bluuebunny/crossref_metadata_2025 huggingface dataset:
Determination of Kinetic Constants for the Interaction Between the Platelet Glycoprotein IIb‐IIIa and Fibrinogen by Means of Surface Plasmon Resonance
Authors: Huber Walter , Hurst Jürg , Schlatter Daniel , Barner Richard , Hübscher Josef , Kouns William C. , Steiner Beat
Published: July 2010

The binding reaction between purified human platelet glycoprotein IIb‐IIIa and fibrinogen was investigated by real‐time measurements using the surface‐plasmon‐resonance sensor technology. In these experiments, either glycoprotein IIb‐IIIa or fibrinogen was immobilized on a sensor surface. The time‐dependent change in surface coverage that occurred immediately upon contact with a solution of the complementary protein was then detected. The ability to record this dynamic event from its initiation allowed the collection of kinetic and thermodynamic data over an extended time period. These data indicated that initially, in fast reaction, a reversible low‐affinity complex with an equilibrium dissociation constant, Kd, of 155–180 nM was formed. In a subsequent slower reaction this complex was transformed into a more stable high‐affinity complex with a Kd of 20–70 nM. Efficient dissociation of the high‐affinity complex could only be induced in the presence of a competitive inhibitor such as RGDV. These data demonstrate that the binding between glycoprotein IIb‐IIIa and fibrinogen is not a single monophasic reaction, but is composed of at least two consecutive processes both with their own kinetics.
Expression of the AAC2 Gene Encoding the Major Mitochondrial ADP/ATP Carrier in Saccharomyces cerevisiae is Controlled at the Transcriptional Level by Oxygen, Heme and HAP2 Factor
Authors: Betina Svätopluk , Gavurníková Gabriela , Haviernik Peter , Šabová L'udmila , Kolarov Jordan
Published: July 2010

Expression of the Saccharomyces cerevisiae AAC2 gene encoding the major mitochondrial ADP/ATP carrier was examined. The intracellular level of the carrier protein, as well as the level of the AAC2 ‐gene‐specific mRNA, is influenced by the presence or absence of oxygen or of heme, and it is subject to carbon‐source control. In addition, the expression of AAC2 gene requires the products of the HAP2 and HAP3 genes, but not that of the HAP1 gene. The 5′‐flanking region of the gene was isolated, sequenced and fused to the lacZ reporter gene in order to study the effect of carbon sources and of specific deletion mutations on expression of the gene in yeast transformants. The expression of the reporter gene reveals that the AAC2 gene possesses a strong inducible promoter. The promoter analysis, combined with expression studies in the wild‐type as well as in various mutant strains, identified an upstream activation site (UAS) contained within a sequence between −393 bp and −268 bp, and several major initiation sites of AAC2 mRNA between −105 bp and −95 bp. Deletion analysis also shows that the TATA boxes located 45 bp and 104 bp upstream of the 5′‐ends of AAC2 mRNA are not essential for the transcription. The UAS of the AAC2 gene is required for activation by HAP2 and heme and for release from glucose repression. A restriction fragment containing the UAS conferred oxygen and carbon source regulation when placed upstream of another yeast gene encoding ADP/ATP carrier (AAC3), deleted of its regulatory sequences. The UAS of the AAC2 gene contains at least two distinct motifs for DNA‐binding transcriptional activators, including one which is identical with the core HAP2/3/4 binding motif, and a second one with the ABF1 consensus binding sequence. Our results indicate that these sequences mediate the effects of the respective transactivator on the oxygen‐ and carbon‐source‐dependent transcription of the AAC2 gene.
Substrate Specificity and Detailed Characterization of a Bifunctional Amylase‐Pullulanase Enzyme from Bacillus circulans F‐2 Having Two Different Active Sites on One Polypeptide
Authors: Kim Cheorl‐Ho , Kim Yong Sung
Published: July 2010

Bacillus circulans F‐2 amylase‐pullulanase enzyme (APE) displayed dual activity with respect to glycosidic bond cleavage. The enzyme was active on α‐1,6 bonds in pullulan, amylopectin, and glycogen, while it showed α‐1,4 activity against malto‐oligosaccharides, amylose, amylopectin, and soluble starch, but not pullulan. Kinetic analysis of the purified enzyme in a system which contained both pullulan and amylose as two competing substrates was used to distinguish the dual specificity of the enzyme from the single‐substrate specificity known for pullulanases and α‐amylases. Enzyme activities were inhibited by some metal ions, and by metal‐chelating agents with a different mode. The enzyme‐inhibitory results of amylase and pullulanase with Hg2+ and Co2+ ions were different, indicating that the activation mechanisms of both enzyme activities are different. Cyclomaltoheptaose inhibited both α‐amylase and pullulanase activities with inhibition constants (Ki) of 0.029 and 0.06 mg/ml, respectively. Modification with 1‐(3‐dimethylaminopropyl)‐3‐ethylcarbodiimide confirmed a carboxy group at the active sites of both enzymes. The N‐terminal sequence of the enzyme was: Ala‐Asp‐Ala‐Lys‐Lys‐Thr‐Pro‐Gln‐Gln‐Gln‐Phe‐Asp‐Ala‐Leu‐Trp‐Ala‐Ala‐Gly‐ILe‐Val‐Trp‐Gly‐Thr‐Pro‐Asp‐Gly‐Phe. The purified enzyme displayed Michaelis constant (Km) values of 0.55 mg/ml for amylose, and 0.71 mg/ml for pullulan. When both amylose and pullulan were simultaneously present, the observed rate of product formation closely fitted a kinetic model in which the two substrates are hydrolyzed at different active sites. These results suggest that amylopullulanases, which possess both α‐1,6 and α‐1,4 cleavage activities at the same active site, should be distinguished from APEs, which contain both activities at different active sites on the same polypeptide. Also, it is proposed that the Enzyme Commission use the term ‘amylase‐pullulanase enzyme’ to refer to enzymes which act on starch and cleave both α‐1,6‐bonds in pullulan and α‐1,4 bonds in amylose at different active sites.
Insights into Thermal Stability from a Comparison of the Glutamate Dehydrogenases from Pyrococcus furiosus and Thermococcus litoralis
Authors: Britton K. Linda , Baker Patrick J. , Borges Kimberley M. M. , Engel Paul C. , Pasquo Alessandra , Rice David W. , Robb Frank T. , Scandurra Roberto , Stillman Timothy J. , Yip Kitty S. P.
Published: July 2010

In the light of the solution of the three‐dimensional structure of the NAD+‐linked glutamate dehydrogenase from the mesophile Clostridium symbiosum, we have undertaken a detailed examination of the alignment of the sequences for the thermophilic glutamate dehydrogenases from Thermococcus litoralis and Pyrococcus furiosus against the sequence and the molecular structure of the glutamate dehydrogenase from C. symbiosum, to provide insights into the molecular basis of their thermostability. This homology‐based modelling is simplified by the relatively small number of amino acid substitutions between the two thermophilic glutamate dehydrogenase sequences. The most frequent amino acid exchanges involve substitutions which increase the hydrophobicity and sidechain branching in the more thermostable enzyme; particularly common is the substitution of valine to isoleucine. Examination of the sequence differences suggests that enhanced packing within the buried core of the protein plays an important role in maintaining stability at extreme temperatures. One hot spot for the accumulation of exchanges lies close to a region of the molecule involved in its conformational flexibility and these changes may modulate the dynamics of this enzyme and thereby contribute to increased stability.
A Single Point Mutation in the Splice Donor Site of the Low‐Density‐Lipoprotein‐Receptor Gene Produces Intron Read‐Through, Exon‐Skipped and Cryptic‐Site‐Utilized Transcripts
Authors: Maruyama Takao , Miyake Yasuko , Tajima Shoji , Funahashi Tohru , Matsuzawa Yuji , Yamamoto Akira
Published: July 2010

Familial hypercholesterolemia is a genetic disorder caused by mutations of the low‐density‐lipoprotein (LDL) receptor gene. We characterized the structures of LDL receptor mRNA transcripts in the fibroblasts of a homozygous patient carrying a single base substitution (T→C) at the 5′ splice donor site of intron 12 of the LDL receptor gene. We identified three aberrant transcripts as a consequence of intron‐12 read‐through, exon‐12 skipping and utilization of a cryptic splice donor site. Only a point mutation at the 5′ splice donor site caused the production of three alternatively spliced products. None of these transcripts produced a functional LDL receptor protein in this patient.
Comparative Study on the Conformation of Phalloidin, Viroisin, and Related Derivatives in Aqueous Solution
Authors: Kobayashi Naohiro , Endo Satoshi , Kobayashi Hideyuki , Faulstich Heinz , Wieland Theodor , Munekata Eisuke
Published: July 2010

We investigated the conformations of toxic phalloidin and viroisin in aqueous solution using 500–MHz 1H‐NMR spectroscopy in conjunction with molecular modeling. The conformations of two non‐toxic phalloidin derivatives, secophalloidin and dethiophalloidin, were also correspondingly studied for comparison purposes. Results indicate that the non‐toxic peptides have a multiple conformation, whereas the toxic peptides are comprised of a rigid molecule. It was found that the conformation of phalloidin partially resembles that of viroisin in the region of Cys3‐Pro4‐Ala5‐Trp6, being different from that of the non‐toxic peptides; thereby suggesting this region plays an important role leading to their toxicity.
Influence of Ca2+ on Conformation and Stability of Three Bacterial Hybrid Glucanases
Authors: Welfle Karin , Misselwitz Rolf , Welfle Heinz , Politz Oliver , Borriss Rainer
Published: July 2010

The three hybrid glucanases (1–12)AMY · MAC(13–214), (1–12)AMY · des‐Tyr13MAC(14–214); (1–16)AMY · MAC(17–214) are composed of short N‐terminal segments of 12 or 16 amino acid residues derived from the Bacillus amyloliquefaciens glucanase (AMY) and of residues 13–214, 14–214 and 17–214, respectively, derived from the Bacillus macerans enzyme (MAC). The three proteins have similar conformational features as shown by the similar characteristics of their CD spectra in the far‐ and near‐ultraviolet region. A metal‐ion‐binding site was identified in the hybrid glucanase (1–16)AMY · MAC(17–214) by a crystal structure analysis [Keitel, T., Simon, O., Borriss, R. & Heinemann, U. (1993) Proc. Natl Acad. Sci. USA 90, 5287–5291]. Only minor conformational changes of the three hybrid glucanases were observed depending on the presence or absence of Ca2+ ions but for (1–16)AMY · MAC(17–214) and (1–12)AMY · des‐Tyr13MAC(14–214) the occupation of this metal‐binding site by a Ca2+ ion is connected with a large increase of the stability against thermal and chemical unfolding. Surprisingly, for (1–12)AMY · MAC(13–214), which differs from (1–12)AMY · des‐Tyr13MAC(14–214) by only one additional amino acid in an N‐terminal loop region, the effect of Ca2+ ions on the stability is small. The exchange of a few amino acid residues near the N‐terminus of the B. macerans glucanase against amino acids found at comparable positions in the B. amyloliquefaciens glucanase seems to influence very strongly the strength of the Ca2+ binding site and concomitantly the stability of the hybrid glucanases.
Relationship of Subunit 8 of Yeast ATP Synthase and the Inner Mitochondrial Membrane
Authors: Papakonstantinou Theo , Law Ruby H. P. , Manon Stephen , Devenish Rodney J. , Nagley Phillip
Published: July 2010

A molecular genetic approach has been used to test the proposition that the central hydrophobic domain of yeast mitochondrial ATP synthase subunit 8 represents a transmembrane stem in contact with the lipid bilayer. The rationale for this approach is the general inability of membrane bilayers to accomodate unshielded charged residues of polypeptide chains. Non‐polar residues at several positions within the central hydrophobic domain of subunit 8 were replaced with the positively charged amino acid lysine. This was done in an attempt to disrupt subunit 8 function, and thereby determine the boundaries of the putative transmembrane stem. Each subunit 8 variant was allotopically expressed in vivo as a mitochondrial import precursor encoded by a nuclear gene. It was found that all variants, which included proteins carrying two lysines at various positions in the hydrophobic domain, exhibited the ability to restore growth of subunit‐8‐deficient cells on the non‐fermentable substrate ethanol. This indicated that the function of none of these subunit 8 variants was severely compromised. There was also no detectable change in the proteolipid characteristics of subunit 8, as defined by the chloroform/methanol solubility properties of variant proteins extracted from membranes following import into isolated mitochondria. These data suggest that subunit 8 is located in a hydrophobic niche in the mitochondrial ATP synthase, probably in contact with other protein subunits of the complex. We conclude that the function of subunit 8 does not necessarily require it to be integrated within the inner mitochondrial membrane, in contact with the lipid bilayer. Our findings also suggest that hydropathy plots, indicating hydrophobic domains within polypeptides, cannot reliably be interpreted as transmembrane helices in the absence of independent evidence.
Characterization and Expression of Multiple Alternatively Spliced Transcripts of the Goodpasture Antigen Gene Region
Authors: Penadés José R. , Bernal Dolores , Revert Fernando , Johansson Charlott , Fresquet Vicente J. , Cervera Javier , Wieslander Jorgen , Quinones Susan , Saus Juan
Published: July 2010

Collagen IV, the major component of basement membranes, is composed of six distinct α chains (α1–α6). Atypically among the collagen IV genes, the exons encoding the carboxyl‐terminal region of the human α3(IV) chain undergo alternative splicing. This region has been designated as the Goodpasture antigen because of its reactivity in the kidney and lung with the pathogenic autoantibodies causing Good‐pasture syndrome. The data presented in this report demonstrate that, in human kidney, the gene region encompassing the Goodpasture antigen generates at least six alternatively spliced transcripts predicting five distinct proteins that differ in their carboxyl‐terminus and retain, except in one case, the exon that harbors the characteristic amino‐terminus of the antigen. Goodpasture antibodies specifically recognize recombinant proteins representing the antigen and the alternative form that retains the amino‐half of the antigen, suggesting that this moiety could be involved in the in vivo binding of the pathogenic antibodies. Furthermore, the sera of control individuals contain autoantibodies against the antigen that can be differentiated from those causing the syndrome based on their specific reactivities, suggesting that the binding of the pathogenic autoantibodies to a specific determinant likely trigger a distinct and unique cascade of events causing the disease.
C‐Terminus or Juxtamembrane Deletions in the Insulin Receptor do not Affect the Glucose‐Dependent Inhibition of the Tyrosine Kinase Activity
Authors: Mosthaf Luitgard , Berti Lucia , Kellerer Monika , Mushack Joanne , Seffer Eva , Bossenmaier Birgit , Coghlan Mathew , Siddle Kenneth , Ullrich Axel , Häring Hans Ulrich
Published: July 2010

We have previously shown, in rat‐1 fibroblasts which stably overexpress high levels of human insulin receptor (HIR), that high glucose levels induce an inhibition of insulin receptor tyrosine kinase (IRK) activity [Berti, L., Mosthaf, L., Kellerer, M., Tippmer, S., Mushack, J., Seffer, E., Seedorf, K., Häring, H. (1994) J. Biol. Chem. 269, 3381–3386]. This effect appears to be mediated through activation of protein kinase C and phosphorylation of the receptor β‐subunit on threonine or serine residues. The aim of the present study was to determine whether the juxtamembrane region or the C‐terminus tail of the receptor are involved in the IRK modulation by glucose. In these domains increased serine and threonine phosphorylation was observed after phorbol ester or insulin stimulation of cells, and a regulatory function for IRK activity seems conceivable. We used an antibody directed against one potential regulatory site in the C‐terminus tail, i.e. PSer1315, to study the effect of glucose. An increased signal was detected in HIR from rat‐1 fibroblasts treated with phorbol 12‐myristate 13‐acetate or glucose (25 mM). To investigate whether this site in the C‐terminus is essential for glucose‐dependent IRK inhibition, rat‐1 fibroblasts stably overexpressing a C‐terminus‐truncated human insulin receptor lacking 43 amino acids (HIRΔCT) were studied in parallel with cells expressing the wild‐type receptor. As described earlier, HIRΔCT has lost the ability to stimulate glucose uptake. Glucose (25 mM) inhibited the insulin effect on the autophosphorylation of both receptors to a similar extent. Thus, glucose (25 mM) stimulates phosphorylation of Ser1315, however, this appears not to mediate the inhibitory effect on IRK. To test whether serine residues 955/956 and 962/964 in the juxtamembrane region of the insulin receptor are involved in the inhibitory effect of glucose, 293 cells transiently transfected either with wild‐type HIR or HIR with a juxtamembrane deletion spanning amino acids 954–965 [des‐(954–965)‐HIR] were studied in parallel. As described earlier, the des‐(954–965)‐HIR has lost the ability to stimulate PI‐3 kinase. However, 25 mM glucose equally inhibited the insulin effect on tyrosine phosphorylation of the receptor. Together, the data suggest that the regulatory serine or threonine phosphorylation site(s) involved in the inhibitory effect of hyperglycemia are neither located in the C‐terminus nor in the juxtamembrane region of the insulin receptor β subunit.
Evidence for Transcriptional Induction of the Liver Fatty‐Acid‐Binding‐Protein Gene by Bezafibrate in the Small Intestine
Authors: Mallordy Anne , Poirier Hélène , Besnard Philippe , Niot Isabelle , Carlier Hélène
Published: July 2010

The effect of bezafibrate on cytosolic fatty‐acid‐binding‐protein (FABPc) production along the small intestine has been investigated in mice. This drug increased the intestinal fatty‐acid‐binding‐protein (I‐FABPc) and liver fatty‐acid‐binding‐protein (L‐FABPc) mRNA levels in the duodenum. The extents of induction in the duodenum and in the liver are similar. However, the degree of stimulation gradually decreases along the length of the gut, no effect being found in the ileum. An efficient absorption of this drug as early as the proximal part of the small intestine may explain this phenomenon. The L‐FABPc gene is silent in terminal ileum of mice, but a direct infusion of bezafibrate into the ileum switches it on. We used this original model to follow the time course of induction of the L‐FABPc gene by bezafibrate. L‐FABPc mRNA was first detected 4 h after fibrate infusion, reached a maximum level at 16 h and subsequently decreased at 24 h. This induction was totally blocked by cycloheximide. Sunflower oil also caused small increases in the L‐FABPc mRNA levels. The transcriptional origin of the induction triggered both by bezafibrate and sunflower oil was demonstrated by run‐on assays. These data indicate that (a) the transcription of the L‐FABPc gene is induced by bezafibrate via de novo protein synthesis and (b) components of sunflower oil can transcriptionally activate the L‐FABPc gene. Our results also demonstrate that the mouse terminal ileum is a useful system for studying the regulation of L‐FABPc gene expression both in vivo and in vitro.
Evidence for Sodium Dodecyl Sulfate/Protein Complexes Adopting a Necklace Structure
Authors: Samsó Montserrat , Daban Joan‐Ramon , Hansen Steen , Jones Gareth R.
Published: July 2010

Structural analysis by cryo‐electron microscopy and small‐angle X‐ray scattering of ten sodium dodecyl sulfate/protein complexes in 25 mM Tris/HCl, 0.192 M glycine, pH 8.3, showed necklace‐like structures of spherical micelles dispersed along the unfolded peptide chain. The micelles of most SDS/protein complexes had a constant diameter (≈ 6.2nm), slightly larger than pure SDS micelles (≈ 5.7 nm), all micelles possessing a degree of surface roughness. The micelle‐associated polypeptide is mostly situated at the interface of the sulfate head groups and hydrocarbon core, intruding into the core rather than outward from the surface. Proteins with a molecular mass less than about 20 kDa formed complexes with a single SDS micelle. Multi‐micellar SDS/protein complexes had centre‐to‐centre intermicellar distances in the range 7.0–12.0 nm. Our findings on the constancy of micellar size, number of micelles/complex, and the relationship between the degree of occupancy of micelles and a polypeptide's molecular mass, have enabled us to speculate on the correlation between the electrophoretic mobility of a polypeptide in SDS/PAGE and its molecular mass. The anomalous electrophoretic behaviour observed for the sodium dodecyl sulfate/histone H5 complex is accounted for by the large micelle of its complex.
The Mutation Asp69→Ser Affects the Chaperone‐Like Activity of αA‐Crystallin
Authors: Smulders Ronald H. P. H. , Merck Karin B. , Aendekerk John , Horwitz Joseph , Takemoto Larry , Slingsby Christine , Bloemendal Hans , De Jong Wilfried W.
Published: July 2010

α‐Crystallins are members of the family of small heat‐shock proteins. The conformation and mode of action of these ‘junior chaperones’ are unknown. To investigate the structure and chaperone‐like activity, four mutants of bovine αA‐crystallin were generated by site‐directed mutagenesis. In comparison with wild‐type αA‐crystallin, the D69S mutant, in which a highly conserved charged residue has been replaced, forms larger multimers and displays a threefold reduced heat‐protection capacity. The conformation and thermal stability of this mutant are not noticeably affected. Three other mutations, replacing hydrophobic by uncharged hydrophilic residues, were aimed at disturbing hydrophobic intersubunit interactions. None of these mutations resulted in major structural perturbations and only minor differences in heat‐protective capacity were observed. Although it is assumed that small heat‐shock proteins interact with denaturing proteins via their hydrophobic surfaces, this study clearly shows that charged residues in α‐crystallin can also influence the efficiency of substrate binding.
Crystal and Molecular Structure at 0.16‐nm Resolution of the Hybrid Bacillus Endo‐1,3‐1,4‐β‐D‐Glucan 4‐Glucanohydrolase H(A16‐M)
Authors: Hahn Michael , Keitel Thomas , Heinemann Udo
Published: July 2010

H(A16‐M) is a hybrid endo‐1,3‐1,4‐β‐D‐glucan 4‐glucanohydrolase from Bacillus. Its crystal structure was refined using synchrotron X‐ray diffraction data up to a maximal resolution of 0.16 nm. The R value of the resulting model is 14.3% against 21032 reflections >2σ. 93% of the amino acid residues are in the most favorable regions of the Ramachandran diagram, and geometrical parameters are in accordance with other proteins solved at high resolution. As shown earlier [Keitel, T., Simon, O., Borriss, R. & Heinemann, U. (1993) Proc. Natl Acad. Sci. USA 90, 5287–5291], the protein folds into a compact jellyroll‐type β‐sheet structure. A systematic analysis of the secondary structure reveals the presence of two major antiparallel β‐sheets and a three‐stranded minor mixed sheet. Amino acid residues involved in catalysis and substrate binding are located inside a deep channel spanning the surface of the protein. To investigate the stereochemical cause of the observed specificity of endo‐1,3‐1,4‐β‐D‐glucan 4‐glucanohy‐drolases towards β‐1,4 glycosyl bonds adjacent to β‐1,3 bonds, the high‐resolution crystal structure has been used to model an enzyme‐substrate complex. It is proposed that productive substrate binding to the subsites p1, p2 and p3 of H(A16‐M) requires a β‐1,3 linkage between glucose units bound to p1 and p2.
Purification and Characterization of the Periplasmic Nickel‐Binding Protein NikA of Escherichia coli K12
Authors: De Pina Karinne , Navarro Clarisse , Mcwalter Laura , Boxer David H. , Price Nicholas C. , Kelly Sharon M. , Mandrand‐Berthelot Marie‐Andrée , Wu Long‐Fei
Published: July 2010

The nik operon of Escherichia coli encodes a periplasmic binding‐protein‐dependent transport system specific for nickel. In this report, we describe the overproduction of the periplasmic nickel‐binding protein NikA by cloning the nikA gene into an overexpression vector, pRE1. NikA was purified free of nickel to near homogeneity from the periplasm by hydrophobic and ion‐exchange chromatography. N‐terminal amino acid sequencing confirmed that the leader peptide of NikA had been removed. The nickel‐binding properties of the protein has been studied by monitoring the quenching of intrinsic protein fluorescence. NikA binds one atom of nickel/molecule of protein with a dissociation constant (Kd) of less than 0.1 μM. Other metals (cobalt, copper, iron) are bound at least 10‐fold less tightly. The high specificity for Ni2+ is also demonstrated by high‐performance immobilized‐metal‐ion affinity chromatography. Biosynthesis of NikA occurred only under anaerobic conditions and was dependent on the general anaerobic regulator FNR. It was repressed by the presence of 250 μM Ni2+ in the growth medium and was not affected by either 30 mM formate or 100 mM nitrate. Anaerobically grown wild‐type strain MC4100 contains about 23 000 molecules of NikA/cell. In addition to the effect on nickel transport, nikA mutation affects also the nickel sensing in Tar‐dependent repellent chemotaxis.
Inhibitory Effect of Annexin V on Protein Kinase C Activity in Mesangial Cell Lysates
Authors: Rothhut Bernard , Dubois Thierry , Feliers Denis , Russo‐Marie Françoise , Oudinet Jean‐Paul
Published: July 2010

Annexin V belongs to a large family of calcium‐binding and phospholipid‐binding proteins and may act as an endogenous regulator of the protein kinase C (PKC) activity. This study examines the effect of annexin V on the in vitro PKC activity in cultured mesangial cells using histone H1, the peptide [Ser25]PKC‐(19–31), or endogenous proteins as substrates. The SDS/PAGE pattern of 32P‐labeled mesangial proteins showed that the calcium‐independent PKC [(n+a)PKC] phosphorylated several proteins from 70 kDa to 40 kDa and 22 kDa to 15 kDa. Three additional proteins from 34 kDa to 29 kDa, including annexin I and its proteolytic forms, were detected after activation of calcium‐dependent PKC (cPKC). Increasing concentrations of annexin V did not alter the phosphorylation of (n+a)PKC substrates. By contrast, specific phosphorylation of proteins and annexin I by cPKC, was reduced in a dose‐dependent manner. Addition of high concentration of calcium and phosphatidylserine did not reverse the inhibitory effect of annexin V. Annexin V also inhibited the phosphorylation of histone H1 or peptide [Ser25]PKC‐(19–31) by cPKC. Moreover, removal of annexin V from cytosols increased the annexin I phosphorylation by these isoforms. From these results, we propose that annexin V may regulate the signal‐transduction pathway involving the activation of cPKC, as they act in vitro as an inhibitor of these kinases.
Structure of the N‐Linked Oligosaccharides from Tridacnin, a Lectin Found in the Haemolymph of the Giant Clam Hippopus Hippopus
Authors: Puanglarp Narongsak , Oxley David , Currie Graeme J. , Bacic Antony , Craik David J. , Yellowlees David
Published: July 2010

Tridacnin, a glycoprotein lectin, was isolated from the symbiotic marine clam Hippopus hippopus and the structure of its major N‐glycan chains determined. Tridacnin contains only N‐linked glycans which were quantitatively cleaved by peptide‐N4‐(N‐acetyl‐β‐glucosaminyl)asparagine amidase F. Following purification by anion‐exchange HPLC, the structures of the oligosaccharides were established using a combination of electrospray ionisation mass spectrometry, 1H‐NMR spectroscopy and linkage analysis. The N‐glycans are primarily of the oligomannose type but, in addition, some contain a novel 6‐O‐Me group on the terminal mannose residue of the chain. The N‐glycan chains had the following structures.

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Structural Characterization of a Short Peptide Fragment that Mediates Estrogen‐Receptor Dimerization
Authors: Baumann Herbert , Härd Torleif
Published: July 2010

In a recent study it is suggested that a short peptide fragment within the estrogen receptor ligand‐binding and dimerization domain might act as a constitutively active dimerization motif [Lees, J. A., Fawell, S. E., White, R. & Parker, M. G. (1990) Mol. Cell. Biol. 10, 5529–5531]. We used NMR and CD spectroscopies to characterize the structure and biophysical properties of a synthetic peptide comprising residues Thr500–His528 of the mouse estrogen receptor, including the putative dimerization motif. We found that residues Leu501–Asn523 form a nascent helix in water solution, whereas the C‐terminal (Lys524–His528) has no propensity for α‐helical conformation. We found no evidence for a strong homodimerization activity of the peptide. However, we observed concentration‐dependent NMR chemical shifts of several residues that would be located on the same face of an α‐helix. This observation suggests a weak, but specific dimerization/oligomerization of the peptide at millimolar concentrations.
Enzymic Remodelling of the N‐ and O‐Linked Carbohydrate Chains of Human Chorionic Gonadotropin
Authors: Nemansky Martin , De Leeuw Renato , Wijnands Robert A. , Van Den Eijnden Dirk H.
Published: July 2010

The effects of altered terminal sequences in human chorionic gonadotropin (hCG) N‐ and O‐linked glycans on receptor binding and signal transduction were analyzed using forms of hCG with remodelled carbohydrate chains. hCG derivatives were obtained by enzymic removal of the α3‐linked sialic acid residues followed by α6‐sialylation, α3‐galactosylation or α3‐fucosylation of uncovered Galβ1→4GlcNAc (LacNAc) termini, or α3‐sialylation of Galβ1→3GalNAc sequences. Also a form that carried GalNAcβ1→4‐GlcNAc units, which are typical for pituitary hormone oligosaccharides, was derived by enzymic desialylation and degalactosylation followed by β4‐N‐acetylgalactosaminylation. The potency to stimulate testosterone production and the binding to the lutotropin/choriogonadotropin receptor of the preparations were compared with those of native and desialylated hCG (as‐hCG). The decrease in bioactivity caused by desialylation of hCG was only restored upon α6‐sialylation of the Galβ1→4GlcNAcβ1→2Manα1→3Man branch of the N‐linked glycans. This was without a major effect on receptor binding. Further α6‐sialylation, occurring at the Galβ1→4GlcNAcβ1→2Manα1→6Man branch, resulted in a bioactivity below a level found with as‐hCG, concomitant with a decreased receptor binding affinity. Similarly α3‐galactosylation of the Galβ1→4GlcNAcβ1→2‐Manα1→6Man branch yielded a hCG derivative that showed decreased bioactivity and receptor binding. α3‐Fucosylation of native as well as as‐hCG also led to a decreased activity. Re‐α3‐sialylation of the O‐linked chains on as‐hCG had little effect on the bioactivity and receptor binding. Hormone preparations with GalNAcβ→4GlcNAc termini showed lower bioactivity and receptor affinity than as‐hCG. It is concluded that the Galβ1→4GlcNAcβ1→2Manα1→3Man‐ rather than the Galβ1→ 4GlcNAcβ1→2‐Manα1→6Man branch of the N‐linked glycans on hCG plays an essential role in signal transduction, whereas the latter branch can potentially interfere with receptor binding. Furthermore attachment of sialic acid, but not of other sugars, to the first branch fulfils the requirement for the full expression of bioactivity, while sialylation of the O‐linked chains is of minor importance.
Kinetics of the two‐step hydrolysis of triacylglycerol by pancreatic lipases
Authors: Lykidis Athanasios , Mougios Vassilis , Arzoglou Pantelis
Published: July 2010

Pancreatic lipases catalyze the hydrolysis of triacylglycerol in a sequential manner. First, triacylglycerol is hydrolyzed to 1,2‐diacylglycerol, which is subsequently converted to 2‐monoacylglycerol. We studied the kinetics of trioleoylglycerol hydrolysis by rabbit and human pancreatic lipases. The products (acylglycerols and fatty acid) were analyzed by extraction from the reaction mixture, separation by thin‐layer chromatography, and quantification by capillary gas chromatography. The first‐order rate constants of trioleoylglycerol and dioleoylglycerol hydrolysis were calculated showing that both enzymes hydrolyze dioleoylglycerol faster than trioleoylglycerol. Using rabbit pancreatic lipase, we found that deoxycholate enhanced dioleoylglycerol hydrolysis to a higher degree than trioleoylglycerol hydrolysis. Colipase increased both rate constants similarly at high deoxycholate concentrations (35 mM), while at low concentrations (5 mM) a selectivity toward trioleoylglycerol was observed. From the variation of the rate constants with respect to temperature, we calculated the apparent activation energies of trioleoylglycerol and dioleoylglycerol hydrolysis to be 59.8 kJ • mol−1 and 53.5 kJ · mol−1, respectively. Upon storage, both rabbit and human pancreatic lipases showed a greater loss of activity toward dioleoylglycerol as compared to trioleoylglycerol, suggesting that different conformational elements of the enzyme molecule are responsible for the interaction with each substrate.
An Unusual Polar Lipid from the Cell Membrane of Mycoplasma fermentans
Authors: Deutsch Joseph , Salman Michael , Rottem Shlomo
Published: July 2010

The major unidentified polar lipid (compound X), recently demonstrated in the cell membrane of Mycoplasma fermentans, was purified by preparative silicic acid column chromatography. Chemical analyses of acid‐hydrolyzed compound X revealed that, in addition to fatty acids, it contains glycerol, choline and phosphate in a molar ratio of approximately 1:1:2, and an amino acid that has a retention time similar to that of homoserine. The methylated fatty acid fraction of compound X was subjected to gas‐liquid chromatography and revealed methyl palmitate and methyl stearate in a 4.6:1 molar ratio. The structure of compound X was further analyzed by combining mass spectrometry, 31P‐NMR and 1H‐NMR. The positive and negative fast atom bombardment spectra showed a major component of Mr 1048 and a minor component of Mr, 1076. Two different phosphate groups were identified in each of the components by 31P‐NMR. Fast atom bombardment, tandem mass spectrometry, negative and positive chemical ionization mass spectrometry together with mass spectra analyses of the water‐soluble and ether‐soluble products obtained by methanolysis has shown that, in addition to palmitic and stearic acid residues, the presence of glycerol, ribitol, cholinephosphate and homoserinephosphate residues. It is suggested that the apparent structure of compound X is either a phosphatidylcholine attached via a phosphotriester bond to a ribitolphosphohomoserine moiety or a phosphatidylhomoserine attached via a phosphotriester bond to a ribitolphosphocholine moiety. The major molecular species is the dipalmitoylderivative (Mr 1048), whereas the minor molecular species is a stearoyl palmitoyl derivative (Mr 1076).
Quinohaemoprotein Ethanol Dehydrogenase from Comamonas testosteroni
Authors: De Jong Govardus A. H. , Geerlof Arie , Stoorvogel Joke , Jongejan Jaap A. , De Vries Simon , Duine Johannis A.
Published: July 2010

Pyrroloquinoline‐quinone(PQQ)‐free quinohaemoprotein ethanol dehydrogenase (QH‐EDH) apoenzyme was isolated from ethanol‐grown Comamonas testosteroni. The purified apoenzyme, showing a single band of 71 kDa on native gel electrophoresis, could be only partially converted into active holoenzyme by addition of PQQ in the presence of calcium ions. In addition to a band with a molecular mass of 71 kDa, additional bands of 51 kDa and 25 kDa were observed with SDS/PAGE. Analysis of the N‐terminal sequences of the bands and comparison with the DNA sequence of the gene, suggested that the latter two originate from the former one, due to scission occurring at a specific site between two vicinal residues in the protein chain. The extent of scission appeared to increase during growth of the organism. After addition of PQQ to apoenzyme, holoenzyme and nicked, inactive enzyme could he separated. Holoenzyme prepared in this way was found to contain equimolar amounts of PQQ, Ca2+ and covalently bound haem. EPR spectra of fully oxidized apo‐QH‐EDH and holo‐QH‐EDH showed g values typical for low‐spin haem c proteins. In partially oxidized holo‐QH‐EDH an organic radical signal attributed to the semiquinone form of PQQ was observed. Binding of PQQ leads to conformational changes, as reflected by changes of spectral and chromatographic properties. Reconstitution of apoenzyme with PQQ analogues resulted in a decreased activity and enantioselectivity for the oxidation of chiral alcohols. Compared with PQQ, analogues with a large substituent had a lower affinity for the apoenzyme. Results with other analogues indicated that possession of the o‐quinone/o‐quinol moiety is not essential for binding but it is for activity.
Detection and Partial Purification of a Cruciform‐Resolving Activity (X‐solvase) from Nuclear Extracts of Mouse B‐Cells
Authors: Solaro Patricia , Greger Beate , Kemper Börries
Published: July 2010

We have identified a cruciform‐resolving enzyme (X‐solvase) in nuclear extracts from mouse B‐cells, called EMX1, by using an exonuclease‐resistant cruciform DNA as a substrate. The cruciform was a 104‐nt oligonucleotide that spontaneously adopted a branched conformation with four arms, each arm protected by a terminal loop of five T residues. A ligatable nick was left in one arm. After ligation, the covalently closed substrate was used to follow an 1800‐fold purification of the mouse X‐solvase (EMX1) from crude nuclear extracts by chromatography on DEAE‐cellulose, MonoQ and heparin‐Sepharose. The purest fractions containing EMX1 show high specificity for cruciform DNA. The cleavage pattern is indistinguishable from that found in the same substrates after treatment with endonuclease VII from phage T4 or endonuclease X3 from the yeast Saccharomyces cerevisiae. EMX1 and yeast endonuclease X3 were also found to be sensitive to anti‐(endonuclease VII) antibodies which inhibited their reactions with cruciform DNAs in vitro.
Characterization of a Novel Epidermal‐Growth‐Factor‐Receptor‐Related 200‐kDa Tyrosine Kinase in Tumor Cells
Authors: Panneerselvam Krishnasamy , Kanakaraj Palanisamy , Raj Sanjiv , Das Manjusri , Bishayee Subal
Published: July 2010

We have detected a tyrosine‐phosphorylated 200‐kDa protein in two human tumor cell lines, A1235 glioma and A172 glioblastoma. The protein is an integral plasma membrane sialoglycoprotein with tyrosine kinase activity. The interesting characteristic of this protein (gp200) is that it is recognized by a number of monoclonal and polyclonal antibodies to the 170‐kDa epidermal‐growth‐factor (EGF) receptor; however, it lacks detectable EGF‐binding activity. gp200 differs from three other EGF‐receptor‐related proteins, erb‐B‐2, erb‐B‐3 and erb‐B‐4 gene products, and hence appears to be yet another member of the EGF‐receptor family of proteins. This is further strengthened by the fact that both gp200 and the EGF receptor contain a common epitope which is recognized by an anti‐peptide IgG to the β‐type platelet‐derived‐growth‐factor (PDGF) receptor. Our previous studies [Bishayee, S., Majumdar, S., Scher, C. D. & Khan, S. (1988) Mol. Cell. Biol. 8, 3696–3702] have demonstrated that this epitope in the PDGF receptor is highly susceptible to the phosphorylation state of the receptor and that such a conformational change appears to be important in biological message transmission. The expression of gp200, which appears to have tyrosine kinase activity and is immununologically related to the EGF receptor in tumor cells, suggests its possible involvement in cell growth.
Site‐Directed Mutagenesis Studies on the Lima Bean Lectin
Authors: Jordan Elizabeth T. , Goldstein Irwin J.
Published: July 2010

The wild‐type seed lima bean lectin (LBL), and recombinant LBL expressed in Escherichia coli show specificity for the human blood group A immunodominant trisaccharide GalNAcal‐3[Fucα1‐2]Galβ1‐R. We have generated four site‐specific mutants of LBL, two of which show altered specificity for extended carbohydrate structures. Four mutants, [C127Y]LBL, [H128P]LBL, [H128R]LBL and [W132F]LBL were expressed in E. coli. Two mutants show altered specificity for the substituent at the C2 hydroxy group of the penultimate Gal in the wild‐type ligand which is α‐l‐fucose in the A trisaccharide. The mutant [C127Y]LBL showed specificity for the A disaccharide (GalNAcα1‐3Gal) and GalNAcα1‐4Gal, with free hydroxyl groups at the C2 position of Gal. The mutant [H128P]LBL bound the Forssman disaccharide structure GalNAcα1‐3GalNAc, in which the C2 hydroxyl group is substituted with an acetamido group. The third and fourth mutants, [H128R]LBL and [W132F]LBL, exhibited wild‐type specificities, both recognizing the A trisaccharide. All of these mutant lectins bound the terminal GalNAc residues exposed on asialoovine submaxillary mucin, thus indicating that the monosaccharide‐binding site had not been altered. We also determined that all but one mutant ([C127Y]LBL) retained the high‐affinity binding site for N6 derivatives of adenine, indicative of tetramer formation; each mutant also expressed the low‐affinity binding site for 8‐anilinonaphthalene 1‐sulfonate (1/monomer). Thus, by targeting two residues in LBL, we have identified a region of the protein that is part of the extended carbohydrate‐binding site and which is specifically involved in the binding/recognition of substituents at the C2 position of the penultimate Gal of the A disaccharide. We have determined, by site‐directed mutagenesis, that an essential Cys residue is involved in the specificity of LBL for the A trisaccharide.
Phosphorylation and Activation of Protein Kinase Ck2 by p34cdc2 are Independent Events
Authors: Meggio Flavio , Boldyreff Brigitte , Marin Oriano , Issinger Olaf‐G. , Pinna Lorenzo A.
Published: July 2010

Recombinant isolated β‐subunit of protein kinase CK2 is readily phosphorylated by p34cdc2/cyclin B kinase at Ser209 with favourable kinetic constants (Km= 1.7 μM, Vmax= 20 nmol · min−1· mg−1). Two synthetic peptides reproducing the 170–215 and the 206–215 C‐terminal fragments of the β‐subunit are also phosphorylated though with tenfold higher Km values (19.5 and 28.0 μM, respectively). In contrast, both the β‐subunit associated with the α‐subunit to give the heterotetrameric holoenzyme and the native CK2 are not appreciably phosphorylated by p34cdc2. These data suggest that the Ser209 β–subunit phosphorylation observed in intact cells occurs prior to β‐subunit incorporation into the holoenzyme. The isolated CK2 α‐subunit is not phosphorylated to any appreciable extent by p34cdc2 kinase. Its catalytic activity is nevertheless increased up to fivefold upon incubation with p34cdc2/cyclin B kinase complex. Such a stimulation of activity is comparable to that induced by the β‐subunit and it is paralleled by a 40% decrease of p34cdc2/cyclin B catalytic activity. Similar to β‐subunit, p34cdc2/cyclin B also protects the α‐subunit against thermal inactivation. CK2 holoenzyme is also stimulated by p34cdc2/cyclin B, albeit less dramatically than the isolated α‐subunit. Such an effect is also evident with CK2 holoenzyme reconstituted with a mutated β‐subunit lacking the p34cdc2 phosphorylation site and it is not accompanied by any appreciable phosphorylation of either the β or the α‐subunit. These data indicate that in vitro CK2 α‐subunit interacts with and is activated by p34cdc2/cyclin B kinase by a mechanism that does not imply the phosphorylation of CK2.
Vitamin‐B12‐Independent Methionine Synthase from a Higher Plant (Catharanthus Roseus)
Authors: Eichel Johannes , González Julio C. , Hotze Michael , Matthews Rowena G. , Schröder Joachim
Published: July 2010

Methionine synthases catalyze the formation of methionine by the transfer of a methyl group from 5‐methyltetrahydrofolate to homocysteine. This reaction is the last step in l‐methionine biosynthesis, and it also serves to regenerate the methyl group of 5‐adenosylmethionine, a cofactor required for biological methylation reactions. We describe the cloning, expression and characterization of a methionine synthase from the higher plant Catharanthus roseus. cDNAs were identified that encoded a protein of 85 kDa sharing 50 % identity with the cobalamin‐independent methionine synthase from Escherichia coli (MetE) and 41 % identity with a partial sequence of a yeast homolog of MetE. The C. roseus protein was expressed at high levels in E. coli. The enzyme accepts the triglutamate form of methyltetrahydrofolate as a methyl donor but not the monoglutamate form, and it does not require 5‐adenosylmethionine or cobalamin for activity. The properties indicate that the enzyme is a cobalamin‐independent methionine synthase (EC 2.1.1.14). In contrast to the E. coli MetE, the plant protein does not require phosphate or magnesium ions for activity. Immunoblots of plant extracts showed that the protein was localized in the cytosol, and was present in a variety of plant species. A nutritional downshift of the C. roseus cell culture revealed a strong, transient transcriptional activation, but no significant increment in the total level of the protein. The availability of the protein and the cDNA now provide tools to investigate the complexities of methionine biosynthesis in plants.
The Relative Contributions of Various Transcription Factors to the Overall Promoter Strength of the Mouse Ribosomal Protein L30 Gene
Authors: Sáfrány Géza , Perry Robert P.
Published: July 2010

The promoter of the mouse gene encoding ribosomal protein L30 contains binding sites for four transcription factors; α (RFX‐1), β (GABP), γ and δ (YY‐1/NF‐E1/UCRBP). The relative contributions of these factors to the strength of the rpL30 promoter in vivo and the degree of synergism among the factors was evaluated by transfection experiments using a series of mutant promoters in which one or more of the binding sites was drastically altered to prevent recognition by its cognate factor. Our results indicated that GABP and RFX‐1 are the major determinants of the rpL30 promoter strength, acting synergistically to boost activity more than eightfold over that which occurs in their absence. The contributions of γ and δ became evident only when the promoter was weakened by eliminating the participation of the other factors. Indeed, as the promoter strength was progressively reduced, the contribution of each individual factor increased, implying that the capacity of the general transcription machinery to be stimulated by these factors is saturable. The activity of the rpL30 promoter was significantly diminished when three pyrimidine residues spanning the start site were converted to purines, indicating that the integrity of the oligopyrimidine tract is also a determinant of the transcriptional efficiency. These studies reveal the hierarchy of importance of four transcription factors that govern the expression of the rpL30 gene. Moreover, they define the graduated levels of promoter activity that would result from deficiencies of these factors in any particular cell type. This information may provide a useful paradigm for understanding the transcriptional regulation of other ubiquitously expressed genes.