bluuebunny/crossref_metadata_2025 huggingface dataset:
Transport of β‐alanine and biosynthesis of carnosine by skeletal muscle cells in primary culture
Authors: Bakardjiev Anastasia , Bauer Karl
Published: March 2005

Uptake of β‐alanine and synthesis of carnosine (β‐alanyl‐histidine) could be demonstrated in primary cell cultures derived from embryonic chick pectoral muscle. Concomitant with the morphological changes, cessation of cell division and the induction of creatine kinase, a rapid increase in the rate of β‐alanine uptake and also in the rate of carnosine synthesis could be observed. The uptake of β‐alanine is sodium and chloride dependent and obeys Michaelis‐Menten kinetics with Km values of about 40 μM that are essentially identical for myoblasts and myotubes. In contrast, Vmax increases considerably during differentiation. The β‐alanine transport system is highly specific for β‐amino acids and exhibits a substantial anion dependency (Cl‐ > J‐ > CSN‐ > SO2‐4). Stoichiometric studies suggest that the transport of one β‐alanine molecule involves two sodium ions and one chloride ion. This ratio is not altered by the process of cell differentiation.
Characterization, quantification and subcellular localization of inositol‐containing sphingolipids of the yeast, Saccharomyces cerevisiae
Authors: Hechtberger Petra , Zinser Erwin , Saf Robert , Hummel Klaus , Paltauf Fritz , Daum Günther
Published: March 2005

In yeast, as in higher eukaryotic cells, sphingolipids are essential membrane components. The yeast, Saccharomyces cerevisiae, contains three classes of sphingolipids, inositolphosphorylceramide (InsP Cer), mannosylinositolphosphorylceramide (ManInsP Cer) and mannosyldiinositolphosphorylceramide (ManP Ins2P Cer). As a prerequisite to localize these sphingolipids in subcellular membranes, authentic standards of the respective lipids were isolated and characterized using biochemical methods and electrospray ionization mass spectrometry. The complete set of yeast subcellular membranes was isolated at high purity, and sphingolipids were extracted. InsP Cer, ManInsP Cer, and ManP Ins2P Cer were separated by thin‐layer chromatography, stained and densitometrically scanned along with the respective standards. These methods enable a complete overview of the subcellular distribution of yeast sphingolipids to be obtained, as far as is known, for the first time. InsP Cer was highly enriched in Golgi and vacuolar membranes, whereas the largest amounts of ManInsP Cer and ManPIns2PCer were found in the plasma membrane. The presence of inositol‐containing sphingolipids in organelles of the protein‐secretory pathway strongly supports the notion that protein secretion and intracellular trafficking of sphingolipids are linked processes.
Effects of mifepristone (RU‐486) on heme metabolism and cytochromes P ‐450 in cultured chick embryo liver cells
Authors: Cable Edward E. , Pepe Joyce A. , Donohue Susan E. , Lambrecht Richard W. , Bonkovsky Herbert L.
Published: March 2005

Mifepristone (RU‐486), a potent progesterone receptor antagonist and inducer of cytochromes P ‐450, is currently in use in Europe, particularly as a post‐coital oral contraceptive. Soon it will be available in the United States, as well. Since progesterone has been implicated in the pathogenesis of acute attacks of porphyria, the use of RU‐486 or related compounds might be considered in porphyric patients. However, as with other cytochrome P ‐450 inducers, RU‐486 may have the ability to precipitate or exacerbate attacks of acute porphyria. The acute porphyrias in relapse are associated with an increase in activity of δ‐aminolevulinic acid synthase, the first and normally rate‐controlling enzyme in heme biosynthesis. We have used primary cultures of chick embryo liver cells to test the ability of RU‐486 to induce δ‐aminolevulinic acid synthase activity and mRNA, cytochromes P ‐450, porphyrin accumulation, and heme oxygenase. We found that RU‐486, at concentrations observed in human plasma after a single oral dose, induced the mRNA and activity of δ‐aminolevulinic acid synthase, both by itself and in the presence of deferoxamine, a potent iron chelator that inhibits ferrochelatase. RU‐486 and deferoxamine together also produced significant accumulations of protoporphyrin. These results indicate that RU‐486 may pose a risk in patients with known acute porphyria and should be used with caution. RU‐486 increased the concentration of total cytochrome P ‐450, and the activity of erythromycin demethylase, an activity specifically catalyzed by cytochrome P ‐450 3 A. Unlike several other porphyrogens (e.g. hydantoins, barbiturates), RU‐486 does not produce accumulation of uroporphyrin or induction of heme oxygenase in chick embryo liver cells.
Design of human interleukin‐4 antagonists inhibiting interleukin‐4‐dependent and interleukin‐13‐dependent responses in T‐cells and B‐cells with high efficiency
Authors: Tony Hans‐Peter , Shen Bo‐Jiang , Reusch Petra , Sebald Walter
Published: March 2005

Human interleukin‐4 possesses two distinct sites for receptor activation. A signalling site, comprising residues near the C‐terminus on helix D, determines the efficacy of interleukin‐4 signal transduction without affecting the binding to the interleukin‐4 receptor α subunit. A complete antagonist and a series of low‐efficacy agonist variants of human interleukin‐4 could be generated by introducing combinations of two or three negatively charged aspartic acid residues in this site at positions 121, 124, and 125. One of the double variants, designated [R121D,Y124D]interleukin‐4, with replacements of both Arg121 and Tyr124 by aspartic acid residues was completely inactive in all analysed cellular responses. The loss of efficacy in [R121D,Y124D]interleukin‐4 is estimated to be larger than 2000‐fold. Variant [R121D,Y124D]interleukin‐4 was also a perfect antagonist for inhibition of interleukin‐13‐dependent responses in B‐cells and the TF‐1 cell line with a Ki value of approximately 100 pM. In addition, inhibition of both interleukin‐4‐induced and interleukin‐13‐induced responses could be obtained by monoclonal antibody X2/45 raised against interleukin‐4Rex, the extracellular domain of the interleukin‐4 receptor α subunit. These results indicate that efficient interleukin‐4 antagonists can be designed on the basis of a sequential two‐step activation model. In addition, the experiments indicate the functional participation of the interleukin‐4 receptor α subunit in the interleukin‐13 receptor system.
Purification of a Novel 43‐kDa Protein (p43) Intermediary in the Activation of Steroidogenesis from Rat Adrenal Gland
Authors: Paz Cristina , Dada Laura A. , Maciel M. Fabiana Cornejo , Mele Pablo G. , Cymeryng Cora B. , Neuman Isabel , Mendez Carlos F. , Finkielstein Carla V. , Solano Angela R. , Park Minkyu , Fischer Wolfgang H. , Towbin Harry , Scartazzini Ricardo , Podestá Ernesto J.
Published: March 2005

In previous reports we have demonstrated the presence of a soluble factor that responds to cAMP signals to induce steroid synthesis in adrenocortical tissue. Here, we describe the purification of this factor from adrenal zona fasciculata cells by using a five‐step procedure that includes DEAE‐cellulose, gel filtration, Mono Q HPLC and Superose HPLC, and elution of the protein from SDS/PAGE. This procedure results in the purification to homogeneity of a protein of 43‐kDa that retains the capacity to stimulate steroid synthesis in anin vitrorecombination assay. This activity is inhibited by the use of phospholipase A2, inhibitors. Antipeptide antibodies against the N‐terminal region recognize p43 as a double band on SDSPAGE that resolves in different spots on two‐dimensional gel electrophoresis. Adrenocorticotropin treatment of adrenal glands results in the appearence of multiple spots that migrated towards a lower pH compared to controls, suggesting the presence of phosphorylated and dephosphorylated forms of p43. Sequencing of the N‐terminal region and internal peptides reveals no significant similarities with other proteins, suggesting that p43 is a novel protein.

We conclude from our data that the isolated protein (p43) is a novel, soluble protein that acts as intermediary in adrenocorticotropin‐induced stimulation of arachidonic acid release and steroid synthesis.
Crystal Structure Analysis of a Serine Proteinase from Streptomyces fradiae at 0.16‐nm Resolution and Molecular Modeling of an Acidic‐amino‐acid‐specific Proteinase
Authors: Kitadokoro Kengo , Tsuzuki Hiroshige , Okamoto Hiroyuki , Sato Tomohiro
Published: March 2005

We have determined the three‐dimensional structure of a proteinase from Streptomyces fradiae ATCC 14544 (SFase‐2) at 0.16‐nm resolution. SFase‐2, a typical serine proteinase, has broad substrate specificity. The characterization and crystallographic analysis of this enzyme have been reported previously [Kitadokoro, K., Tsuzuki, H., Nakamura, E., Sato, T. & Teraoka, H. (1994) Eur: J. Biochem. 220, 55–61]. In the present study, data were collected to approximately 0.16‐nm resolution on a Rigaku R‐AXIS IIC imaging plate detector system. Preliminary phases were obtained by molecular replacement methods with a search model derived from the previously determined structure of Streptomyces griseus protease A [Sielecki, A. R., Hendrickson, W. A., Broughton, C. G., Delbaere, L. T., Brayer, G. D. & James, M. N. (1979) J. Mol. Biol. 134, 781–8041. The starting model gave an initial crystallographic R factor of 0.443. Refinement with restrained least‐squares converged at a final R factor of 0.182 for 16128 observed reflections. The final model includes 86 water molecules.

The crystal structure showed that the enzyme consists of two domains, each of which is comprised of a β barrel with six‐stranded β sheets and two α helices. The overall tertiary structure of SFase‐2 is similar to the structures of other chyrnotrypsin‐like proteinases from S. griseus, namely proteinase A and proteinase B. The essential residues of the catalytic triad are located on the cleft between the two domains. These two domains have different sequences, but possess similar three‐dimensional structures, indicating that a gene duplication event has occurred to produce these two domains.
Structure elucidation and biosynthesis of 31‐methylhopanoids from Acetobacter europaeus
Authors: Simonin Pascale , Tindall Brian , Rohmer Michel
Published: March 2005

Apart from a mixture of bacteriohopanetetrols already found in other Acetobacter species, four new 3β‐methylhopanoids have been isolated from Acetobacter europaeus. All of them present an ether linkage between a bacteriohopanetetrol or a bacteriohopanepentol and a carbapseudopentose moiety often found in bacterial hopanoids. Three of these ethers were shown by comparison with synthetic reference hopanoids to posess a supplementary methyl group at C31. This novel series of methylhopanoids may be the precursor of yet unidentified molecular fossils found in sediments. [methyl ‐2H3]Methionine was efficiently incorporated into the 31‐methylhopanoids with retention of all three deuterium atoms in the transferred methyl group. This labelling pattern might be consistent with a rather rarely found methylation reaction of an enol.
Molecular Cloning and Tissue‐Specific RNA Processing of a Murine Receptor‐Type Protein Tyrosine Phosphatase
Authors: Wagner John , Boerboom Derek , Tremblay Michel L.
Published: March 2005

The molecular cloning of a murine receptor‐type protein tyrosine phosphatase, termed PTP NU‐3, with an extracellular cell‐adhesion‐molecule‐like domain is reported. NU‐3 was isolated from 11.5‐day total mouse embryonic RNA by reverse‐transcriptase PCR using degenerate oligonucleo‐tides flanking the conserved protein tyrosine phosphatase catalytic domain. This produced a 280‐bp DNA probe which was subsequently employed to screen a mouse embryonic kidney library. Several overlapping cDNA clones were isolated, collectively forming a cDNA of 6.0 kb that encodes a putative 211‐kDa protein. Northern‐blot analysis of total RNA from adult and embryonic mouse tissues indicates the existence of two major PTP NU‐3 transcripts of approximately 6 kb and 7 kb. Both messages are expressed predominantly in brain tissues and neuronal‐derived cell lines, although detectable levels of the 7‐kb message were found in other non‐neuronal tissues. We have identified a unique 132‐bp exon segment that is present in the 7‐kb message but is completely absent in the 6‐kb transcript, suggesting tissue‐specific levels of expression and RNA processing. Analysis of the amino acid sequence encoded by the 132‐bp segment reveals that it completes a partial fibronectin type‐III element resulting in a protein with a total of nine such elements. Bacterial expression of the two catalytic domains demonstrated that only the first domain possesses enzymic activity towards a tyrosine phosphorylated substrate.
Detergent‐Induced Folding of the Outer‐Membrane Protein PhoE, a Pore Protein Induced by Phosphate Limitation
Authors: Van Gelder Patrick , De Cock Hans , Tommassen Jan
Published: March 2005

The folding of the in vitro synthesized outer‐membrane protein PhoE, a protein induced by phosphate limitation, has previously been studied using immunoprecipitation experiments with monoclonal antibodies that recognize conformational epitopes [De Cock, H., Hendriks, R., de Vrije, T. & Tommassen, J. (1990) J. Biol. Chem. 265, 4646–4651]. A folded monomer of the protein was detected in this way, while the addition of outer membranes was required to induce trimerization. In this study, we demonstrate that the folding of the in vitro synthesized PhoE protein did not occur spontaneously, but was dependent on the detergent that was present in the immunoprecipitation buffer. A remarkable specificity of phenyl‐containing detergents on the efficient in vitro folding of PhoE molecules was observed. Furthermore, trimerization was detected in the absence of outer membranes when such detergents were present. However, the rate of trimerization was increased by the addition of crude cell envelopes containing outer membranes. The outer membranes probably enhanced trimerization by concentrating the folded PhoE molecules.
Characterization and Molecular Cloning of a Novel MUC1 Protein, Devoid of Tandem Repeats, Expressed in Human Breast Cancer Tissue
Authors: Zrihan‐Licht Sheila , Vos Hans, L. , Baruch Amos , Elroy‐Stein Orna , Sagiv Dalit , Keydar Iafa , Hilkens John , Wreschner Daniel H.
Published: March 2005

The human breast cancer marker protein, MUC1, is a polymorphic transmembrane molecule containing a large extracellular domain that is primarily composed of a variable number of highly conserved 20‐amino‐acid tandem repeats. We report here the detection of a novel invariantly sized 1.2‐kb MUC1 mRNA, in addition to the large polymorphic mRNAs, by probing Northern blots with MUC1‐cDNA‐unique‐sequence probes. The nucleotide sequence of this novel MUC1 mRNA demonstrates that it is identical to the MUC1 cDNA sequences downstream and upstream to the tandem‐repeat array of the transmembrane form of MUC1. However, it contains neither the central tandem repeat array itself nor its directly flanking sequences that are deleted by a differential splicing event utilizing splice acceptor and donor sequences 5′ and 3′ to the tandem‐repeat array. The splice event retains, downstream to the splice acceptor site, an open reading frame identical to that of the repeat‐array‐containing MUC1 thereby generating the novel MUC1/Y protein. Cells transiently transfected with the novel MUC1/Y cDNA express the MUC1/Y protein that is modified by glycosylation. The MUC1/Y protein is also readily detected in human breast cancer cells grown in vitro. Furthermore, primary breast cancer tissue samples demonstrate significant levels of the MUC1/Y protein whereas expression in tissue adjacent to the tumor is undetectable. Molecular characterization presented here, of the novel MUC1/Y molecule lacking the repeat array, suggests that it is likely to play a role distinct to that of the polymorphic repeat‐array‐positive MUC1 protein and that it may act as a new marker protein for human breast cancer.
GPIIIa(90–102) and GPIIIa(631–653) Epitopes as Markers of Conformational Changes Occurring During the Activation of the Glycoprotein IIb/IIIa Complex
Authors: Niewiarowska Jolanta , Cierniewski Czeslaw Stanislaw
Published: March 2005

To evaluate the range of conformational changes in the GPIIIa subunit upon the interaction of GPIIb/IIIa complex with fibrinogen, we employed two polyclonal antipeptide antibodies against synthetic peptides spanning chain segments (90–102) and (631–653) of GPIIIa. The first peptide fragment derives from the region preceding the putative binding site for the RGD sequence, while the second one is located in the vicinity of membrane lipid bilayer. The resting platelets bound about 13700 and 11500 molecules of anti‐[GPIIIa(90–102)] and anti‐[GPIIIa(631–653)] antibodies, respectively. The anti‐[GPIIIa(90–102)] antibodies preferentially bound to the active form of GPIIb/IIIa complex. This binding was significantly reduced after dissociation of the complex, indicating that the recognized epitopes became partially hidden in the GPIIIa subunit. In contrast to anti‐[GPIIIa(90–102)], the anti‐[GPIIIa(631–653)] antibodies reacted optimally with the EDTA‐treated GPIIb/IIIa. Upon stimulation of platelets with ADP, the number of binding sites for anti‐[GPIIIa(631–653)] was reduced 2.8‐fold. The binding affinity of anti‐[GPIIIa(90–102)] antibodies to the resting and ADP‐stimulated platelets differed 4.7‐fold, indicating that the stimulation of the platelets with ADP resulted in better exposure of this epitope. This binding was inhibited almost totally by fibrinogen in a dose‐dependent fashion. Similarly, fibrinogen inhibited binding of the anti‐[GPIIIa(631–653)] antibodies to platelets. In the reversed system, binding of fibrinogen to platelets was inhibited only to 60% by the anti‐[GPIIIa(90–102)] antibodies. Such inhibition of fibrinogen binding was sufficient to block aggregation of ADP‐stimulated platelets by the anti‐[GPIIIa(90–102)] antibodies in platelet‐rich plasma. In contrast, the anti‐[GPIIIa(631–653)] potentiated fibrinogen binding by 20%, confirming that there were additional interacting sites for fibrinogen in other regions of the GPIIb/IIIa complex.

The presented data indicate that the activation of the fibrinogen receptor is associated with extensive conformational changes in the GPIIIa subunit, detectable by use of antipeptide antibodies recognizing not only the epitopes located next to the putative ligand‐binding site but also the epitopes in the vicinity of the platelet membrane lipid bilayer.
The Sum of Flux Control Coefficients in the Electron‐Transport Chain of Mitochondria
Authors: Brand Martin D. , Vallis Brett P. S. , Kesseler Adolf
Published: March 2005

The sum of the flux control coefficients for group‐transfer reactions such as electron transport has been proposed to be two when the coefficients are calculated from experiments in which the concentrations of the electron carriers are changed (CE) but one when they are calculated from changes in the rates of the electron‐transfer processes (Cv). We tested this proposal using electron transport in uncoupled beef heart, potato tuber and rat liver mitochondria. First, with ascorbate plus N,N,N′,N′′‐tetramethyl‐p‐phenylenediamine as substrate, the CE, flux control coefficients of ascorbate, N,N,N′,N′′‐tetramethyl‐p‐phenylenediamine, mitochondria and oxygen over electron‐transport rate were measured by direct titration of the concentrations of these electron carriers. CE, values were close to zero, one, one and zero, respectively, giving a sum of CE, flux control coefficients of approximately two. At higher concentrations of N,N,N′,N′′‐tetramethyl‐p‐phenylenediamine, its CE, control decreased and the sum decreased towards one as predicted. Secondly, the CE, control coefficients of groups of electron‐transfer processes with succinate or ascorbate plus N,N,N′,N′′‐tetramethyl‐p‐phenylenediamine as substrate were measured. This was achieved by measuring the effects of KCN (or malonate or N,N,N′,N′‐tetramethyl‐p‐phenylenediamine) on system flux when intermediates were allowed to relax and on local flux when intermediates were held constant. The Cv, flux control coefficients were calculated as the ratio of the effects on system flux and on local flux. The sum of the Cv, flux control coefficients was approximately one. Whether a sum of one or a sum of two was obtained depended entirely on the definition of control coefficients that was used, since either sum was obtained from the same set of data depending on the method of calculation. Both definitions are valid, but they give different information. It is important to be aware of which definition is being used when analysing control coefficients in electron‐transport chains and other group‐transfer systems.
Unique Primary Structure of 2‐Nitropropane Dioxygenase from Hansenula Mrakii
Authors: Tchorzewski Marek , Kurihara Tatsuo , Esaki Nobuyoshi , Soda Kenji
Published: March 2005

We have isolated the gene encoding 2‐nitropropane dioxygenase from Hansenula mrakii, an FAD enzyme that catalyzes the oxygaenative denitrification of various anionic nitroalkanes. The gene contained an open reading frame consisting of 1122 nucleotides corresponding to 314 amino acid residues. The protein molecular mass was estimated to be 41466 Da, which was similar to the subunit molecular mass of the enzyme determined by SDSPAGE. Several FAD enzymes such as D‐amino acid oxidase and glucose oxidase also catalyze the oxidation of nitroalkanes as a side‐reaction, although not so efficiently [Kido, T. & Soda, K. (1984) Arch. Biochem. Biophys. 234, 468–475]. However, we found no proteins in the databases (GenBank, EMBL, PIR and SWISSPROT) which are homologous to 2‐nitropropane dioxygenase of H. mrakii in primary structure. No protein motifs, including a nucleotide‐binding motif, GXGXXG, were found in PROSITE, a database of biologically significant protein sites and patterns. Accordingly, 2‐nitropropane dioxygenase is a new type of flavoprotein with a unique structure.
1H‐NMR Assignments and Secondary Structure of Dendroaspin, an RGD‐Containing Glycoprotein IIb‐IIIa (αIIb‐β3) Antagonist with a Neurotoxin Fold
Authors: Jaseja Mahesh , Lu Xinjie , Williams Janice A. , Sutcliffe Michael J. , Kakkar Vijay V. , Parslow Rosemary A. , Hyde Eva I.
Published: March 2005

Dendroaspin, also referred to as mambin, was originally isolated from the venom of the Elapidae snake Dendroaspis jamesoni kaimose. It shares a high level of sequence similarity with the short‐chain neurotoxins found in other Elapidae but displays approximately 1000‐fold lower neurotoxin activity than the closely related protein erabutoxin b. However, unlike neurotoxins, it contains an RGD (Arg‐Gly‐Asp) motif and functions as an antagonist of platelet aggregation and cell‐cell adhesion of comparable potency to the disintegrins from the venoms of Viperidae. We have determined the secondary structure of dendroaspin using 1H‐NMR spectroscopy. Its structure resembles that of the short‐chain neurotoxins, with three loops extending from a disulphide‐bridged core; however, the strands of the triple‐stranded β‐sheet are shorter and the loop containing the RGD sequence is moved away from this sheet. The structure bears little resemblance to that of the disintegrins, except in the RGD‐containing loop, suggesting that this loop may be of prime importance in its inhibitory function. Comparison of this preliminary structure with that of the neurotoxins and disintegrins furthers our understanding of the mechanism of integrin antagonists and shows how the neurotoxin fold can be manipulated to give a variety of inhibitors.
Characterization of the Murine Plasma Fibrinolytic System
Authors: Lijnen H. Roger , Van Hoef Berthe , Beelen Veerle , Collen Désiré
Published: March 2005

The main components of the murine plasma fibrinolytic system, including fibrinogen, plasminogen, α2‐antiplasmin, tissue‐type plasminogen activator and plasminogen activator inhibitor‐1, were purified to homogeneity and their interactions were quantitated and compared with those of the human counterparts.

Initial activation rates of murine and human plasminogen by autologous tissue‐type plasminogen activator were comparable (catalytic efficiencies, k2/Km, of 0.4 and 0.6 mM−1 s−1, respectively), but murine plasminogen appeared to be resistant to activation by human tissue‐type plasminogen activator (k2/Km=0.01 mM−1 s−1). Plasminogen activation by tissue‐type plasminogen activator was stimulated 100‐ and 160‐fold in autologous murine and human systems, respectively, with saturating concentrations of 0.45 and 0.32 μM, respectively, of CNBr‐digested fibrinogen. Nearly quantitative binding (85–90%) of tissue‐type plasminogen activator to fibrin was observed both in autologous and heterologous systems.

Murine and human plasmin were very rapidly inhibited by autologous and heterologous α2‐antiplasmin (second‐order inhibition rate constants, k1,app, of 2.1–2.3×107 M−1 s−1) and murine and human tissue‐type plasminogen activator were very rapidly inhibited by autologous or heterologous plasminogen activator inhibitor‐1 (k1,app of 1.8–4.9×107M−1 s−1). Two‐chain murine tissue‐type plasminogen activator (added at a concentration of 1 μg/ml) was inhibited in normal or plasminogen activator inhibitor‐1‐deficient murine plasma with half‐lives of 6.5 min and 4.2 min, respectively, as compared to 80 min for human tissue‐type plasminogen activator, suggesting that murine plasma contains proteinase inhibitors other than plasminogen activator inhibitor‐1 which efficiently inhibit autologous tissue‐type plasminogen activator.

Clot lysis experiments in autologous plasma revealed that the murine plasma fibrinolytic system is more resistant to activation than the human system (20–30% clot lysis in 2 h with 100 nM tissue‐type plasminogen activator in the murine system, as compared to 50% clot lysis in 2 h with 3.5 nM tissue‐type plasminogen activator in the human system). Several mechanisms appear to be involved in this relative resistance observed in the murine system, including resistance of murine plasminogen to quantitative activation and short plasma half‐life of murine tissue‐type plasminogen activator.

Thus, although these quantitative interactions between purified components of the murine fibrinolytic system appear to be comparable to those between the human counterparts, murine plasma clots are >30‐fold more resistant to lysis with autologous tissue‐type plasminogen activator than human plasma clots.
Phospholipids are Necessary for Calmodulin‐stimulated Activation of the Ca2+‐ATPase of Erythrocytes
Authors: Gazzotti Paolo , Gloor‐Amrein Monika , Adebayo Ronke
Published: March 2005

Treatment of red cell ghosts with increasing concentrations of the non‐ionic detergent Triton X‐100 caused a progressive loss of Ca2+‐ATPase activity. Both the basal activity and the calmodulin‐stimulated activity were affected and could be partially restored by acidic phospholipids. Lipid‐free Ca2+‐ATPase was prepared from solubilized ghosts by calmodulin affinity chromatography and extensive washing of the column with detergent to remove the endogenous phospholipids associated with the enzyme. The phospholipid‐free, solubilized Ca2+‐ATPase had very low activity and was not activated by calmodulin. The tryptic proteolytic pattern of the delipidated ATPase differed from the pattern of the phospholipid‐associated enzyme, indicating that the delipidation had caused conformational changes. The activity was fully restored by phosphatidylserine, but was only partially restored by phosphatidylcholine. The phosphatidylcholine‐activated enzyme was restored to maximal activity in the presence of calmodulin. The delipidated ATPase could be reconstituted in soybean lipid vesicles and was able to actively transport Ca2+.
Evidence for a Loop‐Like Insertion Mechanism of Pro‐Omp A into the Inner Membrane of Escherichia Coli
Authors: Kuhn Andreas , Kiefer Dorothee , Köhne Claudia , Zhu Heng‐Yi , Tschantz William R. , Dalbey Ross E.
Published: March 2005

We have studied the insertion of pro‐OmpA into the Escherichia coli membrane in vivo using various mutants that have either alterations in the amino‐terminal parts of the signal peptide or in the mature region that flanks the signal peptide. A pro‐OmpA mutant with an amino terminal extension of 142 residues derived from ribulokinase (AraB) was analysed for its membrane insertion. The AraB portion, which includes a cluster of seven charged residues close to the signal sequence, did not interfere with the Sec components and allowed efficient export of OmpA. During translocation the AraB portion remained in the cytoplasm. Further mutants of OmpA were constructed in the carboxy‐terminal region flanking the signal sequence. Pro‐OmpA does not translocate across the membrane when a charge cluster, comprised of Lys‐Arg‐Arg‐Glu‐Arg, is introduced after positions 5, 11 or 15 of the mature region, but is translocated when the cluster is introduced after position 22. This defines a region of about 20 residues in the mature part of pro‐OmpA that is crucial for membrane insertion. These results suggest that in the case of the Sec‐dependent pro‐OmpA, as with the Sec‐independent M13 procoat, the precursor assumes a loop‐like structure involving the signal peptide and the early part of the mature region, leaving the amino terminus of the signal peptide at the cytoplasmic face.
Differential Regulation of Chromogranin B and Synapsin I Gene Promoter Activity by cAMP and cAMP‐Dependent Protein Kinase
Authors: Jüngling Susanne , Cibelli Giuseppe , Czardybon Margarete , Gerdes Hans‐Hermann , Thiel Gerald
Published: March 2005

cAMP has neurotrophic effects in the nervous system. We have investigated whether there is a correlation between cAMP‐induced neurite outgrowth and induction of chromogranin B and synapsin I gene expression. These genes encode marker proteins of distinct populations of vesicles in neurons, neuroendocrine and endocrine cells, and in addition, they contain a cAMP response element (CRE) in their upstream regions, making it likely that cAMP‐induced neuronal differentiation might be accompanied by increased transcription of these genes. We increased intracellular cAMP levels in neuronal and neuroendocrine cells and analyzed the levels of chromogranin B and synapsin I mRNA. Our data revealed that, while chromogranin B mRNA was in fact induced following cAMP stimulation, synapsin I mRNA was not affected. To analyze the cis‐acting sequences, we constructed hybrid genes containing the upstream region of the mouse chromogranin B gene fused to a reporter gene. Similar plasmids containing the synapsin I or the glucagon promoter were constructed. Transfections of neuronal and endocrine cells, together with deletion mutagenesis, revealed that the CRE of the chromogranin B gene mediated the effect of cAMP upon transcription. This effect was mimicked by overexpression of the catalytic subunit of the cAMP‐dependent protein kinase. In addition, overexpression of the negative‐acting CRE‐binding protein CREB‐2 revealed that the chromogranin B CRE functions as a bifunctional genetic regulatory element in that it mediates basal as well as cAMP‐stimulated transcription. Synapsin I gene expression, however, was not induced by either elevated intracellular cAMP concentration or by overexpression of protein kinase A, although a similar pattern of proteins, including CREB, bound to the synapsin I and chromogranin B CRE in vitro. Thus while the CRE element in the chromogranin B gene promoter is responsive to cAMP, the same element, when present in the synapsin I promoter, does not confer cAMP inducibility.
The Relationship Between Electron Flux and the Redox Poise of the Quinone Pool in Plant Mitochondria
Authors: Van Den Bergen Corrine W. M. , Wagner Anneke M. , Krab Klaas , Moore Anthony L.
Published: March 2005

The dependence of electron flux through quinone‐reducing and quinol‐oxidizing pathways on the redox state of the ubiquinone (Q) pool was investigated in plant mitochondria isolated from potato (Solanum tuberosum cv. Bintje, fresh tissue and callus), sweet potato (Ipomoea batatas) and Arum italicum. We have determined the redox state of the Q pool with two different methods, the Q‐electrode and Q‐extraction techniques. Although results from the two techniques agree well, in all tissues tested (with the exception of fresh potato) an inactive pool of QH2 was detected by the extraction technique that was not observed with the electrode. In potato callus mitochondria, an inactive Q pool was also found. An advantage of the extraction method is that it permits determination of the Q redox state in the presence of substances that interfere with the Q‐electrode, such as benzohydroxamate and NADH. We have studied the relation between rate and Q redox state for both quinol‐oxidizing and quinone‐reducing pathways under a variety of metabolic conditions including state 3, state 4, in the presence of myxothiazol, and benzohydroxamate. Under state 4 conditions or in the presence of myxothiazol, a non‐linear dependence of the rate of respiration on the Q‐redox state was observed in potato callus mitochondria and in sweet potato mitochondria. The addition of benzohydroxamate, under state 4 conditions, removed this non‐linearity confirming that it is due to activity of the cyanide‐resistant pathway. The relation between rate and Q redox state for the external NADH dehydrogenase in potato callus mitochondria was found to differ from that of succinate dehydrogenase. It is suggested that the oxidation of cytoplasmic NADH in vivo uses the cyanide‐resistant pathway more than the pathway involving the oxidation of succinate. A model is used to predict the kinetic behaviour of the respiratory network. It is shown that titrations with inhibitors of the alternative oxidase cannot be used to demonstrate a pure overflow function of the alternative oxidase.
Reversible Dissociation and Unfolding of Pyruvate Decarboxylase from Zymomonas mobilis
Authors: Pohl Martina , Grötzinger Joachim , Wollmer Axel , Kula Maria‐Regina
Published: March 2005

The denaturation and renaturation process of pyruvate decarboxylase (PDC) from Zymomonas mobilis (ATCC 29191) has been investigated using guanidine hydrochloride and urea as denaturing agents. The quarternary structure of the homotetramer is strongly stabilized by the cofactors Mg2+ and thiamine diphosphate (TDP). The structural transitions were monitored by activity measurements, fluorescence spectroscopy, circular dichroism and gel‐filtration chromatography. A three‐step denaturation process, described as follows, is indicated by non‐coincidental denaturation curves: (a) inactivation of the tetramer upon dissociation of cofactors (>0.4 M guanidine hydrochloride, >1 M urea); (b) dissociation of the tetramer into monomers (>1 M guanidine hydrochloride, >3M urea); (c) complete unfolding of these (>2.5 M guanidine hydrochloride, >5 M urea).

The refolding process initiated by rapid dilution of fully denatured protein in renaturation buffer involves the rapid reassociation of an inactive intermediate followed by the reconstitution of the active site.
Trifluoroethanol Stabilizes a Helix‐Turn‐Helix Motif in Equine Infectious‐Anemia‐Virus Trans ‐Activator Protein
Authors: Sticht Heinrich , Willbold Dieter , Ejchart Andrzej , Rosin‐Arbesfeld Rina , Yaniv Abraham , Gazit Arnona , Rösch Paul
Published: March 2005

The solution structure of the 75‐amino‐acid trans ‐activator (Tat) protein of the equine infectious‐anemia virus in trifluoroethanol‐containing solution was determined by two‐dimensional and three‐dimensional nuclear magnetic resonance spectroscopy, resulting in a total of 838 nuclear‐Over‐hauser‐enhancement distance restraints, and restrained molecular‐dynamics simulations. In contrast to the recently determined structure of this protein in trifluoroethanol‐free pH 6.3 solution, the hydrophobic core and the adjacent basic RNA‐binding region of the protein showed well‐defined α‐helical secondary structure in trifluoroethanol‐containing solution. The helical regions comprise those parts of the molecule whose helix‐forming tendencies were noted earlier in trifluoroethanol‐free solution. Two helices (Gln38–Arg43 and Asp48–Ala64) are connected by a tight type‐II turn centered at the strictly conserved Gly46 leading to a helix‐turn‐helix motif in the core and basic region of the protein. A third helix (Thr9–Asn13) is located in the less well defined N‐terminal part of the protein.

These observations may support the notion that the protein adopts a helical structure in the RNA‐binding region on complex formation. Although the secondary‐structure elements become better defined in trifluoroethanol‐containing solution, the opposite is true for the hydrophobically stabilized tertiary structure. This adds a caveat to studies of protein structures in trifluoroethanol‐containing solution in general.
Complete L‐Alanine Scan of Neuropeptide Y Reveals Ligands Binding to Y1 and Y2 Receptors with Distinguished Conformations
Authors: Beck‐Sickinger Annette G. , Weland Heike A. , Wittneben Helmut , Willim Klaus‐Dieter , Rudolf Klaus , Jung Günther
Published: March 2005

The synthesis of more than fifty 36‐residue oligopeptide analogs of neuropeptide Y (NPY) and their affinity to human Y1, and Y2, receptors is described. Each amino acid of the natural sequence was replaced by L‐alanine, the four alanine residues at position 12, 14, 18 and 23 were replaced by glycine. Additional residues were exchanged to closely related ones in order to characterize the prerequisites for binding. A combination of automated single and multiple peptide synthesis using fluoren‐9‐ylmethoxycarbonyl/tert ‐butoxy strategy was applied. The purified peptides were characterized by electrospray mass spectrometry, analytical HPLC and amino acid analysis. Binding was investigated by displacement of 125I‐labelled neuropeptide Y from human neuroblastoma cell lines SK‐N‐MC and SMS‐KAN.

Whereas Pro2 and the integrity of the neuropeptide Y loop is important for the binding to the Y1, receptor, exchanges within the C‐terminal helix affect the affinity to the Y2, receptor. The C‐terminal pentapeptide amide is important for both receptors and probably represents the binding site. However, Arg33 and Arg35 may not be exchanged by L‐alanine in the Y1, system, whereas Arg35 and Tyr36 are the most susceptible residues in the Y2, system. In order to distinguish between conformational effects and direct hormone/receptor interaction via the side chains of neuropeptide Y, circular dichroic studies of the alanine‐containing peptides were performed and structure affinity relationships are discussed.

Comparing the affinities of the neuropeptide Y analogs to Y1, and Y2, receptors significant differences were found for the two binding sites, which suggests a different active conformation of neuropeptide Y at the two subtypes of receptors. Using molecular dynamics calculations, two distinct conformations were identified which are in good agreement with the data obtained by structure/affinity investigations.
Structure of the Drosophila melanogaster Glutathione‐Dependent Formaldehyde Dehydrogenase/Octanol Dehydrogenase Gene (Class III Alcohol Dehydrogenase)
Authors: Luque Teresa , Atrian Sílvia , Danielsson Olle , Jörnvall Hans , Gonzàlez‐Duarte Roser
Published: March 2005

The glutathione‐dependent formaldehyde dehydrogenase gene (gfd) of Drosophila melanogaster encodes an enzyme that is active toward S ‐hydroxymethylglutathione, an adduct of formaldehyde with glutathione, and also with long‐chain primary alcohols, both properties typical of class III alcohol dehydrogenases. gfd hybridizes at the 86D division of the third chromosome, in agreement with the known location of the Drosophila octanol dehydrogenase gene (odh). gfd/odh was isolated from a λEMBL‐4 genomic library and consists of three exons (with coding segments of 21, 90 and 1029 bp) and two introns (69 bp and 70 bp, respectively). The introns are small in size like the Drosophila interrupting sequences and are located at the 5′ end of the coding region. Comparisons with the homologous genes of Saccharomyces, Candida and humans provide information on the evolution of the class III alcohol dehydrogenases. Moreover, results from analysis of exon/intron distributions in eleven dehydrogenases are compatible with the hypothesis of intron loss accounting for aspects of the present structure of these genes.
Characterisation of Neurotrophin Dimers and Monomers
Authors: Kolbeck Roland , Jungbluth Stefan , Barde Yves‐Alain
Published: March 2005

When recombinant brain‐derived neurotrophic factor (BDNF) and neurotrophin‐3 (NT‐3) are purified by reverse‐phase chromatography, these neurotrophins elute as two distinct peaks. This is also the case when naturally occurring BDNF is purified from brain tissue. As indicated by gel‐filtration experiments, the peaks with shorter retention times correspond to neurotrophin dimers, those with longer retention times to monomers. In contrast, a BDNF mutant with a single amino‐acid replacement (Arg‐1→Lys) in the basic processing site common to all neurotrophin precursors elutes as a single peak. This peak is shown by gel‐filtration chromatography to consist of dimers with a molecular mass almost twice that of wild‐type dimers. N‐terminal sequencing indicates an extension of 19 amino acids, including a glycosylated asparagine residue. The biological activity of the BDNF mutant ([R‐1K]BDNF) is identical with that of wild‐type BDNF when tested in a neuron survival assay. Using this assay, the biological activities of guanidine‐hydrochloride‐denatured neurotrophin monomers were found to be much lower than that of the dimers, and experiments with NT‐3 monomers and NIH3T3 cells expressing trkC suggest that such monomers exist in solution in a conformation that prevents efficient interactions with neurotrophin receptors.
Features of Structural Zinc in Mammalian Alcohol Dehydrogenase
Authors: Jeloková Jana , Karlsson Christina , Estonius Mats , Jörnvall Hans , Höög Jan‐Olov
Published: March 2005

All four cysteine ligands to the structural zinc atom of human class‐I and class‐III alcohol dehydrogenase have been exchanged by site‐directed mutagenesis in order to study the importance of the metal in the mammalian enzymes. The cysteine residues were replaced with Ala and Ser, residues that are not able to ligand zinc. All mutations resulted in inactive, unstable enzymes, in contrast to the non‐mutated human alcohol dehydrogenases that are easily isolated. Northern‐blot analysis revealed the presence of the expected mRNAs from expression plasmids constructed with the different mutated and non‐mutated alcohol dehydrogenases, and Western‐blot analysis gave faint signals for the mutated recombinant proteins from crude extracts. This verifies that the plasmid constructs are correct, but that the translated, mutated proteins lacking the zinc‐stabilized local fold, are subject to rapid degradation. Hence, the results directly illustrate the importance of the structural zinc atom in mammalian alcohol dehydrogenase and confirm it as a component with ‘structural’ properties. The results are compatible with those from sensitivities to proteases and from the structures of other proteins within the super‐family, indicating that the structural role of the zinc atom may involve conservation of interfaces regulating the enzyme quaternary structure.
Dissociation of Pigeon‐Liver Malic Enzyme in Reverse Micelles
Authors: Chang Gu‐Gang , Huang Ter‐Mei , Huang Shih‐Ming , Chou Wei‐Yuan
Published: March 2005

Pigeon‐liver malic enzyme has a tendency to aggregate at a large concentration of protein. The larger aggregates (hexamer and octamer) were demonstrated to be enzymically active with specific activity similar to that of the tetramer. When the enzyme was embedded in a reverse micellar system prepared by dissolving the surfactant sodium bis(2‐ethylhexyl)‐sulfosuccinate (AOT) in isooctane, the tetrameric enzyme dissociated into monomers. The dissociated monomers were also enzymically active but with diminished specific activity relative to the activity in aqueous media. The decreased enzyme activity in reverse micelles was due to interactions of surfactant with the enzyme molecules, suggesting that the cytosolic malic enzyme is located near the plasma membrane. When the dissociation was monitored by altering the degree of hydration of the system (represented by the ratio [H2O]/[AOT]), the detergent and organic solvent slightly affected KTD, the dissociation constant of tetramer to dimers (T⇆2 D), but increased KDM, the dissociation constant of dimer to monomers (D⇆2 M), by 1–2 orders of magnitude; this change caused a 2–3 orders of magnitude increase in the overall dissociation constant KTM (T⇆4 M). The dissociation of the tetrameric malic enzyme to monomers was favored by approximately 16 kJ/mol in AOT/isooctane reverse micelles versus aqueous media. We propose water‐shell and induced‐fit models for the enzyme in AOT/isooctane reverse micelles at large and small [H2O]/[AOT] ratios to explain this data, respectively. The asymmetric quaternary structure of the enzyme [Lee, H. J. & Chang, G. G. (1990) FEBS Lett. 277, 175–179] was re‐evaluated in terms of the subunit interactions and various interconvertible enzyme forms.
Cellular Site of Synthesis and Dynamics of Cell Surface Re‐Expression of Polysialic Acid of the Neural Cell Adhesion Molecule
Authors: Scheidegger Paul , Papay Judit , Zuber Christian , Lackie Peter M. , Roth Jürgen
Published: March 2005

Homopolymers of α‐2,8‐ketosidically linked sialic acid (polysialic acid) represent a posttranslational modification which, in mammals, appears to be unique for the neural cell adhesion molecule and the a subunit of sodium channels in brain. Under steady‐state conditions, polysialic acid is detectable in the plasma membrane of different cell types but not in the cytoplasm. We have studied the site of synthesis and the cell surface re‐expression of polysialic acid in a clonal subline of small cell lung carcinoma using the monoclonal antibody 735 and bacteriophage endosialidase, both specific reagents for polysialic acid. After enzymic removal, cell surface polysialic acid re‐expression reached control levels only after 5 days. When Golgi to plasma membrane transport of endosialidase‐treated cells was blocked by culture at 20°C or in the presence of monensin at 37°C, de‐novo ‐synthesized polysialic acid became detectable in the Golgi apparatus. Our data show that synthesis of polysialic acid of the neural cell adhesion molecule with a degree of polymerization of at least nine occurs intracellular in the Golgi apparatus of a human small cell lung carcinoma cell line.
Purification and Characterization of a Cytosolic Thyroid‐Hormone‐Binding Protein (CTBP) inXenopusLiver
Authors: Yamauchi Kiyoshi , Tata Jamshed R.
Published: March 2005

A variety of cytosolic thyroid‐hormone‐binding proteins with different characteristics have previously been reported. Here, we first describe the thyroid‐hormone‐binding characteristics of adultXenopusliver cytosol, then a novel procedure for purifying cytosolic thyroid‐hormone‐binding protein (CTBP) fromXenopusliver (xCTBP). The procedure consists of combining preparative isoelectrofocusing, FPLC cation‐exchange chromatography, HPLC hydrophobic‐interaction chromatography and ultraviolet light cross‐linking of125I‐labeled 3,3′5–triiodo‐L‐thyronine (T3). The isolated xCTBP thus prepared retained all the characteristics of the major thyroid‐hormone‐(TH)‐binding component of the unfractionated cytosol. It is a monomeric protein of approximately 59 kDa with an isoelectric point of 7.0±0.1, binds T3with a higher affinity than its analogs with aKdof approximately 9 nM, and is sensitive to sulfhydryl agents but not to NADPH. In several respects, xCTBP differs from most CTBP‐like preparations from other sources described hitherto. Microse‐quencing of a 23‐amino‐acid peptide generated from xCTBP by cyanogen bromide digestion revealed 92–100% identity of a 23‐amino‐acid sequence of several mammalian (amino acids 236–258) and avian (amino acids 245–267) cytosolic aldehyde dehydrogenases (ALDH); xCTBP also exhibited significant similarity of amino acid composition with rat ALDH. This novel finding of sequence identity between a CTBP and ALDH, and the diversity of CTBPs from different sources, suggest that a variety of cytosolic proteins, depending on the species and tissue, can function as thyroid‐hormone‐binding proteins.