bluuebunny/crossref_metadata_2025 huggingface dataset:
Inhibition of the vacuolar H+‐ATPase perturbs the transport, sorting, processing and release of regulated secretory proteins
Authors: Schoonderwoert Vincent TH. G. , Holthuis Joost C. M. , Tanaka Shigeyasu , Tooze Sharon A. , Martens Gerard J. M.
Published: March 2003

Vacuolar H+‐ATPases (V‐ATPases) are multisubunit enzymes that acidify various intracellular organelles, including secretory pathway compartments. We have examined the effects of the specific V‐ATPase inhibitor bafilomycin A1 (Baf) on the intracellular transport, sorting, processing and release of a number of neuroendocrine secretory proteins in primary Xenopus intermediate pituitary cells. Ultrastructural examination of Baf‐treated intermediate pituitary cells revealed a reduction in the amount of small dense‐core secretory granules and the appearance of vacuolar structures in the trans‐Golgi area. Pulse–chase incubations in combination with immunoprecipitation analysis showed that in treated cells, the proteolytic processing of the newly synthesized prohormone proopiomelanocortin, prohormone convertase PC2 and secretogranin III (SgIII) was inhibited, and an intracellular accumulation of intact precursor forms and intermediate cleavage products became apparent. Moreover, we found that treated cells secreted considerable amounts of a PC2 processing intermediate and unprocessed SgIII in a constitutive fashion. Collectively, these data indicate that in the secretory pathway, V‐ATPases play an important role in creating the microenvironment that is essential for proper transport, sorting, processing and release of regulated secretory proteins.
The human phosphodiesterase PDE10A gene
Authors: Fujishige Kotomi , Kotera Jun , Yuasa Keizo , Omori Kenji
Published: March 2003

PDE10A is a cyclic nucleotide phosphodiesterase (PDE) exhibiting properties of a cAMP PDE and a cAMP‐inhibited cGMP PDE. The transcripts are specifically expressed in the striatum. The human gene encoding PDE10A was cloned and investigated. The PDE10A gene spanned > 200 kb and contained 24 exons. The exon–intron organization of PDE10A was different from those of PDE5A and PDE6B, although these three PDEs include two GAF domains and have similar amino‐acid sequences. The promoter sequence of PDE10A was highly GC‐rich and did not contain a TATA motif and a CAAT box, suggesting it is a housekeeping gene. In Caenorhabditis elegans, the C32E12.2 gene encoding a probable PDE that is 48% identical to the human PDE10A protein showed similar exon organization to PDE10A but not PDE5A and PDE6B. This, together with the phylogenic tree analysis,
suggested that the ancestral gene for PDE10A existed in a lower organism such as C. elegans.
Effect of fatty acid‐binding proteins on intermembrane fatty acid transport
Authors: Veerkamp Jacques H. , van Moerkerk and Herman T. B. , Zimmerman Aukje W.
Published: March 2003

Liposomes of different charge fixed to nitrocellulose filters were used to study the transfer of fatty acids to rat heart or liver mitochondria in the presence of fatty acid‐binding protein (FABP) or albumin. [14C]Palmitate oxidation was used as a parameter. Different FABP types and heart FABP mutants were tested. The charge of the liposomes did not influence the solubilization and mitochondrial oxidation of palmitate without FABP and the amount of solubilized palmitate in the presence of FABP. Mitochondria did not show a preference for oxidation of FABP‐bound palmitate over their tissue‐specific FABP type. All FABP types increased palmitate oxidation by heart and liver mitochondria with neutral, positive and negative liposomes by 2.5‐fold, 3.2‐fold and twofold, respectively. Ileal lipid‐binding protein and H‐FABP mutants that do not bind fatty acid had no effect. Other H‐FABP mutants had different effects, dependent on the site of mutation. The effect of albumin was similar to, but not dependent on, liposome charge. The ionic strength had only a slight effect. In conclusion, the transfer of palmitate from liposomal membranes to mitochondria was increased by all FABP types to a similar extent. The membrane charge had a large effect in contrast to the origin of the mitochondria.
Synthesis of mono‐ and di‐fucosylated type I Lewis blood group antigens by Helicobacter pylori
Authors: Rasko David A. , Wang Ge , Monteiro Mario A. , Palcic Monica M. , Diane E. Taylor
Published: March 2003

The identification of Helicobacter pylori isolates that expresses exclusively type I Lewis antigens is necessary to determine the biosynthetic pathway of these antigens. Fast‐atom bombardment MS
provides evidence that the H. pylori isolate UA1111 expresses predominantly Leb, with H type I and Lea in lesser amounts. Cloning and expression of the H. pylori fucosyltransferases (FucTs) allow comparisons with previously identified H. pylori enzymes and determination of the enzyme specificities. Although all FucTs, one α(1,2) FucT and two α(1,3/4) FucTs, appear to be functional in this isolate, their activities are lower and enzyme specificities are different to other H. pylori FucTs previously characterized. Studies of the cloned enzyme activities and mutational analysis indicate that Lea acts as the substrate for the synthesis of Leb. This is different from the human Leb biosynthetic pathway, but analogous to the biosynthetic pathway utilized by H. pylori for the production of Ley.
Coordinate up‐ and down‐regulation of glutathione‐dependent prostaglandin E synthase and cyclooxygenase‐2 in A549 cells
Authors: Thorén Staffan , Jakobsson Per‐Johan
Published: March 2003

Recently, a microsomal protein with 38% sequence identity to microsomal glutathione S‐transferase 1 was shown to constitute an inducible, glutathione‐dependent prostaglandin E synthase (PGES). To investigate the relationship between cyclooxygenase and PGES, a time‐course study on protein expression was performed in A549 cells after treatment with interleukin‐1β. The result demonstrated a tandem expression of cyclooxygenase‐2 and PGES. The observed induction of PGES protein correlated with microsomal PGES activity. No comparable PGES activity was observed in the absence of glutathione or in the cytosolic fraction. In addition, tumour necrosis factor‐α was found to induce PGES in these cells. Dexamethasone was found to completely suppress the effect of both cytokines on PGES induction. We also describe a quantitative method, based on RP‐HPLC with UV detection for the measurements of PGES activity. This method was used to screen potential PGES inhibitors. Several nonsteroidal anti‐inflammatory drugs, stable prostaglandin H2 analogues and cysteinyl leukotrienes were screened for inhibition of PGES activity. NS‐398, sulindac sulfide and leukotriene C4 were all found to inhibit PGES activity with IC50 values of 20 µm, 80 µm and 5 µm, respectively. In conclusion, it appears that PGES and cyclooxygenase‐2 are functionally coupled in A549 cells and that a required coordinate expression of these enzymes allows for efficient biosynthesis of prostaglandin E2.
Interferon‐γ and lipopolysaccharide regulate the expression of Nramp2 and increase the uptake of iron from low relative molecular mass complexes by macrophages
Authors: Wardrop S. L. , Richardson D. R.
Published: March 2003

The natural resistance associated macrophage protein 2 (Nramp2) is a transporter that is involved in iron (Fe) uptake from transferrin (Tf) and low molecular mass Fe complexes. Here we describe the effect of the inflammatory mediators interferon‐γ (IFN‐γ) and lipopolysaccharide (LPS) on the expression of Nramp2 mRNA and Fe uptake by cells of the macrophage lineage. After incubation of the RAW264.7 macrophage cell line with LPS there was a sevenfold increase in the expression of the 2.3 kb Nramp2 mRNA transcript when compared with the control, but little effect on the Nramp2 3.1 kb transcript. These results indicate differential regulation of the two transcripts. Treatment with LPS resulted in an increase in 59Fe uptake from 59Fe–nitrilotriacetic acid, while transferrin receptor (TfR) mRNA levels and 59Fe uptake from 59Fe–Tf were decreased. Paradoxically, at the same time, an increase in iron regulatory protein (IRP)1 RNA‐binding activity was observed. Incubation with IFN‐γ (50 U·mL−1) resulted in a marked decrease in TfR mRNA levels but had no effect on Nramp2 mRNA expression. Exposure of RAW264.7 cells to both IFN‐γ and LPS resulted in a fourfold increase in the Nramp2 2.3‐kb transcript and a four to fivefold decrease in the 3.1‐kb transcript when compared with the control. Furthermore, there was a decrease in TfR mRNA levels despite an increase in IRP1 RNA‐binding activity and a marked increase in inducible nitric oxide synthase mRNA expression. Hence, TfR and Nramp2 mRNA expression did not appear to be regulated in a concerted manner. Similar responses to those found above for RAW264.7 cells were also observed in the J774 macrophage cell line and also for primary cultures of mouse peritoneal macrophages. These results are of interest as the TfR and Nramp2 are thought to act together during Fe uptake from Tf. This is the first report to demonstrate regulation of the Nramp2 mRNA transcripts by inflammatory mediators.
A novel type of arabinoxylan arabinofuranohydrolase isolated from germinated barley
Authors: Ferré Henrik , Broberg Anders , Duus Jens Ø. , Thomsen Karl K.
Published: March 2003

An arabinoxylan arabinofuranohydrolase was isolated from barley malt. The enzyme preparation, Ara 1, contained two polypeptides with apparent molecular masses of ≈ 60 and ≈ 66 kDa, a pI of 4.55 and almost identical N‐terminal amino‐acid sequences. With p‐nitrophenyl α‐l‐arabinofuranoside (pNPA) as substrate, Ara 1 exhibited a Km of 0.5 mm and a Vmax of 6.7 µmol·min−1·(mg of protein)−1. Maximum activity was displayed at pH 4.2 and 60 °C, and, under these conditions, the half‐life of the enzyme was 8 min. The Ara 1 preparation showed no activity against p‐nitrophenyl α‐l‐arabinopyranoside or p‐nitrophenyl β‐d‐xylopyranoside. Substrate preference and specificity were investigated using pure oligosaccharides and analysis by TLC and nano‐probe NMR. Ara 1 released arabinose from high‐molecular‐mass arabinoxylan and arabinoxylan‐derived oligosaccharides but was inactive against linear or branched‐chain arabinan. Arabinose was readily released from both singly and doubly substituted xylo‐oligosaccharides. Whereas single 2‐O‐linked and 3‐O‐linked arabinose substituents on non‐reducing terminal xylose were released at similar rates, there was a clear preference for 2‐O‐linked arabinose on internal xylose residues. When Ara 1 acted on oligosaccharides with doubly substituted, non‐reducing terminal xylose, the 3‐O‐linked arabinose group was preferred as the initial point of attack. Oligosaccharides with doubly substituted internal xylose were poor substrates and no preference could be determined. The enzyme described here is the first reported arabinoxylan arabinofuranohydrolase which is able to release arabinose from both singly and doubly substituted xylose, and it hydrolyses p‐nitrophenyl α‐l‐arabinofuranoside at a rate similar to that observed for oligosaccharide substrates.
Differential mechanism‐based labeling and unequivocal activity assignment of the two active sites of intestinal lactase/phlorizin hydrolase
Authors: Arribas Juan C. Díaz , Herrero Alicia G , Martín‐Lomas Manuel , Cañada F. Javier , He Shouming , Withers Stephen G.
Published: March 2003

Milk lactose is hydrolysed to galactose and glucose in the small intestine of mammals by the lactase/phlorizin hydrolase complex (LPH; EC 3.2.1.108/62). The two enzymatic activities, lactase and phlorizin hydrolase, are located in the same polypeptide chain. According to sequence homology, mature LPH contains two different regions (III and IV), each of them homologous to family 1 glycosidases and each with a putative active site. There has been some discrepancy with regard to the assignment of enzymatic activity to the two active sites. Here we show differential reactivity of the two active sites with mechanism‐based glycosidase inhibitors. When LPH is treated with 2′,4′‐dinitrophenyl 2‐deoxy‐2‐fluoro‐β‐d‐glucopyranoside (1) and 2′,4′‐dinitrophenyl‐2‐deoxy‐2‐fluoro‐β‐d‐galactopyranoside (2), known mechanism‐based inhibitors of glycosidases, it is observed that compound 1 preferentially inactivates the phlorizin hydrolase activity whereas compound 2 is selective for the lactase active site. On the other hand, glycals (d‐glucal and d‐galactal) competitively inhibit lactase activity but not phlorizin hydrolase activity. This allows labeling of the phlorizin site with compound 1 by protection with a glycal. By differential labeling of each active site using 1 and 2 followed by proteolysis and MS analysis of the labeled fragments, we confirm that the phlorizin hydrolysis occurs mainly at the active site located at region III of LPH and that the active site located at region IV is responsible for the lactase activity. This assignment is coincident with that proposed from the results of recent active‐site mutagenesis studies [Zecca, L., Mesonero, J.E., Stutz, A., Poiree, J.C., Giudicelli, J., Cursio, R., Gloor, S.M. & Semenza, G. (1998) FEBS Lett.435, 225–228] and opposite to that based on data from early affinity labeling with conduritol B epoxide [Wacker, W., Keller, P., Falchetto, R., Legler, G. & Semenza, G. (1992) J. Biol. Chem.267, 18744–18752].
Intracellular contents and assembly states of all 12 subunits of the RNA polymerase II in the fission yeast Schizosaccharomyces pombe
Authors: Kimura Makoto , Sakurai Hitomi , Ishihama Akira
Published: March 2003

The RNA polymerase II (Pol II) of the fission yeast Schizosaccharomyces pombe is composed of 12 different polypeptides, Rpb1 to Rpb12, of which five, Rpb5, Rpb6, Rpb8, Rpb10 and Rpb12, are shared among three forms of the RNA polymerase. To get an insight into the control of synthesis and assembly of individual subunits, we have measured the intracellular concentrations of all 12 subunits in S. pombe by quantitative immunoblotting. Results indicate that the levels are low for the three large subunits, Rpb1, Rpb2 and Rpb3, which are the homologues of β′, β and α subunits, respectively, of prokaryotic RNA polymerase. On the other hand, the levels of small‐sized subunits were between 2‐ to 15‐fold higher than these three core subunits. The levels of the five common subunits shared among RNA polymerases I, II and III are about 10 times greater than those of the Pol II‐specific core subunits. The assembly state of the Rpb proteins was analyzed by glycerol gradient centrifugation of S. pombe whole cell extracts. The three core subunits are mostly assembled in Pol II, but some of the small subunits were detected in the slowly sedimenting fractions, indicating that at least some of the excess Rpb proteins exist in unassembled forms. Based on the intracellular concentration of the least abundant Rpb3 subunit, the total number of Pol II in a growing S. pombe cell was estimated to be about 10 000 molecules. The intracellular distribution of some Pol II subunits was also analyzed by microscopic observation of the green fluorescent protein (GFP)‐fused Rpb proteins. In agreement with the biochemical analysis, the GFP‐Rpb1 and GFP‐Rpb3 fusions were present in the nuclei but the GFP‐Rpb4 was detected in the cytoplasm as well as the nuclei.
A serpin‐induced extensive proteolytic susceptibility of urokinase‐type plasminogen activator implicates distortion of the proteinase substrate‐binding pocket and oxyanion hole in the serpin inhibitory mechanism
Authors: Egelund Rikke , Petersen Torben E. , Andreasen Peter A.
Published: March 2003

The formation of stable complexes between serpins and their target serine proteinases indicates formation of an ester bond between the proteinase active‐site serine and the serpin P1 residue [Egelund, R., Rodenburg, K.W., Andreasen, P.A., Rasmussen, M.S., Guldberg, R.E. & Petersen, T.E. (1998) Biochemistry37, 6375–6379]. An important question concerning serpin inhibition is the contrast between the stability of the ester bond in the complex and the rapid hydrolysis of the acyl‐enzyme intermediate in general serine proteinase‐catalysed peptide bond hydrolysis. To answer this question, we used limited proteolysis to detect conformational differences between free urokinase‐type plasminogen activator (uPA) and uPA in complex with plasminogen activator inhibitor‐1 (PAI‐1). Whereas the catalytic domain of free uPA, pro‐uPA, uPA in complex with non‐serpin inhibitors and anhydro‐uPA in a non‐covalent complex with PAI‐1 was resistant to proteolysis, the catalytic domain of PAI‐1‐complexed uPA was susceptible to proteolysis. The cleavage sites for four different proteinases were localized in specific areas of the C‐terminal β‐barrel of the catalytic domain of uPA, providing evidence that the serpin inhibitory mechanism involves a serpin‐induced massive rearrangement of the proteinase active site, including the specificity pocket, the oxyanion hole, and main‐chain binding area, rendering the proteinase unable to complete the normal hydrolysis of the acyl‐enzyme intermediate. The distorted region includes the so‐called activation domain, also known to change conformation on zymogen activation.
Human platelets bind and degrade 2‐arachidonoylglycerol, which activates these cells through a cannabinoid receptor
Authors: Maccarrone Mauro , Bari Monica , Menichelli Adriana , Giuliani Elena , Del Principe Domenico , Finazzi‐Agrò Alessandro
Published: March 2003

The endocannabinoid 2‐arachidonoylglycerol (2‐Δ4Ach‐Gro) activates human platelets in platelet‐rich plasma at physiological concentrations. The activation was inhibited by selective antagonists of CB1 and CB2 cannabinoid receptors, but not by acetylsalicylic acid. Human platelets can metabolize 2‐Δ4Ach‐Gro by internalization through a high affinity transporter (Km = 300 ± 30 nm, Vmax = 10 ± 1 pmol·min−1·mg protein−1), followed by hydrolysis by a fatty acid amide hydrolase (Km = 8 ± 1 µm, Vmax = 400 ± 50 pmol·min−1·mg protein−1). The anandamide transport inhibitor AM404, and anandamide itself, were ineffective on 2‐Δ4Ach‐Gro uptake by platelets, whereas anandamide competitively inhibited 2‐Δ4Ach‐Gro hydrolysis (inhibition constant = 10 ± 1 µm). Platelet activation by 2‐Δ4Ach‐Gro was paralleled by an increase of intracellular calcium and inositol‐1,4,5‐trisphosphate, and by a decrease of cyclic AMP. Moreover, treatment of preloaded platelet‐rich plasma with 2‐Δ4Ach‐Gro induced an approximately threefold increase in [3H]2‐Δ4Ach‐Gro release, according to a CB receptor‐dependent mechanism. On the other hand, ADP and collagen counteracted the activation of platelets by 2‐Δ4Ach‐Gro, whereas 5‐hydroxytryptamine (serotonin) enhanced and extended its effects. Remarkably, ADP and collagen also reduced [3H]2‐Δ4Ach‐Gro release from 2‐Δ4Ach‐Gro‐activated platelets, whereas 5‐hydroxytryptamine further increased it. These findings suggest a so far unnoticed interplay between the peripheral endocannabinoid system and physiological platelet agonists.
Structure–function relationships in UCP1, UCP2 and chimeras
Authors: Chomiki Nathalie , Voss John C. , Warden Craig H.
Published: March 2003

Uncoupling proteins (UCPs) are composed of three repeated domains of approximately 100 amino acids each. We have used chimeras of UCP1 and UCP2, and electron paramagnetic resonance (EPR), to investigate domain specific properties of these UCPs. Questions include: are the effects of nucleotide binding on proton transport solely mediated by amino acids in the third C‐terminal domain, and are the amino acids in the first two domains involved in retinoic or fatty acid activation? We first confirmed that our reconstitution system produced UCP1 that exhibited known properties, such as activation by fatty acids and inhibition of proton transport by purine nucleotides. Our results confirm the observations reported for recombinant yeast that retinoic acid, but not fatty acids known to activate UCP1, activates proton transport by UCP2 and that this activation is insensitive to nucleotide inhibition. We constructed chimeras in which the last domains of UCP1 or UCP2 were switched and tested for activation by fatty acids or retinoic acid and inhibition by nucleotides. U1U2 is composed of mUCP1 (amino acids 1–198) and hUCP2 (amino acids 211–309). Fatty acids activated proton transport of U1U2 and GTP mediated inhibition. In the other chimeric construct U2U1, hUCP2 (amino acids 1–210) and mUCP1 (amino acids 199–307), retinoic acid still acted as an activator, but no inhibition was observed with GTP. Using EPR, a method well suited to the analysis of the structure of membrane proteins such as UCPs, we confirmed that UCP2 binds nucleotides. The EPR data show large structural changes in UCP1 and UCP2 on exposure to ATP, implying that a putative nucleotide‐binding site is present on UCP2. EPR analysis also demonstrated changes in conformation of UCP1/UCP2 chimeras following exposure to purine nucleotides. These data demonstrate that a nucleotide‐binding site is present in the C‐terminal domain of UCP2. This domain was able to inhibit proton transport only when fused to the N‐terminal part of UCP1 (chimera U1U2). Thus, residues involved in nucleotide inhibition of proton transport are located in the two first carrier motifs of UCP1. While these results are consistent with previously reported effects of the C‐terminal domain on nucleotide binding, they also demonstrate that interactions with the N‐terminal domains are necessary to inhibit proton transport. Finally, the results suggest that proteins such as UCP2 may transport protons even though they are not responsible for basal or cold‐induced thermogenesis.
Protein 4.1 in forebrain postsynaptic density preparations
Authors: Scott Catherine , Keating Lisa , Bellamy Matthew , Baines Anthony J.
Published: March 2003

4.1 Proteins are a family of multifunctional cytoskeletal components (4.1R, 4.1G, 4.1N and 4.1B) derived from four related genes, each of which is expressed in the nervous system. Using subcellular fractionation, we have investigated the possibility that 4.1 proteins are components of forebrain postsynaptic densities, cellular compartments enriched in spectrin and actin, whose interaction is regulated by 4.1R. Antibodies to each of 4.1R, 4.1G, 4.1N and 4.1B recognize polypeptides in postsynaptic density preparations. Of these, an 80‐kDa 4.1R polypeptide is enriched 11‐fold in postsynaptic density preparations relative to brain homogenate. Polypeptides of 150 and 125 kDa represent 4.1B; of these, only the 125 kDa species is enriched (threefold). Antibodies to 4.1N recognize polypeptides of ≈ 115, 100, 90 and 65 kDa, each enriched in postsynaptic density preparations relative to brain homogenate. Minor 225 and 200 kDa polypeptides are recognized selectively by specific anti‐4.1G antibodies; the 200 kDa species is enriched 2.5‐fold. These data indicate that specific isoforms of all four 4.1 proteins are components of postsynaptic densities. Blot overlay analyses indicate that, in addition to spectrin and actin, postsynaptic density polypeptides of 140, 115, 72 and 66 kDa are likely to be 4.1R‐interactive. Of these, 72 kDa and 66 kDa polypeptides were identified as neurofilament L and α‐internexin, respectively. A complex containing 80 kDa 4.1R, α‐internexin and neurofilament L was immunoprecipitated with anti‐4.1R antibodies from brain extract. We conclude that 4.1R interacts with the characteristic intermediate filament proteins of postsynaptic densities, and that the 4.1 proteins have the potential to mediate the interactions of diverse components of postsynaptic densities.
Stimulation of the ICAM‐1 gene transcription by the peroxovanadium compound [bpV(Pic)] involves STAT‐1 but not NF‐κB activation in 293 cells
Authors: Audette Marie , Larouche Lucie , Lussier Isabelle , Fugère Nathalie
Published: March 2003

Vanadate and peroxovanadium derivatives are potent inhibitors of protein tyrosine phosphatases (PTPs) and exhibit insulinomimetic activities in several cell systems. We have found that in 293 and 293T cells, intercellular adhesion molecule‐1 (ICAM‐1) gene transcription is activated by bpV(Pic), a picolinic acid‐stabilized peroxovanadium derivative. To identify the bpV(Pic)‐responsive element(s), several deletion and site‐specific mutants of the ICAM‐1 gene promoter cloned upstream from the firefly luciferase reporter gene were transiently transfected into both cell lines. Deletion or site‐specific mutation of the NF‐κB site did not affect bpV(Pic) responsiveness, whereas deletion or mutation of the palindromic interferon‐γ‐responsive element (pIγRE)/γ‐interferon activated sequence site greatly decreased bpV(Pic) responsiveness in both cell types. bpV(Pic) synergistically co‐operated with interferon‐γ to increase the transcriptional activity of the ICAM‐1 promoter. Electrophoretic mobility‐shift assays showed that bpV(Pic) induces signal transducers and activators of transcription (STAT)‐1 binding to the ICAM‐1 pIγRE/GAS in 293T cells, suggesting that the peroxovanadium compound specifically inhibits the phosphatase(s) required to regulate the JAK/STAT signal‐transduction pathway.
Formation and properties of hybrid photosynthetic F1‐ATPases
Authors: Tucker Ward C. , Du Ziyun , Gromet‐Elhanan Zippora , Richter Mark L.
Published: March 2003

A hybrid ATPase composed of cloned chloroplast ATP synthase β and γ subunits (βC and γC) and the cloned α subunit from the Rhodospirillum rubrum ATP synthase (αR) was assembled using solubilized inclusion bodies and a simple single‐step folding procedure. The catalytic properties of the assembled

were compared to those of the core

complex of the native chloroplast coupling factor 1 (CF1) and to another recently described hybrid enzyme containing R. rubrumα and β subunits and the CF1γ subunit (

). All three enzymes were similarly stimulated by dithiothreitol and inhibited by copper chloride in response to reduction and oxidation, respectively, of the disulfide bond in the chloroplast γ subunit. In addition, all three enzymes exhibited the same concentration dependence for inhibition by the CF1ε subunit. Thus the CF1γ subunit conferred full redox regulation and normal ε binding to the two hybrid enzymes. Only the native CF1 complex was inhibited by tentoxin, confirming the requirement for both CF1α and β subunits for tentoxin inhibition. However, the

complex, like the

complex, was stimulated by tentoxin at concentrations in excess of 10 µm. In addition, replacement of the aspartate at position 83 in βC with leucine resulted in the loss of stimulation in the

hybrid. The results indicate that both inhibition and stimulation by tentoxin require a similar structural contribution from the β subunit, but differ in their requirements for α subunit structure.
Influence of cyclodextrin ring substituents on folding‐related aggregation of bovine carbonic anhydrase
Authors: Sharma Loretta , Sharma Ajit
Published: March 2003

A common obstacle to proper renaturation of an unfolded protein is aggregation, an intermolecular side reaction of immense importance in biotechnology and in the pathogenesis of several neurodegenerative diseases. Cyclic sugars known as cyclodextrins have been used as protein‐folding aids. The effect of cyclodextrin chemistry on aggregation and refolding of carbonic anhydrase was evaluated in this study. Size‐exclusion HPLC showed that cyclodextrins inhibit aggregate formation without interfering with the correct renaturation of carbonic anhydrase. PAGE of refolded enzyme provides further evidence of inhibition of folding‐related aggregation by natural and chemically modified cyclodextrins. Although the amount of aggregate formed and recovery of active enzyme was dependent on cavity size, the nature of the chemical substituents found on the rims of the sugar molecule seems to play a more important role in cyclodextrin‐assisted refolding of carbonic anhydrase. In general, neutral or cationic cyclodextrins with small cavities were found to be better folding aids than anionic cyclodextrins with larger holes. Although the exact prediction of the effect of a cyclodextrin substitution on protein refolding is not possible at present, these results clearly show that modified cyclodextrins can be designed that effectively inhibit protein aggregation.
Identification of catalytically important residues in the active site of Escherichia coli transaldolase
Authors: Schörken Ulrich , Thorell Stina , Schürmann Melanie , Jia Jia , Sprenger Georg A. , Schneider Gunter
Published: March 2003

The roles of invariant residues at the active site of transaldolase B from Escherichia coli have been probed by site‐directed mutagenesis. The mutant enzymes D17A, N35A, E96A, T156A, and S176A were purified from a talB‐deficient host and analyzed with respect to their 3D structure and kinetic behavior. X‐ray analysis showed that side chain replacement did not induce unanticipated structural changes in the mutant enzymes. Three mutations, N35A, E96A, and T156A resulted mainly in an effect on apparent kcat, with little changes in apparent Km values for the substrates. Residues N35 and T156 are involved in the positioning of a catalytic water molecule at the active site and the side chain of E96 participates in concert with this water molecule in proton transfer during catalysis. Substitution of Ser176 by alanine resulted in a mutant enzyme with 2.5% residual activity. The apparent Km value for the donor substrate, fructose 6‐phosphate, was increased nearly fivefold while the apparent Km value for the acceptor substrate, erythrose 4‐phosphate remained unchanged, consistent with a function for S176 in the binding of the C1 hydroxyl group of the donor substrate. The mutant D17A showed a 300‐fold decrease in kcat, and a fivefold increase in the apparent Km value for the acceptor substrate erythrose 4‐phosphate, suggesting a role of this residue in carbon–carbon bond cleavage and stabilization of the carbanion/enamine intermediate.
Structural elucidation of a capsular polysaccharide from a clinical isolate of Bacteroides vulgatus from a patient with Crohn’s disease
Authors: Hashimoto Masahito , Kirikae Fumiko , Dohi Taeko , Kusumoto Shoichi , Suda Yasuo , Kirikae Teruo
Published: March 2003

The structure of a capsular polysaccharide (CPS) from a clinical isolate of Bacteroides vulgatus was elucidated. B. vulgatus IMCJ 1204 was isolated from feces of a patient with Crohn’s disease. CPS was prepared by phenol/water extraction of the bacterial cells followed by hydrophobic interaction chromatography and then gel filtration chromatography of the extract. The structure of CPS was determined by chemical analysis and NMR spectroscopy including DQF‐COSY, TOCSY, ROESY, HSQC‐TOCSY, HMQC and HMBC to be a polysaccharide composed of the following repeating unit: →3)β‐d‐Glcp(1→6)[α‐d‐GalpNAc(1→2)β‐d‐Galp(1→4)]β‐d‐GlcpNAc(1→3)α‐d‐Galp(1→4)β‐d‐Manp(1→.
The interaction of human apolipoprotein C‐I with sub‐micellar phospholipid
Authors: Atcliffe Benjamin W. , MacRaild Christopher A. , Gooley Paul R. , Howlett Geoffrey J.
Published: March 2003

Mature human apolipoprotein C‐I (apoC‐I), comprising 57 amino acids, is the smallest member of the plasma apolipoprotein family. Amphipathic helical regions within apoC‐I, common to this class of proteins, are mediators of lipid binding, a process that underlies the functional properties of apoC‐I, including the capacity to activate the plasma enzyme LCAT, to disrupt apoE mediated receptor interactions and to inhibit cholesterol ester transfer protein. To examine apoC‐I/phospholipid interactions, we have developed an expression system in Escherichia coli to obtain purified apoC‐I with yields of approximately 4–5 mg per L of culture. The purified product has properties similar to plasma‐derived apoC‐I including self‐association in the lipid‐free state and induced α‐helical content in the presence of egg‐yolk phosphatidylcholine and dimyristoylglycerophosphocholine vesicles. We chose the short‐chain phospholipid, dihexanoylglycerophosphocholine (Hex2Gro‐PCho), to examine the interaction of apoC‐I with submicellar phospholipid. Circular dichroism spectroscopy and cross‐linking experiments show that apoC‐I acquires helical content and remains self‐associated at submicellar concentrations of Hex2Gro‐PCho (4 mm). Sedimentation equilibrium studies of apoC‐I at submicellar levels of Hex2Gro‐PCho and analysis of the effects of apoC‐I on the 1H NMR spectrum of Hex2Gro‐PCho indicate micelle induction by apoC‐I, and establish the capacity of apoC‐I to assemble individual phospholipid molecules.
Electrodiffusive magnesium transport across the intestinal brush border membrane of tilapia (Oreochromis mossambicus)
Authors: Bijvelds Marcel J. C. , Kolar Zvonimir I. , Flik Gert
Published: March 2003

Mg2+ transport across the brush border of proximal intestinal epithelium of the teleost fish Oreochromis mossambicus was investigated, using 27Mg2+ to trace movement of Mg2+. Mg2+ uptake in brush border membrane vesicles was stimulated by a K+ diffusion potential (inside negative). Electrodiffusive Mg2+ transport obeyed simple Michaelis–Menten kinetics and was strongly temperature dependent, indicative of a carrier mechanism. The metal ion specificity of this electrodiffusive pathway (inhibition potency order: Co > Mn = Ni > La > Ca > Gd > Ba), predicts a specific role in Mg2+ transport. Competitive inhibition by Co(III) hexammine [Co(NH3] suggests that this transport system interacts with the solvated Mg ion. We propose that this novel transport system allows the uptake of Mg2+ across the apical brush border membrane, and is involved in transcellular Mg2+ transport. Consequently, the prevailing potential difference across the apical membrane represents a major driving force for intestinal Mg2+ absorption.
Difference between the E1 and E2 conformations of gastric H+/K+‐ATPase in a multilamellar lipid film system
Authors: Stricht Delphine Vander , Raussens Vincent , Oberg Keith A. , Ruysschaert Jean‐Marie , Goormaghtigh Erik
Published: March 2003

A liquid flow cell was used for an attenuated total reflection‐Fourier transform infrared spectroscopy (ATR‐FTIR) study of conformational changes taking place in the gastric H+/K+‐ATPase. Shifting from E1 to E2 form is induced by replacing Na+ by K+ ions. Introducing ions through a flow passing over a protein multilayer film induced the conformational change without cell manipulations. Measurement sensitivity was thereby improved by about one order of magnitude. The detection threshold allowed the possibility to detect a change affecting five amino acids out of the 1324 that compose the H+/K+‐ATPase molecule. It appeared that fewer than five amino‐acid residues undergo a conformational change upon replacing Na+ by K+ ions in the medium. Evidence that conformational changes occur in an identical system was brought by monitoring the fluorescence of fluorescein isothiocyanate‐labeled H+/K+‐ATPase in similar conditions. Our data suggest that essentially the tertiary structure of the protein is modified.
Purification and structure of the major product obtained by reaction of NADPH and NMNH with the myeloperoxidase/hydrogen peroxide/chloride system
Authors: Auchère Françoise , Bertho Gildas , Artaud Isabelle , Girault Jean Pierre , Capeillère‐Blandin Chantal
Published: March 2003

The first spectrophotometric study of the reaction of the myeloperoxidase/H2O2/Cl− system with NADPH and NMNH showed that the reaction products were not the corresponding oxidized nucleotides and that modifications would take place on the nicotinamide part of the molecule [Auchère, F. & Capeillère‐Blandin, C. (1999) Biochem. J. 343, 603–613]. In this report, in order to obtain more precise information on the structural modifications and mechanism of the reaction, we focus on the purification and isolation of products derived from NADPH and NMNH by RP‐HPLC. Electrospray ionization mass spectra indicated that the relative height of the peaks reflected that of the natural isotopic abundance of 35Cl and 37Cl, providing evidence that the products derived from NADPH and NMNH were monochlorinated. Moreover, calculated masses revealed the 1 : 1 addition of HOCl to the molecule. Various 1D and 2D NMR experiments provided data for the assignments of the chemical shifts of protons and carbons and the coupling constants of the protons of the chlorinated nucleotides. Further NOESY experiments allowed the characterization of the spatial structure of the chlorinated product and showed that trans HOCl addition occurred at the C5=C6 carbon double bond of the nicotinamide ring, leading to a chlorohydrin.
Functional and computer modelling studies of haemoglobin from horse
Authors: Pellegrini Mariagiuseppina , Corda Marcella , Manca Laura , Olianas Alessandra , Sanna Maria Teresa , Fais Antonella , De Rosa M. Cristina , Bertonati Claudia , Masala Bruno , Giardina Bruno
Published: March 2003

A study was made of the haemoglobin (Hb) system from the Sardinian dwarf horse (Equus caballus jara), one of the last surviving wild horse species in Europe. The oxygen binding properties of the whole haemolysate and of the four different horse Hbs, separated by ion‐exchange chromatography, were studied with special regard to the effect of chloride, 2,3‐diphosphoglycerate and lactate. Results indicate that no significant functional differences exist between the four Hb components of horse haemolysate. Moreover, the molecular basis of the intrinsically low oxygen affinity and of the weak interaction of horse Hb with 2,3‐diphosphoglycerate is discussed in the light of the primary structure of the molecule and of the results of a computer modelling approach. On these bases, it is suggested that the A1 (Thr→Ser) and A2 (Pro→Gly) substitutions observed in the β chains from horse Hb may be responsible for the displacement of the A helix that is known to be a key structural feature of those Hbs that display an altered interaction with 2,3‐diphosphoglycerate as compared with human Hb.
Fibrate induction of the adrenoleukodystrophy‐related gene (ABCD2)
Authors: Fourcade Stéphane , Savary Stéphane , Albet Sabrina , Gauthé Delphine , Gondcaille Catherine , Pineau Thierry , Bellenger Jérome , Bentejac Marc , Holzinger Andreas , Berger Johannes , Bugaut Maurice
Published: March 2003

X‐linked adrenoleukodystrophy (X‐ALD) is a neurodegenerative disease due to a defect in the ABCD1 (ALD) gene. ABCD1, and the two close homologues ABCD2 (ALDR) and ABCD3 (PMP70), are genes encoding ATP‐binding cassette half‐transporters of the peroxisomal membrane. As overexpression of the ABCD2 or ABCD3 gene can reverse the biochemical phenotype of X‐ALD (reduced β‐oxidation of very‐long‐chain fatty acids), pharmacological induction of these partially redundant genes may represent a therapeutic approach to X‐ALD. We previously reported that the ABCD2 and ABCD3 genes could be strongly induced by fibrates, which are hypolipidaemic drugs and peroxisome‐proliferators in rodents. We provide evidence that the induction is dependent on peroxisome proliferator‐activated receptor (PPARα) as both genes were not induced in fenofibrate‐treated PPARα–/– knock‐out mice. To further characterize the PPARα pathway, we cloned and analysed the promoter of the ABCD2 gene, the closest homologue of the ABCD1 gene. The proximal region (2 kb) of the rat promoter displayed a high conservation with the human and mouse cognate sequences suggesting an important role of the region in regulation of the ABCD2 gene. Classically, fibrate‐induction involves interaction of PPARα with a response element (PPRE) characterized by a direct repeat of the AGGTCA‐like motif. Putative PPRE motifs of the rat ABCD2 promoter were studied in the isolated form or in their promoter context by gel‐shift assay and transfection of COS‐7 cells. We failed to characterize a functional PPRE, suggesting a different mechanism for the PPARα‐dependent regulation of the ABCD2 gene.
In vitro processing of the proproteins GrdE of protein B of glycine reductase and PrdA of D‐proline reductase from Clostridium sticklandii
Authors: Bednarski Brit , Andreesen Jan R. , Pich Andreas
Published: March 2003

GrdE and PrdA of Clostridium sticklandii are subunits of glycine reductase and d‐proline reductase, respectively, that are processed post‐translationally to form a catalytic active pyruvoyl group. The cleavage occurred on the N‐terminal side of a cysteine residue, which is thus the precursor of a pyruvoyl moiety. Both proproteins could be over‐expressed in Escherichia coli and conditions were developed for in vitro processing. GrdE could be expressed as full‐size protein, whereas PrdA had to be truncated N‐terminally to achieve successful over‐expression. Both proproteins were cleaved at the in vivo observed cleavage site after addition of 200 mm NaBH4 in Tris buffer (pH 7.6) at room temperature as analysed by SDS/PAGE and MS. Cleavage of GrdE was observed with a half‐time of ≈ 30 min. Cys242, as the precursor of the pyruvoyl group in GrdE, was changed to alanine, serine, or threonine by site‐directed mutagenesis. The Cys242→Ser and Cys242→Thr mutant proteins were also cleaved under similar conditions with extended half‐times. However, the Cys242→Ala mutant protein was not cleaved indicating a pivotal role of the thiol group of cysteine or hydroxyl group of serine and threonine during the processing of pyruvoyl group‐dependent reductases.
Overproduction of a functional A1 ATPase from the archaeon Methanosarcina mazei Gö1 in Escherichia coli
Authors: Lemker Thorsten , Ruppert Claudia , Stöger Heidi , Wimmers Sönke , Müller Volker
Published: March 2003

Single subunits of the A1 ATPase from the archaeon Methanosarcina mazei Gö1 were produced in E. coli as MalE fusions and purified, and polyclonal antibodies were raised against the fusion proteins. A DNA fragment containing the genes ahaE, ahaC, ahaF, ahaA, ahaB, ahaD, and ahaG, encoding the hydrophilic A1 domain and part of the stalk of the A1AO ATPase of M. mazei Gö1, was constructed, cloned into an expression vector and transformed into different strains of Escherichia coli. In any case, a functional, ATP‐hydrolysing A1 ATPase was produced. Western blots demonstrated the production of subunits A, B, C, and F in E. coli, and minicell analyses suggested that subunits D, E, and G were produced as well. This is the first demonstration of a heterologous production of a functional ATPase from an archaeon. The A1 ATPase was sensitive to freezing but lost only about 50% of its activity within 18 days on ice. Inhibitor studies revealed that the heterologously produced A1 ATPase is insensitive to azide, dicyclohexylcarbodiimide and bafilomycin A1, but sensitive to diethylstilbestrol and its analogues dienestrol and hexestrol. The expression system described here will open new avenues towards the functional and structural analyses of this unique class of enzymes.
Co‐clustering of Fcγ and B cell receptors induces dephosphorylation of the Grb2‐associated binder 1 docking protein
Authors: Koncz Gábor , Tóth Gábor K. , Bökönyi Gyöngyi , Kéri György , Pecht Israel , Medgyesi David , Gergely János , Sármay Gabriella
Published: March 2003

The immunoreceptor tyrosine‐based inhibitory motif (ITIM) of human type IIb Fcγ receptor (FcγRIIb) is phosphorylated on its tyrosine upon co‐clustering with the B cell receptor (BCR). The phosphorylated ITIM (p‐ITIM) binds to the SH2 domains of polyphosphoinositol 5‐phosphatase (SHIP) and the tyrosine phosphatase, SHP‐2. We investigated the involvement of the molecular complex composed of the phosphorylated SHIP and FcγRIIb in the activation of SHP‐2. As a model compound, we synthesized a bisphosphopeptide, combining the sequences of p‐ITIM and the N‐terminal tyrosine phosphorylated motif of SHIP with a flexible spacer. This compound bound to the recombinant SH2 domains of SHP‐2 with high affinity and activated the phosphatase in an in vitro assay. These data suggest that the phosphorylated FcγRII‐SHIP complexes formed in the intact cells may also activate SHP‐2. Grb2‐associated binder 1 (Gab1) is a multisite docking protein, which becomes tyrosine‐phosphorylated in response to various types of signaling, including BCR. In turn it binds to the SH2 domains of SHP‐2, SHIP and the p85 subunit of phosphatidyl inositol 3‐kinase (PtdIns3‐K) and may regulate their activity. Gab1 is a potential substrate of SHP‐2, thus its binding to FcγRIIb may modify the Gab1‐bound signaling complex. We show here that Gab1 is part of the multiprotein complex assembled by FcγRIIb upon its co‐clustering with BCR. Gab1 may recruit SH2 domain‐containing molecules to the phosphorylated FcγRIIb. SHP‐2, activated upon the binding to FcγRIIb‐SHIP complex, partially dephosphorylates Gab1, resulting in the release of PtdIns3‐K and ultimately in the inhibition of downstream activation pathways in BCR/FcγRIIb co‐aggregated cells.
Drug sequestration in cytoplasmic organelles does not contribute to the diminished sensitivity of anthracyclines in multidrug resistant K562 cells
Authors: Loetchutinat Chatchanok , Priebe Waldemar , Garnier‐Suillerot Arlette
Published: March 2003

Cells that acquire multidrug resistance (MDR) are characterized by a decreased accumulation of a variety of drugs. In addition, sequestration of drugs in intracellular vesicles has often been associated with MDR. However, the nature and role of intracellular vesicles in MDR are unclear. We addressed the relationship between MDR and vesicular anthracycline accumulation in the erythroleukemia cell line K562 and a drug‐resistant counterpart K562/ADR that overexpresses P‐glycoprotein. We used four anthracyclines (all of which are P‐glycoprotein substrates): daunorubicin and idarubicin, which have good affinity for DNA and as weak bases can accumulate inside acidic compartments; hydroxyrubicin, which binds to DNA but is uncharged at physiological or acidic pH and thus cannot accumulate in acidic compartments; and WP900, an enantiomer of daunorubicin, which is a weak DNA binder but has the same pKa and lipophilicity as daunorubicin. The intrinsic fluorescence of anthracyclines allowed us to use macro‐ and micro‐spectrofluorescence, flow cytometry, and confocal microscopy to characterize their nuclear or intravesicular accumulation in living cells. We found that vesicular accumulation of daunorubicin, WP900 and idarubicin, containing a basic 3′‐amine was predominantly restricted to lysosomes in both cell lines, that pH regulation of acidic compartments was not defective in human K562 cells, and that vesicular drug accumulation was much more pronounced in the parental tumor cell line than in the multidrug‐resistant cells. These results indicate that vesicular anthracycline sequestration does not contribute to the diminished sensitivity to anthracyclines in multidrug‐resistant K562 cells.
Increase of the deacylation rate of PBP2x from Streptococcus pneumoniae by single point mutations mimicking the class A β‐lactamases
Authors: Chesnel Laurent , Zapun André , Mouz Nicolas , Dideberg Otto , Vernet Thierry
Published: March 2003

The class A β‐lactamases and the transpeptidase domain of the penicillin‐binding proteins (PBPs) share the same topology and conserved active‐site residues. They both react with β‐lactams to form acylenzymes. The stability of the PBP acylenzymes results in the inhibition of the transpeptidase function and the antibiotic activity of the β‐lactams. In contrast, the deacylation of the β‐lactamases is extremely fast, resulting in a high turnover of β‐lactam hydrolysis, which confers resistance to these antibiotics. In TEM‐1 β‐lactamase from Escherichia coli, Glu166 is required for the fast deacylation and occupies the same spatial location as Phe450 in PBP2x from Streptococcus pneumoniae. To gain insight into the deacylation mechanism of both enzymes, Phe450 of PBP2x was replaced by various residues. The introduction of ionizable side chains increased the deacylation rate, in a pH‐dependent manner, for the acidic residues. The aspartic acid‐containing variant had a 110‐fold faster deacylation at pH 8. The magnitude of this effect is similar to that observed in a naturally occurring variant of PBP2x, which confers increased resistance to cephalosporins.