bluuebunny/crossref_metadata_2025 huggingface dataset:
Characterization of ligand binding by the human p55 tumour‐necrosis‐factor receptor
Authors: CORCORAN Anne E. , BARRETT Kathy , TURNER Martin , BROWN Alex , KISSONERGHIS Anna‐Maija , GADNELL Margaretha , GRAY Patrick W. , CHERNAJOVSKY Yuti , FELDMANN Marc
Published: March 2005

Two soluble tumour‐necrosis‐factor‐α(TNF)‐binding proteins are derived from the extracellular domains of the p55 and p75 TNF receptors. They are considered to play a pivotal regulatory role in TNF‐mediated inflammatory processes, including deseases such as rheumatoid arthritis, by competing with the cell surface receptors for TNF and lymphotoxin (LT, tumour‐necrosis factor β). The extracellular domains of the two receptors each contain four similar cysteine‐rich repeats of about 40 amino acids, in common with several other cell surface proteins including the p75 nerve‐growth‐factor receptor and the CD40 and Fas antigens. The aim of this study was to characterize the involvement of the four cysteine‐rich repeats of the human p55 TNF receptor in TNF and LT binding by both membrane‐bound and soluble forms of the receptor. Individual repeats were systematically deleted by PCR mutagenesis and the variants transiently expressed in COS cells. Immunoprecipitated receptor variants exhibited the expected sizes on SDS/PAGE gels, and bound a panel of comformation‐dependent anti‐(TNF receptor) antibodies. Binding of TNF by the four soluble derivatives was compared with binding by the wild‐type soluble receptor using a TNF‐affinity column and a BIAcoreTM Biosensor, by measurement of their ability to inhibit TNF cytotoxicity on WEHI cells, and 125I‐TNF binding to U937 cells. Δ4, which lacks the fourth cysteine‐rich repeat, bound TNF comparably with the full‐length soluble receptor. TNF‐binding affinity was unaltered by deletion of the fourth membrane‐proximal cysteine‐rich repeat, as determined by Scatchard analysis of the transmembrane derivatives. We conclude that the fourth cysteine‐rich repeat is not required for TNF binding. In contrast, both the soluble and the transmembrane derivatives lacking any one of the first, second or third repeats failed to bind TNF. Although we cannot entirely exclude the possibility that this may be due to indirect conformational change, rather than the removal of essential epitopes, our results suggest that the first three repeats are each required for TNF binding by both the soluble and the cell‐surface receptor.
The specificity of clostripain from Clostridium histolyticum
Authors: ULLMANN Dirk , JAKUBKE Hans‐Dieter
Published: March 2005

The S′ subsite specificity of clostripain from Clostridium histolyticum was investigated by acyl transfer to libraries of amino acid amides, Ala‐Xaa dipeptides, proline derivatives and pentapeptides using Nα‐benzoyl‐l‐arginine ethyl ester as acyl donor. A pentapeptide library consisting of 29 pentapeptides with general structure Xaa‐Ala‐Ala‐Ala‐Gly, Ala‐Xaa‐Ala‐Ala‐Gly and Ala‐Ala‐Xaa‐Ala‐Gly, where Xaa represents Gly, Ala, Pro, Leu, Phe, Asp, Glu, Arg and Lys, was prepared by solid‐phase synthesis. The data analysis was performed by HPLC and evaluated by statistical algorithms. The nucleophile efficiency covers a range of more than three orders of magnitude. In the P′1 position, low specificity for amino acid amides and Xaa‐(Ala)3‐Gly peptides was found, however, in the P′2 position, positively charged amino acid residues are strongly preferred. The negatively charged side chains of aspartic acid and glutamic acid in the P′1 and P′2 positions, respectively, show only poor nucleophilic behaviour. In the case of these amino acids, the S′‐P′ interactions depend significantly on their position of these residues in the peptide chain of the nucleophile. The transfer of aspartic acid and glutamic acid from P′1–P′3 increases the nucleophile efficiency by approximately two orders of magnitude. The aromatic side chains are not well accepted, especially in the case of P′3Phe. Surprisingly, P′1Gly leads to effective P′‐S′ interactions. However, the opposite result was obtained for P′2Gly. The high efficiency for Gly‐NH2 does not fit with the hydrophobicity structure/activity relationships. In most cases, peptide chain elongation does not improve the nucleophile efficiency. The effective interaction of d‐Leu‐NH2 with the S′ subsite of clostripain emphasizes the fact that the nucleophile stereospecificity is not restricted to l‐amino acids. The results with proline derivatives indicate remarkably different specificities of the S′ binding site which can only be explained by conformational restraints. A positive cooperativity between P′1Pro and P′2Gly and a negative cooperativity between P′1Pro and P′2Phe was observed. The arrangement of three proline residues next to each other represents a favourable conformation for effective enzyme‐nucleophile interactions.
(S)‐2‐Amino‐1,3‐propanediol‐3‐phosphate‐carrying diradylglyceroglycolipids
Authors: FISCHER Werner , HARTMANN Rudolf , PETER‐KATALINIĆ Jasna , EGGE Heinz
Published: March 2005

Two novel aminophosphoglycolipids (I, II) were isolated from Clostridium innocuum which constitute 51% (I) and 15% (II) of total polar membrane lipids. The structures, established by quantitative and methylation analyses, fast‐atom‐bombardment mass spectrometry, and one‐ and two‐dimensional NMR spectroscopy, are (I) S‐2‐amino‐1,3‐propanediol‐3‐phospho‐6‐α‐d‐galactopyranosyl(1‐2)α‐d‐glucopyranosyl(1‐3)diradylglycerol and (II) an acylated derivative of (I) that carries an additional fatty acid ester on O6 of the glucosyl moiety. The stereochemical configuration of the 2‐amino‐1,3‐propanediol 3‐phosphate residue was elucidated by conversion to N‐acetylserine 3‐phosphate, with subsequent release and identification of l‐serine by HPLC. In addition to diacylglycerol species, both aminophosphoglycolipids contain 15–32% 1‐O‐(alk‐1‐enyl)‐2‐O‐acyl‐glycerol species in which C14, C16, and C18 vinyl ether are combined predominantly with unsaturated C16 and C18 fatty acid ester. Hydrogenation of the vinyl ether was required to desorb the alkyl, acylsubstituted species in fast‐atom‐bombardment mass spectrometry. Hydrogenation made it further possible to release the alkyl glycerols by acid hydrolysis and to locate the ether bond at O1 of the glycerol moiety.

In contrast to the glycerophosphoglycolipids of other Gram‐positive bacteria, the aminophosphoglycolipids are metabolically not related to the lipoteichoic acid of C. innocuum and serve, therefore, exclusively as major membrane components. Their large abundance among membrane lipids suggests bilayer‐forming physicochemical properties.
Denaturation of free and complexed bovine trypsinogen with the calcium ion, dipeptide Ile‐Val and basic pancreatic trypsin inhibitor (Kunitz)
Authors: BULAJ Grzegorz , OTLEWSKI Jacek
Published: March 2005

Thermal and chemical denaturation has been used to probe changes in the thermodynamic stability of trypsinogen upon complexation with calcium ion and with ligands, which induce the conformational transition of the zymogen to the trypsin‐like form. Chemical and thermal unfolding curves of ligand‐free trypsinogen at pH 5.8 are cooperative and yielded the following stability parameters: the free energy change of denaturation ΔGden= 44.8 kJ/mol, the denaturation temperature = 65.7°C, the enthalpy change of denaturation ΔHden at the denaturation temperature Tden= 607 kJ/mol and the heat capacity change of denaturation ΔCp,den= 12.4 kJ · mol−1· K−1. Fast phases of both unfolding and refolding of trypsinogen proceed on a time scale of seconds and fit to a single exponential. At pH 5.8, the calcium ion increases the conformational stability ΔGden by 7.1 kJ/mol, Tden by 2.6 K and ΔHden by 80 kJ/mol, but does not induce any substantial structural change in the trypsinogen molecule, as revealed by 1H‐NMR spectra. The trypsin‐like form of trypsinogen, generated by complexation of the zymogen with the dipeptide Ile‐Val and/or basic pancreatic trypsin inhibitor (Kunitz), is characterized by increase of ΔHden by 134 kJ/mol and Tden by 2.5 K, which may be attributed to the additional energy required to disrupt the rigidified activation domain in the complexed trypsinogen.
Antibacterial peptides and mitochonrial presequences affect mitochonrial coupling, respiration and protein import
Authors: HUGOSSON Marie , ANDREU David , BOMAN Hans G. , GLASER Elzbieta
Published: March 2005

Cecropins A and P1, antibacterial peptides from insects and from pig and some related peptides released respiratory control, inhibited protein import and at higher concentrations also inhibited respiration. However, PR‐39, an antibacterial peptide from pig intestine, was found to be almost inert towards mitochondria. The concentrations at which the three mitochondrial functions were effected varied for different peptides. Melittin, magainin and Cecropin‐A‐(1,13)–Melittin(1,13)‐NH2, a hybrid between cecropin A and melittin, were most potent, while the two cecropins acted at higher concentrations. The biosynthesis of cecropin A is known and the intermediates are synthesized. We have used four peptides from this pathway to investigate their effects on coupling, respiration and protein import into mitochondria. Mature cecropin A followed by the preproprotein were most aggressive whereas the intermediates were less active or inert. The efficiency of different derivatives of cecropin A as uncouplers correlates well with their capacity to release membrane potential measured as fluorescence quenching of Rhodamine 123. Inhibition of respiration was found to be dependent on membrrane potential and was most pronounced with mature cecropin A, less so with its three precursors. We also found that three peptides derived from mitochonrial presequences showed antibacterial activity. It is concluded that, there are similarities in the functions of antibacterial peptides and mitochondrial presequences, uncoupling activity in mitochondria cannot be correlated with the antibacterial activity (contrary to a previous suggestion), the processing of preprocecropin A may have evolved in such a way that there is a minimum of membrane damage from the intermediates in the pathway, and peptides produced for delivery outside of an animal have evolved to be more aggressive against mitochondria than peptides for delivery inside of the animal.
Heat‐shock proteins as molecular chaperones
Authors: BECKER Jörg , CRAIG Elizabeth A.
Published: March 2005

Functional proteins within cells are normally present in their native, completely folded form. However, vital processes of protein biogenesis such as protein synthesis and translocation of proteins into intracellular compartments require the protein to exist temporarily in an unfolded or partially folded conformation. As a consequence, regions buried when a polypeptide is in its native conformation become exposed and interact with other proteins causing protein aggregation which is deleterious to the cell. To prevent aggregation as proteins become unfolded, heat‐shock proteins protect these interactive surfaces by binding to them and facilitating the folding of unfolded or nascent polypeptides. In other instances the binding of heat‐shock proteins to interactive surfaces of completely folded proteins is a crucial part of their regulation. As heat shock and other stress conditions cause cellular proteins to become partially unfolded, the ability of heat‐shock proteins to protect cells against the adverse effects of stress becomes a logical extension of their normal function as molecular chaperones.
Lead‐ion‐induced cleavage of RNase P RNA
Authors: CIESIOLKA Jerzy , HARDT Wolf‐Dietrich , SCHLEGL Judith , ERDMANN Volker A. , HARTMANN Roland K.
Published: March 2005

Pb2+‐induced hydrolysis of RNase P RNAs from Escherichia coli and the thermophilic eubacterium Thermus thermophilus HB8 revealed one prominent site‐specific cleavage in the two RNAs and several minor cleavage sites in structurally corresponding regions of both RNAs. Data presented here and in a previous study [Kazakov, S. & Altman, S. (1991) Proc. Natl Acad. Sci. USA 88, 9193–9197] provide evidence for several ubiquitous metal‐ion‐binding sites in eubacterial RNase P RNA subunits. With the T. thermophilus RNase P RNA, susceptibility to Pb2+‐induced strand scission at the most prominent site was hypersensitive at the temperature of highest enzyme activity (55°C). Pb2+ hydrolysis at this site was strongly reduced at a temperature of 37°C, where processing is also inefficient. For E. coli RNase P RNA, specific changes in the lead hydrolysis pattern were observed due to the presence of excess tRNA. Thus, Pb2+‐induced hydrolysis seems suitable to sense different conformations of RNase P RNAs. The T. thermophilus RNase P RNA, in particular, displayed significant processing activity after severe fragmentation by Pb2+, and therefore appears to be suited for reconstituting an active enzyme from RNA subfragments.
Dephosphorylation of phosphopeptides by calcineurin (protein phosphatase 2B)
Authors: DONELLA‐DEANA Arianna , KRINKS Marie H. , RUZZENE Maria , KLEE Claude , PINNA Lorenzo A.
Published: March 2005

38 (6–32 residues) enzymically phosphorylated synthetic peptides have been assayed as substrates for calcineurin, a Ca2+/calmodulin‐dependent protein phosphatase (PP‐2B) belonging to the family of Ser/Thr‐specific enzymes but also active on phosphotyrosine residues. Many peptides reproduce, with suitable modifications, naturally occurring phosphoacceptor sites. While protein phosphatases 2A and 2C are also very active on short phosphopeptides, an extended N‐terminal stretch appears to be a necessary, albeit not sufficient, condition for an optimal dephosphorylation, comparable to that of protein substrates, of both phosphoseryl and phosphotyrosyl peptides by calcineurin. This finding corroborates the view that higher‐order structure is an important determinant for the substrate specificity of calcineurin. However, a number of shorter peptides are also appreciably dephosphorylated by this enzyme, their efficiency as substrates depending on local structural features. All the peptides that are appreciably dephosphorylated by calcineurin contain basic residue(s) on the N‐terminal side. A basic residue located at position –3 relative to the phosphorylated residue plays a particularly relevant positive role in determining the dephosphorylation of short phosphopeptides. Acidic residue(s) adjacent to the C‐terminal side of the phosphoamino acid are conversely powerful negative determinants, preventing the dephosphorylation of otherwise suitable peptide substrates. However, calcineurin displays an only moderate preference for phosphothreonyl peptides which are conversely strikingly preferred over their phosphoseryl counterparts by the other classes of Ser/Thr‐specific protein phosphatases. Moreover calcineurin does not perceive as a strong negative determinant the motif Ser/Thr‐Pro in peptides where this motif prevents dephosphorylation by the other classes of Ser/Thr protein phosphatases. Whenever tested on phosphotyrosyl peptides, calcineurin exhibits a specificity which is strikingly different from that of T‐cell protein tyrosine phosphatase, a bona fide protein tyrosine phosphatase. In particular while the latter enzyme is especially active toward a number of phosphopeptides reproducing the phosphoacceptor sites of src products and of calmodulin whose N‐terminal moieties are predominantly acidic, the artificial substrate phospho‐angiotensin II, bearing an arginine residue at position –2, is far preferred by calcineurin over all phosphotyrosyl peptides of similar size. Collectively taken these results show that the specificity of calcineurin, rather than resting on a given consensus sequence, is determined by a variety of primary and higher‐order structural features conferring to it an overall selectivity that is different from those of any other known protein phosphatase.
Polymorphism in the lipase genes of Geotrichum candidum strains
Authors: BERTOLINI Maria Célia , LARAMÉE Louise , THOMAS David Y. , CYGLER Miroslaw , SCHRAG Joseph D. , VERNET Thierry
Published: March 2005

The fungus Geotrichum candidum produces extracellular lipases. Purification and characterization of different lipase isoforms from various G. candidum strains is difficult due to the close physical and biochemical properties of the isoforms. Consequently, the characterization of these enzymes and their substrate specificites has been difficult. We have determined the lipase genes present in four strains of G. candidum (ATCC 34614, NRCC 205002, NRRL Y‐552 and NRRL Y‐553) by molecular cloning and DNA sequencing. Each strain contains two genes similar to the previously identified lipase I and lipase II cDNAs. Our data suggest that no other related lipase genes are present in these strains. Each lipase‐gene family shows sequence variation (polymorphism) that is confirmed by Southern‐blot analysis. This polymorphism and the sequence differences between lipase I and lipase II have been localized within the previously determined three‐dimensional structure of lipase II. Although most of the amino acid substitutions are located on the protein surface, some are present in structural features possibly involved in determining substrate specificity.
Stimulation of phospholipase C‐β2 by recombinant guanine‐nucleotide‐binding protein βγ dimers produced in a baculovirus/insect cell expression system
Authors: DIETRICH Alexander , MEISTER Michael , BRAZIL Derek , CAMPS Montserrat , GIERSCHIK Peter
Published: March 2005

Recombinant wild‐type β1γ1 dimers of signal‐transducing guanine nucleotide‐binding proteins (G proteins) and β1γ1 dimers carrying a mutation known to block γ‐subunit isoprenylation (β1γ1C71S) were expressed in baculovirus‐infected insect cells. Both wild‐type and mutant β1γ1 dimers were found in soluble fractions of infected cells upon subcellular fractionation. Anion exchange chromatographic and metabolic‐radiolabeling studies revealed that the soluble β1γ1 preparation contained approximately equal amounts of non‐isoprenylated and isoprenylated β1γ1 dimers. Soluble wild‐type and mutant β1γ1 dimers and native β1γ1 dimers purified from bovine retina were reconstituted with recombinant phospholipase C‐β2. Only isoprenylated β1γ1 dimers were capable of stimulating phospholipase C‐β2. The results show that γ‐subunit isoprenylation and/or additional post‐translational processing of the protein are required for βγ subunit stimulation of phospholipase C.
The role of trehalose synthesis for the acquisition of thermotolerance in yeast
Authors: DE VIRGILIO Claudio , HOTTIGER Thomas , DOMINGUEZ Judith , BOLLER Thomas , WIEMKEN Andres
Published: March 2005

In the yeast Saccharomyces cerevisiae, accumulation of the non‐reducing disaccharide trehalose is triggered by various stimuli that activate the heat‐schock response. Several studies have shown a close correlation between trehalose levels and tolerance to heat stress, suggesting that trehalose may be a protectant which contributes to thermotolerance. In this study, we have examined mutants defective in genes coding for key enzymes involved in trehalose metabolism with respect to the heat‐induced and stationary‐phase‐induced accumulation of trehalose and the acquisition of thermotolerance. Inactivation of either TPS1 or TPS2, encoding subunits of the trehalose‐6‐phosphate synthase/phosphatase complex, caused an inability to accumulate trehalose upon a mild heat‐shock or upon initiation of the stationary phase and significantly reduced the levels of heat‐induced and stationary‐phase‐induced thermotolerance. Deletion of NTH1, the gene coding for the neutral trehalase, resulted in a defect in trehalose mobilization during recovery from a heat shock which was paralleled by an abnormally slow decrease of thermotolerance. Our results provide strong genetic evidence that heat‐induced synthesis of trehalose is an important factor for thermotolerance induction. In an accompanying study [Hottiger, T., De Virgilio, C., Hall, M. N., Boller, T. & Wiemken, A. (1993) Eur. J. Biochem. 219, 187–193], we present evidence that the function of heat‐induced trehalose accumulation may be to increase the thermal stability of proteins.
Surface potential regulation of phospholipid composition and in‐out translocation in yeast
Authors: CERBÓN Jorge , CALDERÓN Victor
Published: March 2005

In yeast cells the anionic phospholipids, phosphatidylinositol and phosphatidylserine, determine to a large extent the magnitude of the negative surface charge density (σ) [Cerbón, J. & Calderón, V. (1990) Biochim. Biophys. Acta 1028, 261–267]. We now report further findings. (a) When the yeast ϕout was reduced by increasing the concentration of monovalent (C–) or divalent (C2+) cations in the culture medium, the relative amount of anionic phospholipids increased (45–52%). (b) For each such increment, a corresponding increase in the external surface charge density (σ) was found, due to the translocation from the cytoplasmic side to the exoplasmic side of the plasma membrane. (c) These changes were reversed when the ϕout was increased by reducing the concentration of cations in the culture medium. (d) When the ϕout was reduced and phosphatidylserine decarboxylation or phosphatidylinositol degradation were inhibited, to measure synthesis of anionic phospholipids, a 1.4 times further increase in the anionic/zwitterionic phospholipid ratio occurred. As a consequence, a similar increase in the external surface charge (σ) was found. (e) Under all the conditions studied, the percentage of anionic phospholipid at the external surface of the plasma membrane calculated from the σ values was 2.3–3.0 times less than that in the cells, indicating that the asymmetric composition (more inside) was maintained. A model for the regulation of the anionic phospholipid composition of the yeast membranes is proposed.
The 100‐kDa chain of nicein/kalinin is a laminin B2 chain variant
Authors: VAILLY Joëlle , VERRANDO Patrick , CHAMPLIAUD Marie‐France , GERECKE Donald , WAGMAN D. Wolf , BAUDOIN Christian , ABERDAM Daniel , BURGESON Robert , BAUER Eugene , ORTONNE Jean‐Paul
Published: March 2005

We have isolated the basement membrane component nicein and performed rotary‐shadow analyses using electron microscopy that showed the presence of two forms (I and II) of the protein. Molecular cloning of the cDNA that codes for the 100‐kDa chain of the protein revealed that the sequence matches those independently identified for the 105–155‐kDa subunit of kalinin, a recently identified basement membrane component. These data demonstrate that nicein and kalinin contain an identical chain. The length of the open reading frame in the cDNA (∼5200 nucleotides) and amino acid sequences obtained from the N‐terminus of the 105‐kDa kalinin chain showed the occurrence of a precursor polypeptide. This immature polypeptide is probably related to form I, observed by rotary shadowing, while the mature form is related to form II. It is noteworthy that nicein/kalinin subunits share‐discrete sequence similarities with the B2 chain of human laminin, but with a cleavage occurring within domain III that eliminates domains IV and V from the final product. The sequence of this subunit is nearly identical to that of B2t, a recently described polypeptide supposed to be related to a new laminin variant. Since nicein/kalinin expression is specifically impaired in the severe genodermatosis Herlitz junctional epidermolysis bullosa, the role and structure of this tissue‐restricted laminin variant is crucial for the understanding of epidermal‐dermal adhesion.
Structure determination of extracellular fragments of amyloid proteins involved in Alzheimer's disease and Dutch‐type hereditary cerebral haemorrhage with amyloidosis
Authors: SORIMACHI Kay , CRAIK David J.
Published: March 2005

Amyloid deposition is a biochemical and histopathologic hallmark of various clinical forms of amyloidoses including Alzheimer's disease and the Dutch‐type hereditary cerebral haemorrhage with amyloidosis. The self‐aggregating peptides responsible for these irreversible deposits have been sequenced but the mechanisms involved in the aggregation processes are not well understood. In order to gain an understanding of the possible structures prior to self‐association, the extracellular fragment of the Alzheimer amyloid protein (βA4) responsible for the deposits (the ‘native’ fragment) and a mutant of this with a single residue substitution (which is responsible for deposits in the Dutch‐type amyloidosis) were examined by 1H‐NMR spectroscopy. Interproton distance constraints were derived from NMR experimental data and incorporated into tertiary structure calculations using a simulated annealing protocol. Solution conformations of the fragment peptides associated with the two forms of amyloidoses are presented and compared. Although in both peptides the existence of a mixture of conformations in equilibrium is likely, one such population of structures possesses a flexible N‐terminus and a well defined C‐terminal region. The latter region includes a helical segment and a terminal turn‐like structure. These structural features may be important for the basis of amyloid formation. Comparison of the calculated structures of the two peptides revealed a conformationally different region arising from the conservative substitution of Gln22 for Glu22. This region may be responsible for altered binding in the mutant peptide, giving rise to the clinically different form of amyloidosis.
Metallobiochemistry of the magnesium ion
Authors: HUANG H.‐W. , COWAN J. A.
Published: March 2005

Ribonuclease H (Escherichia coli) contains one strong magnesium‐binding site, as determined by metal‐titration experiments monitored by high field 1H‐NMR and also by direct titration calorimetry. Kinetic and thermodynamic parameters were evaluated by 25Mg‐NMR and were as follows: dissociation constant Kd, ∼60±10 μM; activation free energy ΔG*, ∼49.8±0.9 kJ; on/off‐rate for magnesium binding kon, ∼ 1.8×108M−1 s−1, koff, ∼ 1.1×104 s−1; quadrupole coupling constant χB, 1.2±0.2 MHz. The dissociation constant was independently determined by standard analysis of 1H chemical shifts in magnesium‐titration experiments and by microcalorimetry (Kd∼ 200±20 μM). Cobalt hexaamine, which also activates RNase H [Jou, R. & Cowan, J. A. (1991) J. Am. Chem. Soc. 113, 6685–6686], appears to bind at the same location as Mg2+(aqueous). Assignments of C2H and C4H protons to specific histidine residues have been made by two‐dimensional correlated spectroscopy experiments. Direct 25Mg‐NMR pH titrations show that an ionizable residue (pKa∼ 5.8), most likely one of the carboxylates in the active site, influences magnesium binding. On the basis of the magnesium coordination chemistry elucidated herein, recent proposals on active‐site chemistry are critically assessed and general physicochemical aspects of magnesium‐binding sites on proteins and enzymes are discussed.
The involvement of polyamines in the activation of vitamin D receptor from porcine intestinal mucosa
Authors: MORISHIMA Yoshihiro , INABA Masaaki , NISHIZAWA Yoshiki , MORII Hirotoshi , HASUMA Tadayoshi , MATSUI‐YUASA Isao , OTANI Shuzo
Published: March 2005

In the intracellular process of the action of calcitriol, vitamin D receptor is thought to undergo some kind of physicochemical change, called activation, before the receptor binds to the vitamin D response element of the gene. In this paper, the effects of polyamines and their analogues on the sedimentation properties of vitamin D receptor prepared from porcine intestinal mucosa, and on DNA binding activity of the receptor, were studied. In sucrose density gradient analysis, polyamines decreased the sedimentation coefficient of vitamin D receptor in a dose‐dependent fashion. Polyamines increased DNA binding activity of vitamin D receptor dose‐dependently. These findings show that polyamines can activate vitamin D receptor in vitro. Among naturally existing polyamines, spermidine and spermine, but not putrescine, were effective within their physiological intracellular concentrations, suggesting that both spermidine and spermine can activate vitamin D receptor in vivo as well. Sucrose density gradient analysis using various kinds of polyamine analogues having various numbers of cations showed that the number of cation of polyamines is important for the efficiency to change the sedimentation coefficient of vitamin D receptor, and that the distance between two cationic charges does not play an important role.
Hordothionins inhibit protein synthesis at the level of initiation in the wheat‐germ system
Authors: BRÜMMER Janna , THOLE Hubert , KLOPPSTECH Klaus
Published: March 2005

The inhibitory effect of pure α and β hordothionins on protein synthesis directed by pea mRNA has been studied in the wheat‐germ translation system. It is demonstrated that a component of the wheat germ counteracts the thionin effect. Formation of polysomes in vitro in the presence of thionin was inhibited to the same extent as the total translation system while run‐off translation of isolated polysomes from pea plants was not affected by thionin. These data are consistent with an effect of thionin on the initiation reaction. Analyses of the formation of initiation complexes in the presence and absence of mRNA support this view and show that thionin interferes with the formation of the 43S complex. In accordance with this observation and in contrast to earlier studies no evidence has been obtained for a direct interaction between mRNAs and thionins. The analysis of the translation products also gave no indication for preferential translation of individual mRNAs by the thionin‐inhibited translation system. Compared to translation in vitro, exposure of barley protoplasts to thionins showed a less dramatic effect on protein synthesis as measured by incorporation of [35S]methionine into proteins. These data are discussed with respect to the effects of thionins on the plasma membranes as shown previously with animal cell cultures. It is concluded that at least in barley such effects would need higher concentrations of thionins than are required for the inhibition of protein synthesis.
Expression, purification and characterization of the recombinant kringle 2 and kringle 3 domains of human plasminogen and analysis of their binding affinity for ω‐aminocarboxylic acids
Authors: MARTI Daniel , SCHALLER Johann , OCHENSBERGER Brigitte , RICKLI Egon E.
Published: March 2005

The kringle 2 (E161T/C162S/EEE[K2HPg/C169S]TT) and the kringle 3 (TYQ[K3HPg]DS) domains of human plasminogen (HPg) were expressed in Escherichia coli in an expression vector with the phage T5 promotor/operator element N250PSN250P29 and the cDNA sequence for a hexahistidine tail to facilitate the isolation of the recombinant protein. A coagulation factor Xa (FXa)‐sensitive cleavage site was introduced to remove the N‐terminal histidine tag. In r‐K2, mutations E161T and C162S were introduced to enhance the FXa cleavage yield and C169S to replace the cysteine residue, participating in the inter‐kringle disulfide bridge between kringles 2 and 3. Recombinant proteins were isolated by affinity chromatography on Ni2+‐nitrilotriacetic acid/agarose and refolded under denaturing and reducing conditions followed by a non‐denaturing and oxidising environment. The free thiol group in position 297 in r‐K3 was selectively alkylated with iodoacetamide. The hexahistidine tail was successfully removed with FXa. The N‐terminal sequence, the amino acid composition and the molecular mass analyses are in agreement with the expected data. The correct arrangement of the disulfide bonds was verified by sequence analysis of the corresponding thermolytic and subtilisin fragments. r‐K2 exhibits weak binding to lysine‐Bio‐Gel. The weak binding affinity of r‐K2 for ω‐aminocarboxylic acids is confirmed by intrinsic fluorescence titration with 6‐aminohexanoic acid (NH2C5COOH) indicating a Kd of approximately 401 μM. In contrast, r‐K3 seems to be devoid of a binding affinity for ω‐aminocarboxylic acids. Considering earlier determined Kd values of kringle 1, kringle 4 and kringle 5, the binding affinity of HPg kringle domains for NH2C5COOH is proposed to decrease in the following order, kringle 1 > kringle 4 > kringle 5 > kringle 2 > kringle 3.
The mercury(II) and high‐salt‐induced conformational B↔Z transitions of poly[d(G‐m5C) · d(G‐m5C)] as studied by non‐polarized (ultraviolet) and circularly polarized (CD) ultraviolet spectroscopy
Authors: GRUENWEDEL Dieter W.
Published: March 2005

The B↔Z transition of poly[d(G‐m5C) · d(G‐m5C)] in buffered solution (0.002 M sodium cacodylate, pH 7) was studied by CD and ultraviolet spectroscopy as a function of the supporting electrolyte concentration (0.002–1.1 M NaClO4) in the absence of Hg(ClO4)2 [Hg(II)], and as a function of the Hg(II) concentration at a given NaClO4 level. NaClO4 alone changes the conformation of the polymer from B↔Z at approximately 0.7 M NaClO4. The spectral changes caused by Hg(II) in the B‐form polymer (e.g. at 0.002 M ≤ [Na] ≤ 0.7) resemble those generated by salt alone during the B↔Z transition; the changes generated by Hg(II) in the Z‐form polymer (e.g. in 1.1 M [Na]) leave principally intact the Z‐form spectrum obtained at the higher levels of NaClO4 (e.g. at [Na] > 0.7 M) in the absence of Hg(II). It is concluded that particularly the long‐wavelength positive‐CD band, located at 274 nm, is a correct indicator of duplex DNA right↔left‐screwness inversion. According to generally accepted criteria, the NaClO4‐induced left‐handed form is Z DNA; Hg(II) generates a left‐handed form termed here ZHg(II). This form is close to (but not identical with) the salt‐induced Z‐form. All Hg(II)‐induced spectral changes are reversible upon removal of Hg(II) with a suitable complexing reagent (e.g. NaCN).
Magnetic‐circular‐dichroism studies of Escherichia coli cytochrome bo
Authors: CHEESMAN Myles R. , WATMOUGH Nicholas J. , GENNIS Robert B. , GREENWOOD Colin , THOMSON Andrew J.
Published: March 2005

Room‐temperature (295 K) magnetic‐circular‐dichroism spectra at 280–2500 nm have been recorded for Escherichia coli cytochrome bo in its fast form (which has a g= 3.7 EPR signal and reacts rapidly with cyanide) and for its formate, fluoride, cyanide and hydrogen‐peroxide derivatives. The spectra of all forms are dominated by signals from low‐spin ferric heme b. These include a porphyrin‐to‐ferric ion charge‐transfer transition in the near‐infrared region (the near‐infrared charge‐transfer band) at 1610 nm. High‐spin ferric heme o gives rise to a negative magnetic‐circular‐dichroism feature at 635, 642 and 625 nm (corresponding to a shoulder observed in the electronic absorption spectra) and a derivative charge‐transfer feature at 1100, 1180 and 940 nm for the fast, formate and fluoride forms, respectively. The energies of these bands confirm that fluoride and formate are ligands to heme o. The energies of the analogous bands in the spectrum of fast cytochrome bo are typical for high‐spin ferric hemes with histidine and water axial ligands. Addition of cyanide ion to fast cytochrome bo causes a red shift in the position of the Soret absorption peak, from 406.5 nm to 413 nm, and results in the loss of the 635‐nm feature from the magnetic‐circular‐dichroism spectrum and of the corresponding shoulder in the electronic absorption spectrum. In the magnetic‐circular‐dichroism spectrum, the intensities of the Soret and α,β bands are significantly increased. New near‐infrared charge‐transfer intensity is observed at 1000–2300 nm with a peak near 2050 nm. These changes are interpreted as resulting from a high‐spin to low‐spin transition at ferric heme o brought about by the binding of cyanide ion. The energy of the near‐infrared charge‐transfer band suggests that the cyanide ion is bridged to the CuB of the binuclear site. Treatment of fast cytochrome bo with hydrogen peroxide also causes a red shift in the position of the Soret absorbance, to 412 nm, and a loss of the 625‐nm absorption shoulder. Changes in the magnetic‐circular‐dichroism spectrum at 450–600 nm are observed, but there is no significant increase in the intensity of the magnetic‐circular‐dichroism Soret band and no new near‐infrared charge‐transfer bands are detected, ruling out a similar high‐spin to low‐spin transition at heme o. Subtraction of the contribution of low‐spin ferric heme b from the magnetic‐circular‐dichroism spectrum reveals a spectrum characteristic of ferryl [Fe(IV)] heme o.
1H resonance assignments and secondary structure of the carbon monoxide complex of soybean leghemoglobin determined by homonuclear two‐dimensional and three‐dimensional NMR spectroscopy
Authors: MORIKIS Dimitrios , LEPRE Christopher A. , WRIGHT Peter E.
Published: March 2005

Homonuclear two‐dimensional and three‐dimensional 1H‐NMR spectroscopy has been utilized to study the 15.9‐kDa protein soybean leghemoglobin. NMR experiments were performed on the diamagnetic carbon monoxide complex at two temperatures and two pH values. Sequence‐specific assignments have been made for 94% of the backbone and approximately 70% of the expected side‐chain resonances. The secondary structure of leghemoglobin in solution has been determined on the basis of NOE connectivity patterns, hydrogen exchange and chemical‐shift analyses. Leghemoglobin consists of seven helices and, unlike mammalian myoglobins, is missing the D helix. Instead an extended loop, the CE loop, is observed which might have importance for ligand entry into and exit from the protein interior. The hydrogen exchange behavior for the F helix and at the beginning of the A helix suggests different dynamic stability compared to other helical regions in leghemoglobin. Population of a second protein conformation, in which there is perturbation at the A‐G‐H helix interface, is observed at low pH.
Two binding sites of inhibitors in NADH:ubiquinone oxidoreductase (complex I)
Authors: FRIEDRICH Thorsten , VAN HEEK Petra , LEIF Hans , OHNISHI Tomoko , FORCHE Edgar , KUNZE Brigitte , JANSEN Rolf , TROWITZSCH‐KIENAST Wolfram , HÖFLE Gerhard , REICHENBACH Hans , WEISS Hanns
Published: March 2005

The effect of ten naturally occurring and two synthetic inhibitors of NADH:ubiquinone oxidoreductase (complex I) of bovine heart, Neurospora crassa and Escherichia coli and glucose:ubiquinone oxidoreductase (glucose dehydrogenase) of Gluconobacter oxidans was investigated. These inhibitors could be divided into two classes with regard to their specifity and mode of action. Class I inhibitors, including the naturally occuring piericidin A, annonin VI, phenalamid A2, aurachins A and B, thiangazole and the synthetic fenpyroximate, inhibit complex I from all three species in a partially competitive manner and glucose dehydrogenase in a competitive manner, both with regard to ubiquinone. Class II inhibitors including the naturally occuring rotenone, phenoxan, aureothin and the synthetic benzimidazole inhibit complex I from all species in an non‐competitive manner, but have no effect on the glucose dehydrogenase. Myxalamid PI could not be classified as above because it inhibits only the mitochondrial complex I and in a competitive manner. All inhibitors affect the electron‐transfer step from the high‐potential iron‐sulphur cluster to ubiquinone. Class I inhibitors appear to act directly at the ubiquinone‐catalytic site which is related in complex I and glucose dehydrogenase.
The mode of action of SA‐FF22, a lantibiotic isolated from Streptococcus pyogenes strain FF22
Authors: JACK Ralph , BENZ Roland , TAGG John , SAHL Hans‐Georg
Published: March 2005

SA‐FF22 is a lanthionine‐containing bacteriocidal peptide isolated from Streptococcus pyogenes strain FF22. The peptide interacts closely with non‐energised artificial phospholipid vesicles, as evidenced by a ‘blue shift’ in the fluorescent emissions associated with a tryptophan residue within the peptide sequence. Furthermore, SA‐FF22 induced efflux of radiolabelled amino acids from artificially energised cytoplasmic membrane vesicles and arrested uptake of amino acids by intact cells. By measuring the decrease in membrane potential of both starved and energised SA‐FF22‐treated cells, and through the use of artificial planar membranes, a potential of approximately 100 mV was deduced as the minimum required to induce pore formation by SA‐FF22. This threshold potential is independent of the orientation of the applied voltage (i.e. trans or cis orientations are equally effective). Single channel conductance measurements suggested that the pores formed by SA‐FF22 are relatively unstable, short‐lived and approximately 0.5–0.6 nm in diameter. This is somewhat smaller than those of the previously described, pore‐forming lantibiotics and should not allow significant efflux of large molecules such as ATP. Thus, death of affected cells seems to result from membrane‐potential disruption and subsequent exhaustion of the cells.
Functional Interference Between AP‐1 and the Vitamin D Receptor on Osteocalcin Gene Expression in Human Osteosarcoma Cells
Authors: Jääskeläinen Tiina , Pirskanen Asta , Ryhänen Sanna , Palvimo Jorma J. , Deluca Hector F. , Mäenpää Pekka H.
Published: March 2005

The binding of transcription factor AP‐1 and vitamin D receptor (VDR) to the composite AP‐1 plus vitamin‐D‐responsive promoter region (AP‐1+VDRE) of the human osteocalcin gene was characterized in osteocalcin‐producing (MG‐63) and non‐producing (U2‐Os, SaOs‐2) human osteosarcoma cell lines. In mobility‐shift assays with AP‐1+VDRE, AP‐1, and VDRE probes and nuclear extracts from these cells, one AP‐1‐specific and two VDR‐specific (fast and slow mobility) interactions were observed. Characterization of the complexes indicated that AP‐1 and VDR do not bind simultaneously to the AP‐1 + VDRE oligonucleotide. Intensity of the complexes was greatly influenced by cell density: in MG‐63 and SaOs‐2 cells, AP‐1 binding was strong during the proliferative period disappearing at confluency whereas, in U2‐Os cells, AP‐1 binding was prominent also at the confluent stage. Furthermore, MG‐63 cells possessed the faster migrating VDR complex at all stages of confluency whereas, in U2‐Os and SaOs‐2 cells, it was very weak or absent. There were no detectable differences in the levels of VDR protein between these cell lines. In U2‐Os cells, the level of c‐Jun mRNA was higher than in the other two cell lines, whereas none of these cell lines exhibited detectable levels of c‐fos mRNA at the confluent stage. Exogenous c‐Jun protein effectively blocked the VDR‐DNA interaction. Further, all these cell lines expressed mRNA for retinoid X receptor α (RXRα), the factor suggested to be required for the VDR‐DNA interaction. The presence of an accessory factor in the VDR‐DNA complexes was indirectly shown by treatment of the cells with 9‐cis retinoic acid and by cycloheximide. Both treatments reduced VDR binding without affecting the VDR protein level. These results suggest that AP‐1 interferes with VDR binding to the AP‐1+VDRE element and that the vitamin D responsiveness of the osteocalcin gene correlates with weak AP‐1 binding and strong binding of the faster migrating VDR complex.
Structural Organization and Chromosomal Localization of a Human Gene (HIP/PAP) Encoding a C‐type Lectin Overexpressed in Primary Liver Cancer
Authors: Lasserre Chantal , Simon Marie‐Thérèse , Ishikawa Hitoshi , Diriong Sylvie , Nguyen Van Cong , Christa Laurence , Vernier Philippe , Brechot Christian
Published: March 2005

We previously identified, through differential screening of a human primary liver cancer library, a novel gene (named HIP)the expression of which is markedly increased in 25% of human primary liver cancers. HIP mRNA expression is tissue specific since it is restricted to pancreas and small intestine. HIP protein consists in a signal peptide linked to a carbohydrate‐recognition domain (CRD), typical of C‐type lectins without other binding domains. We have proposed that HIP and related proteins belong to a new family of C‐type lectins. Drickamer [Drickamer, K. (1993) Curr Opin. Struct. Biol. 3, 393–400] included this group of proteins in his classification of C‐type lectins as the free CRD (group VII) lectins. In the present report we describe the genomic organization and the chromosomal localization of HIP. We have shown that HIP is in fact the pancreatitis‐associated protein (PAP) and provided a phylogenetic analysis of the free CRD lectins. Furthermore, the analysis of HIP/PAP gene indicates that the HIP/PAP CRD is encoded by four exons, a pattern shared with all members of this group of proteins. This common intron‐exon organization indicates an ancient divergence of the free CRD‐lectin group from other groups of C‐type lectins. We provide evidence for the localization of HIP/PAP on chromosome 2, suggesting previous duplication of HIP/PAP and the related reg Iα and reg Iβ genes from the same ancestral gene. Finally, the sequence of the 5′ upstream region of the HIP gene shows several potential regulatory elements which might account for the enhanced expression of the gene during pancreatic inflammation and liver carcinogenesis.
Poly(ADP‐Ribose) Polymerase in Plant Nuclei
Authors: Chen Yi‐Min , Shall Sydney , O'farrell Minnie
Published: March 2005

We show that poly(ADP‐ribose) polymerase is present in maize, pea and wheat nuclei. We have identified the enzyme product as poly(ADP‐ribose) by purification and electrophoresis on a DNA sequencing gel. This reveals a polymer ladder consisting of up to 45 residues. The polymer product from maize, after digestion with snake venom phosphodiesterase, gave only 5′‐AMP and (phosphoribosyl)‐AMP; the mean chain length of the polymer was 5 and 11 residues in two separate experiments. The optimum pH of the plant enzyme is greater than pH 7.0 in pea, wheat and maize; the optimum temperature for enzyme activity is approximately 15°C. The Km for NAD+ for the enzyme from maize is estimated to be approximately 50 μM under optimal conditions. Several compounds (nicotinamide, deoxythymidine, 3‐aminobenzamide, 3‐methoxybenzamide and 5‐bromodeoxyuridine) that specifically inhibit the animal enzyme also inhibit the enzyme from plants. The ratio of the IC50 for 5‐bromodeoxyuridine to the IC50 for 3‐aminobenzamide in maize is similar to that of the animal enzyme indicating that the enzyme involved is poly(ADP‐ribose) polymerase and not mono(ADP‐ribosyl) transferase. SDS gel electrophoresis and gel filtration analysis of a crude extract of maize nuclei indicate a molecular mass for poly(ADP‐ribose) polymerase of approximately 114 kDa.
Structural Studies of the O‐Antigenic Polysaccharides of Escherichia coli O3 and the Enteroaggregative Escherichia coli Strain 17‐2
Authors: Medina Ernesto C. , Widmalm Göran , Weintraub Andrej , Vial Pablo A. , Levine Myron M. , Lindberg Alf A.
Published: March 2005

The polysaccharide part of the lipopolysaccharide isolated from an enteroaggregative Escherichia coli isolated from a young child with diarrhoea in Santiago, Chile (strain 17‐2), has been investigated. Sugar and methylation analyses of native and partially degraded polysaccharide together with 1H‐NMR and 13C‐NMR spectroscopies revealed that the polysaccharide is composed of pentasaccharide repeating units. The structure of the repeating unit of E. coli strain 17‐2 O‐polysaccharide is:

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The structure of the O‐polysaccharide from E. coli O3 was shown to be identical to that of E. coli strain 17‐2 by sugar and methylation analyses and by 1H‐NMR and 13C‐NMR spectroscopies.