bluuebunny/crossref_metadata_2025 huggingface dataset:
Involvement of EF hand motifs in the Ca2+‐dependent binding of the pleckstrin homology domain to phosphoinositides
Authors: Yamamoto Tada‐aki , Takeuchi Hiroshi , Kanematsu Takashi , Allen Victoria , Yagisawa Hitoshi , Kikkawa Ushio , Watanabe Yutaka , Nakasima Akihiko , Katan Matilda , Hirata Masato
Published: March 2003

The pleckstrin homology (PH) domains of phospholipase C (PLC)‐δ1 and a related catalytically inactive protein, p130, both bind inositol phosphates and inositol lipids. The binding to phosphatidylinositol 4,5‐bisphosphate [PtdIns(4,5)P2] by PLC‐δ1 is proposed to be the critical interaction required for membrane localization to where the substrate resides; it is also required for the Ca2+‐dependent activation of PLC‐δ1 observed in the permeabilized cells. In the proximity of the PH domain, both PLC‐δ1 and p130 possess the EF‐hand domain, containing classical motifs implicated in calcium binding. Therefore, in the present study we examined whether the binding of the PH domain to PtdIns(4,5)P2 is regulated by changes in free Ca2+ concentration within the physiological range. A Ca2+ dependent increase in the binding to PtdIns(4,5)P2 was observed with a full‐length PLC‐δ1, while the isolated PH domain did not show any Ca2+ dependence. However, the connection of the EF‐hand motifs to the PH domain restored the Ca2+ dependent increase in binding, even in the absence of the C2 domain. The p130 protein showed similar properties to PLC‐δ1, and the EF‐hand motifs were again required for the PH domain to exhibit a Ca2+ dependent increase in the binding to PtdIns(4,5)P2. The isolated PH domains from several other proteins which have been demonstrated to bind PtdIns(4,5)P2 showed no Ca2+ dependent enhancement of binding. However, when present within a chimera also containing PLC‐δ1 EF‐hand motifs, the Ca2+ dependent binding was again observed. These results suggest that the binding of Ca2+ to the EF‐hand motifs can modulate binding to PtdIns(4,5)P2 mediated by the PH domain.
Studies of the Escherichia coli Trp repressor binding to its five operators and to variant operator sequences
Authors: Jeeves Mark , Evans Paul D. , Parslow Rosemary A. , Jaseja Mahesh , Hyde Eva I.
Published: March 2003

The Escherichia coli Trp repressor binds to promoters of very different sequence and intrinsic activity. Its mode of binding to trp operator DNA has been studied extensively yet remains highly controversial. In order to examine the selectivity of the protein for DNA, we have used electromobility shift assays (EMSAs) to study its binding to synthetic DNA containing the core sequences of each of its five operators and of operator variants. Our results for DNA containing sequences of two of the operators, trpEDCBA and aroH are similar to those of previous studies. Up to three bands of lower mobility than the free DNA are obtained which are assigned to complexes of stoichiometry 1 : 1, 2 : 1 and 3 : 1 Trp repressor dimer to DNA. The mtr and aroL operators have not been studied previously in vitro. For DNA containing these sequences, we observe predominantly one retarded band in EMSA with mobility corresponding to 2 : 1 complexes. We have also obtained retardation of DNA containing the trpR operator sequence, which has only been previously obtained with super‐repressor Trp mutants. This gives bands with mobilities corresponding to 1 : 1 and 2 : 1 complexes. In contrast, DNA containing containing a symmetrized trpR operator sequence, trpRs, gives a single retarded band with mobility corresponding solely to a 1 : 1 protein dimer–DNA complex. Using trpR operator variants, we show that a change in a single base pair in the core 20 base pairs can alter the number of retarded DNA bands in EMSA and the length of the DNase I footprint observed. This shows that the binding of the second dimer is sequence selective. We propose that the broad selectivity of Trp repressor coupled to tandem 2 : 1 binding, which we have observed with all five operator sequences, enables the Trp repressor to bind to a limited number of sites with diverse sequences. This allows it to co‐ordinately control promoters of different intrinsic strength. This mechanism may be of importance in a number of promoters that bind multiple effector molecules.
An active ribonucleotide reductase from Arabidopsis thaliana
Authors: Sauge‐Merle Sandrine , Falconet Denis , Fontecave Marc
Published: March 2003

In all living organisms, deoxyribonucleotides, the DNA precursors, are produced by reduction of the corresponding ribonucleotides catalyzed by ribonucleotide reductase. In mammals as in Escherichia coli, the enzyme consists of two proteins. Protein R1 is the proper reductase as it contains, in the substrate binding site, the reducing active cysteine pair. Protein R2 provides a catalytically essential organic radical. Here we report the cloning, expression, purification and characterization of protein R1 from Arabidopsis thaliana. Expression in E. coli was made possible by coexpression of tRNAArg4 which is required for the utilization of AGA and AGG as codons for arginines. Protein R1 shows extensive similarities with protein R1 from mammals: (a) it shows 69% amino‐acid sequence identity to human and mouse R1 protein; (b) it is active during CDP reduction by dithiothreitol, in the presence of protein R2 [Sauge‐Merle, S., Laulhère, J.‐P., Coves, J., Ménage, S., Le Pape, L. & Fontecave, M. (1997) J. Biol. Inorg. Chem.2, 586–594]; (c) activity is stimulated by thioredoxin and ATP and is inhibited by dATP, showing that as in the mammalian enzyme, the plant ribonucleotide reductase seems to be allosterically regulated by positive (ATP) and negative (dATP) effectors.
Differential characteristics of human 15‐lipoxygenase isozymes and a novel splice variant of 15S‐lipoxygenase
Authors: Kilty Iain , Logan Alison , Vickers Philip J.
Published: March 2003

The lipoxygenases (LOs) are a family of nonheme iron dioxygenases that catalyse the insertion of molecular oxygen into polyunsaturated fatty acids. Five members of this gene family have been described in man, 5‐LO, 12S‐LO, 12R‐LO, 15‐LO and 15S‐LO. Using partially purified recombinant 15S‐LO enzyme and cells constitutively expressing this protein, we have compared the activity, substrate specificity, kinetic characteristics and regulation of this enzyme to that previously reported for 15‐LO. 15S‐LO has a threefold higher Km, similar Vmax and increased specificity of oxygenation for arachidonic acid, and a similar Km but decreased Vmax for linoleic acid in comparison to 15‐LO. Unlike 15‐LO, 15S‐LO is not suicide inactivated by the products of fatty acid oxygenation. However, in common with other LOs, 15S‐LO activity is regulated through calcium‐dependent association of the enzyme with the membrane fraction of cells.

In addition, whilst independently cloning the recently described 15S‐LO, we identified a splice variant containing an in‐frame 87‐bp deletion corresponding to amino acids 401–429 inclusive. Modelling of the 15S‐LO and subsequent studies with partially purified recombinant protein suggest that the deleted region comprises a complete α‐helix flanking the active site of the enzyme resulting in decreased specificity of oxygenation and affinity for fatty acid substrates.

Alternative splicing of 15S‐LO would therefore provide a further level of regulation of fatty acid metabolism. These results demonstrate that there are substantial differences in the enzyme characteristics and regulation of the 15‐LO isozymes which may reflect differing roles for the proteins in vivo.
Structural analysis of 13 neutral oligosaccharide‐alditols released by reductive β‐elimination from oviducal mucins of Rana temporaria
Authors: Coppin Alexandra , Maes Emmanuel , Morelle Willy , Strecker Gérard
Published: March 2003

Amphibian eggs are always surrounded by an extracellular matrix, named the jelly coat. This is mainly composed of a highly O‐glycosylated, mucin‐type glycoprotein. This work has consisted of isolating O‐linked neutral oligosaccharides from oviducal mucin of Rana temporaria, with a view to determining their primary structure. Hence, these carbohydrate chains have been released by alkaline borohydride treatment leading to stable glycans. The oligosaccharide‐alditols have been purified by ion‐exchange chromatography and separated by HPLC. The primary structure of 13 of these carbohydrate chains have been obtained by 1D/2D 1H‐NMR spectroscopy and methylation analyses, in combination with MALDI‐TOF mass spectroscopy. The results confirm what has been observed for six other amphibians about the species‐specificity of the carbohydrate moieties and their likely involvement in the species‐specific gamete recognition.
Ca2+‐induced p38/SAPK signalling inhibited by the immunosuppressant cyclosporin A in human peripheral blood mononuclear cells
Authors: Kreideweiß Stefan , Ahlers Christoph , Nordheim Alfred , Rühlmann Andreas
Published: March 2003

To understand the effects of the immunosuppressant cyclosporin A (CsA) on Ca2+‐mediated intracellular signalling pathways in human peripheral blood mononuclear cells (PBMCs), we investigated its effects on the activity profiles of mitogen‐activated protein kinase (MAPK) cascades. PBMCs, or subpopulations thereof, were simultaneously stimulated with a phorbol ester and the calcium ionophore ionomycin, in the presence or absence of therapeutic concentrations of CsA. In these primary human cells, CsA significantly inhibited PMA/ionomycin‐mediated and ionomycin‐mediated activation of the MAPK kinase MKK6, as well as its downstream kinases SAPK2a (p38α) and MAPKAP‐K2. PMA/ionomycin treatment also mediated activation of SAPK1 (JNKs) which was inhibited by CsA. Treatment with ionomycin alone also resulted in CsA‐sensitive activation of SAPK1. With regard to transcription factors targeted by the Ca2+‐induced MAPK signalling network, we found CsA to inhibit the ionomycin‐mediated phosphorylation of ATF2 at Thr71. We identified the heterodimeric transcription factor ATF2/CREB as constitutively binding to the essential cAMP response element (CRE) site within the Ca2+‐regulated DNA polymerase β promoter and contributing to the activation of this promoter. Our data implicate ATF2 phosphorylation status as a nuclear sensor within PBMCs that monitors converging intracellular Ca2+‐signalling pathways.
Lack of oxygen sensing by mitochondria in platelets
Authors: Arthur Peter G , Ngo Cam‐Tu , Moretta Paul , Guppy Michael
Published: March 2003

The range over which cells are sensitive to changes in oxygen concentration remains uncertain. Wilson and colleagues [Wilson, D.F. (1994) Med. Sci. Sports Exerc.26, 37–43] have suggested that cytochrome oxidase is sensitive to oxygen concentrations below about 40 µm, but proposed that this sensitivity is obscured in intact cells because an increase in reduction state of cytochrome c acts to maintain oxygen consumption. We have tested this hypothesis in platelets, which are small cells (2–4 µm diameter, < 0.5 µm thick) that do not decrease their rate of oxygen consumption until oxygen concentrations fall below 2.5 µm. Contrary to the expectations of the hypothesis, the reduction state of cytochrome c, the concentration of NADH and the rate of glycolytic output are not changed as oxygen concentration declines from 40 µm down to 5 µm. Therefore, we conclude that at least some cell types contain mitochondria that are not capable of sensing oxygen above 5 µm by the mechanism proposed by Wilson and colleagues.
Determination of the sites required for the allosteric inhibition of serine acetyltransferase by L‐cysteine in plants
Authors: Inoue Kenji , Noji Masaaki , Saito Kazuki
Published: March 2003

Serine acetyltransferase (SATase; EC 2.3.1.30) catalyzes the formation of O‐acetylserine from l‐Ser and acetyl‐CoA in plants and bacteria. In plants, two types of SATase have been described. One is allosterically inhibited by l‐Cys, and the second is not sensitive to l‐Cys inhibition. However, the allosteric site in SATase has not been identified. To understand better the mechanism of l‐Cys inhibition of plant SATases, we constructed several chimeric SATase enzymes from watermelon SATase (WaSATase) (sensitive type) and Arabidopsis SAT‐p (insensitive type). These enzymes were expressed in Escherichia coli, and inhibition of the mutated SATase activity by l‐Cys was analyzed. Mutated WaSATase, in which Met280 was changed to Ile, was no longer inhibited by l‐Cys. Analysis of the inhibition the chimeric enzymes indicated that the C‐terminal region of WaSATase from Pro276 to Phe285, in which five amino acids are different from those of SAT‐p, was responsible for the determination of the sensitivity to l‐Cys. In particular, Gly277 in the C‐terminal region of WaSATase was primarily responsible for the l‐Cys inhibition. The N‐terminal half of the protein, which does not contain the catalytic domain, was also important for the sensitivity to l‐Cys. These results indicate that the sensitivity of SATase to l‐Cys is due to the N‐terminal and C‐terminal regions rather than to the catalytic domain.
Role of N‐glycosylation in cathepsin E
Authors: Yasuda Yoshiyuki , Ikeda Shinobu , Sakai Hideaki , Tsukuba Takayuki , Okamoto Kuniaki , Nishishita Kazuhisa , Akamine Akifumi , Kato Yuzo , Yamamoto Kenji
Published: March 2003

Cathepsin E (CE), a nonlysosomal, intracellular aspartic proteinase, exists in several molecular forms that are N‐glycosylated with high‐mannose and/or complex‐type oligosaccharides. To investigate the role of N‐glycosylation on the catalytic properties and molecular stability of CE, both natural and recombinant enzymes with distinct oligosaccharides were purified from different sources. An N‐glycosylation minus mutant, that was constructed by site‐directed mutagenesis (by changing asparagine residues to glutamine and aspartic acid residues at positions 73 and 305 in potential N‐glycosylation sites of rat CE) and expressed in normal rat kidney cells, was also purified to homogeneity from the cell extracts. The kinetic parameters of the nonglycosylated mutant were found to be essentially equivalent to those of natural enzymes N‐glycosylated with either high‐mannose or complex‐type oligosaccharides. In contrast, the nonglycosylated mutant showed lower pH and thermal stabilities than the glycosylated enzymes. The nonglycosylated mutant exhibited particular sensitivity to conversion to a monomeric form by 2‐mercaptoethanol, as compared with those of the glycosylated enzymes. Further, the high‐mannose‐type enzymes were more sensitive to this agent than the complex‐type proteins. A striking difference was found between the high‐mannose and complex‐type enzymes in terms of activation by ATP at a weakly acidic pH. At pH 5.5, the complex‐type enzymes were stabilized by ATP to be restored to the virtual activity, whereas the high‐mannose‐type enzymes as well as the nonglycosylated mutant were not affected by ATP. These results suggest that N‐glycosylation in CE is important for the maintenance of its proper folding upon changes in temperature, pH and redox state, and that the complex‐type oligosaccharides contribute to the completion of the tertiary structure to maintain its active conformation in the weakly acidic pH environments.
α‐Complemented β‐galactosidase
Authors: Abbas‐Terki Toufik , Picard Didier
Published: March 2003

Intracistronic complementation of N‐terminally truncated β‐galactosidase mutants such as M15 by coexpressed α‐peptide was originally discovered in Escherichia coli and exploited for plasmid cloning as the well‐known blue–white screening method. We show here that α‐complementation also works in the budding yeast Saccharomyces cerevisiae, and that it can be used as a simple nonselective enzymatic marker for a variety of in vivo studies, for example, on the role of molecular chaperones in protein folding and assembly. To be able to induce α‐complementation post‐translationally, we have constructed a hormone‐inducible α‐peptide by fusion of the DNA encoding the α‐peptide to that of to the hormone binding domain of the estrogen receptor. The accumulation of both subunits, the α‐peptide and M15, is severely compromised when they are expressed separately, presumably because their hydrophobic surfaces remain exposed. Moreover, α‐complementation is defective in a strain of S. cerevisiae carrying a point mutant of the molecular chaperone heat‐shock protein 90. Heat‐shock protein 90, which coprecipitates with M15, might be required in vivo to prevent the degradation of unassembled M15 and to hold it in an interaction‐competent conformation.
Structural requirements for V2 vasopressin receptor proteolytic cleavage
Authors: Kojro Elzbieta , Postina Rolf , Gilbert Sandra , Bender Frank , Krause Gerd , Fahrenholz Falk
Published: March 2003

The ligand‐induced proteolytic cleavage of the V2 vasopressin receptor transiently expressed in COS cells was investigated. After incubation of the cell membranes with a photoreactive ligand possessing full agonistic properties for V2 receptors, approximately 90% of the porcine and bovine V2 vasopressin receptors were cleaved in the upper part of transmembrane helix 2 at a heptapeptide sequence conserved in both vasopressin and oxytocin receptors. The oxytocin receptor was completely resistant to proteolysis after binding the same photoreactive ligand, which is only a partial agonist for this receptor. Chimeric V2/oxytocin receptors obtained by transfer of extracellular domains of the oxytocin receptor into the V2 receptor showed an increase in binding affinity for oxytocin versus vasopressin and a diminished cleavage. The proteolysis‐resistant chimeric V2/oxytocin receptor, which contains the first three extracellular domains of the oxytocin receptor, stimulated cAMP accumulation to a larger extent in response to vasopressin than the wild‐type receptor and showed impaired desensitization of the adenylate cyclase system. Our data indicate that the proteolytic cleavage of the V2 receptor requires a defined conformation, especially of the first two extracellular domains that is induced by agonist binding. Furthermore, the results suggest that the proteolytic V2 receptor cleavage might play a role in signal termination at elevated hormone concentrations.
Chemical synthesis, antibacterial activity and conformation of diptericin, an 82‐mer peptide originally isolated from insects
Authors: Cudic Mare , Bulet Philippe , Hoffmann Ralf , Craik David J. , Otvos Jr Laszlo
Published: March 2003

The small amounts of antibacterial peptides that can be isolated from insects do not allow detailed studies of their range of activity, side‐chain sugar requirements, or their conformation, factors that frequently play roles in the mode of action. In this paper, we report the solid‐phase step‐by‐step synthesis of diptericin, an 82‐mer peptide, originally isolated from Phormia terranovae. The unglycosylated peptide was purified to homogeneity by conventional reversed‐phase high performance liquid chromatography, and its activity spectrum was compared to that of synthetic unglycosylated drosocin, which shares strong sequence homology with diptericin's N‐terminal domain. Diptericin appeared to have antibacterial activity for only a limited number of Gram‐negative bacteria. Diptericin's submicromolar potency against Escherichia coli strains indicated that, in a manner similar to drosocin, the presence of the carbohydrate side chain is not necessary to kill bacteria. Neither the N‐terminal, drosocin‐analog fragment, nor the C‐terminal, glycine‐rich attacin‐analog region was active against any of the bacterial strains studied, regardless of whether the Gal‐GalNAc disaccharide units were attached. This suggested that the active site of diptericin fell outside the drosocin or attacin homology domains. In addition, the conformation of diptericin did not seem to play a role in the antibacterial activity, as was demonstrated by the complete lack of ordered structure by two‐dimensional nuclear magnetic resonance spectroscopy and circular dichroism. Diptericin completely killed bacteria within 1 h, considerably faster than drosocin and the attacins; unlike some other, fast‐acting antibacterial peptides, diptericin did not lyse normal mammalian cells. Taken together, these data suggest diptericin does not belong to any known class of antibacterial peptides.
Purification, cDNA cloning and modification of a defensin from the coconut rhinoceros beetle, Oryctes rhinoceros
Authors: Ishibashi Jun , Saido‐Sakanaka Hisako , Yang Jun , Sagisaka Aki , Yamakawa Minoru
Published: March 2003

A novel member of the insect defensins, a family of antibacterial peptides, was purified from larvae of the coconut rhinoceros beetle, Oryctes rhinoceros, immunized with Escherichia coli. A full‐size cDNA was cloned by combining reverse‐transcription PCR (RT‐PCR), and 5′‐ and 3′‐rapid amplification of cDNA ends (RACE). Analysis of the O. rhinoceros defensin gene expression showed it to be expressed in the fat body and hemocyte, midgut and Malpighian tubules. O. rhinoceros defensin showed strong antibacterial activity against Staphylococcus aureus. A 9‐mer peptide amidated at its C‐terminus, AHCLAICRK‐NH2 (Ala22–Lys30‐NH2), was synthesized based on the deduced amino‐acid sequence, assumed to be an active site sequence by analogy with the sequence of a defensin isolated from larvae of the beetle Allomyrina dichotoma. This peptide showed antibacterial activity against S. aureus, methicillin‐resistant S. aureus, E. coli and Pseudomonas aeruginosa. We further modified this oligopeptide and synthesized five 9‐mer peptides, ALRLAIRKR‐NH2, ALLLAIRKR‐NH2, AWLLAIRKR‐NH2, ALYLAIRKR‐NH2 and ALWLAIRKR‐NH2. These oligopeptides showed strong antibacterial activity against Gram‐negative and Gram‐positive bacteria. The antibacterial effect of Ala22–Lys30‐NH2 analogues was due to its interaction with bacterial membranes, judging from the leakage of liposome‐entrapped glucose. These Ala22–Lys30‐NH2 analogues did not show haemolytic activity and did not inhibit the growth of murine fibroblast cells or macrophages, except for AWLLAIRKR‐NH2.
Solution structure of a new hypothalamic neuropeptide, human hypocretin‐2/orexin‐B
Authors: Lee Jung‐Hoon , Bang Eunjung , Chae Kyeong‐Jun , Kim Jin‐Young , Lee Dai Woon , Lee Weontae
Published: March 2003

Orexin‐A and orexin‐B (also called hypocretin‐1 and hypocretin‐2, respectively) are novel hypothalamic neuropeptides encoded by a single mRNA transcript; they stimulate food intake. We have determined the three‐dimensional solution structure of human hypocretin‐2/orexin‐B using two‐dimensional 1H‐NMR data and dynamical simulated annealing calculations. On the basis of NOEs, 3JHNα coupling constants and hydrogen–deuterium exchange rates together with chemical shift indices, human hypocretin‐2/orexin‐B was deduced to consist of two α‐helices connected with a short linker in both H2O and 30% trifluoroethanol solutions. The helical axis of helix I is oriented about 60–80° relative to helix II. Hybrid distance geometry and simulated‐annealing protocols were used to generate an ensemble of 30 structures with no constraint violations greater than 0.03 nm for distances and 3° for angles. In addition, human hypocretin‐2/orexin‐B shares a similar secondary‐structural motif with human neuropeptide Y. This result can form the basis for further study on ligand–receptor recognition of human orexin receptors.
Structural and functional implications of a proline residue in the antimicrobial peptide gaegurin
Authors: Suh Jeong‐Yong , Lee Young‐Tae , Park Chan‐Bae , Lee Keun‐Hyeung , Kim Sun‐Chang , Choi Byong‐Seok
Published: March 2003

Although it is commonly known as a helix breaker, proline residues have been found in the α‐helical regions of many peptides and proteins. The antimicrobial peptide gaegurin displays α‐helical structure and has a central proline residue (P14). The structure and activity of gaegurin and its alanine derivative (P14A) were determined by various spectroscopic methods, restrained molecular dynamics, and biological assays. Both P14 and P14A exhibited cooperative helix formation in solution, but the helical stability of P14 was reduced substantially when compared to that of P14A. Chemical‐shift analysis indicated that both of the peptides formed curved helices and that P14 showed diminished stability in the region around the central proline. However, hydrogen‐exchange data revealed remarkable differences in the location of stable amide protons. P14 showed a stable region in the concave side of the curved helix, while P14A exhibited a stable region in the central turn of the helix. The model structure of P14 exhibited a pronounced kink, in contrast to the uniform helix of P14A. Both peptides showed comparable binding affinities for negatively charged lipids, while P14 had a considerably reduced affinity for a neutral lipid. With its destabilized α‐helix, P14 exhibited greater antibacterial activity than did P14A. Hence, electrostatic interaction between helical peptides and lipid membranes is believed to be the dominant factor for antibacterial activity. Moreover, helical stability can modulate peptide binding to membranes that is driven by electrostatic interactions. The observation that P14 is a more potent antibacterial agent than P14A implies that the helical kink of P14 plays an important role in the disruption of bacterial membranes.
Extensive regulation compromises the extent to which DNA gyrase controls DNA supercoiling and growth rate of Escherichia coli
Authors: Ruhdal Jensen Peter , van der Weijden Coen C. , Bogø Jensen Lars , Westerhoff Hans V. , Snoep Jacky L.
Published: March 2003

As DNA gyrase is the only enzyme to supercoil DNA actively, we address here the question of whether it does play the expected dominant role in controlling the level of DNA supercoiling and growth rate in Escherichia coli. We modulated the expression of DNA gyrase around its wild‐type level, and measured the effect on plasmid supercoiling and growth rate. As both the activity and the transcription rate of DNA gyrase are sensitive to DNA supercoiling we distinguish two types of control (with control defined as the percentage change observed on a 1% modulation of a parameter). The first type of control, here named inherent control, quantifies the effect of a sustained modulation of the transcription rate of gyrase. At its wild‐type expression level this inherent control exerted by DNA gyrase on growth rate was very low, i.e.

, as was the inherent control on DNA supercoiling,

0.2. The second type of control, here named global control, quantifies the effect of a change in gyrase activity whilst allowing the cell to respond by readjusting gyrase transcription. Both types of control are linked via the sensitivity of gyrase transcription to DNA supercoiling, as determined from the inherent control by gyrase of the gyrase promoter activity using a chromosomal gyrB::lacZ fusion. As expected, the latter control was negative, but small, i.e.

. The global control by gyrase of active linking number was 0.1. These results show that although gyrase is an essential enzyme it does not have a high control, on either growth rate or DNA supercoiling. Homeostatic regulation of physiological DNA structure appears to dominate. At low degrees of DNA supercoiling, the control by DNA gyrase and by the other topoisomerases is much stronger.
Analysis of natural and synthetic sphingomyelins using high‐performance thin‐layer chromatography
Authors: Ramstedt Bodil , Leppimäki Petra , Axberg Maria , Slotte J. Peter
Published: March 2003

The chromatographic behaviour of molecular species of sphingomyelin on HPTLC was investigated. Sphingomyelin gave a double band pattern on HPTLC plates developed using chloroform/methanol/acetic acid/water (25 : 15 : 4 : 2, v/v) or chloroform/methanol/water (25 : 10 : 1.1, v/v). HPTLC analysis of acyl chain‐defined sphingomyelins showed that the Rf values increased linearly with the length of the N‐linked acyl chain. A double‐banded pattern was therefore seen for natural sphingomyelins with a bimodal fatty acid composition. Racemic sphingomyelins also gave a double band pattern on HPTLC, where the lower band represented the derythro and the upper band the lthreo isomer. We also showed that derythro‐N‐16:0‐dihydrosphingomyelin migrated faster on HPTLC than derythro‐N‐16:0‐sphingomyelin. The upper and lower band sphingomyelins from two different cell lines (human skin fibroblasts and baby hamster kidney cells) were separately scraped off the HPTLC plates and the fatty acid and long‐chain base profiles were studied using GC‐MS. The lower bands contained short‐chain fatty acids and most of the fatty acids in the upper bands were long. The predominant long‐chain base was sphingosine, which was found in both upper and lower bands, but sphinganine was found only in the upper bands. To conclude, there are at least three possible reasons for the sphingomyelin double bands on HPTLC; acyl chain length, long‐chain base composition and stereochemistry. These reasons might sometimes overlap and, therefore, HPTLC alone is insufficient for complete analysis of the molecular species of sphingomyelin.
Prediction of protein (domain) structural classes based on amino‐acid index
Authors: Bu Wei‐Shu , Feng Zhi‐Ping , Zhang Ziding , Zhang Chun‐Ting
Published: March 2003

A protein (domain) is usually classified into one of the following four structural classes: all‐α, all‐β, α/β and α + β. In this paper, a new formulation is proposed to predict the structural class of a protein (domain) from its primary sequence. Instead of the amino‐acid composition used widely in the previous structural class prediction work, the auto‐correlation functions based on the profile of amino‐acid index along the primary sequence of the query protein (domain) are used for the structural class prediction. Consequently, the overall predictive accuracy is remarkably improved. For the same training database consisting of 359 proteins (domains) and the same component‐coupled algorithm [Chou, K.C. & Maggiora, G.M. (1998) Protein Eng.11, 523–538], the overall predictive accuracy of the new method for the jackknife test is 5–7% higher than the accuracy based only on the amino‐acid composition. The overall predictive accuracy finally obtained for the jackknife test is as high as 90.5%, implying that a significant improvement has been achieved by making full use of the information contained in the primary sequence for the class prediction. This improvement depends on the size of the training database, the auto‐correlation functions selected and the amino‐acid index used. We have found that the amino‐acid index proposed by Oobatake and Ooi, i.e. the average nonbonded energy per residue, leads to the optimal predictive result in the case for the database sets studied in this paper. This study may be considered as an alternative step towards making the structural class prediction more practical.
The role of cysteine residues in structure and enzyme activity of a maize β‐glucosidase
Authors: Rotrekl Vladimír , Nejedlá Eliška , Kučera Igor , Abdallah Fuad , Palme Klaus , Brzobohatý Břetislav
Published: March 2003

The maize Zm‐p60.1 gene encodes a β‐glucosidase that can release active cytokinins from their storage forms, cytokinin‐O‐glucosides. Mature catalytically active Zm‐p60.1 is a homodimer containing five cysteine residues per a subunit. Their role was studied by mutating them to alanine (A), serine (S), arginine (R) or aspartic acid (D) using site‐directed mutagenesis, and subsequent heterologous expression in Escherichia coli. All substitutions of C205 and C211 resulted in decreased formation and/or stability of the homodimer, manifested as accumulation of high levels of monomer in the bacterial expression system. Examination of urea‐ and glutathione‐induced dissociation patterns of the homodimer to the monomers, HPLC profiles of hydrolytic fragments of reduced and oxidized forms, and a homology‐based three‐dimensional structural model revealed that an intramolecular disulfide bridge formed between C205 and C211 within the subunits stabilized the quaternary structure of the enzyme. Mutating C52 to R produced a monomeric enzyme protein, too. No detectable effects on homodimer formation were apparent in C170 and C479 mutants. Given the Km values for C170A/S mutants were equal to that for the wild‐type enzyme, C170 cannot participate in enzyme–substrate interactions. Possible indirect effects of C170A/S mutations on catalytic activity of the enzyme were inferred from slight decreases in the apparent catalytic activity, k′cat. C170 is located on a hydrophobic side of an α‐helix packed against hydrophobic amino‐acid residues of β‐strand 4, indicating participation of C170 in stabilization of a (β/α)8 barrel structure in the enzyme. In C479A/D/R/S mutants, Km and k′cat were influenced more significantly suggesting a role for C479 in enzyme catalytic action.
Chicken cystatin stimulates nitric oxide release from interferon‐γ‐activated mouse peritoneal macrophages via cytokine synthesis
Authors: Verdot Ludovic , Lalmanach Gilles , Vercruysse Vincent , Hoebeke Johan , Gauthier Francis , Vray Bernard
Published: March 2003

Cystatins are natural tight‐binding, reversible inhibitors of cysteine proteases. We have shown that cystatins also stimulate nitric oxide (NO) production by interferon‐γ‐activated mouse peritoneal macrophages [Verdot, L., Lalmanach, G., Vercruysse, V., Hartman, S., Lucius, R., Hoebeke, J., Gauthier F. & Vray, B. (1996) J. Biol. Chem. 271, 28077–28081]. The present study was undertaken to further document this new function. Macrophages activated with interferon‐γ and then stimulated with interferon‐γ plus chicken cystatin generated increased amounts of NO in comparison with macrophages only activated with interferon‐γ. Interferon‐γ‐activated macrophages must be incubated with chicken cystatin for at least 8 h to upregulate NO production. NO induction was due to increased inducible nitric oxide synthase protein synthesis. Macrophages incubated with chicken cystatin alone or with interferon‐γ plus chicken cystatin produced increased amounts of both tumor necrosis factor α and interleukin 10. The addition of recombinant murine tumor necrosis factor α alone or in combination with recombinant murine interleukin‐10 mimicked the effect of chicken cystatin. The addition of neutralizing anti‐(tumor necrosis factor α) antibodies reduced sharply NO production by chicken cystatin/interferon‐γ‐activated mouse peritoneal macrophages. Taken together, these data suggest that chicken cystatin induces the synthesis of tumor necrosis factor α and interleukin 10. In turn, these two cytokines stimulate the production of NO by interferon‐γ‐activated macrophages. The findings point to a new relationship between cystatins, cytokines, inflammation and the immune response.
Insulin‐like growth factor‐I (IGF‐I)‐dependent activation of pp42/44 mitogen‐activated protein kinase occurs independently of IGF‐I receptor kinase activation and IRS‐1 tyrosine phosphorylation
Authors: Yau Lorraine , Lukes Helena , McDiarmid Heather , Werner Julieta , Zahradka Peter
Published: March 2003

The proliferation and metabolism of H4IIE hepatoma cells is apparently mediated through the insulin receptor. These cells, however, also have high‐affinity binding sites for insulin‐like growth factor‐I (IGF‐I). Addition of insulin to H4IIE cells increased RNA synthesis, DNA synthesis and cell number. IGF‐I, on the other hand, was ineffective at concentrations equivalent to the lowest effective insulin dose, although stimulation was observed with concentrations 100‐fold higher. Similar results were obtained when glucose uptake was measured. Western blot analysis demonstrated that tyrosine phosphorylation patterns produced by insulin and IGF‐I differed. In particular, phosphorylation of insulin receptor substrate‐1 (IRS‐1) was evident after treatment with insulin, but not after treatment with IGF‐I. Correspondingly, insulin, but not IGF‐I, stimulated receptor tyrosine kinase activity. In contrast with these results, both insulin and IGF‐I induced mitogen‐activated protein (MAP) kinase phosphorylation and activity at a concentration of 10 nm. The correlation between insulin‐dependent and IGF‐I‐dependent MAP kinase activation was confirmed by Western blot analysis of phosphorylated MAP kinase kinase (MEK). These results suggest that phosphorylation of IRS‐1 is essential for both cell proliferation and glucose metabolism, but is uncoupled from the MAP kinase cascade. Furthermore, stimulation of MEK and MAP kinase is independent of receptor tyrosine kinase activity.
Determination of solution conformations of PrP106–126, a neurotoxic fragment of prion protein, by 1H NMR and restrained molecular dynamics
Authors: Ragg Enzio , Tagliavini Fabrizio , Malesani Paolo , Monticelli Luca , Bugiani Orso , Forloni Gianluigi , Salmona Mario
Published: March 2003

Experimental two‐dimensional 1H NMR data have been obtained for PrP106–128 under the following solvent conditions: deionized water/2,2,2‐trifluoroethanol 50 : 50 (v/v) and dimethylsulfoxide. These data were analyzed by restrained molecular mechanics calculations to determine how changes in solvation affect the conformation of the peptide. In deionized water at pH 3.5, the peptide adopted a helical conformation in the hydrophobic region spanning residues Met112–Leu125, with the most populated helical region corresponding to the Ala115–Ala119 segment (≈ 10%). In trifluoroethanol/H2O, the α‐helix increased in population especially in the Gly119–Val122 tract (≈ 25%). The conformation of this region was found to be remarkably sensitive to pH, as the Ala120–Gly124 tract shifted to an extended conformation at pH 7. In dimethylsulfoxide, the hydrophobic cluster adopted a prevalently extended conformation. For all tested solvents the region spanning residues Asn108–Met112 was present in a ‘turn‐like’ conformation and included His111, situated just before the starting point of the α‐helix. Rather than by conformational changes, the effect of His111 is exerted by changes in its hydrophobicity, triggering aggregation. The amphiphilic properties and the pH‐dependent ionizable side‐chain of His111 may thus be important for the modulation of the conformational mobility and heterogeneity of PrP106–126.
Purification and primary structure of a macromolecular‐translocation inhibitor II of glucocorticoid‐receptor binding to nuclei from rat liver
Authors: Okamoto Kazuki , Isohashi Fumihide
Published: March 2003

The nuclear binding of the activated glucocorticoid‐receptor (GR) is inhibited by endogenous macromolecules in vitro. Previously, we have separated the inhibitors into three species (MTI‐I, MTI‐II and MTI‐III). In this study, we purified the most potent of the three species (MTI‐II) from the livers of adrenalectomized rats to apparent homogeneity as judged by two‐dimensional PAGE. Purified MTI‐II inhibits GR binding to DNA containing glucocorticoid‐response elements. To obtain the amino acid sequence of MTI‐II, we digested the MTI‐II with endopeptidases. The N‐terminal amino acid sequences of the four digested fragments indicated that MTI‐II is an 11.5‐kDa Zn2+‐binding protein (ZnBP, also known as parathymosin). Furthermore, we purified ZnBP to apparent homogeneity and found that it also inhibits GR binding to nuclei. ZnBP is known to be an abundant acidic protein involved in cell proliferation, and interacts with histone H1 or key enzymes of carbohydrate metabolism via its acidic domain. We also showed that the inhibition of GR binding to nuclei is mediated by the acidic domain of MTI‐II (ZnBP, parathymosin) and that GR binds to the MTI‐II affinity matrix. Our findings add a new biological function, i.e. the inhibition of GR binding to nuclei and DNA, to this ZnBP. Moreover, our findings suggest that the abundant acidic protein is involved in glucocorticoid action.
A spectroscopic assay for the analysis of carbohydrate transport reactions
Authors: Heuberger Esther H. M. L. , Poolman Bert
Published: March 2003

A carbohydrate‐transport assay was developed that does not require isotopically labelled substrates, but allows transport reactions to be followed spectrophotometrically. It makes use of a membrane system (hybrid membranes or proteoliposomes) bearing the transport system of interest, and a pyrroloquinoline quinone‐dependent aldose dehydrogenase [soluble glucose dehydrogenase (sGDH)] and the electron acceptor 2,6‐dichloroindophenol (Cl2Ind) enclosed in the vesicle lumen. After transport across the vesicular membrane, the sugar is oxidized by sGDH. The accompanying reduction of Cl2Ind results in a decrease in A600. The assay was developed and optimized for the lactose carrier (LacS) of Streptococcus thermophilus, and both solute/H+ symport and exchange types of transport could be measured with high sensitivity in crude membranes as well as in proteoliposomes. To observe exchange transport, the membranes were preloaded with a nonoxidizable substrate analogue and diluted in assay buffer containing an oxidizable sugar. Transport rates measured with this assay are comparable with those obtained with the conventional assay using isotopically labelled substrates. The method is particularly suited for determining transport reactions that are not coupled to any form of metabolic energy such as uniport reactions, or for characterizing mutant proteins with a defective energy‐coupling mechanism or systems with high‐affinity constants for sugars.
NMR solution structure of a novel thioredoxin from Bacillus acidocaldarius
Authors: Nicastro Giuseppe , de Chiara Cesira , Pedone Emilia , Tatò Marco , Rossi Mosè , Bartolucci Simonetta
Published: March 2003

The thioredoxin (Trx) from Bacillus acidocaldarius (BacTrx), an eubacterium growing optimally at 333 K, is the first Trx described to date from a moderate thermophilic source. To understand the molecular basis of its thermostability, the three‐dimensional structure in the oxidized form was determined by NMR methods. A total of 2276 1H‐NMR derived distance constraints along with 23 hydrogen‐bonds, 72 φ and 27 χ1 torsion angle restraints, were used in a protocol employing simulated annealing followed by restrained molecular dynamics and restrained energy minimization.

BacTrx consists of a well‐defined core region of five strands of β‐sheet, surrounded by four exposed α‐helices, features shared by other members of the thioredoxin family. The BacTrx 3D structure was compared with the Escherichia coli Trx (EcTrx) determined by X‐ray crystallographic diffraction, and a number of structural differences were observed that may contribute to its thermostabilty. The results of structural analysis indicated that protein stability is due to cumulative effects, the main factor being an increased number of ionic interactions cross‐linking different secondary structural elements and clamping the C‐terminal α‐helix to the core of the protein.
Photo‐induced cyclic electron transfer involving cytochrome bc1 complex and reaction center in the obligate aerobic phototroph Roseobacter denitrificans
Authors: Schwarze Claudia , Carluccio Anna V. , Venturoli Giovanni , Labahn Andreas
Published: March 2003

Flash‐induced redox changes of b‐type and c‐type cytochromes have been studied in chromatophores from the aerobic photosynthetic bacterium Roseobacter denitrificans under redox‐controlled conditions. The flash‐oxidized primary donor P+ of the reaction center (RC) is rapidly re‐reduced by heme H1 (Em,7 = 290 mV), heme H2 (Em,7 = 240 mV) or low‐potential hemes L1/L2 (Em,7 = 90 mV) of the RC‐bound tetraheme, depending on their redox state before photoexcitation. By titrating the extent of flash‐induced low‐potential heme oxidation, a midpoint potential equal to −50 mV has been determined for the primary quinone acceptor QA. Only the photo‐oxidized heme H2 is re‐reduced in tens of milliseconds, in a reaction sensitive to inhibitors of the bc1 complex, leading to the concomitant oxidation of a cytochrome c spectrally distinct from the RC‐bound hemes. This reaction involves cytochrome c551 in a diffusional process. Participation of the bc1 complex in a cyclic electron transfer chain has been demonstrated by detection of flash‐induced reduction of cytochrome b561, stimulated by antimycin and inhibited by myxothiazol. Cytochrome b561, reduced upon flash excitation, is re‐oxidized slowly even in the absence of antimycin. The rate of reduction of cytochrome b561 in the presence of antimycin increases upon lowering the ambient redox potential, most likely reflecting the progressive prereduction of the ubiquinone pool. Chromatophores contain approximately 20 ubiquinone‐10 molecules per RC. At the optimal redox poise, approximately 0.3 cytochrome b molecules per RC are reduced following flash excitation. Cytochrome b reduction titrates out at Eh < 100 mV, when low‐potential heme(s) rapidly re‐reduce P+ preventing cyclic electron transfer. Results can be rationalized in the framework of a Q‐cycle‐type model.
Pigments and proteins in green bacterial chlorosomes studied by matrix‐assisted laser desorption ionization mass spectrometry
Authors: Persson Søren , Sönksen Carsten P. , Frigaard Niels‐Ulrik , Cox Raymond P. , Roepstorff Peter , Miller Mette
Published: March 2003

We have used matrix‐assisted laser desorption ionization time‐of‐flight mass spectrometry (MALDI‐TOF‐MS) for mass determination of pigments and proteins in chlorosomes, the light‐harvesting organelles from the photosynthetic green sulfur bacterium Chlorobium tepidum. By applying a small volume (1 µL) of a concentrated suspension of isolated chlorosomes directly to the target of the mass spectrometer we have been able to detect bacteriochlorophyll a and all the major homologs of bacteriochlorophyll c. The peak heights of the different bacteriochlorophyll c homologs in the MALDI spectra were proportional to peak areas obtained from HPLC analysis of the same sample. The same result was also obtained when whole cells of Chl. tepidum were applied to the target, indicating that MALDI‐MS can provide a rapid method for obtaining a semiquantitative determination or finger‐print of the bacteriochlorophyll homologs in a small amount of green bacterial cells. In addition to information on pigments, the MALDI spectra also contained peaks from chlorosome proteins. Thus we have been able with high precision to confirm the molecular masses of the chlorosome proteins CsmA and CsmE which have been previously determined by conventional biochemical and genetic methods, and demonstrate the presence of truncated versions of CsmA and CsmB.
Thermal unfolding of G‐actin monitored with the DNase I‐inhibition assay
Authors: Schüler Herwig , Lindberg Uno , Schutt Clarence E. , Karlsson Roger
Published: March 2003

Actin is one of the proteins that rely on chaperonins for proper folding. This paper shows that the thermal unfolding of G‐actin, as studied by CD and ultraviolet difference spectrometry, coincides with a loss in DNase I‐inhibiting activity of the protein. Thus, the DNase I inhibition assay should be useful for systematic studies of actin unfolding and refolding. Using this assay, we have investigated how the thermal stability of actin is affected by either Ca2 + or Mg2 + at the high affinity divalent cation binding site, by the concentration of excess nucleotide, and by the nucleotide in different states of phosphorylation (ATP, ADP.Pi, ADP.Vi, ADP.AlF4, ADP.BeFx, and ADP). Actin isoforms from different species were also compared, and the effect of profilin on the thermal stability of actin was studied. We conclude that the thermal unfolding of G‐actin is a three‐state process, in which an equilibrium exists between native actin with bound nucleotide and an intermediate free of nucleotide. Actins in the Mg‐form were less stable than the Ca‐forms, and the stability of the different isoforms decreased in the following order: rabbit skeletal muscle α‐actin =  bovine cytoplasmic γ‐actin >  yeast actin >  cytoplasmic β‐actin. The activation energies for the thermal unfolding reactions were in the range 200–290 kJ·mol− 1, depending on the bound ligands. Generally, the stability of the actin depended on the degree with which the nucleotide contributed to the connectivity between the two domains of the protein.