDa on SDS/PAGE which was strongly labeled. A second 32.5‐kDa band was also consistently labeled, although not to the same extent. The binding sites were solubilized in functional form and a purification scheme was developed based on affinity and ion‐exchange procedures. The purified fraction contains two polypeptides of apparent molecular masses of 30.5 kDa and 31.6 kDa. A detailed molecular analysis of the fusicoccin‐binding complex is now possible.
Characterization of a cDNA clone encoding molluscan insulin‐related peptide II of Lymnaea stagnalis
Authors: SMIT August B. , GERAERTS Wijnand P. M. , MEESTER Irene , van HEERIKHUIZEN Harm , JOOSSE Joos
Published: March 2005

A cDNA clone encoding molluscan insulin‐related peptide (MIP) II was isolated from a cDNA library of the central nervous system (CNS) of the freshwater snail, Lymnaea stagnalis, using a heterologous screening with a previously identified MIP cDNA (renamed MIP‐I cDNA). The MIP‐II cDNA encodes a preprohormone resembling the organization of preproinsulin, with a putative signal sequence, and A and B chains; however, in this case connected by two distinct C peptides, Cα and Cβ, instead of a single C peptide, a phenomenon which represents a new development in the prohormone organization of peptides belonging to the insulin superfamily. The A and B chains of MIP II and I differ remarkably in primary structure; in contrast, the Cα peptide domains are fully identical. MIP II has only limited sequence similarity with insulins and related peptides. Both MIP II and I exhibit structural features, which make them a unique class of the insulin superfamily. The MIP I and II genes are expressed in a single type of neuron: the growth‐controlling neuroendocrine light green cells of the Lymnaea CNS.
Two different aa3‐type cytochromes can be purified from the bacterium Bacillus cereus
Authors: GARCIA‐HORSMAN J. Arturo , BARQUERA Blanca , ESCAMILLA Jose E.
Published: March 2005

Two aa3‐type cytochromes were purified from membranes of sporulating Bacillus cereus. One of them, an aa3 complex, was found to be composed of two subunits (51 and 31 kDa), two a hemes and three copper atoms, thus being similar to the cytochrome aa3 previously purified from vegetative B. cereus [García‐Horsman, J. A., Barquera, B., González‐Halphen, D. & Escamilla, J. E. (1991) Mol. Microbiol. 5, 197–205]. The second isoform, a caa3 complex, was expressed in sporulating cells only, and was found to be composed of two subunits (51 and 37 kDa). The 37‐kDa subunit (subunit II) is a heme‐c‐containing polypeptide as shown by its peroxidase activity in SDS/PAGE gels and by its spectral features. Both subunits of the caa3 complex immunologically cross‐reacted with antiserum raised against B. cereus cytochrome aa3, suggesting homology between the two enzymes. Also, the heme‐c‐containing subunit of the caa3 complex was reactive with anti‐(bovine cytochrome c) antiserum, but not with anti‐(bovine cytochrome c1) antiserum. In addition to one heme c and two hemes a, the caa3 complex contained three copper atoms.

Kinetic comparison of aa3 and caa3 complexes revealed that the latter is slightly more active (k= 150 s−1) and has a lower affinity to yeast cytochrome c (Km= 76 μM) and to oxygen (Km= 2 μM) as compared with cytochrome aa3 (100 s−1, 10 μM, and 5 μM, respectively).