bluuebunny/crossref_metadata_2025 huggingface dataset:
An NMR Study of the Interaction of 15N‐Labelled Bradykinin with an Antibody Mimic of the Bradykinin B2 Receptor
Authors: Ottleben Holger , Haasemann Martina , Ramachandran Ramadurai , Görlach Matthias , Müller‐Esterl Werner , Brown Larry R.
Published: July 2004

An isotope‐edited NMR study of the peptide hormone bradykinin (RPPGFSPFR) bound to the Fab fragment of a monoclonal antibody against bradykinin (MBK3) is reported. MBK3 was previously shown to provide a binding site model of the B2 bradykinin receptor [Haasemann, M., Buschko, J., Faussner, A., Roscher, A. A., Hoebeke, J., Burch, R. M. & Müller‐Esterl, W. (1991) Anti‐idiotypic antibodies bearing the internal image of a bradykinin epitope, J. Immunol. 147, 3882–3892]. Bradykinin was obtained in a uniformly 15N‐labelled form using recombinant expression of a fusion protein consisting of the glutathione‐binding domain of glutathione S‐transferase fused to residues 354–375 of the high‐molecular‐mass kininogen from which bradykinin was released by proteolytic digestion with its natural protease plasma kallikrein. Bradykinin forms a complex with the Fab fragment of MBK3 which exchanges slowly on the NMR time scale. The 15N and 1H resonances of the tightly bound residues of bradykinin show appreciable changes in chemical shift with respect to the free form, while the 15N and 1H linewidths indicate that the hydrodynamic behaviour of bound bradykinin is dominated by the high‐molecular‐mass Fab fragment. The NMR data indicate that essentially the entire nonapeptide is involved in binding. The kinetics of the ligand‐exchange process, together with resonance assignments obtained via exchange spectroscopy, indicate that bradykinin binds to MBK3 only in the all‐trans conformation at all three Xaa‐Pro amide bonds. NH‐NH NOE connectivities suggest that bradykinin is bound in an extended conformation. The spectroscopic data obtained from this study are compared to recently proposed computational models of the conformation of bradykinin bound to the B2 receptor.
Heparin Binding of Protein‐C Inhibitor Analysis of the Effect of Heparin on the Interaction of Protein‐C Inhibitor with Tissue Kallikrein
Authors: Ecke Sonja , Geiger Margarethe , Binder Bernd R.
Published: July 2004

The non‐specific serine‐protease inhibitor protein‐C inhibitor (PCI) inactivates its target enzymes by forming stable 1:1 complexes. Heparin stimulates most PCI/protease reactions, but interferes with the inhibition of tissue kallikrein by PCI by a hitherto unknown mechanism. In this study we analyzed the inhibitory effect of heparin on the tissue‐kallikrein–PCI interaction. Free PCI and tissue‐kallikrein – PCI complexes but not free tissue kallikrein bound to heparin‐Sepharose, implying that the inhibitory effect of heparin cannot be caused by a tissue‐kallikrein–heparin interaction. Heparin did not dissociate tissue‐kallikrein – PCI complexes, making it unlikely that in the presence of heparin PCI becomes a substrate for, rather than an inhibitor of, tissue kallikrein. However, heparin‐bound PCI, which was able to form complexes with 125I‐urokinase, did not form complexes with 125I‐tissue‐kallikrein. This suggests that the inhibitory effect of heparin is either based on the neutralization of positive charges in the PCI molecule, which might be required for the interaction of PCI with the acidic protease tissue kallikrein, or on a change in reactivity of PCI upon heparin binding, making heparin‐bound PCI no longer a tissue‐kallikrein inhibitor. Neutralization of basic amino acids in the PCI molecule by glutamic acid, which prevented in a dose‐dependent way the inhibitory effect of heparin, did not have any effect on the tissue‐kallikrein–PCI interaction. Therefore, direct involvement of basic amino acid residues present in the heparin‐binding site of PCI in the tissue‐kallikrein–PCI interaction can be excluded. Heparin binding might rather cause a change in reactivity of PCI (e.g. by inducing a conformational change or by steric interference), thereby preventing its interaction with tissue kallikrein.
Demonstration of the Presence of α‐l,6‐Branched Side Chains in the Mannan of Candida Stellatoidea
Authors: Shibata Nobuyuki , Senbongi Nobutaka , Hosoya Tomoko , Kawahara Kumi , Akagi Rieko , Suzuki Akifumi , Kobayashi Hidemitsu , Suzuki Shigeo , Okawa Yoshio
Published: July 2004

A mild acetolysis of the mannans of Candida stellatoidea was performed after acetylation to yielded an α‐1,6‐branched mannohexaose, the presence of which had been predicted from the appearance of a specific H1‐H2‐correlated cross‐peak in two‐dimensional homonuclear Hartmann‐Hahn spectroscopy. In this study, we found that the de‐O‐acetylation of a 4‐O‐acetyl group at the branching point, the 3,6‐di‐O‐substituted mannose unit, of an acetylated oligosaccharide by sodium methoxide is significantly slower than that of other acetyl groups. We could separate the 4‐O‐acetylated branching oligosaccharide from linear isomer using high‐performance liquid chromatography. Before and after the de‐O‐acetylation of the purified branching oligosaccharide, their 1H‐NMR signals were sequentially assigned by means of the nuclear Overhauser effect. In the sequential NMR assignment study, we showed that the α‐1,6‐linked mannose unit is attached to the 3‐O‐substituted unit based on the presence of NOE cross‐peak between HI of the branching mannose unit and H6 of the 3‐0‐substituted mannose unit. An enzyme‐linked immu‐nosorbent inhibition assay of the reactivity of factor 4 serum to C. stellatoidea mannan by several oligo‐saccharides indicated that the α‐1,6‐branched oligosaccharide and the β‐1,2 linkage‐containing oligosac‐charides showed inhibitory activity. This result indicates that factor 4 serum, as well as factor 5 and 6 sera, contains antibodies against β‐1,2‐linked mannose units which have been reported to participate in pathogenicity via cytokine production and/or adherence. From the assignment results of H1‐H2‐correlated cross‐peaks of oligosaecharides and mannans, the molar ratio of the mannan side chains was proposed. In this study, we demonstrated that the epitope structure of the C. stellatoidea type I strains was the same as that of the C. albicam NIH B‐792 (serotype B) strain.
Identification, Isolation and Biochemical Characterization of a Phosphopantetheine:Protein Transferase that Activates the Two Type‐I Fatty Acid Synthases of Brevibacterium Ammoniagenes
Authors: Stuible Hans‐Peter , Meier Sandra , Schweizer Eckhart
Published: July 2004

Upon heterologous expression of the Brevibacterium ammoniagenes type‐I fatty acid synthase FAS‐A in Escherichia coli, only the pantetheine‐free apoenzyme is synthesized. Activation of FAS‐A to its holoform was achieved by transformation with a second B. ammoniagenes gene, PPTI, encoding a type‐I FAS‐specific phosphopantetheine transferase. PPTI was identified as a coding sequence located immediately downstream of the second FAS gene present on the B. ammoniagenes genome, fasB. Due to this linkage, PPT1 was part of the cloned fasB DNA region and, consequently, FAS‐B but not FAS‐A was synthesized as holoFAS in E. coli. PPTI encodes a protein of 153 amino acids and has a calculated molecular mass of 16884 Da. The PPT1 gene product contains 25% identical and 42 % conserved amino acids compared with the type‐II acyl‐carrier‐protein‐ activating enzyme of E. coli. Although there is essentially no intergenic region between jusB and PPT1, the PPTase gene is autonomously expressed in E. coli if flanked by 200 bp of its endogenous 5′ DNA. The structural independence of Ppt1p was confirmed immunologically, as specific antibodies react with the purified PPTase but not with FAS‐B. Overexpression and purification of the His‐tagged Ppt1p allowed the in vitro activation of apoFAS‐A. This holoenzyme synthesis requires, in addition to Ppt1p, CoA and Mg2+ and leads to a specific FAS activity comparable to that of natural B. ammoniugenes FAS‐A. The reactivity of the in vitro‐activated FAS‐A was verified by the optical FAS assay and by analysis of its in vitro products. In agreement with the known overall colinearity of B. ammoniagenes FAS‐B and the Saccharomyces cerevisiae FAS1 and FAS2 gene products, a PPT1‐like sequence is also observed at the C terminus of FAS2. However, in contrast to B. ammioniagenes PPT1, this sequence is an integral part of the yeast FAS2 gene. Thus, activation of type‐I fatty acid synthases may be accomplished by distinct trans‐acting PPTase enzymes and by intrinsic cis‐acting PPTase domains.
Expression of Bovine β‐Lactoglobulin/Human Erythropoietin Fusion Protein in the Milk of Transgenic Mice and Rabbits
Authors: Korhonen Veli‐Pekka , Tolvanen Minna , Hyttinen Juha‐Matti , Uusi‐Oukari Mikko , Sinervirta Riitta , Alhonen Leena , Jauhiainen Matti , Jänne Olli A. , Jänne Juhani
Published: July 2004

We have generated several transgenic mouse lines and rabbits expressing efficiently (up to 0.3 mg/ml in mice and up to 0.5 mg/ml in rabbits) human erythropoietin in their milk as bovine β‐lactoglobulin fusion protein. Human erythropoietin cDNA was inserted in frame into exon 5 of the bovine β‐lactoglobu‐lin gene with a linker oligonucleotide encoding the cleavage site for bacterial IgA protease. RNA analysis performed on one lactating transgenic mouse and one transgenic rabbit revealed that the fusion gene was expressed almost exlusively in the mammary gland, although low amounts of transgene‐derived RNA were detectable in salivary glands and uterus or in the kidney. The fusion protein was specifically cleaved with IgA protease. The erythropoietin part obtained upon digestion had a lower molecular mass than recombinant erythropoietin produced in Chinese hamster ovary cells. By deglycosylation analysis it was shown that the difference in size was due to a different type of glycosylation. Biological activity of the fusion protein, as determined by growth stimulation of TF‐1 erythroleukemia cells, was less than 15 % of that of human recombinant erythropoietin. Upon digestion of the fusion protein with IgA protease, biological activity comparable to that of the recombinant erythropoietin was recovered. Transgenic males and virgin females did not show signs of enhanced erythropoiesis, but lactating females expressing the transgene displayed transient increases in their hematocrit values.
Substrate Binding and Catalysis by L‐arginine: Glycine Amidinotransferase — A Mutagenesis and Crystallographic Study
Authors: Fritsche Erich , Humm Andreas , Huber Robert
Published: July 2004

L‐Arginine:glycine amidinotransferase catalyzes the committed step in the biosynthesis of creatine. Eight active‐site mutants, D170N, D254N, H303V, D305A, R322E, S355A, C407S, and C410A of recom‐binant human L‐arginine : glycine amidinotransferase were prepared by site‐directed mutagenesis and enzymatically characterized. The crystal structures of the three mutants D170N, D254N, and C407S have been determined at 0.28‐nm, 0.29‐nm and 0.236‐nm resolution, respectively. The mutation of active‐site residues which are involved in substrate‐binding yielded inactive mutants. Substitution of Asp254, which is not directly involved in substrate binding but is thought to transfer protons in concert with the His303 imidazole group, results in a strongly (2000‐fold) reduced activity. However, the substitution of Cys410, a residue near the active site but not involved in catalysis or substrate binding, by Ala does not change the kinetic properties with respect to the wild‐type enzyme. The loss of enzymatic activity of the D170N, D254N, C407S and likely all other mutants is solely due to the inserted point mutations, affecting substrate binding or transition‐state stabilization, and not due to major conformational rearrangements of the protein.

These results show that a His‐Asp pair on one side of the substrate and a Cys on the other side are key residues for activity and are part of a disjoint triad. The imidazole ring of the His is proposed to act as a general acid/general base during catalysis whereas the Cys acts as a nucleophile analogous to Cys25 of papain‐like cysteine proteinases.
A point Mutation Responsible for Defective Function of the Aryl‐Hydrocarbon‐Receptor Nuclear Translocator in Mutant Hepa‐1c1c7 Cells
Authors: Numayama‐Tsuruta Keiko , Kobayashi Akira , Sogawa Kazuhiro , Fujii‐Kuriyama Yoshiaki
Published: July 2004

A 3,4‐benzopyrene‐resistant mutant clone (c4) of the mouse hepatoma Hepa‐1c1c7 cell line was examined for the mutation that causes the defective function of aryl‐hydrocarbon receptor (AHR) nuclear translocator (Arnt). Arnt dimerizes with AHR and mediates the induction signal of aryl‐hydrocarbon hydroxylase activity. The Arnt cDNAs of c4 cells were cloned by reverse‐transcription/PCR to compare the sequences with that of wild‐type Arnt cDNA. The Arnt cDNA of c4 cells was found to have a single point mutation, leading to replacement of Gly326 with Asp between two internal repeats in the highly conserved Per‐Arnt Sim (PAS) domain, PAS A and PAS B. The inability of [Asp326]Arnt/AHR heterodimers to enhance reporter gene transcription under the control of the CYP1A1gene promoter and enhancer confirmed that the G326→D substitution was a causative mutation. While fluorescence microscopy and coimmunoprecipitation experiments showed that this mutant form of Arnt was not changed from wild‐type Arnt in terms of nuclear localization or heterodimer formation with AHR, the binding activity of the [Asp326]Arnt. AHR heterodimer to the xenobiotic‐responsive element was reduced markedly. Determination of the turnover rate in COS‐7 cells transfected with expression plasmids for mutant Arnt or normal Arnt showed that the mutant protein turned over with an accelerated rate compared with that of the normal. Moreover, the mutant protein displayed increased proteolytic digestibility in vitro with various proteases.
Visualization of a Covalent Intermediate between Microsomal Epoxide Hydrolase, but not Cholesterol Epoxide Hydrolase, and their Substrates
Authors: Müller Frank , Arand Michael , Frank Heinz , Seidel Albrecht , Hinz Willy , Winkler Lars , Hänel Karen , Blée Elizabeth , Beetham Jeffrey K. , Hammock Bruce D. , Oesch Franz
Published: July 2004

Mammalian soluble and microsomal epoxide hydrolases have been proposed to belong to the family of αlβ‐hydrolase‐fold enzymes. These enzymes hydrolyse their substrates by a catalytic triad, with the first step of the enzymatic reaction being the formation of a covalent enzyme‐substrate ester. In the present paper, we describe the direct visualization of the ester formation between rat microsomal epoxide hydrolase and its substrate. Microsomal epoxide hydrolase was precipitated with acetone after brief incubation with |l‐14C]epoxystearic acid. After denaturing SDS gel electrophoresis the protein‐bound radioactivity was detected by fluorography. Pure epoxide hydrolase and crude microsomes showed a single radioactive signal of the expected molecular mass that could be suppressed by inclusion of the competitive inhibitor 1,1,1‐trichloropropene oxide in the incubation mixture. In a similar manner, 4‐fluorochalcone‐oxide‐sensitive binding of epoxystearic acid to rat soluble epoxide hydrolase could be demonstrated in rat liver cytosol. Under similar conditions, no covalent binding of [26‐14C]cholesterol‐5α,6α‐epoxide to microsomal proteins or solubilized fractions tenfold enriched in cholesterol epoxide hydrolase activity could be observed. Our data provide definitive proof for the formation of an enzyme‐substrate‐ester intermediate formed in the course of epoxide hydrolysis by microsomal epoxide hydrolase, show no formation of a covalent intermediate between cholesterol epoxide hydrolase and its substrate under the same conditions as those under which an intermediate was shown for both microsomal and soluble epoxide hydrolases and therefore indicate that the cholesterol epoxide hydrolase apparently does not act by a similar mechanism and is probably not structurally related to microsomal and soluble epoxide hydrolases.
Regulatory Mechanisms Involved in Activator‐Protein‐1 (AP‐1)‐Mediated Activation of Glutathione‐S‐Transferase Gene Expression by Chemical Agents
Authors: Ainbinder Elena , Bergelson Svetlana , Pinkus Ron , Daniel Violet
Published: July 2004

Induction of murine glutathione‐S‐transferase (GST) Ya gene expression by a variety of chemical agents is mediated by a regulatory element, EpRE, composed of an Ets and two adjacent activator protein‐1 (AP‐1)‐like sites and activated by the Fos/Jun heterodimeric complex (AP‐1). The mechanism of this induction was examined in the present study. We find that the regulation of EpRE‐mediated GST Ya gene expression by 3‐methylcholanthrene, tert‐butylhydroquinone and β‐naphthoflavone is associated with an induction of AP‐1 DNA‐binding activity and that the AP‐1 complex induced in hepatoma cells by these chemicals contains members of the Fos and Jun protein families. We show that tert‐butylhydroquinone induces c‐fos gene expression and indicate the formation of a transcriptionally active AP‐1 complex that contains Fos/Jun heterodimer. In F9 cells, which are considered to lack AP‐1 complex, a careful examination reveals that tert‐butylhydroquinone induces a low level of an AP‐1‐related activity responsible for the enhanced expression of EpRE as well as of AP‐1 reporter constructs. We find that protein phosphorylations mediate the activation of the GST Ya gene by chemical agents since okadaic acid, an inhibitor of protein phosphatases, can mimic this activation while protein kinase inhibitors abolish it. Evidence is presented that 3‐methylcholanthrene, tert‐butylhydroquinone and β‐naphthoflavone use a signal transduction pathway to Fos/Jun‐dependent GST Ya gene expression via Ras and protein‐tyrosine kinase activity. Furthermore, we find that activation by phorbol 12‐myristate 13‐acetate, which uses both protein kinase C and protein‐tyrosine kinase activities, may share a common pathway with these chemicals downstream of Ras.
Widespread Tissue Expression of Gastrin‐Binding‐Protein Mrna
Authors: Monstein Hans‐Jürg , Nylander Anna‐Greta , Håkanson Rolf
Published: July 2004

Glycine‐extended forms of gastrin (gastrin‐Gly) are thought to be involved in the autocrine growth control of colorectal carcinomas. The recently described gastrin‐binding protein has been suggested to be a gastrin‐Gly accepting receptor. Northern blot analysis demonstrated the expression of gastrin‐binding‐protein mRNA in many tissues of mouse, rat, and man. The gastrin‐binding‐protein mRNA expression was confirmed by reverse‐transcribed PCR analysis. Analysis of the cDNA and the deduced ammo acid sequence of the PCR‐amplified rat gastrin‐binding‐protein DNA fragments revealed sequence identity (except in a single position) with the corresponding human and pig gastrin‐binding protein and with the a‐subunit of a rat and human mitochondrial trifunctional enzyme, involved in fatty acid oxidation. The widespread and abundant tissue expression of gastrin‐binding‐protein mRNA and its sequence identity with a fatty‐acid‐oxidizing enzyme do not support the view that it represents a genuine gastrin receptor.
Regulation of Human Interstitial Collagenase (Matrix Metalloproteinase‐1) Promoter Activity by Fibroblast Growth Factor
Authors: Aho Sirpa , Rouda Susan , Kennedy Susan H. , Qin Huiping , Tan Elaine M. L.
Published: July 2004

Basic fibroblast growth factor (bFGF) is a pleiotropic factor that is implicated in tissue remodeling. The growth factor is capable of up‐regulating the expression of the interstitial collagenase (matrix metalloproteinase‐1 or MMP‐1) gene. In this study, the full‐length human MMP‐1 promoter, spanning 4.3 kb, was sequenced and the regulatory control of its activity by bFGF was examined in NIH3T3 fibroblasts. Several regulatory sequences, including five activator protein‐1 (AP‐1), five activator protein‐2 (AP‐2), five glucocorticoid‐response elements and multiple ets/polyoma enhancer‐binding 3 elements, were identified. Deletion constructs were prepared and transiently transfected into fibroblast cultures incubated with and without bFGF. The results showed that bFGF enhanced the activity of the deletion promoter fragments and the full‐length MMP‐1 promoter by sixfold or more in the cell cultures. Stimulation of the MMP‐1 promoter activity by bFGF was reflected in substantial increase of the collagenase mRNA levels. A bFGF‐responsive element appeared to be the AP‐1 consensus sequence. Mutation of the first AP‐1 site resulted in major reduction of the basal level of the MMP‐1 promoter activity, supporting the notion that the AP‐1 consensus sequence is essential for the constitutive expression of the MMP‐1 gene. Furthermore, bFGF induction of the activity of the promoter constructs containing a mutant AP‐1 site was essentially absent, suggesting that the regulatory element is necessary for the induction of the promoter activity by the growth factor. Thus, bFGF up‐regulates MMP‐1 gene expression in NIH3T3 fibroblasts via induction of its promoter activity that is dependent on an AP‐1 consensus sequence.
DNA Interactions of Bifunctional Dinuclear Platinum(II) Antitumor Agents
Authors: Žaludová Renata , Žákovská Alena , Kašpárková Jana , Balcarová Zdenka , Kleinwächter Vladimír , Vrana Oldřich , Farrell Nicholas , Brabec Viktor
Published: July 2004

Modifications of natural DNA in a cell‐free medium by dinuclear bisplatinum complexes with equivalent coordination spheres, represented by the general formula [{trans‐PtCl(NH3)2}2(H2N‐R‐NH2)]2+, where R is a propane or hexane, were studied by various methods of biochemical analysis or molecular biophysics. These methods include binding studies by means of differential‐pulse polarography, measurements of melting curves with the aid of absorption spectrophotometry, measurements of CD spectra, ELISA with specific antibodies that recognize DNA modified by platinum complexes, interstrand cross‐linking assay employing gel electrophoresis under denaturing conditions and mapping of DNA adducts by means of transcription assays. The results indicated that the major adduct of [{trans‐PtCl(NH3)2}2(H2N‐R‐NH2)]2+ in DNA was an interstrand cross‐link which was formed with a relatively short half‐time (≈1 h). At least some types of these interstrand cross‐links induced local denaturational changes in the DNA. The results of analyses of interactions of [{trans‐PtCl(NH3)2}2(H2N‐R‐NH2)]2+ with linear DNA at relatively higher levels of the modification could be interpreted to mean that these dinuclear platinum complexes were also capable of intrastrand‐cross‐link formation between adjacent base residues in DNA. However, these intrastrand adducts of [{trans‐PtCl(NH3)2}2(H2N‐R‐NH2)]2+ distorted DNA conformation in a way different from the DNA intrastrand adducts of cisplatin. In addition, the DNA adducts of the dinuclear platinum complexes inhibited DNA transcription in vitro. The length of the aliphatic linker chain affected the DNA‐binding mode of [{trans‐PtCl(NH3)2}2(H2N‐R‐NH2)]2+ and the resulting conformational changes in DNA. The extensive analysis of DNA interactions with [{trans‐PtCl(NH3)2}2(H2N‐R‐NH2)]2+ described in this communication has provided further experimental support for previous suggestions [Farrell, N. (1991) in Platinum and other metal coordination compounds in cancer chemotherapy (Howell, S. B., ed.) pp. 81–91, Plenum Press, New York] that the binding of the dinuclear platinum complexes modifies DNA in a way that is different from the modification by antitumor cisplatin. Thus, the results of this work are consistent with the hypothesis that platinum drugs that bind to DNA in a manner fundamentally different from that of cisplatin can exhibit altered biological properties, including a different spectrum and intensity of antitumor activity.
Preferential Esterification of Endogenously Formed 5‐Hydroxyeicosatetraenoic Acid to Phospholipids in Activated Polymorphonuclear Leukocytes
Authors: Arai Masayoshi , Imai Hirotaka , Metori Atsuko , Nakagawa Yasuhito
Published: July 2004

The esterification of endogenously formed 5‐hydroxyeicosatetraenoic acid (5‐HETE) to cellular lipids in rat polymorphonuclear leukocytes (PMNL) was studied quantitatively by a fluorometric method using HPLC. Rapid and maximal production of free 5‐HETE was observed after a 5‐min stimulation of PMNL with A23187. The amount of free 5‐HETE then declined rapidly, while that of 5‐HETE esterified to phospholipids and triacylglycerol concomitantly increased in a time‐dependent manner. Stimulation by A23187 yielded approximately 100 ng/107 cells esterified 5‐HETE in 60 min, which corresponded to the decrease in the amount of free 5‐HETE from 5 min to 60 min and indicated that free 5‐HETE, which was formed endogenously, was metabolized predominantly by esterification to cellular lipids.

The esterification profile of exogenous 5‐HETE was different from that of endogenous 5‐HETE. 5‐[3H]HETE, which was added exogenously to the culture medium, was rapidly incorporated into PMNL and almost 80% of the total radioactivity was located in triacylglycerol. A quantitative study revealed that endogenous 5‐HETE was esterified equally to phospholipids and triacylglycerol.

Like PMNL, peritoneal macrophages treated with A23187 released significant amounts of 5‐HETE. However, less 5‐HETE was esterified to cellular lipids than in PMNL. Negligible amounts of 12‐HETE, produced by activated peritoneal macrophages or activated platelets after a challenge with A23187, were esterified during the entire incubation.

Exogenous 5‐HETE was rapidly taken up by PMNL, but was incorporated into macrophages much more slowly than into PMNL. No uptake of 12‐HETE into macrophages was observed. The rapid uptake of exogenous 5‐HETE was strongly inhibited by the suppression of acylation of 5‐HETE by triacsin C. These results suggest that esterification might be one of the factors that regulate the rate of incorporation of 5‐HETE.
Cloning and Expression of the fadH Gene and Characterization of the Gene Product 2,4‐Dienoyl Coenzyme A Reductase from Escherichia Coli
Authors: He Xue‐Ying , Yang Song‐Yu , Schulz Horst
Published: July 2004

The fadH gene coding for an NADPH‐dependent 2,4‐dienoyl‐CoA reductase from Escherichia coli has been cloned by the polymerase chain reaction. This gene is located at 67.65 min on the E. coli chromosome. The complete open reading frame contains 2019 bp coding for the processed protein of 671 amino acid residues, with a calculated molecular mass of 72.55 kDa, which lacks the N‐terminal methio‐nine. Construction and expression of the plasmid pNDH, which contained the fadH gene under the control of the T7 promoter, resulted in a 110‐fold increase in the reductase activity above the level detected in E. coli cells containing the control vector. The kinetic parameters of the purified reductase were determined to be 50 μM and 2.3 μM for the Km, values of NADPH and 2‐trans, 4‐trans‐decadienoyl‐CoA, respectively, and 16 s−1 for the kcat value. Analysis of the kinetic data revealed that the reaction catalyzed by this enzyme proceeds via a ping‐pong mechanism. The observed dissimilarity between the E. coli and mammalian 2,4‐dienoyl‐CoA reductase sequences suggests that they have evolved from distinct ancestral genes. Sequence analysis also suggests that the N‐terminal part of the E. coli reductase contains the FAD‐binding domain whereas the NADPH‐binding domain is located in the C‐terminal region of the protein.
Post‐Translational Activation of Non‐Homologous DNA End‐Joining in Xenopus Oocyte Extracts
Authors: Aoufouchi Said , Patrick Tina , Lindsay Howard D. , Shall Sydney , Ford Chris C.
Published: July 2004

We have analysed the recircularisation of plasmid DNA, cut with two different endonucleases to generate non‐homologous DNA ends, in extracts of unfertilised eggs and oocytes of Xenopus. We found that the capacity to join non‐homologous DNA ends, generating diagnostic covalently closed monomer circles, appeared during oocyte maturation at the time of germinal vesicle breakdown. This enzyme function was post‐translationally activated in oocyte extracts incubated with unfertilised egg extract containing active cdc2kyclin B, or by incubation with purified cdc2/cyclin B. Dephosphorylation of egg proteins by alkaline phosphatase inhibited the ability to join non‐homologous DNA endr. We show that most linear non‐homologous DNA ends repaired to form closed‐circular supercoiled monomers, are joined without loss of nucleotides. Following partial purification, the activity was inhibited by inhibitors of poly(ADP‐Rib) polymerase, an enzyme that is inactive in oocytes, but phosphorylated and activated during maturation. Competitive inhibition of poly(ADP‐Rib) polymerase by > 50 μM 3‐aminobenzamide prevented the joining of both matched and non‐homologous DNA ends. We conclude that post‐translational phosphorylation provides one route by which end‐joining of non‐homologous DNA can be regulated.
Biosynthesis of the Proteoglycan Decorin Transient 2‐Phosphorylation of Xylose during Formation of the Trisaccharide Linkage Region
Authors: Moses Jonatan , Oldberg Åke , Cheng Fang , Fransson Lars‐Åke
Published: July 2004

Phosphorylation of decorin was investigated by incubating a rat fibroblast cell line with radiolabelled phosphate and carbohydrate precursors. There was a transient phosphorylation of the linkage‐region sac‐charides in intracellular decorin prior to assembly of the galactosaminoglycan chain. Phosphorylation gradually increased from xylosylated, galactosyl‐xylosylated to galactosyl‐galactosyl‐xylosylated core protein where all trisaccharide stubs were phosphorylated. Addition of the first glucuronate residue was accompanied by rapid dephosphorylation. Brefeldin A treatment resulted in segregation of galactosaminoglycan synthesis and dephosphorylation. Enzymatic degradation of brefeldin‐A‐arrested immature proteoglycan with incomplete galactosaminoglycan chain [Moses, J., Oldberg, Å., Eklund, E. & Fransson, L.‐Å. (1997)Eur. J. Biochem., in the press] by using chondroitin AC lyase and chondro‐glycuronidase, followed by β‐galactosidase treatment, demonstrated the sequence galactosyl‐galactosyl‐phosphoxylose. The xylose was resistant to direct periodate oxidation, but sensitive after treatment with alkaline phospha‐tase, showing that the phosphate was located at C2 of xylose. The transient 2‐phosphorylation of xylose may be involved in intracellular transport and/or in the control of modifications of the glycan chain.
The Pipecolate‐Incorporating Enzyme for the Biosynthesis of the Immunosuppressant Rapamycin — Nucleotide Sequence Analysis, Disruption and Heterologous Expression of Rap P from Streptomyces Hygroscopicus
Authors: König Ariane , Schwecke Torsten , Molnár Istvin , Böhm Günter A. , Lowden Philip A. S. , Staunton James , Leadlay Peter F.
Published: July 2004

An open reading frame (rapP) encoding the putative pipecolate‐incorporating enzyme (PIE) has been identified in the gene cluster for the biosynthesis of rapamycin in Streptomyces hygroscopicus. Conserved amino acid sequence motifs for ATP binding, ATP hydrolysis, adenylate formation, and 4′‐phosphopantetheine attachment were identified by sequence comparison with authentic peptide synthetases. Disruption of rup P by phage insertion abolished rapamycin production in S. hygroscopicus, and the production of the antibiotic was specifically restored upon loss of the inserted phage by a second recombination event. rup P was expressed in both Escherichia coli and Streptomyces coelicolor, and recombinant PIE was purified to homogeneity from both hosts. Although low‐level incorporation of [14C]β‐alanine into recombinant PIE isolated from E. coli was detected, formation of the covalent acylenzyme intermediate could only be shown with the PIE from S. coelicolor, suggesting that while the recombinant PIE from S. coelicolor was phosphopantetheinylated, only a minor proportion of the recombinant enzyme from E. coli was post‐translationally modified.
Partial Characterization and Enrichment of a Membrane‐Bound Sialidase Specific for Gangliosides from Human Brain Tissue
Authors: Kopitz Jürgen , Sinz Karin , Brossmer Reinhard , Cantz Michael
Published: July 2004

Gangliosides, constituents of surfaces of vertebrate cells, modulate important cellular functions. Ganglioside‐specific sialidases that possibly control these processes have been observed in a number of tissues, but their characterization has proved difficult due to their low abundance and lability. Here we describe the partial isolation and characterization of a ganglioside sialidase from human brain grey matter. After membrane extraction with octylglucoside, the enzyme was purified about 1300‐fold by ion‐exchange, affinity and gel‐permeation chromatographies. Although PAGE still showed several protein bands, specific photoaffinity labelling with iodinated 5‐Af‐acetyl‐9‐(4‐azidosalicoylamido)‐2,9‐dideoxy‐2,3‐didehydroneuraminic acid identified a single polypeptide of 60 kDa likely to contain the active site of the sialidase. In the presence of 0.4% octylglucoside, the purified sialidase desialylated gangliosides GM3, GD1a, GD1b and GT1b, but was inactive towards GM1, GM2, colominic acid, sialyl‐(α2–3)‐lactose, 2‐(4‐methylumbelliferyl)‐neuraminate, or the glycoprotein fetuin. The ganglioside sialidase activity was strongly inhibited by 2‐deoxy‐2,3‐didehydro‐N‐acetylneuraminic acid, heparin and heparan sulfate. Because of its substrate and inhibitor profiles, the purified enzyme resembles the activity characterized previously in the plasma membrane of human neuroblastoma cells, but is distinct from a lysosomal activity. The purified brain sialidase thus appears to function in the selective desialylation of gangliosides with terminal sialic acid residues.
Sequence of a Gene Cluster from Klebsiella Pneumoniae Encoding Malonate Decarboxylase and Expression of the Enzyme in Escherichia Coli
Authors: Hoenke Stefan , Schmid Markus , Dimroth Peter
Published: July 2004

Malonate decarboxylase of Klebsiella pneumoniae consists of four different subunits and catalyzes the conversion of malonate plus H+ to acetate and CO2. The catalysis proceeds via acetyl and malonyl thioester residues with the phosphribosyl‐dephospho‐CoA prosthetic group of the acyl carrier protein (ACP) subunit. From a cosmid library of K. pneumoniae, a gene cluster of 9 kb has been isolated and sequenced that included the structural genes for the malonate decarboxylase. The cluster consisted of the eight consecutive genes mdcABCDEFGH and the divergently oriented mdcR gene. The intergenic regions were short (usually < 17 bp, 136 bp between mdcE and mdcF) and ribosome binding sites were found 4‐lObp before each gene. According to N‐terminal protein sequencing, the mdcA, C, D and E genes encoded subunits α, δ, β and γ of malonate decarboxylase. Data bank searches for related proteins with known function revealed that MdcA represents the ACP‐transferase and that MdcD and E together probably function as malonyl‐S‐ACP decarboxylase. MdcC is the (apo) ACP subunit. MdcB and MdcG could be involved in the synthesis and attachment of the prosthetic group. MdcH is similar to various malonyl‐CoA:ACP‐SH transacylases and therefore probably involved in the initial activation of the enzyme by malonylation. MdcF is a membrane protein that could function as a malonate carrier. The mdcR gene encodes a protein of the LysR regulator family. Malonate decarboxylase was functionally expressed in Escherichia coli from plasmids harbouring the entire gene cluster including mdcR. As partial deletion of the mdcR gene impaired growth of the transformants on malonate, MdcR is probably a transcriptional regulator of the mdc genes.
An Angiogenic Protein from Bovine Serum and Milk — Purification and Primary Structure of Angiogenin‐2
Authors: Strydom Daniel J. , Bond Michael D. , Vallee Bert L.
Published: July 2004

Bovine serum and milk contain a basic angiogenic protein that binds tightly to placental ribonuclease inhibitor. It was purified from both sources by ion‐exchange and reversed‐phase chromatographies. Its amino acid sequence revealed that it is a member of the ribonuclease superfamily. It contains 123 amino acids in a single polypeptide chain, is cross‐linked by three disulfide bonds, is glycosylated at Asn33, and is 57% identical to bovine angiogenin. The amino‐terminal and carboxyl terminal residues are pyroglutamic acid and proline, respectively. The protein has ribonucleolytic activity that is similar to, but somewhat lower than, that of bovine angiogenin, i.e. very low relative to RNase. It is angiogenically potent on chicken chorioallantoic membrane, but less so than angiogenin. The sequence and activities demonstrate that this protein is a second, distinct, member of the angiogenin sub‐family of pancreatic ribonucleases, and is referred to as angiogenin‐2.
General Structure/Function Properties of Microbial Methionyl‐Trna Synthetases
Authors: Schmitt Emmanuelle , Panvert Michel , Mechulam Yves , Blanquet Sylvain
Published: July 2004

Alignment of the sequences of methionyl‐tRNA synthetases from various microbial sources shows low levels of identities. However, sequence identities are clustered in a limited number of sites, most of which contain peptide patterns known to support the activity of the Escherichia coli enzyme. In the present study, site‐directed mutagenesis was used to probe the role of these conserved residues in the case of the Bacillus stearothermophilus methionyl‐tRNA synthetase. The B. stearothermophilus enzyme was chosen in this study because it can be produced as an active truncated monomeric form, similar to the monomeric derivative of E. coli methionyl‐tRNA synthetase produced by mild proteolysis. The two core enzyme molecules share only 27% identical residues. The results allowed the identification of the binding sites for ATP, methionine and tRNA, as well as that responsible for the tight binding of the zinc ion to the enzyme. It is concluded that the thermostable synthetase adopts a three‐dimensional folding very similar to that of the E. coli one. Therefore, the two methionyl‐tRNA synthetase sequences, although significantly different, maintain a common scafold with the functionally important residues exposed at constant positions. Sequence alignments suggest that the above conclusion can be generalized to the known methionyl‐tRNA synthetases from various sources.
Dissection of the Domain Architecture of the α2macroglobulin‐Receptor‐Associated Protein
Authors: Ellgaard Lars , Holtet Thor Las , Nielsen Peter Reinholt , Etzerodt Michael , Gliemann Jørgen , Thøgersen Hans Christian
Published: July 2004

The α2macroglobulin‐receptor‐associated protein (RAP) binds to the α2macroglobulin receptor/low‐density lipoprotein receptor‐related protein (α2MR/LRP), a multi‐functional cell surface receptor known to bind and internalize several macromolecular ligands. RAP has been shown to inhibit binding of all known α2MR/LRP ligands. Mutational studies have implicated distinct parts of RAP as specifically involved in inhibition of binding of a multitude of ligands.

In the present paper we provide experimental evidence allowing assignment of elements of triplicate internal sequence similarity in RAP, noted previously [Warshawsky, I., Bu, G. & Schwartz, A. L. (1995) Sites within the 39‐kDa protein important for regulating ligand binding to the low‐density lipoprotein receptor‐related protein, Biochemistry 34, 3404–3415], to three structural domains, 1, 2 and 3, comprising residues 18–112, 113–218 and 219–323 of RAP, respectively. Structural analysis by 1H‐NMR spectroscopy shows that domains 1 and 2 as separate domains have similar secondary structures, consisting almost exclusively of α‐helices, whereas domain 3 as a separate domain appears only to be marginally stable.

Ligand competition titration of recombinant RAP domains 1, 2 and 3 and double domains 1+2 and 2+3 against 125I‐RAP and 125I‐α2M* (methylamine‐activated α2M) for binding to α2MR*LRP demonstrated (a) that functional integrity in single domains is largely preserved, and (b) that important determinants for the inhibition of test ligands reside in the C‐terminal regions of domains 1 and 3.
The Solution Structure and Activity of Caerin 1.1, an Antimicrobial Peptide from the Australian Green Tree Frog, Litoria Splendida
Authors: Wong Herbert , Bowie John H. , Carver John A.
Published: July 2004

Caerin 1.1 is one of the major antimicrobial peptides isolated from the skin of the Australian green tree frog, Litoria splendida. Two‐dimensional 1H‐1H and 1H‐13C NMR spectroscopy in trifluoroethanol/ H2O (50:50, by vol.) have been used to assign the 1H and 13C‐NMR spectra of this 25‐amino‐acid peptide. From an examination of these data, and using distance geometry and molecular dynamics calculations, the solution conformation of caerin 1.1 has been determined. The peptide adopts two well‐defined helices from Leu2 to Lys11 and from Val17 to His24 separated by a region of less‐defined helicity and greater flexibility. Overall, the peptide has a distinct amphipathic charge distribution. The solution structure of caerin 1.1 is compared with activity data against a variety of micro‐organisms for the parent peptide and some naturally occurring and synthetic variants of caerin 1.1. The structural and activity data are consistent with caerin 1.1 interacting with membranes in a similar manner to other antimicrobial peptides, i.e. via a carpet‐like mechanism whereby the individual peptides aggregate in a helical manner and orient themselves parallel to the membrane in a sheet‐like arrangement [Shai, Y. (1995) Trends Biochem. Sci. 20, 460–464].
Expression and Characterisation of Plasmepsin I from Plasmodium falciparum
Authors: Moon Richard P. , Tyas Lorraine , Certa Ulrich , Rupp Katharina , Bur Daniel , Jacquet Catherine , Matile Hugues , Loetscher Hansruedi , Grueninger‐Leitch Fiona , Kay John , Dunn Ben M. , Berry Colin , Ridley Robert G.
Published: July 2004

Two aspartic proteinases, plasmepsins I and II, are present in the digestive vacuole of the human malarial parasite Plasmodium falciparum and are believed to be essential for parasite degradation of haemoglobin. Here we report the expression and kinetic characterisation of functional recombinant plasmepsin I. In order to generate active plasmepsin I from its precursor, an autocatalytic cleavage site was introduced into the propart of the zymogen by mutation of Lys110P to Val (P indicates a propart residue). Appropriate refolding of the mutated zymogen then permitted pH‐dependent autocatalytic processing of the zymogen to the active mature proteinase. A purification scheme was devised that removed aggregated and misfolded protein to yield pure, fully processable, proplasmepsin I. Kinetic constants for two synthetic peptide substrates and four inhibitors were determined for both recombinant plasmepsin I and recombinant plasmepsin II. Plasmepsin I had 5–10–fold lower Kcat/Km values than plasmepsin II for the peptide substrates, while the aspartic proteinase inhibitors, selected for their ability to inhibit P. falciparum growth, were found to have up to 80‐fold lower inhibition constants for plasmepsin I compared to plasmepsin II. The most active plasmepsin I inhibitors were antagonistic to the antimalarial action of chloroquine on cultured parasites. Northern blot analysis of RNA, isolated from specific stages of the erythrocytic cycle of P. falciparum, showed that the proplasmepsin I gene is expressed in the ring stages whereas the proplasmepsin II gene is not transcribed until the later trophozoite stage of parasite growth. The differences in kinetic properties and temporal expression of the two plasmepsins suggest they are not functionally redundant but play distinct roles in the parasite.
A conditional‐Lethal Translation Termination Defect in a sup45 Mutant of the Yeast Succhuromyces Cerevisiue
Authors: Stansfield Ian , Kushnirov Vitaly V. , Jones Kerrie M. , Tuite Mick F.
Published: July 2004

Genetic studies have indicated that the product of the yeast SUP45 gene encodes a component of the translational‐termination machinery. In higher eukaryotes, genes similar to SUP45 encode eukaryote release factor 1 (eRFI), which has a stop‐codon‐dependent peptidyl‐release activity. Using a conditional‐lethal mutant allele of SUP45 (sup4.5‐2) and a combination of in vivo and in vitro approaches, we demonstrate that the product of the SUP45 gene (Sup45p or eRF1) is a factor required for translation termination in yeast. A homologous in vitro assay based on suppressor‐tRNA‐mediated readthrough of stop codons is used to show that a translating lysate from a sup45–2 mutant strain exhibits a termination defect when heated for short periods to greater than the non‐permissive temperature (37°C). This defect can be complemented with a purified preparation of Sup45p (eRF1) expressed in Eschericha coli. The termination defect in this strain appears to be due to an inability of the Sup45p protein to bind the ribosome, resulting in vivo in a reduced ability of Sup45p to release nascent polypeptides from the ribosome at the non‐permissive temperature. Cell‐free translation lysates from the sup45‐2 strain do not show a defect in sense‐codon translation at the non‐permissive temperature. These data demonstrate that yeast eRFl plays a role in translation termination and is functionally equivalent to its higher eukaryotic homologues.
Cloning and Expression of cDNA for a Human Gal(β1‐3)GalNAc α2,3‐Sialyltransferase from the CEM T‐Cell Line
Authors: Giordanengo Valérie , Bannwarth Sylvie , Laffont Catherine , Van Miecem Vincent , Harduin‐Lepers Anne , Delannoy Philippe , Lefebvre Jean‐Claude
Published: July 2004

Complementary DNA encoding a human Gal(β1‐3)GalNAc α2,3‐sialyltransferase type II (hST3Gal II) was cloned from a CEM T‐cell cDNA library using a 23‐base oligonucleotide probe. The sequence of this probe was established on the basis of a slightly divergent sialylmotif L that was obtained by polymerase chain reaction with degenerate oligonucleotide primers based on the conserved sialylmotif L of mammalian Gal(β1‐3)GalNAc α2,3‐sialyltransferases. It was thus confirmed that a short oligonucleotide probe may be sensitive and highly specific. The nucleotide and amino acid sequences of hST3Gal II show, respectively, 56.3% and 49.3% similarity to hST3Gal I [Kitagawa, H. & Paulson, J. C. (1994) J. Biol. Chem. 269, 17872–17878] and 88.1% and 93.7% similarity to inurine ST3Gal II [Lee, Y. C., Kojima, N., Wada, E., Kurosawa, N., Nakaoka, T., Hamamoto, T. & Tsuji, S. (1994) J. Biol. Chem. 269, 10028–10033]. hST3Gal II mRNA was highly expressed in heart, liver, skeletal muscle and various lymphoid tissues but not in brain and kidney. A soluble form of hST3Gal II expressed in COS‐7 cells was tested in vitro for substrate specificity and kinetic properties. Asialofetuin and asialo‐bovine submaxillary mucin appeared better substrates for hST3Gal II than for its murine counterpart as previously reported [Kojima, N., Lee, Y.‐C., Hamamoto, X, Kurosawa, N. & Tsuji, S. (1994) Biochemistry 33, 5772–57761. In previous studies, we have shown hyposialylation of O‐glycans attached to two major lymphocyte CD43 and CD45 cell surface molecules in human‐immunodeficiency‐virus‐1(H1V‐1) –infected T‐cell lines. Since comparable levels of hST3Gal 1 and hST3Gal II mRNA and enzymatic activity were observed in parental and HIV‐1‐infected CEM T‐cell lysates, the sialylation defect associated with HIV infection of this cell line is probably due to a mechanism different from a simple altered catalytic activity of these sialyltransferases.