bluuebunny/crossref_metadata_2025 huggingface dataset:
Mechanism of Inactivation of a Catalytic Antibody by P‐Nitrophenyl Esters
Authors: Gigant Benoit , Charbonnier Jean‐Baptiste , Golinelli‐Pimpaneau Béatrice , Zemel Romy R. , Eshhar Zelig , Green Bernard S. , Knossow Marcel
Published: July 2004

Antibody CNJ206 catalyses the hydrolysis of p‐nitrophenyl esters with significant rate enhancement; however, after a few cycles, 90% of the catalytic activity of CNJ206 is irreversibly lost. This report investigates the properties of the inactivated Fab (fragment antigen binding). After inactivation, the residual esterase activity of CNJ206 is similar to that of the catalytic antibody inhibited by the transition‐state analogue (TSA) used to elicit it; the affinity of CNJ206 for the TSA is also dramatically lowered. Here we propose a simple scheme that accounts for the steady‐state kinetics of inactivation. The following lines of evidence, when taken together, suggest that stable acylated tyrosine side chains within or close to the Fab combining site are involved in the inactivation process: isoelectric focusing and matrix‐as‐sisted‐laser‐desorption‐ionisation–time‐of‐flight (MALDI‐TOF) mass spectrometry show that incubation with substrate results in several acylated Fab species; inactivation is stable at pH 8, is reversed by mild hydroxylamine treatment and follows the same kinetics as inhibition of binding, which is slowed down by the presence of the TSA hapten. Analysis of the Fab‐TSA X‐ray structure shows that three tyrosine residues are potential candidates for the inactivation of CNJ206 by its substrates, Tyr L96 being the most likely one; this also suggests that site‐directed mutation of one or more of these residues might prevent substrate inactivation and significantly improve catalysis.
Structural and Serological Characterisation of the O‐Antigenic Polysaccharide of the Lipopolysaccharide from Acinetobacter Junii Strain 65
Authors: Haseley Simon R. , Pantophlet Ralph , Brade Lore , Holst Otto , Brade Helmut
Published: July 2004

A polysaccharide containing rhamnose (Rha) and Gal was isolated by acetic acid hydrolysis, followed by gel‐permeation chromatography, from the water‐soluble lipopolysaccharide (phenol/water extracted) from Acinetobacter junii strain 65. The polysaccharide was characterised by means of monosaccharide analyses, Smith degradation, and NMR studies, and was shown to have a linear pentasaccharide repeating unit, as depicted below. This structure was specifically recognised in western blots and enzyme immuno‐assays by polyclonal rabbit antisera.
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Differential Characterization of Neutrophil Cytochrome p30 and Cytochrome b‐558 by Low‐Temperature Absorption and Resonance Raman Spectroscopies
Authors: Escriou Virginie , Laporte François , Vignais Pierre V. , Desbois Alain
Published: July 2004

Cytochrome p30, a novel hemoprotein isolated from rabbit peritoneal neutrophils [Escriou, V., Laporte, R, Garin, J., Brandolin, G. & Vignais, P. V. (1994)J. Biol. Chem. 269, 14007–14014] has been characterized by low‐temperature (77 K) absorption and resonance Raman spectroscopies. The spectral data have been compared with those obtained with neutrophil cytochrome b‐558. At room temperature, the absorption difference spectra (reduced minus oxidized) of cytochrome p30 and cytochrome b‐558 could not been distinguished from each other. However, at 77 K, significant differences were observed. In particular, the α band of cytochrome p30 was split whereas that of cytochrome b‐558 was symmetrical, but particularly broad. The resonance Raman spectra of cytochrome p30 provided evidence for the presence of two hemes both in the ferric and ferrous states. One of them was a six‐coordinated low‐spin heme either oxidized or reduced whereas the other one was a high‐spin heme, five‐coordinated in the reduced state and six‐coordinated in the oxidized state. It is probable that two histidine residues constitute the axial ligands of the six‐coordinated low‐spin heme of cytochrome p30. The resonance Raman spectra of cytochrome b‐558 allowed the detection of a six‐coordinated low‐spin heme, similar to that found in cytochrome p30. The component typical of the high‐spin heme of cytochrome p30 was however absent in the spectra of oxidized and reduced cytochrome b‐558.
Copper A of Cytochrome c Oxidase, A Novel, Long‐Embattled, Biological Electron‐Transfer Site
Authors: Beinert Helmut
Published: July 2004

This review traces the history of understanding of the CUA, site in cytochrome c oxidase (COX) from the beginnings, when few believed that there was any significant Cu in COX, to the verification of three atoms Cu/monomer and to the final identification of the site as a dinuclear, Cys‐bridged average valence CU1,5+… Cu1.5+ structure through spectroscopy, recombinant DNA techniques, and crystallography. The critical steps forward in understanding the nature of the CuA, site are recounted and the present state (as of the end of 1996) of our knowledge of the molecular and electronic structure is discussed in some detail. The contributions made through the years by the development of methodology and concepts for solving the enigma of CuA, are emphasized and impediments, often rooted in contemporary preconceptions and attitudes rather than solid data, are mentioned, which discouraged the exploitation of early valuable clues. Finally, analogies in construction principles of polynuclear Cu‐S and Fe‐S proteins are pointed out.
Theoretical Approaches to the Evolutionary Optimization of Glycolysis
Authors: Meléndez‐Hevia Enrique , Waddell Thomas G. , Heinrich Reinhart , Montero Francisco
Published: July 2004

In the first part of this work [Heinrich, R., Montero, F., Klipp, E., Waddell, T. G. & Meléndez‐Hevia, E. (1997)Eur. J. Biochem. 243, 191–201] the kinetic and thermodynamic constraints under which an optimal glycolysis must be designed have been analysed. In this second part, we present a chemical analysis of the glycolytic pathway in order to determine if its design is chemically optimized according the possibilities that a glycolytic design can have. Our results demonstrate that glycolysis in modern‐day cells (from glucose to lactate) has an optimized design for maximizing the flux of ATP production, and a thermodynamic profile which guarantees a high kinetic efficiency. We also discuss some cases of paleometabolism for this pathway as alternative metabolic pathways, less optimized, that exist in some bacteria. Our analysis relates mainly to metabolism designed under constant chemical affinity (substrates and products of the pathway constant), where the target of optimization can be the flux of ATP production. We also discuss the case of an externally imposed input flux, whose target of optimization is the stoichio‐metric yield of ATP.
Molecular Cloning and Functional Expression of an Insect High‐Affinity Na+‐Dependent Glutamate Transporter
Authors: Donly B. Cameron , Richman Alex , Hawkins Erika , Mclean Heather , Caveney Stan
Published: July 2004

Excitatory amino acid transporters in the central and peripheral nervous systems of insects are thought to assist in maintaining glutamate concentrations in the resting synapse below the activation threshold of glutamate receptors. We have isolated a cDNA from the caterpillar Trichoplusia ni which encodes a high‐affinity Na+‐dependent glutamate transporter, designated TrnEAAT1. The deduced amino acid sequence shows strong identity with known members of the vertebrate Na+–and K+‐dependent amino acid transporter family. Expression of the insect transporter mRNA was predominantly localized in the caterpillar brain. The function of the TrnEAAT1 protein was analyzed in cultured insect cells using a baculovirus expression system. Cells infected with the recombinant virus were found to exhibit a 50‐fold increase in ability to accumulate labeled L‐glutamate compared to mock‐infected cultures, and this activity was shown to be Na+‐dependent. Transport activity was further demonstrated by chromatographic identification of various glutamate analogues accumulated by infected cells. Various glutamate uptake inhibitors were used to outline the pharmacological properties of the cloned transporter and to compare it with known mammalian transporters. Despite the significant differences between insect and vertebrate physiology, the characteristics of the respective transporters were found to be remarkably similar.
Animal Lectins
Authors: Gabius Hans‐Joachim
Published: July 2004

Protein and lipid glycosylation is no longer considered as a topic whose appeal is restricted to a limited number of analytical experts perseveringly pursuing the comprehensive cataloguing of structural variants. It is in fact arousing curiosity in various areas of basic and applied bioscience. Well founded by the conspicuous coding potential of the sugar part of cellular glycoconjugates which surpasses the storage capacity of oligonucleotide‐ or oligopeptide‐based code systems, recognition of distinct oligosaccharide ligands by endogenous receptors, i.e. lectins and sugar‐binding enzymes or antibodies, is increasingly being discovered to play salient roles in animal physiology. Having inevitably started with a descriptive stage, research on animal lectins has now undubitably reached maturity. Besides listing the current categories for lectin classification and providing presentations of the individual families and their presently delineated physiological significance, this review places special emphasis on tracing common structural and functional themes which appear to reverberate in nominally separated lectin and animal categories as well as lines of research which may come to fruition for medical sciences.
The Effect of High Pressure on Thermolysin
Authors: Kunugi Shigeru , Kitayaki Moto , Yanagi Yuuichi , Tanaka Naoki , Lange Reinhard , Balny Claude
Published: July 2004

The effects of high pressure on thermolysin activity and spectroscopic properties were studied. Thermolysin showed distinct pressure‐induced activation with a maximum observed at 200–250 MPa for a dipeptide amide substrate and at 100–120 MPa for a heptapeptide substrate. By examining the pressure dependence of the hydrolytic rate for the former substrate using a high pressure stopped‐flow apparatus as a mixing device under elevated pressures, the activation volume of the reaction was ‐71 ml mol−1 at 25°C. ΔV‡ was accompanied by a negative activation expansibility and a value of ‐95 ml mol−1 was obtained at 45°C. A prolonged incubation of thermolysin under high pressure, however, caused a time‐dependent deactivation.

These changes due to pressure were monitored by several spectroscopic methods. The fourth‐derivative absorbance spectrum showed an irreversible change, mostly in the tyrosine and tryptophan regions, at a pressure higher than 300 MPa. Intrinsic fluorescence and circular dichroism measurements of thermolysin in solution also detected irreversible changes. All these measurements indicated that a change occurred at higher pressures and are explained by a simple two‐state transition model accompanied by a large, negative change in the volume of reaction.
Secondary Structure and Tertiary Fold of the Human Immunodeficiency Virus Protein U (Vpu) Cytoplasmic Domain in Solution
Authors: Willbold Dieter , Hoffmann Silke , Rösch Paul
Published: July 2004

The human immunodeficiency virus type 1 Vpu protein enhances virus particle release from infected cells, decreases the tendency of syncytia formation and induces degradation of human CD4 receptor. It is known that the cytoplasmic part of Vpu is responsible for direct interaction to and degradation of CD4. The tertiary fold of the Vpu cytoplasmic domain in aqueous solution was determined employing NMR spectroscopy and molecular‐dynamics simulated‐annealing protocols. We found a very well defined amphipathic α‐helix in the membrane proximal part (40–50), a less well defined helix (60–68), and a short α‐helix at the C‐terminus (75–79). We further determined the overall tertiary structure based on long‐range nuclear Overhauser enhancement effects. Correlation of results from mutation experiments of Vpu and the structure data is discussed.
Examination of the Thiamin Diphosphate Binding Site in Yeast Transketolase by Site‐Directed Mutagenesis
Authors: Meshalkina Ludmilla , Nilsson Ulrika , Wikner Christer , Kostikowa Tatiana , Schneider Gunter
Published: July 2004

The role of two conserved amino acid residues in the thiamin diphosphate binding site of yeast transketolase has been analyzed by site‐directed mutagenesis. Replacement of El62, which is part of a cluster of glutamic acid residues at the subunit interface, by alanine or glutamine results in mutant enzymes with most catalytic properties similar to wild‐type enzyme. The two mutant enzymes show, however, significant increases in the K0.5 values for thiamin diphosphate in the absence of substrate and in the lag of the reaction progress curves. This suggests that the interaction of E162 with residue E418, and possibly E167, from the second subunit is important for formation and stabilization of the transketolase dimer. Replacement of the conserved residue D382, which is buried upon binding of thiamin diphosphate, by asparagine and alanine, results in mutant enzymes severely impaired in thiamin diphosphate binding and catalytic efficiency. The 25 ‐80‐fold increase in K0.5 for thiamin diphosphate suggests that D382 is involved in cofactor binding, probably by electrostatic compensation of the positive charge of the thiazolium ring and stabilization of a flexible loop at the active site. The decrease in catalytic activities in the D382 mutants indicates that this residue might also be important in subsequent steps in catalysis.
Aminopeptidase N from Bombyx Mori as a Candidate for the Receptor of Bacillus Thuringiensis Cry1Aa Toxin
Authors: Yaoi Katsuro , Kadotani Tomoyuki , Kuwana Hisanaga , Shinkawa Ayaka , Takahashi Tsuyoshi , Iwahana Hidenori , Sato Ryoichi
Published: July 2004

Cry1Aa toxin‐binding proteins from the midgut brush border membrane vesicles of Bombyx mori, a toxin‐susceptible silkworm, were analyzed to find candidates for the toxin receptors. Ligand blotting showed that Cry1Aa toxin bound to a 120‐kDa protein. A part of the 120‐kDa protein was solubilized from the membrane vesicles with phosphatidylinositol‐specific phospholipase C, resulting in a 110‐kDa protein which therefore may be linked to a glycosylphosphatidylinositol anchor. The 120‐kDa and 110‐kDa Cry1Aa toxin‐binding proteins were solubilized with detergent or pohosphatidylinositol‐specific phospholipase C, respectively, and purified using anion‐exchange chromatography. Scatchard plot analysis for the specific binding of purified 110‐kDa protein to Cry1Aa toxin yielded a Kd value of 7.6 nM, which was similar to that for the binding of intact brush border membrane vesicles to the toxin. N‐terminal and internal amino acid sequences of the 120‐kDa and 110‐kDa proteins showed high degrees of similarity to those of aminopeptidase N, a putative Cry1Ac toxin receptor, reported in Manduca sexta and Heliothis virescens. On this basis, the 120‐kDa Cry1Aa toxin‐binding protein from B. mori was identified as a member of the aminopeptidase family.
Methanol: Coenzyme M Methyltransferase from Methanosarcina Barkeri
Authors: Sauer Karin , Harms Ulrike , Thauer Rudolf K.
Published: July 2004

In Methanosarcina barkeri, methanogenesis from methanol is initiated by the formation of methyl‐coenzyme M from methanol and coenzyme M. This methyl transfer reaction is catalyzed by two enzymes, designated MT1 and MT2. Transferase MT1 is a corrinoid protein. The purification, catalytic properties and encoding genes of MT2 (MtaA) have been described previously [Harms, U. and Thauer, R. K. (1996) Eur. J. Biochem. 235, 653–659], We report here on the corresponding analysis of MT1. The corrinoid protein MT1 was purified to apparent homogeneity and showed a specific activity of 750 μmol min−1 mg−1. The enzyme catalyzed the methylation of its bound corrinoid in the cob(I)amide oxidation state by methanol. In addition to this automethylation, the purified enzyme was found to catalyze the methylation of free cob(I)alamin to methylcob(III)alamin. It was composed of two different subunits designated MtaB and MtaC, with apparent molecular masses of 49 kDa and 24 kDa, respectively. The subunit MtaC was shown to harbour the corrinoid prosthetic group. The genes mtaB and mtaC were cloned and sequenced. They were found to be juxtapositioned and to form a transcription unit mtaCB. The corrinoid‐harbouring subunit MtaC exhibits 35% sequence similarity to the cobalamin‐binding domain of methionine synthase from Escherichia coli.
Critical Glutamic Acid Residues Affecting the Mechanism and Nucleotide Specificity of Vibrio Harveyi Aldehyde Dehydrogenase
Authors: Vedadi Masoud , Meighen Edward
Published: July 2004

Fatty aldehyde dehydrogenase (ALDH) from the luminescent marine bacterium, Vibrio harveyi, differs from other ALDHs in its unique specificity and high affinity for NADP+. Two glutamic acid residues, Glu253 and Glu377, which are highly conserved in ALDHs, were investigated in the present study. Mutation of Glu253 to Ala decreased the kcat for ALDH activity by over four orders of magnitude without a significant change in the Km values for substrates or the ability to interact with nucleotides. Both thioesterase activity and a pre‐steady‐state burst of NAD(P)H were also eliminated, implicating Glu253 in promoting the nucleophilicity of the cysteine residue(Cys289) involved in forming the thiohemiacetal intermediate in the enzyme mechanism. Mutation of Glu377 to Gin (E377Q mutant) selectively decreased the kcat for NAD+‐dependent ALDH activity (>102‐fold) compared to only a 6‐fold loss in NADP+‐dependent activity without comparable changes to the Km values for substrates. Consequently, the E377Q mutant had a very high specificity for NADP+(kcat/km>103 of that for NAD+) which was over 20 times higher than that of the wild‐type ALDH. Although a pre‐steady‐state burst of NAD(P)H was eliminated by this mutation, thioesterase activity was completely retained. Using [1‐2H]acetaldehyde as a substrate, a significant deuterium isotope effect was observed, implicating Glu377 in the hydride transfer step and not in acylation or release of the acyl group from the cysteine nucleophile. The increase in specificity of the E377Q mutant for NADP+ is consistent with a change in the rate‐limiting step determining kcat from nucleotide‐dependent NAD(P)H dissociation to hydride transfer. The results provide biochemical evidence that the two highly conserved Glu residues are involved in different functions in the active site of V. harveyi ALDH.
Functional Expression of a Tobacco Gene Related to the Serine Hydrolase Family
Authors: Baudouin Emmanuel , Charpenteau Martine , Roby Dominique , Marco Yves , Ranjeva Raoul , Ranty Benoit
Published: July 2004

We have recently reported the isolation of a tobacco gene, hsr 203J, whose transcripts accumulate during the hypersensitive reaction, a plant response associated with resistance to pathogens. We present and discuss here some structural and biochemical properties of the gene product. Nucleotide sequence analysis has shown that the hsr 203J gene contains an open reading frame coding for a polypeptide of 335 amino acids. The predicted amino acid sequence contains the GXSXG motif characteristic of serine hydrolases, and displays limited but significant similarity to lipases and esterases of prokaryotic origin. The hsr 203J gene was expressed in Escherichia coli, and the recombinant protein, purified to near homogeneity, was able to degrade p‐nitrophenylbutyrate, a general substrate for carboxylesterases. The enzyme was unable to hydrolyze lipids, and was active on short‐chain acyl esters only. The hydrolytic activity was abolished by diisopropyl fluorophosphate and a derivative of isocoumarin, as expected for a member of the serine hydrolase family. Sequence similarities between the tobacco esterase and expressed sequence tags in databases suggest the existence of members of this enzyme family in various plant species.
Specific Stimulation of c‐Fgr Kinase by Tyrosine‐Phosphorylated (Poly)Peptides
Authors: Ruzzene Maria , Brunati Anna Maria , Donella‐Deana Arianna , Marin Oriano , Pinna Lorenzo A.
Published: July 2004

Hematopoietic lineage cell‐specific HS1 protein is converted into a substrate for c‐Fgr by previous Syk‐mediated phosphorylation, at site(s) that bind to the SH2 domain of c‐Fgr [Ruzzene, M., Brunati, A. M., Marin, O., Donella‐Deana, A. & Pinna, L. A. (1996)Biochemistry 35, 5327–53321. Here we show that a phosphopeptide derived from one such site, HS1‐(320–329)‐phosphopeptide (PEGDYpEEVLE), enhances up to tenfold, in a dose‐dependent manner, the catalytic activity of c‐Fgr either assayed with peptide substrates or evaluated as intermolecular autophosphorylation of c‐Fgr itself. The dephosphorylated HS1‐(320‐329)‐peptide is totally ineffective, while the stimulatory efficacy of other phosphopeptides derived from the polyoma virus middle T antigen‐(393–402) sequence, c‐Src, and c‐Fgr autophosphorylation sites, and the C‐terminal c‐Src site (Tyr527) is variable and correlates reasonably well with the predicted affinity for the c‐Fgr SH2 domain. Stimulation of c‐Fgr catalytic activity is also promoted by the full‐length HS1 protein, previously tyrosine phosphorylated by Syk, and is accounted for by an increased Vmax while the Km values are unchanged. If the normal activator of c‐Fgr kinase, Mg2+, is replaced by Mn2+, stimulation by HS1‐(320–329)‐phosphopeptide is still observable with peptide substrates, while autophosphorylation is, in contrast, inhibited by the phosphopeptide. These findings, in conjunction with the ability of previously autophosphorylated c‐Fgr to be stimulated by HSL(320–329)‐phosphopeptide, support the view that stimulation of c‐Fgr by phosphopeptide is not or is not entirely a consequence of increased autophosphorylation. Interestingly, neither Syk and C‐terminal Src kinase nor three other members of the Src family (Lyn, Lck, and Fyn) are susceptible to stimulation by phosphopeptide, as observed with c‐Fgr. These data support the notion that c‐Fgr undergoes a unique mechanism of activation promoted by tyrosine‐phosphorylated polypeptide that binds to its SH2 domain. This suggests that such a mode of regulation is peculiar of protein‐tyrosine kinases committed to the secondary phosphorylation of sequentially phosphorylated proteins.
Molecular Cloning and Nucleotide Sequence of an Endo‐1,5‐α‐L‐Arabinase Gene from Bacillus Subtilis
Authors: Sakamoto Tatsuji , Yamada Masako , Kawasaki Haruhiko , Sakai Takuo
Published: July 2004

The nucleotide sequence of the gene encoding an endo‐1,5‐α–arabinase (protopectinase C) of Bacillus subtilis was determined by sequencing fragments amplified by the cassette‐ligation‐mediated PCR (CLM‐PCR). The gene covering the start and stop codon was amplified by PCR with two specific primers, which were designed from the sequence data determined by CLM‐PCR. An approximately 1.5‐kb amplification product was cloned into the vector pUC119, forming a plasmid termed pPPC. An ORF that encodes the arabinase composed of 324 amino acids including a 33‐amino‐acid signal peptide was assigned. Comparison of the deduced amino acid sequence of the enzyme with that of an Aspergillus niger endoarabinase showed 37% identity in a 207‐amino‐acid overlap. The optimal nucleotide sequence for catabolite repression of B. subtilis was found upstream of the structural gene. In a culture of Escherichia coli DH5u cells harboring pPPC, no arabinase activity was detected, either intracellularly or extracellularly, suggesting that the B. subtilis promotor is not functional in this transformant.

In B. subtilis IFO 3134 strain, production of protopectinase C was repressed by readily metabolizable carbohydrates. In contrast, productivity (total enzyme activityhacterial growth) of the enzyme was increased about fourfold in the presence of 0.75 M potassium phosphate in the culture medium. The phosphate anion seemed to be involved in the stimulation of protopectinase C production in this stain.
Molecular Cloning and Expression of a Hexamerin cDNA from the Malaria Mosquito, Anopheles Gambiae
Authors: Zakharkin Stanislav O. , Gordadze Alexey V. , Korochkina Svetlana E. , Mathiopoulos Kostas D. , Della Torre Alessandra , Beneš Helen
Published: July 2004

During the last larval instar, dipteran insects synthesize two hexamerins rich in aromatic residues, typified by the larval serum proteins 1 and 2 (LSP‐1 and LSP‐2) of Drosophila melanogaster. We report here the characterization of a complete cDNA sequence encoding a LSP‐1‐like protein from a lower dipteran insect, the malaria mosquito Anopheles gambiae. The cDNA encodes the subunit of a homohexamer, A. gambiae hexamerin‐1.1 (AgHex‐1.1), which is a major pupal protein but only a minor constituent of late larval hemolymph. AgHex‐1.1 is moderately rich in methionine (3.9%) and particularly rich in aromatic residues (21% Phe + Tyr). Cytogenetic analysis reveals AgHex‐1.1 to be encoded by a single‐copy gene localized to division 22F within the proximal 2La inversion breakpoint of chromosome 2 of A. gambiae. The AgHex‐1.1 transcript is first detected in fourth‐instar larvae (L4) and disappears abruptly in early pupae. In situ hybridization shows accumulation of the transcript uniquely in the larval fat body. AgHex‐1.1 mRNA is re‐expressed in male and female adults at about 10% of the L4 level, with no effect of bloodfeeding in females. The potential roles of AgHex‐1.1 in Anopheles development and reproductive maturation are discussed.
Undersulfation of Cartilage Proteoglycans Ex Vivo and Increased Contribution of Amino Acid Sulfur to Sulfation In Vitro in McAlister Dysplasia/Atelosteogenesis Type 2
Authors: Rossi Antonio , Bonaventure Jacky , Delezoide Anne‐Lise , Superti‐Furga Andrea , Cetta Giuseppe
Published: July 2004

Mutations in the diastrophic dysplasia sulfate transporter gene cause a family of chondrodysplasias including, in order of increasing severity, diastrophic dysplasia, atelosteogenesis type 2 and achondrogenesis type 1B. McAlister dysplasia is a lethal chondrodysplasia considered on the basis of minor radiographic features to be a disorder different from atelosteogenesis type 2. Here, we demonstrate that McAlister dysplasia arises from mutations in the diastrophic dysplasia sulfate transporter gene and that this disorder essentially coincides on molecular and biochemical grounds with atelosteogenesis type 2. The fetus affected by McAlister dysplasia we have studied is a compound heterozygote for mutations leading to R279W and N425D substitutions in the diastrophic dysplasia sulfate transporter. Proteoglycan sulfation was studied in epiphyseal cartilage and in chondrocyte cultures of the patient by high performance liquid chromatography of chondrotinase digested proteoglycans; a high amount of non‐sulfated disaccharide was observed as a consequence of the alteration of the transporter function caused by the mutations. However, sulfated disaccharides were detectable even if in low amounts, both in cultured cells and tissue. Functional impairment of the sulfate transporter was demonstrated in vitro by reduced incorporation of [35S]sulfate relative to [3H]glucosamine in proteoglycans synthesized by chondrocytes and by sulfate‐uptake assays in fibroblasts. Parallel in vitro studies in a patient with achondrogenesis 1B indicated that the severity of the clinical phenotype seems to be correlated to the residual activity of the sulfate transporter. The capacity of fibroblasts to use cysteine as an alternative source of sulfate was evaluated by double‐labeling experiments. Relative incorporation of [35S]cysteine‐derived sulfate in the glycosaminoglycan chains was increased in the patient's cells, indicating that, in vitro, the catabolism of sulfur‐containing amino acids can partially compensate for intracellular sulfate deficiency. Residual sulfation observed in proteoglycans extracted from cartilage suggests that this mechanism may be operating also in vivo.
Drosophila Laminin Binds to Mammalian Nidogen and to Heparan Sulfate Proteoglycan
Authors: Mayer Ulrike , Mann Karlheinz , Fessler Liselotte I. , Fessler John H. , Timpl Rupert
Published: July 2004

A Drosophila laminin that has the chain composition α5β1γ1, relative to mammalian laminins, bound human and mouse nidogen almost as strongly as mouse laminin‐1 (α1βγ1) in solid‐phase assays, and had only a fourfold lower affinity in a radioligand competition test. This is due to a short, highly conserved sequence that occurs in both laminin γ1 chains and which binds nidogen. When the single conservative amino acid difference between the two sequences (Tyr→His) was introduced into the mouse laminin binding module γ1III4 it failed to cause any change of binding. A high affinity between Drosophila laminin and mouse nidogen resulted in the formation of a stable complex in solution. Drosophila laminin also bound to the mouse heparan sulfate proteoglycan perlecan and the formation of this complex was inhibited by heparin, but not by chondroitin sulfate. In addition, a weaker connection between the core protein of mouse perlecan and Drosophila laminin can be mediated through nidogen. Elastase and other proteases degraded Drosophila laminin to a restricted number of larger fragments (40‐300 kDa), almost all of which were bound to a heparin affinity column. Three fragments could be displaced at low salt concentration and were derived from the short arms of the Drosophila laminin, as shown by sequence analysis. A more strongly bound 50‐kDa fragment apparently comprised the globular domains LG2 and LG3 derived from the C‐terminal part of its α chain. Therefore, Drosophila laminin and mouse laminin‐1 differ in certain aspects of protease stability and heparin‐binding sites that, in part, can be attributed to their different α chains. The data suggest the existence of a nidogen analog and heparan sulfate proteoglycans in Drosophila, which remain to be identified.
Phosphorylation and Activation of Cytosolic Phospholipase A2 by 38‐kDa Mitogen‐Activated Protein Kinase in Collagen‐Stimulated Human Platelets
Authors: Börsch‐Haubold Angelika G. , Kramer Ruth M. , Watson Steve P.
Published: July 2004

Phosphorylation and activation of cytosolic phospholipase A2 (PLA2) can occur independently of the activation of 42/44‐kDa mitogen‐activated protein (MAP) kinase in human platelets. We have investigated the hypothesis that the stress‐activated p38 MAP kinase plays a role in the regulation of cytosolic PLA2 The specific inhibitor of p38 MAP kinase, SB 203580 [4‐(4‐fluorophenyl)‐2‐(4‐methylsulfinylphenyl)‐5‐(4‐pyridyl) imidazole], completely blocked the collagen‐stimulated phosphorylation of cytosolic PLA2 in the presence of a cyclooxygenase blocker, and reduced the release of [3H]arachidonic acid by low concentrations of collagen. Stimulation of platelets with collagen (100 pg/ml) enhanced in vitro PLA2 activity of platelet lysates twofold over basal levels. in vitro PLA2 activity was reduced to basal levels when platelets were stimulated in the presence of SB 203580, but not in the presence of an inhibitor of the kinase that activates p42/p44 MAP kinase. SB 203580 only partially inhibited phosphorylation of cytosolic PLA2 in platelets that had not been treated with a cyclooxygenase blocker indicating that secondary stimulation by thromboxane A, induces cytosolic PLA, phosphorylation, by kinase(s) other than p38 MAP kinase. Under these conditions, inhibition of p42/p44 MAP kinase did not result in a reduction of cytosolic PLA, phosphorylation, which is in agreement with the results obtained in the presence of cyclooxygenase blockers. In contrast to collagen, both p38 MAP kinase and p42/p44 MAP kinase participated in the phosphorylation of cytosolic PLA, in platelets stimulated by cross‐linking of the low‐affinity receptor for immune complexes, FcyRIIA. The present results demonstrate an important role for p38 MAP kinase in the regulation of cytosolic PLA2 activity in collagen‐stimulated human platelets.
Biosynthesis of the Proteoglycan Decorin
Authors: Moses Jonatan , Oldberg Åke , Eklund Erik , Fransson Lars‐Åke
Published: July 2004

Biosynthesis of decorin was investigated by incubating a rat fibroblast cell line with various radio‐labelled protein and galactosaminoglycan precursors. The following cell‐associated and distinct intermediates were isolated and identified: a pool of non‐glycosylated core protein, two pools of decorin with incomplete chains, one with three sulphated disaccharide repeats and another with five or more sulphated disaccharide repeats, as well as decorin with mature chains. Results of pulse/chase experiments indicated that these pools represented discrete stages in chain growth. Treatment with brefeldin A, which blocks transport from the endoplasmic reticulum to the Golgi, resulted in accumulation of decorin with an incomplete chain containing six or seven largely unsulphated disaccharide repeats. During recovery from drug treatment, 4‐sulfation reappeared earlier than 6‐sulfation. The results suggest that the galactosaminoglycan assembly‐line consists of separate multienzyme complexes that build only a limited section of the chain. Furthermore, brefeldin A causes segregation of compartments involved in separate stages of the assembly line. In an earlier report [Moses, J., Oldberg, Å., Cheng, F. & Fransson, L.‐Å. (1997) Eur. J. Biochem. 248, 521–526] we took advantage of such segregation to identify and characterize a transient 2‐phosphorylation of xylose in the linkage region.
Oligomeric States and Siderophore Binding of the Ligand‐Gated FhuA Protein that Forms Channels Across Escherichia Coli Outer Membranes
Authors: Locher Kaspar P. , Rosenbusch Jurg P.
Published: July 2004

The channel‐forming FhuA protein, which translocates ferrichrome across Escherichia coli outer membranes, binds 1 mol ligand/mol monomer in detergent solution. The protein is homogenous and migrates as a single band with a mobility corresponding to 77 kDa in SDS/PAGE electrophoresis. Analytical ultracentrifugation revealed a monodisperse species (S20,w= 3.8 S) with a mass of 77 800 ± 3200 Da. The properties of ligand binding, determined by two independent methods, revealed one binding site/ monomer, but are complicated by a pronounced convexity of the Scatchard plot and a Hill coefficient calculated to be 2.5. This strongly suggests that oligomeric species are present. Cross‐linking agents revealed the existence of possibly transient, mostly dimeric and trimeric species. The difference between the FhuA protein in detergent solution and in its native membrane environment may be related to the removal of lateral pressure that exists in situ.
Protein and Gene Structure of the NADH‐Binding Fragment of Rhodobacter Capsulatus NADH: Ubiquinone Oxidoreductase
Authors: Herter Stefan M. , Schiltz Emile , Drews Gerhart
Published: July 2004

Membranes of aerobically grown Rhodobacter capsulatus contain only one type of NADH:ubiquinone oxidoreductase which is homologous to the proton‐translocating complex I. The Km value of the enzyme for NADH was determined to be 8 μM. After solubilization of the membranes with an alkylglucoside detergent, two fragments of complex I with molecular masses of 110 kDa and 140 kDa were isolated by chromatographic steps in the presence of detergent. Both fragments contain at least two polypeptides with apparent molecular masses of 46 kDa and 42 kDa. FMN was identified as cofactor in the preparations. Degenerative oligonucleotide primers were used to amplify a part of the sequence coding for the NADH‐binding subunit of complex I by PCR. With the PCR product as probe, a genomic fragment was cloned and sequenced containing the genes encoding the two purified polypeptides and additional reading frames. The two genes are named nuoE and nuoF and are homologous to nqo2 and nqol of Paracoccus denitrificans. However, NuoE contains a C–terminal extension of 149 amino acids compared with Nqo2. NuoE and NuoF have molecular masses of 41259 Da and 47133 Da and contain the NADH‐, FMN‐ and FeS‐cluster‐binding motifs.
A Putative β‐Tubulin Phosphate‐Binding Motif is Involved in Lateral Microtubule Protofilament Interactions
Authors: PÉRez Mar , Aloria Kerman , Zabala Juan Carlos , Avila Jesús
Published: July 2004

We have investigated the role of a putative GTP‐binding β‐tubulin motif in microtubule polymerization. A peptide containing residues 126–142 of the β‐tubulin subunit (peptide G) was synthesised and an antibody against it raised. Peptide G prevents the binding of GTP to tubulin and also microtubule polymerization but not the formation of vinblastine‐induced tubulin spirals, suggesting that it may prevent lateral but not longitudinal tubulin‐tubulin interactions. The antibody to peptide G shows little reaction with the interphase microtubule network, mitotic spindles or midbody of cultured cells, whereas it clearly reacts with vinblastine‐induced paracrystals. These results suggest that this putative phosphate‐binding site present in β‐tubulin could be involved in the lateral tubulin–tubulin interactions along the microtubule structure.
Expression of Natural and Synthetic Genes Encoding Herpes Simplex Virus 1 Protease in Escherichia Coli and Purification of the Protein
Authors: Apeler Heiner , Gottschalk Uwe , Guntermann Diana , Hansen Jutta , Mässen Jutta , Schmidt Elisabeth , Schneider Karl‐Heinz , Schneidereit Marcus , Rübsamen‐Waigmann Helga
Published: July 2004

An attractive target for anti‐herpes chemotherapy is the herpes simplex virus 1 (HSV‐1) protease encoded by the UL26 gene. Studies with HSV‐1 strains that harbour mutations in the protease gene have demonstrated that the protease is essential for DNA packaging and virus maturation.

The UL26 translation product is 635 amino acids long and undergoes autoproteolytic processing between residues Ala247/Ser248 and Ala610/Ser611. The N‐terminal processing product (amino acids 1–247) contains the protease domain.

To perform crystallization studies and high throughput screening for potent inhibitors, large amounts of the HSV‐1 protease are required. However, expression of the natural HSV‐1 protease gene in Escherichia coli using a T7‐promoter‐regulated system is low and does not allow for the efficient production of larger amounts of highly purified enzyme. In this report, we describe the use of a synthetic protease gene with optimized E. coli codon usage. The level of protease expression was at least 20 times higher with the synthetic gene as compared to the natural UL26 gene. The HSV‐1 protease was purified to homogeneity in three steps using mixed‐bed ion‐exchange chromatography, affinity chromatography, and hydroxyapatite chromatography.
Destabilization, Oligomerization and Inhibition of the Mitogenic Activity of Acidic Fibroblast‐Growth Factor by Aurintricarboxylic Acid
Authors: Lozano Rosa M. , Rivas Germán , Giméanez‐Gallego Guillermo
Published: July 2004

The triphenylmethane derivative aurintricarboxylic acid has been used to inhibit angiogenesis, vascular smooth muscle cell proliferation and cell transformation, an effect that has been attributed to its relatively nonspecific inhibitory activity of protein‐nucleic acid interactions. Here, we show that this compound binds to acidic fibroblast growth factor, a prototypic member of a family of protein mitogens activated by heparin, altering its physicochemical properties and decreasing its mitogenic activity. Counteraction of the effects of aurintricarboxylic acid by heparin shows that the two compounds have opposite and reversible effects on acidic fibroblast growth factor structure and biological activity. The studies reported here may contribute to a deeper understanding of the inhibition of fibroblast‐growth factor‐dependent mitogenesis of relevance to future pharmacologic developments.
Influence of theGloeophyllumMetabolite Oosponol and Some Synthetic Analogues on Protein and RNA Synthesis in Target Cells
Authors: Sonnenbichler Johann , Kovács Tibor
Published: July 2004

Oosponol [4‐(hydroxyacetyl)‐8‐hydroxy‐1H–2‐benzopyran‐1‐one], a toxic metabolite of the basidiomyceteGloeophyllum abietinum, and some synthetic analogues were studied to clarify the molecular basis of their fungicidal, bactericidal, and phytotoxic effects. The antibiotic activity was due to a strong inhibition of RNA and protein synthesis in target cells.
Memory and Imprinting Effects in Multienzyme Complexes
Authors: Lebreton Sandrine , Gontero Brigitte , Avilan Luisana , Ricard Jacques
Published: July 2004

Oxidized, free, stable phosphoribuiokinase from Chlamydomonas reinhardtii was almost completely devoid of catalytic activity (0.06 s−1site). However, when it was bound to glyceraldehyde‐3‐phosphate dehydrogenase from the same organism, it displayed significant activity (3.25 s−1/site). Moreover, this complex tended to spontaneously dissociate upon dilution; the isolated phosphoribulokinase activity increased up to 56 s−1site, subsequently decreased, and finally became almost completely inactive. Its intrinsic kinetic properties (Km and kcat) changed with the variation of the overall activity. These effects were paralleled by changes of conformation of the enzyme as revealed by fluorescence analysis. A model is proposed that allows quantitative expression of the dynamics of the dissociation of the oxidized bienzyme complex and the effects of either of the two substrates, ATP and ribulose 5‐phosphate, on this dissociation process. Whereas ATP destabilized the complex and promoted its dissociation, ribulose 5‐phosphate tended to stabilize this complex.

Inactive, stable, oxidized phosphoribulokinase may form a complex with glyceraldehyde‐3‐phosphate dehydrogenase regaining its catalytic activity. In this case, glyceraldehyde‐3‐phosphate dehydrogenase acts in a manner similar, but not identical to a chaperonin. The information content of the phosphoribulokinase gene, as defined by the sequence of its base pairs, was therefore not sufficient to specify full enzyme activity. It needed the presence of glyceraldehyde‐3‐phosphate dehydrogenase to give the oxidized phosphoribulokinase a conformation competent for its activity.

The potential biological significance of these effects remains to be discovered.
Comparison of the Uptake and Processing of Cholesterol from Chylomicrons of Different Fatty Acid Composition in Rats Fed High‐Fat and Low‐Fat Diets
Authors: Bravo Elena , Flora Loredana , Cantafora Alfredo , De Luca Veronica , Tripodi Marco , Avella Michael , Mayes Peter A. , Botham Kathleen M.
Published: July 2004

The fate of [3H]cholesterol carried in chylomicrons prepared from rats given a meal of palm oil (rich in long‐chain saturated fatty acids), olive oil (rich in monounsaturated fatty acids) or corn oil (rich in n–6 polyunsaturated fatty acids) was investigated in vivo in rats fed a low‐fat diet or a diet supplemented with the corresponding oil (to provide 40% of the calories) for 21 days. In the low‐fat‐fed groups, radioactivity was removed from the blood and secreted into bile over 180 min more rapidly when the chylomicrons were derived from corn oil as compared to palm or olive oil. After feeding the corresponding high‐fat diets, however, both parameters were decreased in rats fed palm and corn oil, but not olive oil. As a result of these changes, the rates of removal of radioactivity from the blood and secretion into bile were similar in animals given the olive oil and corn oil diets, and higher than those in rats fed the palm oil diet. All the high‐fat diets tended to increase the proportion of the radioactivity in the plasma found in the 1.006–1.050‐g/ml fraction (low‐density lipoprotein) and decrease that in the 1.050–1.25‐g/ml (high‐density lipoprotein) fraction in comparison to the respective low‐fat diet groups, but the transfer of radioactivity to the plasma high‐density lipoprotein fraction was particularly slow in palm‐oil‐fed rats. These findings indicate that diets high in saturated or n‐6 polyunsaturated fat retard the metabolism of chylomicron cholesterol in comparison to diets low in fat, while those high in monounsaturated fat do not have this effect. As a consequence of this, the rate of removal of cholesterol of dietary origin from the body is slower in animals fed saturated as compared to monounsaturated or n–6 polyunsaturated fat. Thus, differential metabolism of chylomicron cholesterol clearly plays an important role in the hyper‐and hypo‐cholesterolaemic effects of these dietary fats.
Effect of Nucleotides on the Thermal Stability and on the Deuteration Kinetics of the Thermophilic F0F1 ATP Synthase
Authors: Villaverde Joaquim , Cladera Josep , Padrós Esteve , Rigaud Jean‐Louis , Duñach Mireia
Published: July 2004

Differential scanning calorimetry has been used to characterize the influence of specific nucleotide binding on the thermal unfolding of the F0F1,‐type ATP synthase from the thermophilic Bacillus PS3 (TF0F1). The calorimetric trace shows an irreversible and kinetically controlled endothermic transition for TF0F1, in the absence of nucleotides. The thermal denaturation occurs at a transition temperature (tm) of 81.7°C. The remarkable thermostability of this enzyme was decreased upon tight binding of Mg2+. ATP to noncatalytic sites, whereas binding of Mg2+. ADP increased the temperature at which thermal denaturation occurred. At high temperatures, an exothermic transition due to aggregation processes was also affected by nucleotide binding. With the aim to correlate these thermal effects with possible structural differences among the various forms of TF0F1, Fourier transform infrared spectroscopy was carried out. Hydrogen/deuterium exchange was clearly affected by specific nucleotide occupancy. As illustrated by the total extent of protons exchanged, our results demonstrate that more peptide groups are exposed to the medium in the presence of Mg2+. ATP than in the presence of Mg2+. ADP. Therefore, consistent with microcalorimetric data, binding of Mg2+. ADP induces conformational changes which shield amide protons to more buried hydrogen‐bonded structures, whereas binding of Mg2+. ATP results in a more open or flexible structure.
The Retinoblastoma Protein: A Master Regulator of Cell Cycle, Differentiation and Apoptosis
Authors: Herwig Susanne , Strauss Michael
Published: July 2004

The retinoblastoma susceptibility gene is a tumour suppressor and its product retinoblastoma protein (pRb) has been known for 10 years as a repressor of progression towards S phase. Its major activity was supposed to be sequestration or inactivation of the transcription factor E2F which is required for activation of S phase genes. However, within recent years growing evidence has been accumulating for a more general function of pRb at both the transcriptional level and the cellular level. pRb not only regulates the activity of certain protein‐encoding genes but also the activity of RNA polymerase pol I and pol III transcription. This protein appears to be the major player in a regulatory circuit in the late G1 phase, the so‐called restriction point. Moreover, it is involved in regulating an elusive switch point between cell cycle, differentiation and apoptosis. Here, it seems to cooperate with another major tumour suppressor, p53. Thus, pRb sits at the interface of the most important cell‐regulatory processes and therefore deserves close attention by specialists from different fields of research. This review provides an introduction to the complex functions of pRb.