bluuebunny/crossref_metadata_2025 huggingface dataset:
Thermodynamic stability and solution conformation of tandem G · A mismatches in RNA and RNA · DNA hybrid duplexes
Authors: EBEL Susanne , BROWN Tom , LANE Andrew N.
Published: March 2005

G · A mismatches form a variety of hydrogen‐bonded structures in DNA, most of which destabilise the duplex. Tandem G · A mismatches in the context YGAR (Y = pyrimidine, R = purine), however, form base pairs using the amino group of the guanine residue [Li, Y., Zon, G. & Wilson, W. D. (1991) Proc. Natl Acad. Sci. USA 88, 26–30], which permits extensive base‐base stacking, leading to a slight stabilisation of the helix [Ebel, S., Lane, A. N. & Brown, T. (1992) Biochemistry 31, 12083–12086]. We have measured the thermodynamic stability of several RNA and RNA · DNA hybrid duplexes containing tandem G · A mismatches. The RNA duplexes are intrinsically much more stable than the corresponding DNA duplexes and the mutations are destabilising in all cases.

NOE and coupling‐constant data show that all of the sugars are in the C3′‐endo range of conformations, and glycosidic torsion angles are in the range ‐160° to ‐180° in r(CCACGAGUGG) · r(CCACGAGUGG). Both sequential NOE intensities and circular‐dichroism measurements indicate that the global conformation of the mismatched RNA is A‐like. The N1H group of the mismatched guanine residue is not involved in hydrogen bonding with the adenine residue, indicating the presence of the amino‐pairing scheme. Determination of the structure using ‘loose’ NMR‐derived constraints shows that the potential energies of the imino‐paired and amino‐paired forms are similar, but substantially higher than energy‐minimised RNA. Using tighter constraints derived from more extensive analysis of one‐dimensional and two‐dimensional NOE data showed that the amino‐paired structure agrees with the constraint data better than the imino‐paired structure, and also accounts for unusual chemical shifts and the lack of hydrogen bonding of the guanine N1H group. Resulting molecular models show that the amino‐paired mismatches are not as extensively stacked on the neighbouring part of the duplex as in the B‐DNA analogues, largely accounting for the lower thermodynamic stability in the RNA duplexes.
Escherichia colielongation‐factor‐Tu mutants with decreased affinity for aminoacyl‐tRNA
Authors: ANDERSEN Carsten , WIBORG Ove
Published: March 2005

The two evolutionary well‐conserved histidine residues, His66 and His118, ofEscherichia colielongation factor Tu have been subjected to mutational analysis. The two histidines have each been replaced by alanines, denoted H66A and H118A, respectively. His 118 has also been substituted by glutamate, H118E. The three mutants have been characterized with respect to thermostability, GTPase activity and affinity for aminoacylated tRNA. Most conspicuously, the tRNA affinity is reduced or almost abolished.k−1for dissociation of the ternary complex increases by factors of 14, 40 and 48 for H66A, H118A and H118E, respectively, when compared to the wild type. The half‐lives for the non‐enzymic deacylation of aminoacylated tRNA in the ternary complex are 391, 107, 69, 54 and 61 min for wild type, H66A, H118A, H118E and free aminoacylated tRNA, respectively. TheKdis about 20‐times higher for H66A compared to wild type.

Our results strongly suggest that His66 and His118 play major roles in stabilization of the ternary complex.
Translocation and processing of various human parathyroid hormone peptides in Escherichia coli are differentially affected by protein‐A‐signal‐sequence mutations
Authors: KAREEM B. Najma , ROKKONES Erik , HØGSET Anders , HOLMGREN Erik , GAUTVIK Kaare M.
Published: March 2005

Two staphylococcal protein‐A signal sequences were constructed and tested for function in Escherichia coli, after being linked to human parathyroid hormone (hPTH) cDNAS representing the intact form (1–84 amino acids) and two N‐terminal (1–37 and 1–7 amino acids) peptides. One signal sequence was identical to the wild type, and the other signal contained a deletion of 12 bp at the 3′ end. The truncated hPTH cDNAs were fused at their 3′ ends to IgG‐binding domains (ZZ) derived from protein A in order to facilitate purification and characterization. The expression plasmid pSPTH, containing the wild‐type signal sequence, secreted efficiently the intact recombinant hPTH (1–84) into the medium. Plasmids containing the truncated hPTH genes after the wild‐type signal, gave rise to hPTH‐ZZ hybrid proteins which were correctly processed at the N‐terminal, but the major fractions appeared in the periplasmic compartment. In contrast, the plasmid pS'PTH which harboured the 4‐amino‐acid signal deletion did not promote a uniform secretion of intact hPTH (1–84) to the medium, but released a non‐processed form both into the periplasmic compartment and to the medium. The related plasmids pS'PTH37ZZ and pS'PTH7ZZ with the mutated signal sequence gave rise to small or trace amounts of unprocessed forms of fusion proteins in the medium and periplasm, thus the secretion competence was markedly reduced. Thus, for correct N‐terminal processing, we conclude that the amino acid sequence in the signal adjacent to the expressed protein, is a key determinant. However, release into the medium or periplasmic space appeared to be dependent also on protein folding, irrespective of signal‐sequence cleavage. Furthermore, we observed that the peptides with the wild‐type signal sequence and correct N‐terminal processing, were the only forms that showed internal cleavage of hPTH. Uncleaved signals may contribute to folding characteristics of the ensuing protein and e.g., prevent internal proteolysis.
Comparative studies on the primary structures and inhibitory properties of subtilisin‐trypsin inhibitors from Streptomyces
Authors: TAGUCHI Seiichi , KOJIMA Shuichi , TERABE Mahito , MIURA Kin‐ichiro , MOMOSE Haruo
Published: March 2005

Three novel proteinaceous inhibitors of serine proteases which had been identified as Streptomyces subtilisin inhibitor‐like (SIL) inhibitors were isolated from culture supernatant of Streptomyces; SIL2 from Streptomyces parvulus, SIL3 from Streptomyces coelicolor and SIL4 from Streptomyces lavendulae. They exhibited not only strong inhibitory activity toward subtilisin BPN' but also less strong inhibition of trypsin. Their primary sequences were determined by sequence analysis of peptides obtained by specific cleavage at the reactive site and subsequent proteolytic digestion. Each inhibitor consisted of about 110 amino acids, and was considered to form a dimer. The reactive site of the inhibitors was identified as Arg‐Glu for SIL2 and SIL3, and Lys‐Leu for SIL4, from sequence analysis of modified forms of the inhibitors produced from the inhibitor‐subtilisin complex under acidic conditions. The presence of an arginine/lysine residue at the P1 site was in agreement with their trypsin‐inhibition property. Sequence comparison with other members of the Streptomyces subtilisin inhibitor family revealed that amino acid replacements in the three isolated SIL inhibitors were frequently localized on the surface region, and many of the amino acid residues in β‐sheets and the hydrophobic core were highly conserved. Values of the inhibitor constant (Ki) toward subtilisin BPN' and trypsin were also measured, and the differences were discussed on the basis of the determined structures of the inhibitors.
Characterization of a membrane glycoprotein having pharmacological and biochemical properties of an AT2 angiotensin II receptor from human myometrium
Authors: LAZARD Daniel , VILLAGEOIS Phi , BRIEND‐SUTREN Malène M. , CAVAILLÉ Françoise , BOTTARI Serge , STROSBERG A. Donny , NAHMIAS Clara
Published: March 2005

The angiotensin II receptors of human myometrial tissue were characterized using ligand binding, cross‐linking with radioactive label, detergent solubilization and partial purification by lectinaffinity chromatography. Human myometrial membrane preparations contained variable amounts (5–650 fmol/mg protein) of high affinity (Kd= 44–65 pM) binding sites for 125I‐CGP42112, a ligand specific for the AT2 subtype of angiotensin II receptors. Competition studies with AT1‐specific and AT2‐specific compounds indicated that angiotensin II receptors on these membranes were exclusively of the AT2 subtype. The binding sites for 125I‐CGP42112 were efficiently solubilized by the detergent Chaps, albeit with a marked decrease in affinity (Kd= 1.2 nM). The proteins in the myometrial membrane preparation were cross‐linked to 125I‐[Sar1, Ile8]angiotensin II (Sarile) with disuccinimidyl suberate. When low concentrations of cross‐linker were used, a single radiolabelled band of about 66–70 kDa was revealed on SDS/PAGE. At higher concentrations additional bands of about 105–120 kDa and 200 kDa were labelled. The 66–70‐kDa and 105–120‐kDa bands were separated by electroelution of SDS/PAGE gel slices and submitted to trypsin cleavage. The tryptic‐peptide maps were identical for both products, suggesting that the additional bands are homodimers and trimers of the labelled polypeptide. The Chaps‐solubilized receptor was retained on wheat‐germ‐agglutinin‐Sepharose and specifically eluted by the competing sugar triacetylchitotriose, leading to a fivefold purification factor. Treatment of the 125I‐Sarile‐labelled protein with N‐glycanase caused a shift in its apparent molecular mass on SDS/PAGE from 66–70 kDa to 33 kDa.
Purification of 1,3‐β‐d‐glucan synthase activity from pea tissue
Authors: DHUGGA Kanwarpal Singh , RAY Peter M.
Published: March 2005

From pea plasma membranes isolated by aqueous polymer two‐phase partitioning we have purified 1,3‐β‐d‐glucan synthase [glucan synthase‐II (GS‐II) or callose synthase], an enzyme that several reports have suggested consists of between six and nine different subunits. The procedure involves (a) preliminary removal of peripheral proteins by 0.1% digitonin; (b) solubilization of GS‐II with 0.5% digitonin; (c) precipitation of activity‐irrelevant proteins from the digitonin extract by Ca2+, spermine and cellobiose, which are GS‐II effectors needed in step (d); (d) product entrapment by formation of 1,3‐β‐D‐glucan from UDP‐Glc by GS‐II in the presence of the mentioned effectors, followed by centrifugal sedimentation of product micelles and elution of proteins therefrom with buffer; (e) preparative isoelectric focusing (IEF) of product‐entrapped proteins; and (f) glycerol gradient centrifugation of the fractions of peak GS‐II activity from IEF. The procedure yields 300‐fold enrichment of GS‐II specific activity over that in isolated plasma membranes, and 5500‐fold over that in the original homogenate. Out of approximately six principal polypeptides that occur after the product entrapment step, the glycerol gradient GS‐II activity peak contains only two major polypeptides, one of 55 kDa and another of 70 kDa, plus minor amounts of one or two others whose distribution and occurrence indicate are not responsible for GS‐II activity. Antisera against either the 55‐kDa or the 70‐kDa polypeptide adsorb more than 60% of the GS‐II activity from a productentrapped preparation. After native gel electrophoresis, GS‐II activity is associated with a single protein band of very large molecular mass, whose principal components are the 55‐kDa and 70‐kDa polypeptides, accompanied by minor amounts of a few other polypeptides most of which do not occur in enzyme prepartions purified by the previously described procedure. The 55‐kDa but not the 70‐kDa component can be labeled by ultraviolet irradiation of the plasma membranes in the presence of [α‐32P]UDP‐Glc under GS‐II assay conditions. It seems likely, therefore, that the 55‐kDa and 70‐kDa polypeptides form a large catalytic complex of which the 55‐kDa component is the UDP‐Glc‐binding subunit.
Protein stabilization by hydrophobic interactions at the surface
Authors: VAN DAN BURG Bertus , DIJKSTRA Bauke W. , VRIEND Gert , VAN DAR VINNE Bernard , VENEMA Gerard , EIJSINK Vincent G. H.
Published: March 2005

The contribution of the solvent‐exposed residue 63 to thermal stability of the thermolysin‐like neutral protease of Bacillus stearothermophilus was studied by analyzing the effect of twelve different amino acid substitutions at this position. The thermal stability of the enzyme was increased considerably by introducing Arg, Lys or bulky hydrophobic amino acids. In general, the effects of the mutations showed that hydrophobic contacts in this surface‐located region of the protein are a major determinant of thermal stability. This observation contrasts with general concepts concerning the contribution of surface‐located residues and surface hydrophobicity to protein stability and indicates new ways for protein stabilization by site‐directed mutagenesis.
All potential glycosylation sites of the nicotinic acetylcholine receptor δ subunit from Torpedo californica are utilized
Authors: STRECKER Andreas , FRANKE Peter , WEISE Christoph , HUCHO Ferdinand
Published: March 2005

All possible N‐glycosylation sites of the δ subunit of the nicotinic acetylcholine receptor from Torpedo californica electric tissue are utilized. By a combination of microsequencing and mass spectrometry, it was shown that a high‐mannose‐type oligosaccharide is bound at Asn143 of the δ subunit. The oligosaccharides at positions Asn70 and Asn208 of the δ subunit are probably of the complex type. The utilized glycosylation sites pose restrictions on possible transmembrane folding models of the subunit.
Assignment of the 13C and 13CO resonances for Rhodobacter capsulatus ferrocytochrome c2 using double‐resonance and triple‐resonance NMR spectroscopy
Authors: CAFFREY Michael , BRUTSCHER Bernhard , SIMORRE Jean‐Pierre , FITCH John , CUSANOVICH Michael , MARION Dominique
Published: March 2005

Rhodobacter capsulatus cytochrome c2 uniformly labelled with 13C/15N has been prepared. The 13C resonances of the reduced state, including those of the carbonyl and heme 13C, have been assigned using a combination of various two‐ and three‐dimensional correlated NMR experiments. Assignment of the sidechain 13C resonances facilitated correction of a small number of previously misassigned sidechain 1H and led to the additional assignment of 32 1H. It was found that 13Cα and 13CO secondary shifts were better indicators of secondary structure than 1Hα and 13Cβ secondary shifts. Moreover, it was demonstrated that, despite the significant ring current effects present in heme proteins, 13Cα and 13CO secondary shifts can be employed to accurately identify secondary structure in heme proteins, independently of NOE experiments.
Conformational changes in proteins induced by dynamic associations
Authors: GABELLIERI Edi , STRAMBINI Giovanni B.
Published: March 2005

Random collisions between macromolecules lead to dynamic associations (lengthy encounters) that in principle could affect their conformation and, in the case of enzymes, their binding and catalytic properties. Exploiting the unique sensitivity of the phosphorescence lifetime, τ, of Trp to the internal flexibility of globular proteins we probed the perturbations induced in the structure of the coenzyme‐binding domain of alcohol dehydrogenase (LADH) and glyceraldehyde‐3‐phosphate dehydrogenase (GraPDH) by the presence in solution of other dehydrogenases and of functionally unrelated proteins. With Trp314 of LADH, the results emphasize that while τ is not affected by the concentration of LADH itself, the addition of micromolar quantities of other proteins causes a distinct reduction in it. From the linear increase of 1/τ with protein concentration one obtains values for the apparent second‐order Stern‐Volmer rate constant that range between 2–200 × 103 M−1 s−1, decreasing 2–3‐fold when ternary complexes of LADH with NADH or NAD+ and inhibitors are involved. Similar effects were observed with Trp310 of GraPDH except that with sorbitol dehydrogenase as perturbant the increase of 1/τ is hyperbolic and governed by an apparent dissociation constant of about 1 μM. Finally, glycerol‐3‐phosphate dehydrogenase, the strongest perturber of both LADH and GraPDH, has either no effect on lactic dehydrogenase from pig heart or induces a moderate lengthening of the triplet lifetime of the rabbit muscle enzyme.

Because Stern‐Volmer behaviour is typical also of diffusion‐mediated quenching reactions, a parallel investigation with cysteine, cystine and N‐acetyl‐tryptophanamide demonstrated that among potential, protein‐associated, quenching moieties namely, ‐SH, ‐S‐S‐ and indole groups, only the latter has rate constants approaching the magnitude of protein perturbants. Since considerable evidence rules out the predominance of such quenching reactions, these findings confirm a subtle form of communication between protein molecules in solution. The lack of specificity and the similar effects between dehydrogenases with right and wrong stereospecificity for direct coenzyme transfer suggests that the perturbations monitored are unrelated to this function.
Sequential 1H and 15N nuclear magnetic resonance assignments and secondary structure of the N‐terminal lipoyl domain of the dihydrolipoyl transacetylase component of the pyruvate dehydrogenase complex from Azotobacter vinelandii
Authors: BERG Axel , DE KOK Arie , VERVOORT Jacques
Published: March 2005

The N‐terminal lipoyl domain (79 residues) of the transacetylase component of the pyruvate dehydrogenase complex from Azotobacter vinelandii has been sub‐cloned and produced in Escherichia coli. Over‐expression exceeds the capacity of E. coli cells to lipoylate all expressed lipoyl domain, but addition of lipoic acid to the growth medium results in expression of fully lipoylated domain. A two‐dimensional homo‐ and heteronuclear NMR study of the lipoyl domain has resulted in sequential 1H and 15N resonance assignments of the unlipoylated form of the protein. Small differences in chemical shift values for protons of residues in the vicinity of the lipoyl‐lysine residue are observed for the lipoylated form of the domain, suggesting that the conformation of the lipoyl domain is not altered significantly by the coupled cofactor. From nuclear Overhauser effects, backbone coupling constants and slowly exchanging amide protons, two antiparallel β‐sheets, each containing four strands, were identified. The lipoyl‐lysine residue is exposed to the solvent and located in a type‐I turn between two strands. The N‐ and C‐terminal residues of the folded chain are close together in the other sheet. Preliminary data on the relative three‐dimensional orientation of the two β‐sheets are presented. Comparison with the solution structure of the lipoyl domain of the Bacillus stearothermophilus pyruvate dehydrogenase complex shows resemblance to a large extent, despite the sequence identity of 31%.
Crystal structures of rat acid phosphatase complexed with the transition‐state analogs vanadate and molybdate
Authors: LINDQVIST Ylva , SCHNEIDER Gunter , VIHKO Pirkko
Published: March 2005

The three‐dimensional structures of complexes of recombinant rat prostatic acid phosphatase with the transition‐state analogs vanadate and molybdate were determined to 0.3‐nm resolution using protein crystallographic methods. The overall structure of the enzyme remains unchanged upon binding of the metal oxyanions; only local conformational differences in the positions of some side chains at the active site were found. The metal oxyanions bind in an identical fashion at the active site with trigonal bipyramidal coordination geometry. The metal ion is within coordination distance of the His 12 side chain which is located at one of the axial positions. The three equatorial oxygen atoms interact with the conserved residues Arg11, Arg15, Arg79 and His257, Within hydrogen‐bonding distance of the axial oxygen atom is the side chain of the conserved residue Asp258. The implications of these results for the catalytic mechanism of acid phosphatase are discussed.
Recombinant expression and structural and binding properties of α1(VI) and α2(VI) chains of human collagen type VI
Authors: TILLET Emmanuelle , WIEDEMANN Hanna , GOLBIK Ralph , PAN Te‐Cheng , ZHANG Rui‐Zhu , MANN Karlheinz , CHU Mon‐Li , TIMPL Rupert
Published: March 2005

Full‐length α1(VI) and α2(VI) cDNAs in an eukaryotic expression vector were used to obtain stably transfected human kidney cell clones and to purify these collagen‐VI chains in substantial quantities from the culture medium. Both chains appeared mainly as monomers together with some dimers that were disulfide linked through their C‐terminal globular domains. Despite sufficient hydroxylation of proline and lysine residues, the chains did not form a triple‐helix, as shown by electronmicroscopy, CD spectra and pepsin sensitivity. Digestion of the chains with bacterial collagenase released the N‐terminal and C‐terminal globular domains, which were identified by their size and partial sequences. They showed a substantial content of α‐helical conformation and a distinct globular structure after rotary shadowing. Antibodies could be raised that distinguished between the two chains and reacted with the globular domains. The α2(VI) but not the α1(VI) chain showed binding to a heparan sulfate proteoglycan (perlecan), fibronectin and pepsin‐solubilized collagen VI. Purified globular domains did not bind these ligands indicating the localization of binding sites within the triple‐helical domain. Both chains showed a distinct affinity for heparin but failed to bind to various collagen types.
Role of cyclic GMP in atrial‐natriuretic‐peptide stimulation of erythrocyte Na+/H+ exchange
Authors: PETROV Victor , AMERY Antoon , LIJNEN Paul
Published: March 2005

Human atrial natriuretic peptide (ANP) fragments ANP‐(127–150) or ANP‐III and ANP‐(127–149) or ANP‐II activate Na+/H+ exchange in human erythrocytes at concentrations as low as 1 pM. Both ANP‐(127–147) or ANP‐I and ANP‐(129–150) or des‐Ser5, Ser6‐ANP‐III have no effect on erythrocyte Na+/H+ exchange. ANP‐III also produces a time‐dependent increase of intraerythrocyte guanosine 3′,5′‐phosphate (cGMP) concentration. M&B 22,948, a specific inhibitor of cGMP phosphodiesterase, increases Na+/H+ exchange and the intracellular concentration of cGMP. Both 8‐bromoguanosine 3′,5′‐phosphate (8‐Br‐cGMP) and dibutyryl‐cGMP mimic the effect of ANP‐III on erythrocyte Na+/H+ exchange. Our data suggest that human erythrocytes possess guanylate‐cyclase activity stimulated by ANP‐III and that activation of Na+/H+ exchange by this peptide is mediated by cGMP. Human erythrocytes display a high degree of sensitivity to ANP‐III or ANP‐II and a specificity for ANP‐fragment structures just as cells with established ANP‐specific receptors.
Characterization and transcriptional profiles of a Drosophila gene encoding an insect defensin
Authors: DIMARCQ Jean‐Luc , HOFFMANN Daniéle , MEISTER Marie , BULET Philippe , LANOT René , REICHHART Jean‐Marc , HOFFMANN Jules A.
Published: March 2005

Insect defensins are a family of 4‐kDa, cationic, inducible antibacterial peptides which bear six cysteine residues engaged in three intramolecular disulfide bridges. They owe their name to certain sequence similarities with defensins from mammalian neutrophiles and macrophages. We report the characterization of a novel defensin isoform from Drosophila and the cloning of the gene encoding a preprodefensin. The gene, which is intronless and present in a single copy/haploid genome, maps at position 46CD on the right arm of the second chromosome. The analysis of the upstream region of the gene reveals the presence of multiple putative cis‐regulatory sequences similar to mammalian regulatory motifs of acute‐phase‐response genes. Transcriptional profiles indicate that the Drosophila defensin gene is induced by bacterial challenge with acute‐phase kinetics. It is also expressed in the absence of immune challenge during metamorphosis. These and other data on the Drosophila defensin gene lead us to suggest that insect and mammalian defensins have evolved independently.
Cloning, expression and tissue distribution of rat platelet‐activating‐factor‐receptor cDNA
Authors: BITO Haruhiko , HONDA Zen‐ichiro , NAKAMURA Motonao , SHIMIZU Takao
Published: March 2005

The biological functions of platelet‐activating factor (PAF) have been extensively studied in the rat. However, the precise structure and distribution of rat PAF receptor has not been reported. To address this question, we isolated a rat PAF‐receptor cDNA from a size‐fractionated rat spleen cDNA library. The deduced amino acid sequence of the rat PAF receptor showed 80% and 79% identity with guinea pig and human PAF receptors, respectively. Pharmacological properties (ED50, inhibition by WEB2086) of rat PAF receptors expressed in Xenopus oocytes were similar to those for PAF receptors expressed from guinea pig or human cDNAs. Northern blot analysis showed a widespread distribution of PAF‐receptor mRNA in almost all organs including spleen, small intestine, kidney, lung, liver and brain. Considerable difference in the PAF‐receptor distribution detected among species suggests the existence of a species‐specific and tissue‐specific regulatory mechanism for PAF‐receptor‐mRNA expression. Isolation of rat PAF‐receptor cDNA should facilitate further analysis of PAF‐receptor function and pharmacology in diverse pathophysiological processes.
An ADP‐sensitive cyclosporin‐A‐binding protein in rat liver mitochondria
Authors: ANDREEVA Larisa , CROMPTON Martin
Published: March 2005

Mitochondria contain a structure which forms a large aqueous pore in the inner membrane after Ca2+ overload in the presence of Pi. In the present study, pore activation in liver mitochondria was monitored using the collapse of the inner membrane potential (ΔΨ). Ca2+‐induced pore opening (ΔΨ collapse) was prevented by the immunosuppressant cyclosporin A, but cyclosporin A did not reverse pore opening (i.e. allow ΔΨ regeneration) unless ADP was also added. At concentrations that produced substantial pore blockade, [3H]cyclosporin partitioned more or less equally between membrane and soluble fractions, but the distribution was shifted slightly to the membranes in the presence of ADP. ADP also increased the binding of [3H]cyclosporin A to membranes washed free of soluble components. The indication that cyclosporin A inhibition of the pore is mediated by an ADP‐sensitive membrane component was examined using a tritiated photoactivatable derivative of cyclosporin A. ADP selectively increased covalent binding of this derivative to a membrane component. This component eluted from molecular‐sizing columns as a 13–17‐kDa‐protein in the presence of 0.5% Chaps as detergent and migrated as a 10‐kDa (approximately) protein in SDS/PAGE. These findings provide the first evidence that a protein of approximately 10 kDa may be part of the cyclosporin‐A receptor of the Ca2+‐activated pore. The possible implications of these findings are discussed.
Rapid alterations in lateral mobility of lipids in the plasma membrane of activated human megakaryocytes
Authors: SCHOOTEMEIJER Annelies , VAN BEEKHUIZEN André E. , GORTER Gertie , TERTOOLEN Leon G. J. , DE LAAT Siegfried W. , AKKERMAN Jan‐Willem N.
Published: March 2005

In the present study we measured membrane fluidity as the lateral mobility of the lipid probe 1,1′‐ditetradecyl‐3,3,3′,3′‐tetramethylindocarbocyanine perchlorate by fluorescence recovery after photobleaching in the plasma membrane of a single megakaryocyte, the progenitor cell of platelets. Megakaryocytes after 13 days in culture (maturation stage III) had a lateral diffusion coefficient (D) of (4.56±0.10)×10−9 cm2/s and a mobile fraction of 65±2% (means ± SEM, n= 140). Megakaryocytes isolated from rib had a similar D and mobile fraction. Stimulation with α‐thrombin (1–10 U/ml) induced a dose‐dependent decrease in D to (3.40±0.22)×10−9 cm2/s between 1–5 min after stimulation (P < 0.001). The mobile fraction did not change. A similar decrease in D was found following stimulation with ADP (20 μM) and ionomycin (100 nM). Modulation of calpain I activity with calpain I inhibitor or tetracain had no effect. Pretreatment with cytochalasin B or colchicine decreased D to (3.64±0.29)×10−9 cm2/s (P < 0.003) and (3.96±0.18)×10−9 cm2/s (P < 0.013) respectively. After stimulation D decreased further in cytochalasin‐treated cells (3.37±0.16)×10−9 cm2/s (P < 0.020) but remained at the same level in colchicine‐treated cells. Both treatments increased the mobile fraction to 73–75% in stimulated megakaryocytes (P < 0.03). These data indicate that the diffusion velocity of lipids in megakaryocytes is low and decreases further after stimulation. These changes are independent of calpain I. Treatments that decrease the cytoskeletal mass and thereby increase the mobility of proteins in the plasma membrane increase the number of lipids that participate in this process.
Correlation between the inhibition of cell growth by bis(ethyl)polyamine analogues and the decrease in the function of mitochondria
Authors: HE Yong , SUZUKI Toshikazu , KASHIWAGI Keiko , KUSAMA‐EGUCHI Kuniko , SHIRAHATA Akira , IGARASHI Kazuei
Published: March 2005

The antiproliferating effect of nine kinds of bis(ethyl)polyamine analogues [three kinds each of bis(ethyl)triamine, bis(ethyl)tetraamine and bis(ethyl)pentaamine] was compared using FM3A cells. The inhibitory effect was in the order BE4444 > BE3443 > BE4334≥BE444 > BE343 > BE333 > BE44 > BE34 > BE33. Our results indicate that not only polyamine deficiency but also the accumulation of polyamine analogues is involved in the inhibition of cell growth. Accumulation of bis(ethyl)polyamine analogues caused the inhibition of protein synthesis and the decrease in the ATP content. The protein synthetic system in mitochondria was more strongly inhibited by bis(ethyl)polyamine analogues than that in the cytoplasm. Under conditions such that cytoplasmic protein synthesis was inhibited by 50% by bis(ethyl)polyamine analogues, mitochondrial protein synthesis was almost completely inhibited. Mitochondrial Ile‐tRNA formation was inhibited by bis(ethyl)polyamine analogues at the concentrations that cytoplasmic Ile‐tRNA formation was stimulated. This may be one of the reasons for the selective inhibition of mitochondrial protein synthesis. This inhibition was followed by the decrease in ATP content, swelling of mitochondria and depletion of mitochondrial DNA. These results suggest that the early event of metabolic change caused by bis(ethyl)polyamine analogues in cells is the inhibition of protein synthesis, especially of mitochondrial protein synthesis.
Single‐stranded‐DNA‐binding proteins from human mitochondria and Escherichia coli have analogous physicochemical properties
Authors: CURTH Ute , URBANKE Claus , GREIPEL Joachim , GERBERDING Holger , TIRANTI Valeria , ZEVIANI Massimo
Published: March 2005

The gene for the mature human mitochondrial single‐stranded‐DNA binding protein (HsmtSSB) has been transferred into a protein‐overproducing vector and expressed in Escherichia coli. The protein was purified to homogeneity and its physicochemical properties were investigated. From sequence comparison, HsmtSSB shows some similarities to the N‐terminal part of the single‐stranded DNA‐binding protein (SSB) from E. coli (EcoSSB). Hydrodynamic measurements show the protein to be tetrameric and give a sedimentation coefficient of 4.1 S corresponding to a C‐terminally shortened EcoSSB. Electron‐microscopic images of the free protein show a globular tetrahedral structure. Binding of poly(desoxythymidylic acid) [poly(dT)] leads to a reduction of the tryptophan fluorescence of the protein up to 96%. Fluorescence titrations with poly(dT) show apparent binding‐site sizes of 50–70 nucleotides/tetramer between 0.05 M and 2 M NaCl. Binding to poly(dT) proceeds in a nearly diffusion‐controlled reaction with an association‐rate constant kass of 4 · 108 M−1S−1. The rate‐limiting step is the formation of a transient complex where less than four binding sites on the protein are involved and the reshuffling of the protein of the protein on the linear matrix is fast. Electron microscopy of the complex with poly(dT) using negative staining shows a nearly random distribution of the protein between the individual poly(dT) strands. This leads to the conclusion that the binding cooperativity is low (ω < 150). The two tryptophans of HsmtSSB were replaced by threonine and tyrosine. The environment of both residues is influenced by nucleic acid binding with mutations of Trp68 strongly reducing the DNA‐binding affinity of the protein.
The solution structures of the first and second transmembrane‐spanning segments of band 3
Authors: GARGARO Angelo R. , BLOOMBERG Graham B. , DEMPSEY Christopher E. , MURRAY Martin , TANNER Michael J. A.
Published: March 2005

We have studied the structures of synthetic peptides which correspond to the proposed first and second membrane‐spanning segments of the human red cell anion transporter (band 3). The peptides, which were acetylated at their N‐termini and amidated at the C‐termini, comprise the 20 amino acids of residues 405–424 and 21 amino acids of residues 436–456 of the human band 3 sequence. The solution structures of the peptides in trifluoroethanol were studied by two‐dimensional NMR spectroscopy. Characteristic NOEs were observed indicating that the peptides adopted a predominantly α‐helical structure in trifluoroethanol solution. Dynamical simulated annealing using the program XPLOR was employed for the structure calculations. The amide exchange rates in trifluoroethanol have also been measured and are consistent with an α‐helical structure for the peptides.
Influence on proline‐specific enzymes of a substrate containing the thioxoaminoacyl–prolyl peptide bond
Authors: SCHUTKOWSKI Mike , NEUBERT Klaus , FISCHER Gunter
Published: March 2005

Dipeptidyl peptidase IV from porcine kidney and aminopeptidase P fromEscherichia colican utilize thioxoalanyl‐proline 4‐nitroanilide but with decreased kinetic constants compared to the normal substrates. Product analysis showed that exclusively thioxoalanyl‐proline was liberated in the case of dipeptidyl peptidase IV catalysis and thioxo‐alanine in the case of aminopeptidase‐P‐mediated thioxo peptide bond hydrolysis. For the proline‐specific aminopeptidase P thekcat/Kmvalue for the thioxo peptide is 1100‐fold lower than for the corresponding oxo peptide. This difference is entirely due tokcat. Because the rotation about the thioxo amide bond is about 12.5 kJ mol−1more difficult than rotation about an amide bond, these data support a mechanism involving rate‐limiting rotation about the scissile peptide bond. It was found that the specificity rate constant for the reaction of thioxoalanyl‐proline 4‐nitroanilide and dipeptidyl peptidase IV is 100–1000‐fold lower compared to the corresponding rate constant for alanyl‐proline 4‐nitroanilide. This remarkable effect is interpreted in terms of a distorted binding of the transition state for the thioxo substrate. The hydrolysis of the thioxo substrate by dipeptidyl peptidase IV is isomer‐specific. The conformation about the nonscissile P2‐P1thioxo amide bond has to bein transfor successful cleavage of the scissile peptide bond. We can now directly compare the rotational energy barrier of the prolyl peptide bond for the oxo and the thioxo form.
Inhibition of human secretory class II phospholipase A2 by heparin
Authors: DUA Rajiv , CHO Wonhwa
Published: March 2005

By means of kinetic analyses using Triton X‐100/deoxycholic acid/dilauroylglycerophosphoethanolamine (4 : 2 : 1, molar ratio) mixed micelles we examined the effects of heparin on the activity of several phospholipases A2 (PLA2). Heparin avidly bound cationic PLA2s including human secretory class II PLA2 and thereby inhibited their hydrolysis of phospholipids in the mixed micelles. Initial velocity measurements indicated that heparin behaved as a competitive inhibitor for human secretory class II PLA2 and closely related A.h. blomhoffii PLA2 and A.p. piscivorus PLA2. In particular, heparin showed the highest specificity for human secretory class II PLA2. In the absence of deoxycholic acid in mixed micelles, A.h. blomhoffii PLA2 was also strongly inhibited by heparin. The observed inhibition was not due to the interaction of heparin with the active site of PLA2 because heparin did not inhibit the hydrolysis of monomeric substrates by PLA2s. Both kinetic measurements and fluorescence measurements of PLA2‐bound 8‐anilino‐1‐naphthalene sulfonate in the presence of varying amounts of heparin showed that a heparin molecule bound about seven molecules of PLA2. When positive charges of four lysines in the amino‐terminal region of A.h. blomhoffii PLA2 were neutralized by limited carbamoylation, heparin neither bound the carbamoylated A.h. blomhoffii PLA2 nor inhibited the hydrolysis of Triton X‐100/dilauroylglycerophosphocholine mixed micelles by the carbamoylated A.h. blomhoffii PLA2 that retained 50% activity of native A.h. blomhoffii PLA2. Also, heparin did not inhibit the hydrolysis of mixed micelles by 7,10‐bis(octanoyl)ated A.p. piscivorus PLA2 in which two lysines in the amino‐terminal α‐helix are acylated. These results indicate that the inhibition of human secretory class II PLA2 and related cationic PLA2s by heparin originates from the interaction of heparin with cationic residues in the amino‐terminal region that forms a part of interfacial binding site. In addition, unique structural features of human secretory class II PLA2, together with its unique mode of interaction with heparin, suggest that this PLA2 might have an additional heparin‐binding site. Although the heparin‐PLA2 binding diminished as the ionic strength of reaction medium increased, the inhibition of human secretory class II PLA2 by heparin remained significant at the physiological ionic strength. An estimated value of inhibition constant (Ki) was 0.1 μM under physiological conditions, which suggests that a normal pharmaceutical dose of heparin might inhibit human secretory class II PLA2 and regulate its biological effects.
In vitro synthesis of disialoganglioside (Gd1α) from asialo‐Gm1 using sialyltransferases in rat liver Golgi vesicles
Authors: HIDARI Kazuya I.‐P. Jwa , KAWASHIMA Ikuo , TAI Tadashi , INAGAKI Fuyuhiko , NAGAI Yoshitaka , SANAI Yutaka
Published: March 2005

Two gangliosides were efficiently synthesized from asialo‐Gm1 (Galβ1‐3GalNAcβ1‐4Galβ1‐4Glcβ1‐1 Cer) and cytidine 5′‐phosphate‐N‐acetylneuraminic acid (CMP‐NeuAc) by using sialyltransferases in rat liver Golgi vesicles in vitro. These gangliosides were rapidly purified by a combination of anion exchange and reverse‐phase column chromatographies. The ganglioside structures were determined by TLC analysis, treatment with a sialidase from Salmonella typhimurium LT2, which specifically hydrolyzes α2‐3 N‐acetylneuraminic acid (NeuAcα2‐3) linkages, TLC immunostaining, and 1H‐NMR spectroscopy. One of the gangliosides was identified as Gd1α [NeuAcα2‐3Galβ1‐3(NeuAcα2‐6)GalNAcβ1‐4Galβ1‐4Glcβ1‐1 Cer]. The other ganglioside was determined to be Gm1b (NeuAcα2‐3Galβ1‐3GalNAcβ1‐4Galβ1‐4Glcβ1‐1 Cer), which has been reported in a previous study [Pohlentz, G., Klein, D., Schmitz, D., Schwarzmann, G., Peter‐Katalinic, J. & Sandhoff, K. (1988) Biol. Chem. Hoppe‐Seyler 369, 55–63]. Finally, Gm1b and Gd1α were obtained from asialo‐Gm1 as a starting material in 8.1% and 1.2% overall yields, respectively. This study also suggests that the novel synthetic pathway asialo‐Gm1→ Gm1b→ Gd1α may exist in rat liver.
Transcriptional activation by amphipathic carboxylic peroxisomal proliferators is induced by the free acid rather than the acyl‐CoA derivative
Authors: HERTZ Rachel , BERMAN Ina , BAR‐TANA Jacob
Published: March 2005

Most peroxisomal proliferators consist of a carboxylic group attached to a hydrophobic backbone yielding an amphipathic carboxylate molecule. The respective CoA derivatives of peroxisomal proliferators, formed by ATP‐dependent CoA thioesterification catalyzed by long‐chain‐acyl‐CoA synthase, have been repeatedly considered as the immediate inducers of peroxisome and other genes. In this study, the putative requirement for prior CoA thioesterification of peroxisomal proliferators was evaluated by analyzing the induced expression of a reporter plasmid promoted by the peroxisomal acyl‐CoA‐oxidase promoter in cells transiently cotransfected with expression vectors for the peroxisome‐proliferator‐activated receptor and the long‐chain‐acyl‐CoA synthase. Transcriptional activation of peroxisomal acyl‐CoA oxidase by peroxisomal proliferators was inhibited in the presence of transfected functional acyl‐CoA synthase. The inhibitory effect was negatively correlated with the capacity of the acyl‐CoA synthase to catalyze CoA thioesterification of the respective proliferator. Hence, the immediate inducer is the peroxisomal proliferator free acid rather than the respective CoA derivative or a metabolite derived from the peroxisomal‐proliferator‐CoA intermediate.
Binding of transforming growth factor‐β (TGF‐β) to pregnancy zone protein (PZP)
Authors: PHILIP Anie , BOSTEDT Lena , STIGBRAND Torgny , O'CONNOR‐McCOURT Maureen D.
Published: March 2005

Pregnancy zone protein (PZP) is quantitatively the most important pregnancy‐associated plasma protein and it has strong similarity to α2‐macroglobulin. Since α2‐macroglobulin is a binding protein for transforming growth factors‐β (TGF‐β), it was of interest to test whether the related protein, PZP, also binds to these growth‐regulatory proteins. Using affinity‐labelling methods, we demonstrate that PZP binds both TGF‐β1 and TGF‐β2 and that the binding characteristics are similar to those of the TGF‐β–α2‐macroglobulin interaction. TGF‐β2 and TGF‐β1 bind to PZP in a predominantly noncovalent manner in vitro. TGF‐β1 and TGF‐β2 bind to both the dimeric and tetrameric forms of PZP. Our studies also indicate that PZP binds TGF‐β2 with higher affinity than TGF‐β1. Finally, we demonstrate that PZP inhibits the binding of TGF‐β1 and TGF‐β2 to their cell surface receptors. The increased level of PZP during pregnancy may affect the action of TGF‐β by regulating the distribution, clearance and/or general availability of TGF‐β. The preferential binding of TGF‐β2 over TGF‐β1 by PZP implies that PZP may differentially regulate the action of TGF‐β1 and TGF‐β2.
Primary structure and binding characteristics of locust and human muscle fatty‐acid‐binding proteins
Authors: MAATMAN Ronald G. H. J. , DEGANO Massimo , VAN MOERKERK Herman T. B. , VAN MARREWIJK Wil J. A. , VAN DER HORST Dick J. , SACCHETTINI James C. , VEERKAMP Jacques H.
Published: March 2005

The conservation between muscle fatty‐acid‐binding proteins (M‐FABP) of Locusta migratoria flight muscle and human skeletal muscle was investigated. The locust M‐FABP cDNA (632 bp) was isolated by 5′ and 3′ rapid amplification of cDNA ends. The identities of the locust and human M‐FABP on the cDNA and protein levels were 54% and 42%, respectively. The predicted amino acid sequence of locust M‐FABP indicated a molecular mass of 14935 Da and isoelectric point 6.1. The locust M‐FABP was expressed in Escherichia coli, purified by (NH4)2SO4 precipitation, anion‐exchange and gel‐filtration chromatographies and compared with the recombinant human M‐FABP with respect to immunological and binding properties. In spite of the high sequence similarity, the proteins did not show immunological cross‐reactivity. The binding parameters of locust M‐FABP were analyzed with radiolabeled oleic acid by the Lipidex assay and titration microcalorimetry. Both methods revealed a Kd for oleic acid of 0.5 μM and a binding stoichiometry of 1 mol fatty acid/mol FABP. The ΔH, ΔG and ΔS for oleic acid binding were −146 kJ · mol−1 and −36 J · mol−1 and −369 J · mol−1· K−1 respectively. All the information obtained from binding, fluorescence and displacement studies indicated that locust M‐FABP has binding characteristics similar to human M‐FABP. Finally the recombinant locust M‐FABP was crystallized with and without oleic acid. All crystals were trigonal in the P3121 space group. The unit cell dimensions were a=b= 5.89 nm and c= 14.42 nm.
Purification of a two‐subunit cytochrome‐b‐containing heterodisulfide reductase from methanol‐grown Methanosarcina barkeri
Authors: HEIDEN Stefanie , HEDDERICH Reiner , SETZKE Edgar , THAUER Rudolf K.
Published: March 2005

Heterodisulfide reductase catalyzes the terminal step in the energy‐conserving electron‐transport chain in methanogenic Archaea. The heterodisulfide reductase activity of the membrane fraction of methanol‐grown Methanosarcina barkeri was solubilized by Chaps. Chromatography on Q‐Sepharose and Superdex‐200 yielded a high‐molecular‐mass fraction (> 700 kDa) which was dissociated by dodecyl β‐D‐maltoside. After chromatography on Q‐Sepharose, an active heterodisulfide reductase preparation was obtained which was composed of only two different subunits of apparent molecular masses 46 kDa and 23 kDa. For each 69 kDa, the enzyme contained 0.6 mol cytochrome b, 0.2 mol FAD, 20 mol non‐heme iron and 20 mol acid‐labile sulfur. The 23‐kDa subunit possessed heme‐derived peroxidase activity, showing that this polypeptide is the cytochrome b. The purified enzyme contained the cytochrome b in the reduced form. Upon addition of the heterodisulfide of coenzyme M and N‐7‐mercaptoheptanoylthreonine phosphate the cytochrome was instantaneously oxidized, indicating that the cytochrome b served as electron donor for heterodisulfide reduction.
Chondroitin sulphate covalently cross‐links the three polypeptide chains of inter‐α‐trypsin inhibitor
Authors: MORELLE Willy , CAPON Calliope , BALDUYCK Malika , SAUTIERE Pierre , KOUACH Mostapha , MICHALSKI Catherine , FOURNET Bernard , MIZON Jacques
Published: March 2005

Inter‐α‐trypsin inhibitor (ITI) is a tight complex of three different proteins: bikunin and two heavy chains H1 and H2. In order to demonstrate that the three chains are covalently linked by a chondroitin sulphate chain as previously proposed [Enghild, J. J., Salvesen, G., Hefta, S. A., Thogersen, I. B., Rutherford, S. and Pizzo, S. V. (1991) J. Biol. Chem. 266, 747–751], ITI was extensively digested with thermolysin and the glycosaminoglycan‐containing fragment was isolated from the digest by ion‐exchange chromatography. Its peptide structural determination and mass spectrometry analysis both provide evidence that the different peptide chains constituting ITI are associated by the new cross‐link described as the protein‐glycosaminoglycan‐protein cross‐link.
Mutational analysis of human endothelin receptors ETA and ETB
Authors: BECKER Andreas , HAENDLER Bernard , HECHLER Ulrike , SCHLEUNING Wolf‐Dieter
Published: March 2005

Two endothelin(ET)‐receptor subtypes have been identified in mammals. They differ in their affinity towards the ET isopeptides with ETA displaying an ET‐1‐selective profile and ETB a non‐selective one. To identify the regions responsible for the differential selectivity, chimeric forms were engineered by sequentially exchanging extracellular regions together with their flanking transmembrane domains. Two sets of reciprocal receptor mutants were thereby generated and analysed by expression in COS‐7 cells. The recombinant receptor chimeras were characterised by direct and competitive radioligand‐binding analysis. COS‐7 cells transfected with vectors for the mutant receptors exhibited specific saturable [3‐125I]iodotyrosyl ET‐1 (125I‐ET‐1) binding, with affinities comparable to those of the wild‐type receptors (apparent Ki∼ 1–6 × 10−9 M). An average of 105–106 binding sites/cell was calculated for the wild‐type and mutant forms. In competition experiments using 125I‐ET‐1 and unlabeled ET‐3, an ETB‐selective agonist, we detected a clear switch from an ET‐1‐selective profile to a non‐isopeptide‐selective profile in ETA chimeras where the second extracellular loop and the flanking transmembrane domains IV and V, or the third extracellular loop and the flanking transmembrane domains VI and VII, had been exchanged for the corresponding parts of ETB. The opposite effect, namely a switch from a non‐isopeptide‐selective to an ET‐1‐selective binding, was observed for the mirror ETB chimeras where the symmetrical exchange had been operated. Using 125I‐ET‐1 and the ETA‐specific antagonist cyclo‐(d‐Trp‐d‐Asp‐Pro‐d‐Val‐Leu) (BQ123), we were able to map the main determinants responsible for this selectivity to the N‐terminal moiety of this receptor. Therefore, the ability for the interaction with ET‐3 or BQ123 is governed by two different regions of the ET receptors.