bluuebunny/crossref_metadata_2025 huggingface dataset:
Synthesis, characterization and application of two nucleoside triphosphate analogues, GTPγNH2 and GTPγF
Authors: Stumber Michael , Herrmann Christian , Wohlgemuth Sabine , Kalbitzer Hans Robert , Jahn Werner , Geyer Matthias
Published: March 2003

Guanosine triphosphate nucleotide analogues such as GppNHp (also named GMPPNP) or GTPγS are widely used to stabilize rapidly hydrolyzing protein‐nucleotide complexes and to investigate biochemical reaction pathways.Here we describe the chemical synthesis of guanosine 5′‐O‐(γ‐amidotriphosphate) (GTPγNH2) and a new synthesis of guanosine 5′‐O‐(γ‐fluorotriphosphate) (GTPγF). The two nucleotides were characterized using NMR spectroscopy and isothermal titration calorimetry. Chemical shift data on 31P, 19F and 1H NMR resonances are tabulated. For GTPγNH2 the enthalpy of magnesium coordination is ΔH° = 3.9 kcal·mol−1 and the association constant Ka is 0.82 mm−1. The activation energy for GTPγNH2·Mg2+ complex formation is ΔH‡ = 7.8 ± 0.15 kcal·mol−1, similar to that for the natural substrate GTP. For GTPγF we obtained a similar enthalpy of ΔH° = 3.9 kcal·mol−1 while the magnesium association constant is only Ka = 0.2 mm−1. The application of both guanine nucleotide analogues to theGTP‐binding protein Ras was investigated. The rate of hydrolysis of GTPγNH2 bound to Ras protein lay between the rates found for Ras‐bound GTPγS and GppNHp, while Ras‐catalysed hydrolysis of GTPγF was almost as fast as for GTP. The two compounds extend the variety of nucleotide analogues and may prove useful in structural, kinetic and cellular studies.
Structure of human immunodeficiency virus type 1 Vpr(34–51) peptide in micelle containing aqueous solution
Authors: Engler Andrea , Stangler Thomas , Willbold Dieter
Published: March 2003

Human immunodeficiency virus type 1 protein R (HIV‐1 Vpr) promotes nuclear entry of viral nucleic acids in nondividing cells, causes G2 cell cycle arrest and is involved in cellular differentiation and cell death. Vpr subcellular localization is as variable as its functions. It is known, that consistent with its role in nuclear transport, Vpr localizes to the nuclear envelope of human cells. Further, a reported ion channel activity of Vpr is clearly dependent on its localization in or at membranes. We focused our structural studies on the secondary structure of a peptide consisting of residues 34–51 of HIV‐1 Vpr. This part of Vpr plays an important role in Vpr oligomerization, contributes to cell cycle arrest activity, and is essential for virion incorporation and binding to HHR23A, a protein involved in DNA repair. Employing NMR spectroscopy we found this part of Vpr to be almost completely α helical in the presence of micelles, as well as in trifluoroethanol containing and methanol/chloroform solvent. Our results provide structural data suggesting residues 34–51 of Vpr to contain an amphipathic, leucine‐zipper‐like α helix, which serves as a basis for oligomerization of Vpr and its interactions with cellular and viral factors involved in subcellular localization and virion incorporation of Vpr.
The cold‐active lipase of Pseudomonas fragi
Authors: Alquati Claudia , De Gioia Luca , Santarossa Gianluca , Alberghina Lilia , Fantucci Piercarlo , Lotti Marina
Published: March 2003

A recombinant lipase cloned from Pseudomonas fragi strain IFO 3458 (PFL) was found to retain significant activity at low temperature. In an attempt to elucidate the structural basis of this behaviour, a model of its three‐dimensional structure was built by homology and compared with homologous mesophilic lipases, i.e. the Pseudomonas aeruginosa lipase (45% sequence identity) and Burkholderia cepacia lipase (38%). In this model, features common to all known lipases have been identified, such as the catalytic triad (S83, D238 and H260) and the oxyanion hole (L17, Q84). Structural modifications recurrent in cold‐adaptation, i.e. a large amount of charged residues exposed at the protein surface, have been detected. Noteworthy is the lack of a disulphide bridge conserved in homologous Pseudomonas lipases that may contribute to increased conformational flexibility of the cold‐active enzyme.
Expression of the aspartate/glutamate mitochondrial carriers aralar1 and citrin during development and in adult rat tissues
Authors: del Arco Araceli , Morcillo Julián , Martínez‐Morales Juan Ramon , Galián Carmen , Martos Vera , Bovolenta Paola , Satrústegui Jorgina
Published: March 2003

Aralar1 and citrin are members of the subfamily of calcium‐binding mitochondrial carriers and correspond to two isoforms of the mitochondrial aspartate/glutamate carrier (AGC). These proteins are activated by Ca2+ acting on the external side of the inner mitochondrial membrane. Although it is known that aralar1 is expressed mainly in skeletal muscle, heart and brain, whereas citrin is present in liver, kidney and heart, the precise tissue distribution of the two proteins in embryonic and adult tissues is largely unknown. We investigated the pattern of expression of aralar1 and citrin in murine embryonic and adult tissues at the mRNA and protein levels. In situ hybridization analysis indicates that both isoforms are expressed strongly in the branchial arches, dermomyotome, limb and tail buds at early embryonic stages. However, citrin was more abundant in the ectodermal components of these structures whereas aralarl had a predominantly mesenchymal localization. The strong expression of citrin in the liver was acquired postnatally, whereas the characteristic expression of aralar1 in skeletal muscle was detected at E18 and that in the heart began early in development (E11) and was preferentially localized to auricular myocardium in late embryonic stages. Aralar1 was also expressed in bone marrow, T‐lymphocytes and macrophages, including Kupffer cells in the liver, indicating that this is the major AGC isoform present in the hematopoietic system. Both aralar1 and citrin were expressed in fetal gut and adult stomach, ovary, testis, and pancreas, but only aralar1 is enriched in lung and insulin‐secreting β cells. These results show that aralar1 is expressed in many more tissues than originally believed and is absent from hepatocytes, where citrin is the only AGC isoform present. This explains why citrin deficiency in humans (type II citrullinemia) only affects the liver and suggests that aralar1 may compensate for the lack of citrin in other tissues.
Identification of mammalian‐type transglutaminase in Physarum polycephalum
Authors: Wada Fumitaka , Nakamura Akio , Masutani Tomohiro , Ikura Koji , Maki Masatoshi , Hitomi Kiyotaka
Published: March 2003

Transglutaminase (TGase) catalyses the post‐translational modification of proteins by transamidation of available glutamine residues. While several TGase genes of fish and arthropods have been cloned and appear to have similar structures to those of mammals, no homologous gene has been found in lower eukaryotes. We have cloned the acellular slime mold Physarum polycephalum TGase cDNA using RT‐PCR with degenerated primers, based on the partial amino acid sequence of the purified enzyme. The cDNA contained a 2565‐bp ORF encoding a 855‐residue polypeptide. By Northern blotting, an mRNA of ≈ 2600 bases was detected. In comparison with primary sequences of mammalian TGases, surprisingly, significant similarity was observed including catalytic triad residues (Cys, His, Asn) and a GTP‐binding region. The alignment of sequences and a phylogenetic tree also demonstrated that the structure of P. polycephalum TGase is similar to that of TGases of vertebrates. Furthermore, we observed that the purified TGase had GTP‐hydrolysing activity and that GTP inhibited its transamidating activity, as in the case of mammalian tissue‐type TGase (TGase 2).
Interaction of the GTS1 gene product with glyceraldehyde‐ 3‐phosphate dehydrogenase 1 required for the maintenance of the metabolic oscillations of the yeast Saccharomyces cerevisiae
Authors: Liu Weidong , Wang Jinqing , Mitsui Kazuhiro , Shen Hua , Tsurugi Kunio
Published: March 2003

We previously reported that GTS1 is involved in regulating ultradian oscillations of the glycolytic pathway induced by cyanide in cell suspensions as well as oscillations of energy metabolism in aerobic continuous cultures. Here, we screened a yeast cDNA library for proteins that bind to Gts1p using the yeast two‐hybrid system and cloned multiple TDH cDNAs encoding the glycolytic enzyme glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH). We found that the zinc‐finger and dimerization sites of Gts1p were required for full ability to bind GAPDH, and Gts1ps mutated at these sites lost the ability to regulate both aerobic and unaerobic ultradian oscillations of energy metabolism. Of the three TDH genes, only TDH1 fluctuated at the mRNA level in continuous culture and its deletion resulted in the disappearance of the oscillation without any affect on growth rate. This loss of biological rhythms in the TDH1‐deleted mutant was rescued by the expression of TDH1 but not of TDH2 or TDH3 under the control of the TDH1 promoter. Thus, we hypothesized that Gts1p plays a role in the regulation of metabolic oscillation by interacting with the TDH1 product, GAPDH1, in yeast.
Action of palytoxin on apical H+/K+‐ATPase in rat colon
Authors: Scheiner‐Bobis Georgios , Hübschle Thomas , Diener Martin
Published: March 2003

Palytoxin stimulated a cation‐dependent short‐circuit current (Isc) in rat distal and proximal colon in a concentration‐dependent fashion when applied to the mucosal surface of the tissue. The distal colon exhibited a higher sensitivity to the toxin. The palytoxin‐induced Isc was blocked by vanadate but was resistant to ouabain or scilliroside, suggesting the conversion of a vanadate‐sensitive H+/K+‐ATPase into an electrogenic cation transporter. Cation substitution experiments with basolaterally depolarized tissues suggested an apparent permeability of the palytoxin‐induced conductance of Na+>K+>Li+. Immunohistochemical control experiments confirmed the absence of the Na+/K+‐ATPase in the apical membrane. Consequently, the pore‐forming action of palytoxin is not restricted to Na+/K+‐ATPase but is also observed with the colonic H+/K+‐ATPase.
Differential effects of arachidonoyl trifluoromethyl ketone on arachidonic acid release and lipid mediator biosynthesis by human neutrophils
Authors: Fonteh Alfred N.
Published: March 2003

The goal of this study was to determine the effects of a putative specific cytosolic phospholipase A2 inhibitor, arachidonyl trifluoromethyl ketone (AACOCF3), on arachidonic acid (AA) release and lipid mediator biosynthesis by ionophore‐stimulated human neutrophils. Initial studies indicated that AACOCF3 at concentrations 0–10 µm did not affect AA release from neutrophils. In contrast, AACOCF3 potently inhibited leukotriene B4 formation by ionophore‐stimulated neutrophils (IC50 ≈ 2.5 µm). Likewise, AACOCF3 significantly inhibited the biosynthesis of platelet activating factor. In cell‐free assay systems, 10 µm AACOCF3 inhibited 5‐lipoxygenase and CoA‐independent transacylase activities. [3H]AA labeling studies indicated thatthe specific activities of cell‐associated AA mimicked that of leukotriene B4 and PtdCho/PtdIns, while the specific activities of AA released into the supernatant fluid closely mimicked that of PtdEtn. Taken together, these data argue for the existence of segregated pools of arachidonate in human neutrophils. One pool of AA is linked to lipid mediator biosynthesis while another pool provides free AA that is released from cells. Additionally, the data suggest that AACOCF3 is also an inhibitor of CoA‐independent transacylase and 5‐lipoxygenase. Thus, caution should be exercised in using AACOCF3 as an inhibitor of cytosolic phospholipase A2 in whole cell assays because of the complexity of AA metabolism.
In vitro and in vivo self‐cleavage of Streptococcus pneumoniae signal peptidase I
Authors: Zheng Feng , Angleton Eddie L. , Lu Jin , Peng Sheng‐Bin
Published: March 2003

We have previously demonstrated that Streptococcus pneumoniae signal peptidase (SPase) I catalyzes a self‐cleavage to result in a truncated product, SPase37–204 [Peng, S.B., Wang, L., Moomaw, J., Peery, R.B., Sun, P.M., Johnson, R.B., Lu, J., Treadway, P., Skatrud, P.L. & Wang, Q.M. (2001) J. Bacteriol.183, 621–627]. In this study, we investigated the effect of phospholipid on invitro self‐cleavage of S. pneumoniae SPase I. In the presence of phospholipid, the self‐cleavage predominantly occurred at one cleavage site between Gly36–His37, whereas the self‐cleavage occurred at multiple sites in the absence of phospholipid, and two additional self‐cleavage sites, Ala65–His66 and Ala143–Phe144, were identified. All three self‐cleavage sites strongly resemble the signal peptide cleavage site and follow the (−1, −3) rule for SPase I recognition. Kinetic analysis demonstrated that self‐cleavage is a concentration dependent and intermolecular event, and the activity in the presence of phospholipid is 25‐fold higher than that in the absence of phospholipid. Biochemical analysis demonstrated that SPase37–204, the major product of the self‐cleavage totally lost activity to cleave its substrates, indicating that the self‐cleavage resulted in the inactivation of the enzyme. More importantly, the self‐cleavage was demonstrated to be happening in vivo in all the growth phases of S. pneumoniae cells. The bacterial cells keep the active SPase I at the highest level in exponential growth phase, suggesting that the self‐cleavage may play an important role in regulating the activity of the enzyme under different conditions.
Prediction of protein structural class by amino acid and polypeptide composition
Authors: Luo Rui‐yan , Feng Zhi‐ping , Liu Jia‐kun
Published: March 2003

A new approach of predicting structural classes of protein domain sequences is presented in this paper. Besides the amino acid composition, the composition of several dipeptides, tripeptides, tetrapeptides, pentapeptides and hexapeptides are taken into account based on the stepwise discriminant analysis. The result of jackknife test shows that this new approach can lead to higher predictive sensitivity and specificity for reduced sequence similarity datasets. Considering the dataset PDB40‐B constructed by Brenner and colleagues, 75.2% protein domain sequences are correctly assigned in the jackknife test for the four structural classes: all‐α, all‐β, α/β and α + β, which is improved by 19.4% in jackknife test and 25.5% in resubstitution test, in contrast with the component‐coupled algorithm using amino acid composition alone (AAC approach) for the same dataset. In the cross‐validation test with dataset PDB40‐J constructed by Park and colleagues, more than 80% predictive accuracy is obtained. Furthermore, for the dataset constructed by Chou and Maggiona, the accuracy of 100% and 99.7% can be easily achieved, respectively, in the resubstitution test and in the jackknife test merely taking the composition of dipeptides into account. Therefore, this new method provides an effective tool to extract valuable information from protein sequences, which can be used for the systematic analysis of small or medium size protein sequences. The computer programs used in this paper are available on request.
Inactivation of annexin II tetramer by S‐nitrosoglutathione
Authors: Liu Lin , Enright Edward , Sun Peng , Tsai Shwu Yar , Mehta Pragna , Beckman David L , Terrian David M
Published: March 2003

We investigated the effect of nitric oxide (NO) donors on the activities of annexin II tetramer (AIIt), a member of the Ca2+‐ dependent phospholipid‐binding protein family. Incubation of purified AIIt with S‐nitrosoglutathione (GSNO) led to the inhibition of AIIt‐mediated liposome aggregation. This effect was dose‐dependent with an IC50 of approximately 100 µm. Sodium nitroprusside, another NO donor also inhibited AIIt‐mediated liposome aggregation, whereas reduced glutathione, nitrate, or nitrite had no effects. GSNO also inhibited AIIt‐mediated membrane fusion, but not the binding of AIIt to the membrane. GSNO only has a modest effect on liposome aggregation mediated by annexins I, III or IV. The binding of AIIt to the membrane protected the reactive sites of GSNO on AIIt. GSNO did not inhibit AIIt‐mediated liposome aggregation in the presence of dithiothreitol. Taken together, our results suggest that GSNO inactivates AIIt possibly via S‐nitrosylation and/or the formation of disulfide bonds.
Identification of two cysteine residues involved in the binding of UDP‐GalNAc to UDP‐GalNAc:polypeptide N‐acetylgalactosaminyltransferase 1 (GalNAc‐T1)
Authors: Tenno Mari , Toba Shinya , Kézdy Ferénc J , Elhammer Åke P. , Kurosaka Akira
Published: March 2003

Biosynthesis of mucin‐type O‐glycans is initiated by a family of UDP‐GalNAc:polypeptide N‐acetylgalactosaminyltransferases, which contain several conserved cysteine residues among the isozymes. We found that a cysteine‐specific reagent, p‐chloromercuriphenylsulfonic acid (PCMPS), irreversibly inhibited one of the isozymes (GalNAc‐T1). Presence of either UDP‐GalNAc or UDP during PCMPS treatment protected GalNAc‐T1 from inactivation, to the same extent. This suggests that GalNAc‐T1 contains free cysteine residues interacting with the UDP moiety of the sugar donor. For the functional analysis of the cysteine residues, several conserved cysteine residues in GalNAc‐T1 were mutated individually to alanine. All of the mutations except one resulted in complete inactivation or a drastic decrease in the activity, of the enzyme. We identified only Cys212 and Cys214, among the conserved cysteine residues in GalNAc‐T1, as free cysteine residues, by cysteine‐specific labeling of GalNAc‐T1. To investigate the role of these two cysteine residues, we generated cysteine to serine mutants (C212S and C214S). The serine mutants were more active than the corresponding alanine mutants (C212A and C214A). Kinetic analysis demonstrated that the affinity of the serine‐mutants for UDP‐GalNAc was decreased, as compared to the wild type enzyme. The affinity for the acceptor apomucin, on the other hand, was essentially unaffected. The functional importance of the introduced serine residues was further demonstrated by the inhibition of all serine mutant enzymes with diisopropyl fluorophosphate. In addition, the serine mutants were more resistant to modification by PCMPS. Our results indicate that Cys212 and Cys214 are sites of PCMPS modification, and that these cysteine residues are involved in the interaction with the UDP moiety of UDP‐GalNAc.
High‐pressure effects on horse heart metmyoglobin studied by small‐angle neutron scattering
Authors: Loupiac Camille , Bonetti Marco , Pin Serge , Calmettes Patrick
Published: March 2003

Small‐angle neutron scattering experiments were performed on horse azidometmyoglobin (MbN3) at pressures up to 300 MPa. Other spectroscopic techniques have shown that a reorganization of the secondary structure and of the active site occur in this pressure range. The present measurements, performed using various concentrations of MbN3, show that the compactness of the protein is not altered as the value of its radius of gyration remains constant up to 300 MPa. The value of the second virial coefficient of the protein solution indicates that the interactions between the molecules are always strongly repulsive even if their magnitude decreases with increasing pressure. Taking advantage of the pressure‐induced contrast variation, these experiments allow the partial specific volume of MbN3 to be determined as a function of pressure. Its value decreases by 5.4% between atmospheric pressure and 300 MPa. In this pressure range the isothermal compressibility of hydrated MbN3 is found to be almost constant. Its value is (1.6 ± 0.1) 10−4 MPa−1.
Vibrio cholerae hemolysin
Authors: Chattopadhyay Kausik , Bhattacharyya Debasish , Banerjee Kalyan K
Published: March 2003

Vibrio cholerae hemolysin (HlyA), a water‐soluble protein with a native monomeric relative molecular mass of 65 000, forms transmembrane pentameric channels in target biomembranes. The HlyA binds to lipid vesicles nonspecifically and without saturation; however, self‐assembly is triggered specifically by cholesterol. Here we show that the HlyA partitioned quantitatively to amphiphilic media irrespective of their compositions, indicating that the toxin had an amphiphilic surface. Asialofetuin, a β1‐galactosyl‐terminated glycoprotein, which binds specifically to the HlyA in a lectin‐glycoprotein type of interaction and inhibits carbohydrate‐independent interaction of the toxin with lipid, reduced effective amphiphilicity of the toxin significantly. Resistance of the HlyA to proteases together with the tryptophan fluorescence emission spectrum suggested a compact structure for the toxin. Fluorescence energy transfer from the HlyA to dansyl‐phosphatidylethanolamine required the presence of cholesterol in the lipid bilayer and was synchronous with oligomerization. Phospholipid bilayer without cholesterol caused a partial unfolding of the HlyA monomer as indicated by the transfer of tryptophan residues from the nonpolar core of the protein to a more polar region. These observations suggested: (a) partitioning of the HlyA to lipid vesicles is driven by the tendency of the amphiphilic toxin to reduce energetically unfavorable contacts with water and is not affected significantly by the composition of the vesicles; and (b) partial unfolding of the HlyA at the lipid–water interface precedes and promotes cholesterol‐induced oligomerization to an insertion‐competent configuration.
Effects of a tryptophanyl substitution on the structure and antimicrobial activity of C‐terminally truncated gaegurin 4
Authors: Won Hyung‐Sik , Park Sang‐Ho , Kim Hyung Eun , Hyun Byongkuk , Kim Mijin , Lee Byeong Jae , Lee Bong‐Jin
Published: March 2003

Gaegurin 4 (GGN4), a 37‐residue antimicrobial peptide, consists of two amphipathic α helices (residues 2–10 and 16–32) connected by a flexible loop region (residues 11–15). As part of an effort to develop new peptide antibiotics with low molecular mass, the activities of C‐terminally truncated GGN4 analogues were tested. Δ24−37 GGN4, a peptide analogue with 14 residues truncated from the C‐terminus of GGN4, showed a complete loss of antimicrobial activity. However, the single substitution of aspartic acid 16 by tryptophan (D16W) in the Δ24−37 GGN4 completely restored the antimicrobial activity, without any significant hemolytic activity. In contrast, neither the D16F nor K15W substitution of the Δ24−37 GGN4 allowed such a dramatic recovery of activity. In addition, the D16W substitution of the native GGN4 significantly enhanced the hemolytic activity as well as the antimicrobial activity. The structural effect of the D16W substitution in the Δ24−37 GGN4 was investigated by CD, NMR, and fluorescence spectroscopy. The results showed that the single tryptophanyl substitution at position 16 of the Δ24−37 GGN4 induced an α helical conformation in the previously flexible loop region in intact GGN4, thereby forming an entirely amphipathic α helix. In addition, the substituted tryptophan itself plays an important role in the membrane‐interaction of the peptide.
Isoprenoid biosynthesis via the methylerythritol phosphate pathway
Authors: Hoeffler Jean‐François , Tritsch Denis , Grosdemange‐Billiard Catherine , Rohmer Michel
Published: March 2003

The 1‐deoxyxylulose 5‐phosphate reductoisomerase (DXR, EC 1.1.1.267) catalyzes the conversion of 1‐deoxy‐d‐xylulose 5‐phosphate (DXP) into 2‐C‐methyl‐d‐erythritol 4‐phosphate (MEP). This transformation is a two‐step process involving a rearrangement of DXP into the putative intermediate 2‐C‐methyl‐d‐erythrose 4‐phosphate followed by a NADPH‐dependent reduction of the latter aldehyde. By using [1‐13C]DXP as a substrate, the rearrangement of DXP into [5‐13C]2‐C‐methyl‐d‐erythrose 4‐phosphate was shown to be NADPH dependent, although it does not involve areduction step. The putative aldehyde intermediate, obtained by chemical synthesis, was converted into MEP by the DXR in the presence of NADPH and into DXP in the presence of NADP+, indicating the reversibility of the reaction catalyzed by the DXR. This reversibility was confirmed by the conversion of MEP into DXP in the presence of NADP+. The equilibrium was, however, largely displaced in favour of the formation of MEP. The reduction step required the presence of a divalent cation such as Mg2+ or Mn2+.
NblA from Anabaena sp. PCC 7120 is a mostly α‐helical protein undergoing reversible trimerization in solution
Authors: Strauss Holger , Misselwitz Rolf , Labudde Dirk , Nicklisch Sabine , Baier Kerstin
Published: March 2003

The nblA family of genes encodes for small proteins necessary for the ordered degradation of phycobilisomes under certain stress conditions, a process known as chlorosis. Genes homologous to nblA seem to occur in all phycobilisome‐containing organisms. However, to date, no molecular mechanism is known for the action of NblA, nor have the gene products been characterized to understand the physical properties of the molecule and thus help elucidate the mechanism on a structural basis.

In this study we report on the first characterization of an NblA‐homologous gene product. The chromosomal gene from the cyanobacterium Anabaena sp. PCC 7120 was cloned, heterologously expressed in Escherichia coli and purified to apparent homogeneity. This allowed the protein to be characterized by analytical ultracentrifugation and CD spectroscopy. These experiments show that the NblA protein has a mostly α‐helical structure, undergoing an association reaction of folded monomers to form trimers in solution. No dimers are detectable.
Novel bradykinins and their precursor cDNAs from European yellow‐bellied toad (Bombina variegata) skin
Authors: Chen Tianbao , Orr David F. , Bjourson Anthony J. , McClean Stephen , O'Rourke Martin , Hirst David G. , Rao Pingfan , Shaw Chris
Published: March 2003

Two novel bradykinin‐related peptides (Ala3,Thr6)‐bradykinin and (Val1,Thr3,Thr6)‐bradykinin, were identified by a systematic sequencing study of peptides in the defensive skin secretion of the yellow‐bellied toad, Bombina variegata. These peptides are the first amphibian skin bradykinins to exhibit amino acid substitutions at the Pro3 position of the bradykinin nonapeptide. Previously reported bradykinins from other Bombina species were not detected. Respective precursor cDNAs, designated BVK‐1 and BVK‐2, respectively, were cloned from a skin library by 3′‐ and 5′‐RACE reactions. BVK‐1 contained an open‐reading frame of 97 amino acids encoding a single copy of (Ala3,Thr6)‐bradykinin and similarly, the open‐reading frame of BVK‐2 consisted of 96 amino acids encoding a single copy of (Val1,Thr3,Thr6)‐bradykinin. Synthetic replicates of each novel bradykinin were found to be active on mammalian arterial and small intestinal smooth muscle preparations. The structural diversity of bradykinins in amphibian defensive skin secretions may be related to defence against specific predators.
Molecular modeling of the dimeric structure of human lipoprotein lipase and functional studies of the carboxyl‐terminal domain
Authors: Kobayashi Yoko , Nakajima Toshiaki , Inoue Ituro
Published: March 2003

Lipoprotein lipase (LPL) plays a key role in lipid metabolism. Molecular modeling of dimeric LPL was carried out using insight ii based upon the crystal structures of human, porcine, and horse pancreatic lipase. The dimeric model reveals a saddle‐shaped structure and the key heparin‐binding residues in the amino‐terminal domain located on the top of this saddle. The models of two dimeric conformations – a closed, inactive form and an open, active form – differ with respect to how surface‐loop positions affect substrate access to the catalytic site. In the closed form, the surface loop covers the catalytic site, which becomes inaccessible to solvent. Large conformational changes in the open form, especially in the loop and carboxyl‐terminal domain, allow substrate access to the active site. To dissect the structure–function relationships of the LPL carboxyl‐terminal domain, several residues predicted by the model structure to be essential for the functions of heparin binding and substrate recognition were mutagenized. Arg405 plays an important role in heparin binding in the active dimer. Lys413/Lys414 or Lys414 regulates heparin affinity in both monomeric and dimeric forms. To evaluate the prediction that LPL forms a homodimer in a ‘head‐to‐tail’ orientation, two inactive LPL mutants – a catalytic site mutant (S132T) and a substrate‐recognition mutant (W390A/W393A/W394A) – were cotransfected into COS7 cells. Lipase activity could be recovered only when heterodimerization occurred in a head‐to‐tail orientation. After cotransfection, 50% of the wild‐type lipase activity was recovered, indicating that lipase activity is determined by the interaction between the catalytic site on one subunit and the substrate‐recognition site on the other.
Receptor crosstalk
Authors: Dzimiri Nduna
Published: March 2003

There are at least three well‐defined signalling cascades engaged directly in the physiological regulation of cardiac circulatory function: the β1‐adrenoceptors that control the cardiac contractile apparatus, the renin‐angiotensin‐aldosterone system involved in regulating blood pressure and the natriuretic peptides contributing at least to the factors determining circulating volume. Apart from these pathways, other cardiac receptor systems, particularly the α1‐adrenoceptors, adenosine, endothelin and opioid receptors, whose physiological role may not be immediately evident, are also important with respect to regulating cardiovascular function especially in disease. These and the majority of other cardiovascular receptors identified to date belong to the guanine nucleotide binding (G) protein‐coupled receptor families that mediate signalling by coupling primarily to three G proteins, the stimulatory (Gs), inhibitory (Gi) and Gq/11 proteins to stimulate the adenylate cyclases and phospholipases, activating a small but diverse subset of effectors and ion channels. These receptor pathways are engaged in crosstalk utilizing second messengers and protein kinases as checkpoints and hubs for diverting, converging, sieving and directing the G protein‐mediated messages resulting in different signalling products. Besides, the heart itself is endowed with the means to harmonize these signalling mechanisms and to fend off potentially fatal consequences of functional loss of the essential signalling pathways via compensatory reserve pathways, or by inducing some adaptive mechanisms to be turned on, if and when required. This receptor crosstalk constitutes the underlying basis for sustaining a coherently functional circulatory entity comprising mechanisms controlling the contractile apparatus, blood pressure and circulating volume, both in normal physiology and in disease.
Processing, stability, and kinetic parameters of C5a peptidase from Streptococcus pyogenes
Authors: Anderson Elizabeth T. , Wetherell Michael G. , Winter Laurie A. , Olmsted Stephen B. , Cleary Patrick P. , Matsuka Yury V.
Published: March 2003

A recombinant streptococcal C5a peptidase was expressed in Escherichia coli and its catalytic properties and thermal stability were subjected to examination. It was shown that the NH2‐terminal region of C5a peptidase (Asn32–Asp79/Lys90) forms the pro‐sequence segment. Upon maturation the propeptide is hydrolyzed either via an autocatalytic intramolecular cleavage or by exogenous protease streptopain. At pH 7.4 the enzyme exhibited maximum activity in the narrow range of temperatures between 40 and 43 °C. The process of heat denaturation of C5a peptidase investigated by fluorescence and circular dichroism spectroscopy revealed that the protein undergoes biphasic unfolding transition with Tm of 50 and 70 °C suggesting melting of different parts of the molecule with different stability. Unfolding of the less stable structures was accompanied by the loss of proteolytic activity. Using synthetic peptides corresponding to the COOH‐terminus of human complement C5a we demonstrated that in vitro peptidase catalyzes hydrolysis of two His67‐Lys68 and Ala58‐Ser59 peptide bonds. The high catalytic efficiency obtained for the SQLRANISHKDMQLGR extended peptide compared to the poor hydrolysis of its derivative Ac‐SQLRANISH‐pNA that lacks residues at P2′–P7′ positions, suggest the importance of C5a peptidase interactions with the P′ side of the substrate.
Proteasome‐driven turnover of tryptophan hydroxylase is triggered by phosphorylation in RBL2H3 cells, a serotonin producing mast cell line
Authors: Iida Yoshiko , Sawabe Keiko , Kojima Masayo , Oguro Kazuya , Nakanishi Nobuo , Hasegawa Hiroyuki
Published: March 2003

We previously demonstrated in mast cell lines RBL2H3 and FMA3 that tryptophan hydroxylase (TPH) undergoes very fast turnover driven by 26S‐proteasomes [Kojima, M., Oguro, K., Sawabe, K., Iida, Y., Ikeda, R., Yamashita, A., Nakanishi, N. & Hasegawa, H. (2000) J. Biochem (Tokyo) 2000, 127, 121–127]. In the present study, we have examined an involvement of TPH phosphorylation in the rapid turnover, using non‐neural TPH. The proteasome‐driven degradation of TPH in living cells was accelerated by okadaic acid, a protein phosphatase inhibitor. Incorporation of 32P into a 53‐kDa protein, which was judged to be TPH based on autoradiography and Western blot analysis using anti‐TPH serum and purified TPH as the size marker, was observed in FMA3 cells only in the presence of both okadaic acid and MG132, inhibitors of protein phosphatase and proteasome, respectively. In a cell‐free proteasome system constituted mainly of RBL2H3 cell extracts, degradation of exogenous TPH isolated from mastocytoma P‐815 cells was inhibited by protein kinase inhibitors KN‐62 and K252a but not by H89. Consistent with the inhibitor specificity, the same TPH was phosphorylated by exogenous Ca2+/calmodulin‐dependent protein kinase II in the presence of Ca2+ and calmodulin but not by protein kinase A (catalytic subunit). TPH protein thus phosphorylated by Ca2+/calmodulin‐dependent protein kinase II was digested more rapidly in the cell‐free proteasome system than was the nonphosphorylated enzyme. These results indicated that the phosphorylation of TPH was a prerequisite for proteasome‐driven TPH degradation.
The β‐1,4‐endogalactanase A gene from Aspergillus niger is specifically induced on arabinose and galacturonic acid and plays an important role in the degradation of pectic hairy regions
Authors: de Vries Ronald P. , Pařenicová Lucie , Hinz Sandra W. A. , Kester Harry C. M. , Beldman Gerrit , Benen Jacques A. E. , Visser Jaap
Published: March 2003

The Aspergillus nigerβ‐1,4‐endogalactanase encoding gene (galA) was cloned and characterized. The expression of galA in A. niger was only detected in the presence of sugar beet pectin, d‐galacturonic acid and l‐arabinose, suggesting that galA is coregulated with both the pectinolytic genes as well as the arabinanolytic genes. The corresponding enzyme, endogalactanase A (GALA), contains both active site residues identified previously for the Pseudomonas fluorescensβ‐1,4‐endogalactanase.

The galA gene was overexpressed to facilitate purification of GALA. The enzyme has a molecular mass of 48.5 kDa and a pH optimum between 4 and 4.5. Incubations of arabinogalactans of potato, onion and soy with GALA resulted initially in the release of d‐galactotriose and d‐galactotetraose, whereas prolonged incubation resulted in d‐galactose and d‐galactobiose, predominantly. MALDI‐TOF analysis revealed the release of l‐arabinose substituted d‐galacto‐oligosaccharides from soy arabinogalactan. This is the first report of the ability of a β‐1,4‐endogalactanase to release substituted d‐galacto‐oligosaccharides. GALA was not active towards d‐galacto‐oligosaccharides that were substituted with d‐glucose at the reducing end.
Thermostability of manganese‐ and iron‐superoxide dismutases from Escherichia coli is determined by the characteristic position of a glutamine residue
Authors: Hunter Thérèse , Bannister Joe V. , Hunter Gary J.
Published: March 2003

The structurally homologous mononuclear iron and manganese superoxide dismutases (FeSOD and MnSOD, respectively) contain a highly conserved glutamine residue in the active site which projects toward the active‐site metal centre and participates in an extensive hydrogen bonding network. The position of this residue is different for each SOD isoenzyme (Q69 in FeSOD and Q146 in MnSOD of Escherichia coli). Although site‐directed mutant enzymes lacking this glutamine residue (FeSOD[Q69G] and MnSOD[Q146A]) demonstrated a higher degree of selectivity for their respective metal, they showed little or no activity compared with wild types. FeSOD double mutants (FeSOD[Q69G/A141Q]), which mimic the glutamine position in MnSOD, elicited 25% the activity of wild‐type FeSOD while the activity of the corresponding MnSOD double mutant (MnSOD[G77Q/Q146A]) increased to 150% (relative to wild‐type MnSOD). Both double mutants showed reduced selectivity toward their metal. Differences exhibited in the thermostability of SOD activity was most obvious in the mutants that contained two glutamine residues (FeSOD[A141Q] and MnSOD[G77Q]), where the MnSOD mutant was thermostable and the FeSOD mutant was thermolabile. Significantly, the MnSOD double mutant exhibited a thermal‐inactivation profile similar to that of wild‐type FeSOD while that of the FeSOD double mutant was similar to wild‐type MnSOD. We conclude therefore that the position of this glutamine residue contributes to metal selectivity and is responsible for some of the different physicochemical properties of these SODs, and in particular their characteristic thermostability.
The structures of the lipooligosaccharide and capsule polysaccharide of Campylobacter jejuni genome sequenced strain NCTC 11168
Authors: Michael Frank St. , Szymanski Christine M. , Li Jianjun , Chan Kenneth H. , Khieu Nam Huan , Larocque Suzon , Wakarchuk Warren W. , Brisson Jean‐Robert , Monteiro Mario A.
Published: March 2003

Campylobacter jejuni infections are one of the leading causes of human gastroenteritis and are suspected of being a precursor to Guillain–Barré and Miller–Fisher syndromes. Recently, the complete genome sequence of C. jejuni NCTC 11168 was described. In this study, the molecular structure of the lipooligosaccharide and capsular polysaccharide of C. jejuni NCTC 11168 was investigated. The lipooligosaccharide was shown to exhibit carbohydrate structures analogous to the GM1a and GM2 carbohydrate epitopes of human gangliosides (shown below):

The high Mr capsule polysaccharide was composed of β‐d‐Ribp, β‐d‐GalfNAc, α‐d‐GlcpA6(NGro), a uronic acid amidated with 2‐amino‐2‐deoxyglycerol at C‐6, and 6‐O‐methyl‐d‐glycero‐α‐l‐gluco‐heptopyranose as a side‐branch (shown below):

The structural information presented here will aid in the identification and characterization of specific enzymes that are involved in the biosynthesis of these structures and may lead to the discovery of potential therapeutic targets. In addition, the correlation of carbohydrate structure with gene complement will aid in the elucidation of the role of these surface carbohydrates in C. jejuni pathogenesis.
The inhibitory region of troponin‐I alters the ability of F‐actin to interact with different segments of myosin
Authors: Patchell Valerie B. , Gallon Clare E. , Hodgkin Matthew A. , Fattoum Abdellatif , Perry S. Victor , Levine Barry A.
Published: March 2003

Peptides corresponding to the N‐terminus of skeletal myosin light chain 1 (rsMLC1 1–37) and the short loop of human cardiac β‐myosin (hcM398–414) have been shown to interact with skeletal F‐actin by NMR and fluorescence measurements. Skeletal tropomyosin strengthens the binding of the myosin peptides to actin but does not interact with the peptides. The binding of peptides corresponding to the inhibitory region of cardiac troponin I (e.g. hcTnI128–153) to F‐actin to form a 1 : 1 molar complex is also strengthened in the presence of tropomyosin. In the presence of inhibitory peptide at relatively lower concentrations the myosin peptides and a troponin I peptide C‐terminal to the inhibitory region, rcTnI161–181, all dissociate from F‐actin. Structural and fluorescence evidence indicate that the troponin I inhibitory region and the myosin peptides do not bind in an identical manner to F‐actin. It is concluded that the binding of the inhibitory region of troponin I to F‐actin produces a conformational change in the actin monomer with the result that interaction at different locations of F‐actin is impeded. These observations are interpreted to indicate that a major conformational change occurs in actin on binding to troponin I that is fundamental to the regulatory process in muscle. The data are discussed in the context of tropomyosin's ability to stabilize the actin filament and facilitate the transmission of the conformational change to actin monomers not in direct contact with troponin I.
The reactivity of α‐hydroxyhaem and verdohaem bound to haem oxygenase‐1 to dioxygen and sodium dithionite
Authors: Sakamoto Hiroshi , Omata Yoshiaki , Hayashi Shunsuke , Harada Saori , Palmer Graham , Noguchi Masato
Published: March 2003

Recently we have shown that ferric α‐hydroxyhaem bound to haem oxygenase‐1 can be converted to ferrous verdohaem by approximately an equimolar amount of O2 in the absence of exogenous electrons [Sakamoto, H., Omata, Y., Palmer, G., and Noguchi, M. (1999) J. Biol. Chem.274, 18196–18200]. Contrary to those results, other studies have claimed that the conversion requires both O2 and an electron. More recently, Migita et al. have reported that the major reaction product of ferric α‐hydroxyhaem with O2 is a ferric porphyrin cation radical that can be converted to ferrous α‐hydroxyhaem with sodium dithionite [Migita, C. T., Fujii, H., Matera, K. M., Takahashi, S., Zhou, H., and Yoshida, T. (1999) Biochim. Biophys. Acta1432, 203–213]. To clarify the reason(s) for the discrepancy, we compared the reactions; i.e. α‐hydroxyhaem to verdohaem and verdohaem to biliverdin, under various conditions as well as according to the procedures of Migita. We find that complex formation of α‐hydroxyhaem with haem oxygenase may be small and a substantial amount of free α‐hydroxyhaem may remain, depending on the reconstitution conditions; this could lead to a misinterpretation of the experimental results. We also find that ferrous verdohaem appears to be air‐sensitive and is therefore easily converted to a further oxidized species with excess O2. Finally, we find that dithionite seems to be inappropriate for investigating the haem oxygenase reaction, because it reduces ferrous verdohaem to a further reduced species that has not been seen in the haem degradation system driven by NADPH‐cytochrome P450 reductase.
Cloning and characterization of the mammalian‐specific nicolin 1 gene (NICN1) encoding a nuclear 24 kDa protein
Authors: Backofen Bianca , Jacob Ralf , Serth Katrin , Gossler Achim , Naim Hassan Y , Leeb Tosso
Published: March 2003

We have identified a novel mammalian gene, termed nicolin 1 gene (NICN1), that is present in human, dog and mouse, whereas it is absent from the available genome sequences of nonmammalian organisms. The NICN1 gene consists of six exons and spans about 6 kb of genomic DNA. It encodes a 213 amino acid protein that does not belong to any known protein family. Experiments using green fluorescent protein (GFP)‐tagged nicolin 1 fusion proteins indicate that nicolin 1 is a nuclear protein. Northern analysis and semiquantitative RT‐PCR demonstrated that the 2.5 kb NICN1 mRNA is expressed in a tissue‐specific manner. The highest NICN1 expression levels are found in brain, testis, liver, and kidney. On the other hand the NICN1 expression is weak in spleen, leukocytes, small intestine and colon. The NICN1 gene is also expressed during development.
Hemocyanin from the keyhole limpet Megathura crenulata (KLH) carries a novel type of N‐glycans with Gal(β1–6)Man‐motifs
Authors: Kurokawa Tomofumi , Wuhrer Manfred , Lochnit Günter , Geyer Hildegard , Markl Jürgen , Geyer Rudolf
Published: March 2003

Keyhole limpet (Megathura crenulata) hemocyanin (KLH), an extracellular respiratory protein, is widely used as hapten carrier and immune stimulant. Although it is generally accepted that the sugar constituents of this glycoprotein are likely to be implicated in the antigenicity and biomedical properties of KLH, knowledge of its carbohydrate structure is still limited. Therefore, we have investigated the N‐linked oligosaccharides of KLH. Glycan chains were enzymatically liberated from tryptic glycopeptides, pyridylaminated and separated by two‐dimensional HPLC. Only neutral oligosaccharides were obtained and characterized by carbohydrate constituent and methylation analyses, MALDI‐TOF‐MS, ESI‐ion trap‐MS and sequential exoglycosidase digestion. The results revealed that KLH is carrying high mannose‐type glycans and truncated sugar chains derived thereof. As a characteristic feature, a number of the studied N‐glycans contained a Gal(β1–6)Man‐unit which has not been found in glycoprotein‐N‐glycans so far. Hence, our studies demonstrate that this marine mollusk glycoprotein is characterized by a unique oligosaccharide pattern comprising, in part, novel structural elements.
Co‐existence of two regulatory NADP‐glyceraldehyde 3‐P dehydrogenase complexes in higher plant chloroplasts
Authors: Scheibe Renate , Wedel Norbert , Vetter Susanne , Emmerlich Vera , Sauermann Sonja‐Manuela
Published: March 2003

Light/dark modulation of the higher plant Calvin‐cycle enzymes phosphoribulokinase (PRK) and NADP‐dependent glyceraldehyde 3‐phosphate dehydrogenase (NADP‐ GAPDH‐A2B2) involves changes of their aggregation state in addition to redox changes of regulatory cysteines. Here we demonstrate that plants possess two different complexes containing the inactive forms (a) of NADP‐GAPDH and PRK and (b) of only NADP‐GAPDH, respectively, in darkened chloroplasts. While the 550‐kDa PRK/GAPDH/CP12 complex is dissociated and activated upon reduction alone, activation and dissociation of the 600‐kDa A8B8 complex of NADP‐GAPDH requires incubation with dithiothreitol and the effector 1,3‐bisphosphoglycerate. In the light, PRK is therefore completely in its activated state under all conditions, even in low light, while GAPDH activation in the light is characterized by a two‐step mechanism with 60–70% activation under most conditions in the light, and the activation of the remaining 30–40% occurring only when 1,3‐bisphosphoglycerate levels are strongly increasing. In vitro studies with the purified components and coprecipitation experiments from fresh stroma using polyclonal antisera confirm the existence of these two aggregates. Isolated oxidized PRK alone does not reaggregate after it has been purified in its reduced form; only in the presence of both CP12 and purified NADP‐GAPDH, some of the PRK reaggregates. Recombinant GapA/GapB constructs form the A8B8 complex immediately upon expression in E. coli.