bluuebunny/crossref_metadata_2025 huggingface dataset:
Purification and properties of N‐acetylglucosaminidase from eggs of the ascidian, Halocynthia roretzi
Authors: MATSUURA Katsuyuki , SAWADA Hitoshi , YOKOSAWA Hideyoshi
Published: March 2005

In several ascidians, β‐D‐N‐acetylglucosaminidase (GlcNAcase) released from eggs following fertilization is proposed to play a key role in polyspermy block through its binding to the sperm receptor on the vitelline coat [C. C. Lambert (1989) Development 105, 415–420]. In the ascidian Halocynthia roretzi, GlcNAc‐specific lectins inhibited the fertilization most strongly among various lectins. Furthermore, GlcNAcase activity was released from the eggs in response to the egg activation. The GlcNAcase was purified from the eggs to apparent homogeneity by chromatographies on DEAE‐Toyopearl, SP‐Toyopearl, Toyopearl HW‐65, and Mono S. The purified enzyme gave a single band on isoelectric focusing with an isoelectric point of 7.0. It gave two bands on SDS/PAGE: the molecular masses of the bands were estimated to be 65 kDa/66 kDa, and 84 kDa/85 kDa under reducing/non‐reducing conditions, respectively. The two bands were found to converge to a single band of 56 kDa after deglycosylation, which suggests microheterogeneity in the sugar moiety. The enzyme showed an oligomeric structure with an apparent molecular mass of 520 kDa, estimated by gel filtration. The optimum pH of the activity was around 4.5. The enzyme hydrolyzed both 4‐methylumbelliferyl‐GlcNAc and 4‐methylumbelliferyl‐GalNAc, suggesting that it should be characterized as a β‐D‐N‐acetylhexosaminidase, with Km values of 1.2 mM and 0.52 mM, respectively. The purified enzyme was found to be capable of binding to the vitelline coat in a GlcNAc‐specific manner. Immunoblot analysis using antibody raised against the purified GlcNAcase revealed that the enzyme itself is released from the eggs upon fertilization. Thus, the GlcNAcase purified in this study is released from eggs following fertilization and bound to the vitelline coat in order to function in the polyspermy blocking mechanism.
The structure of the carbohydrate backbone of the core‐lipid‐A region of the lipopolysaccharide from Vibrio cholerae strain H11 (non‐O1)
Authors: VINOGRADOV Evgeny V. , STUIKE‐PRILL Rainer , BOCK Klaus , HOLST Otto , BRADE Helmut
Published: March 2005

Lipopolysaccharide from Vibrio cholerae strain H11 (non‐O1) was de‐O‐acylated, dephosphorylated, reduced, de‐N‐acylated, N‐acetylated, and the products were separated by high‐performance anion‐exchange chromatography (HPAE). A decasaccharide, 1, was isolated as the major product, representing the core oligosaccharide attached to the reduced GlcN‐disaccharide lipid A backbone. Its structure was established by compositional and methylation analyses, and extensive NMR investigations including 1H, 1H correlation spectroscopy (COSY), total correlation spectroscopy (TOCSY), and nuclear Overhauser enhancement spectroscopy (NOESY), as well as heteronuclear 13C, 1H COSY. In another reaction sequence the lipopolysaccharide was hydrolysed with dilute acetic acid and reduced with NaBH4. The resulting core fractions were separated by HPAE giving seven individual octasaccharides differing at the reducing 3‐deoxy‐D‐manno‐octulosonic acid (Kdo) residue. A major product, 2, was isolated and investigated by the same methods as described for the decasaccharide 1. The following structures are proposed for compounds 1 and 2:

image

where R is α‐Kdop‐(2–6)‐β‐D‐GlcNAcp‐(1–6)‐D‐GlcNAcol in 1 and 4,8‐anhydro‐Kdool in 2, and Hep is L‐glycero‐D‐manno‐heptose.

In lipopolysaccharide, the terminal residue of α‐D‐glucosamine possessed a free amino group, as proved by deamination with nitrous acid and the 1H‐NMR spectrum of de‐O‐acylated lipopolysaccharide.

The conformational preferences of the terminal core heptasaccharide was assessed by Monte Carlo simulations combined with restrained calculations of side chains based on experimentally determined proton‐coupling constants. These calculations, confirmed by NOE data, displayed several long‐range interactions, which resulted in a well‐defined three‐dimensional structure of the core oligosaccharide.
Analysis of two mutated vacuolar proteins reveals a degradation pathway in the endoplasmic reticulum or a related compartment of yeast
Authors: FINGER Andreas , KNOP Michael , WOLF Dieter H.
Published: March 2005

The fate of a mutant form of each of the two yeast vacuolar enzymes proteinase yscA (PrA) and carboxypeptidase yscY (CPY) has been investigated. Both mutant proteins are rapidly degraded after entering the secretory pathway. Mutant PrA is deleted in 37 amino acids spanning the processing site region of the PrA pro‐peptide. The mutant enzyme shows no activity towards maturation of itself or other vacuolar hydrolases, a function of wild‐type PrA. Mutant CPY carries an Arg instead of a Gly residue in a highly conserved region, two positions distant from the active‐site Ser. In contrast to wild‐type CPY, the mutant form was quickly degraded by trypsin in vitro, indicating an altered structure. Using antisera specific for α‐1→6 and α‐1→3 outer‐chain mannose linkages, no Golgi‐specific carbohydrate modification could be detected on either mutant protein. Subcellular fractionation studies located both mutant enzymes in the endoplasmic reticulum. Degradation kinetics of both proteins show the same characteristics, indicating similar degradation pathways. The degradation process was shown to be independent of a functional sec18 gene product and takes place before Golgi‐specific carbohydrate modifications occur. The proteasome, the major proteolytic activity of the cytoplasm, is not involved in this degradation event. All degradation characteristics of the two mutant proteins are consistent with a degradation process within the endoplasmic reticulum (‘ER degradation’).
Specific hydrolysis of intact erythrocyte cell‐surface glycosphingolipids by endoglycoceramidase
Authors: ITO Makoto , IKEGAMI Yuko , TAI Tadashi , YAMAGATA Tatuya
Published: March 2005

This study represents the specific hydrolysis of cell‐surface glycosphingolipids (GSLs) of intact cells by endoglycoceramidase (EGCase; EC.3.2.1.123) which cleaves the linkage between oligosaccharides and ceramides of various GSLs. After a 2‐h incubation of horse intact erythrocytes with 20 mU EGCase II in the presence of activator at 37°C, 68% of the N‐glycolylneuraminic‐acid‐containing ganglioside GM3(NeuGc) and 70% of 4‐O‐acetyl GM3(NeuGc) were found to be hydrolyzed without hemolysis, accompanied by a corresponding increase in ceramide but not sphingosine or N,N‐dimethylsphingosine. No hydrolysis was observed for sphingomyelin, phosphatidylcholine, phosphatidylethanolamine, cholesterol or membrane proteins. The decrease in immunoreactivity with GMR8 antibody, specific to NeuGcα2,3Gal‐ of GM3(NeuGc), corresponded to that of cell‐surface GM3(NeuGc) by the enzyme, and almost no immunoreactivity was found when 70% of the GM3(NeuGc) was hydrolyzed. Besides the cell‐surface GM3(NeuGc) of horse erythrocytes, Gg3Cer of guinea pig, GM3(NeuAc) and LcCer of human, and bovine and rabbit erythrocyte IV3Galα‐nLc4Cer were found to be efficiently hydrolyzed by EGCase II even when present in intact cells, while human erythrocyte Gb4Cer is quite resistant to hydrolysis by the enzyme on the cell surface as well as in detergent miscelles. Glucose incorporation via the glucose transporter in erythrocytes was not affected at all by the specific and exhaustive hydrolysis of cell‐surface GSLs by EGCase II. This result strongly suggested that glucose transporter function was not directly modulated by endogenous GSLs. In summary, this paper demonstrates that, together with the assistance of activator protein, EGCase II will become a powerful tool for selectively removing sugar chains from cell‐surface GSLs without damaging other cell membrane components, and will be useful for describing the biological functions of endogenous GSLs.
Nanomolar arachidonic acid influences the respiratory burst in eosinophils and neutrophils induced by GTP‐binding protein
Authors: AEBISCHER Claude‐Pierre , PASCHE Isabelle , JÖRG Andreas
Published: March 2005

To investigate a possible role of phospholipase A2 (PLA2) in the respiratory burst in bovine eosinophilic and neutrophilic leukocytes dependent on GTP‐binding protein (G‐protein), we permeabilized these cells with Staphylococcus aureusα‐toxin and induced NADPH oxidase activity with the non‐hydrolysable GTP analogue GTP[S] or the aluminium tetrafluoro complex AlF4−. Under same experimental conditions, cells responded with different onset times. The onset time for eosinophils was 50–200 s, for neutrophils it was only a few seconds. GTP[S] stimulated in neutrophils only 5% of the respiratory burst compared to eosinophils, whereas AlF4−‐induced comparable responses (neutrophils 120% of eosinophils). GDP inhibited these responses with an IC50 value of 2.4 mM. Arachidonic acid showed, with the exception of AlF4− stimulated neutrophils, on both stimuli and cell types an enhancing effect (150%) that reached its maximum at 0.1–1 μM. The PLA2 inhibitor 4‐bromophenacylbromide reduced the GTP[S]‐ and AlF4−‐induced response almost completely (10 μM) and the inhibition was not significantly different for eosinophils and neutrophils (IC50 1–3 μM). If the respiratory burst was reduced with 4‐bromophenacylbromide to 1–4% of the original value, 10% of the basal NADPH oxidase activity could be restored by addition of only 20–100 nM arachidonic acid. In addition, the PLA2 activator adriamycin enhanced the response in a dose‐dependent manner and in the same order as arachidonic acid did. The results presented above suggest that the respiratory burst may be regulated by different low‐molecular‐mass and/or heterotrimeric G‐proteins and an active role for arachidonic acid or its metabolites in the activation and the maintenance of the direct G‐protein‐stimulated respiratory burst in bovine eosinophils and neutrophils.
Control of sex determination in animals
Authors: McELREAVEY Ken , VILAIN Eric , COTINOT Corinne , PAYEN Emmanuel , FELLOUS Marc
Published: March 2005

CONTENTS –Sex determination in the fruit fly and nematode–Somatic sex determination – Dosage compensation – Germ‐line sex determination–Mammalian sex determination signifies testis determination – The mammalian sex‐determining master‐regulatory gene, SRY–XX and XY sex reversal in man – Sex‐determining region Y chromosome (SRY) –Is SRY the primary testis determinant?– Biochemical properties of SRY – Sequence versus structure recognition – SRY: positive or negative regulation?– Evidence for other mammalian sex‐determining genes–Murine autosomal sex‐determining genes – Wilms' tumour gene WT1 –Chromosomal abnormalities associated with sex reversal – Müllerian inhibiting substance: a target of SRY?–Perspectives
1H‐NMR studies and secondary structure of the RGD‐containing snake toxin, albolabrin
Authors: JASEJA Mahesh , SMITH K. John , LU Xinjie , WILLIAMS Janice A. , TRAYER Hylary , TRAYER Ian P. , HYDE Eva I.
Published: March 2005

Albolabrin is a naturally occurring peptide from snake venom containing the sequence Arg‐Gly‐Asp (RGD). It inhibits platelet aggregation by blocking the binding of fibrinogen to the glycoprotein Gp IIb‐IIIa, on the surface of activated platelets. Albolabrin consists of 73 residues with six intramolecular disulphide bonds. The 1H‐NMR spectrum of albolabrin has been assigned using homonuclear two‐dimensional techniques and its secondary structure determined. Like kistrin and echistatin, two related peptides from snake venom, albolabrin appears to have little regular secondary structure in solution. Several bends and two short distorted β sheets are observed. The RGD sequence, important for binding to the receptor, lies in a mobile loop joining two strands of one of these β sheets. This loop undergoes a pH‐dependent conformational change.
The actin/actin interactions involving the N‐terminus of the DNase‐I‐binding loop are crucial for stabilization of the actin filament
Authors: KHAITLINA Sofia Y. , MORACZEWSKA Joanna , STRZELECKA‐GOŁASZEWSKA Hanna
Published: March 2005

Actin can be specifically cleaved between residues 42 and 43 with a novel protease from Escherichia coli A2 strain (ECP) [Khaitlina, S. Y., Collins, J. H., Kuznetsova, I. M., Pershina, V. P., Synakevich, I. G., Turoverov, K. K. & Usmanova, A. M. (1991) FEBS Lett. 279, 49–51]. The resulting C‐terminal and N‐terminal fragments remained associated to one another in the presence of either Ca2+ or Mg2+. The protease‐treated actin was, however, neither able to spontaneously assemble into filaments nor to copolymerize with intact actin unless its tightly bound Ca2+ was replaced with Mg2+. Substitution of Mg2+ for the bound Ca2+ was also necessary to partially restore the ability of the protease‐treated actin to inhibit the DNase I activity.

The critical concentration for KCl‐induced polymerization of ECP‐treated ATP–Mg–G‐actin, determined by measuring the fluorescence of pyrenyl label, was approximately threefold higher than that for actin cleaved between residues 47 and 48 using subtilisn, and 36‐fold higher than the critical concentration for polymerization of intact actin under the same conditions. Morphologically, the filaments of ECP‐treated actin were indistinguishable from those of intact actin. Comparison of the fluorescence spectra of pyrenyl‐labelled actins and chemical cross‐linking with N,N′‐1,2‐phenylenebismaleimide have, however, revealed structural differences between the filaments assembled from ECP‐treated actin and those of intact as well as subtilisin‐treated actin. Moreover, the filaments of ECP‐treated actin were easily disrupted by centrifugal forces or shearing stress unless they were stabilized by phalloidin. The results are consistent with the direct participation of the region around residues 42 and 43 in the monomer/monomer interactions as predicted from the atomic model of F‐actin [Holmes, K. C., Popp, D., Gebhard, W. & Kabsch, W. (1990) Nature 347, 44–49] and suggest that the interactions involving this region are of primary importance for stabilization of the actin filament. The mechanism of the regulation of actin polymerization by the tightly bound divalent cation is also discussed.
Secretory leucocyte proteinase inhibitor is produced by human articular cartilage chondrocytes and intervertebral disc fibrochondrocytes
Authors: JACOBY Arie S. , MELROSE James , ROBINSON Bruce G. , HYLAND Valentine J. , GHOSH Peter
Published: March 2005

The objective of this study was to examine the expression of the secretory leucocyte proteinase inhibitor (SLPI) gene by human articular cartilage chondrocytes and intervertebral disc fibrochondrocytes. RNA was extracted from human articular cartilage chondrocytes, synovial fibroblasts and fibrochondrocytes of the annulus fibrosus. Expression was analyzed by Northern blotting and hybridisation to a SLPI RNA probe. SLPI was isolated from chondrocyte culture supernatant by gelpermeation and cation‐exchange chromatography and examined by a solid‐phase enzyme‐linked immunofiltration assay for SLPI and by SDS/PAGE using Western blotting with polyclonal IgG samples against bronchial mucus leucocyte proteinase inhibitor and SLPI. This identified biochemically and immunologically the major serine proteinase inhibitory protein synthesised by chondrocytes as SLPI. Secretory leucoyte proteinase inhibitor mRNA was detected in RNA preparations from articular cartilage chondrocytes and annulus fibrosus fibrochondrocytes. The results presented here allowed us to deduce that human articular cartilage chondrocytes and intervertebral disc fibrochondrocytes synthesized SLPI whereas human synovial fibroblasts did not.
Equine infectious anemia virus Tat is a predominantly helical protein
Authors: STICHT Heinrich , WILLBOLD Dieter , BAYER Peter , EJCHART Andrzej , HERRMANN Franz , ROSIN‐ARBESFELD Rina , GAZIT Arnona , YANIV Abraham , FRANK Rainer , RÖSCH Paul
Published: March 2005

Nuclear magnetic resonance (NMR) spectroscopy revealed features of the secondary structure of the equine infectious anemia virus (EIAV) Tat protein in solution. We could show that this protein, which is required in the replication cycle of lentiviruses, forms a predominantly helical structure in trifluoroethanol/water (40% by vol.) solution. In particular, the basic RNA‐binding region and the adjacent core domain, which are highly conserved among lentiviral Tat proteins, show helix‐type secondary structure under these conditions. Our observations, in concert with recent biochemical data from other laboratories, suggest that the core sequence region and the basic sequence region form interdependent structural domains, both possibly necessary for correct RNA binding.
Overproduction, purification and characterization of the Escherichia coli ferritin
Authors: HUDSON Aaron J. , ANDREWS Simon C. , HAWKINS Chris , WILLIAMS John M. , IZUHARA Mika , MELDRUM Fiona C. , MANN Stephen , HARRISON Pauline M. , GUEST John R.
Published: March 2005

Recent studies have indicated that Escherichia coli possesses at least two iron‐storage proteins, the haem‐containing bacterioferritin and ferritin. The ferritin protein has been amplified 600‐fold to 11–14% of total cell protein in a bfr mutant and purified to homogeneity with an overall yield of 13%. The cellular ferritin content remained relatively constant throughout the growth cycle and amplification was accompanied by a 2.5‐fold increase in cellular iron content. The isolated ferritin contained 5–20 non‐haem iron atoms/holomer and resembled the eukaryotic ferritins rather than the prokaryotic bacterioferritins in containing no haem. The 24 subunits of this ferritin (Mr 19400) assemble into a spherical protein shell (12±1 nm diameter, Mr 465000) which sequesters at least 2000 iron atoms in vitro to form an electron‐dense iron core of 7.9±1 nm diameter. Electron‐microscopic and Mössbauer spectroscopic studies with iron‐loaded ferritin showed that the core can be either crystalline (ferrihydrite) or amorphous, depending on the absence or presence of phosphate, respectively. Mössbauer spectroscopy with intact E. coli revealed a novel‐high spin Fe(II) component which is enhanced in bacteria amplified for ferritin but not in the parental strain. Western blotting showed that ferritin and bacterioferritin are immunologically distinct proteins. E. coli is thus an organism containing both a ferritin and a bacterioferritin and the relative roles of the two iron‐storage proteins are discussed in this study.
The complex between ribonuclease T1 and 3′GMP suggests geometry of enzymic reaction path
Authors: HEYDENREICH Andrea , KOELLNER Gertraud , CHOE Hui‐Woog , CORDES Frank , KISKER Caroline , SCHINDELIN Hermann , ADAMIAK Ryszard , HAHN Ulrich , SAENGER Wolfram
Published: March 2005

The crystal structure of the complex between ribonuclease T1 and 3′GMP suggests that (a) a substrate GpN is bound to the active site of ribonuclease T1 in a conformation that actively supports the catalytic process, (b) the reaction occurs in an in‐line process, (c) His40 NɛH+ activates O2′‐H, (d) Glu58 carboxylate acts as base and His92 NɛH+ as acid in a general acid‐base catalysis.

The crystals have the monoclinic space group P21, a= 4.968 nm, b= 4.833 nm, c= 4.048 nm, β= 90.62° with two molecules in the asymmetric unit. The structure was determined by molecular replacement and refined to R= 15.3% with 11338 data ≥1σ(F°) in the resolution range 1.0–0.2 nm; this includes 180 water molecules and two Ca2+. The structure of ribonuclease T1 is as previously observed. 3′GMP is bound in syn conformation; guanine is located in the specific recognition site, the ribose adopts C4′‐exo puckering, the ribose phosphate is extended with torsion angle ɛ in trans. The O2′‐H group is activated by accepting and donating hydrogen bonds from His40 NɛH+ and to Glu58 Oɛ1; the phosphate is hydrogen bonded to Glu58 Oɛ2H, Arg77 NɛH+ and Nη2H+, Tyr38 OηH, His92 NɛH+. The conformation of ribose phosphate is such that O2′ is at a distance of 0.31 nm from phosphorus, and opposite the P‐OP3 bond which accepts a hydrogen bond from His92 NɛH+; we infer from a model building study that this bond is equivalent to the scissile P‐O5′ in a substrate GpN.
Monodehydroascorbate reductase activity in the surface membrane of leukemic cells
Authors: SCHWEINZER Esther , GOLDENBERG Hans
Published: March 2005

A transmembrane monodehydroascorbate reductase activity with a high affinity in the subpicomolar concentration range of the free radical can be measured at the surface of erythroleukemic cells using a ferricyanide‐driven redox cycle. The activity is dependent on the membrane potential and can therefore only be found in intact cells. It is independent of the glutathione content of the cells. Thenoyltrifluoroacetone is an efficient inhibitor of the activity, whereas ouabain, monensin and tetraethylammonium show no effect.

Cells are able to generate ascorbate from dehydroascorbic acid. This explains why both forms of vitamin C show practically the same affinity for the redox cycle but why it does not drive the redox cycle by itself because it is much slower and is not inhibited by thenoyltrifluoroacetone.

The reductase activity is independent of the degree of differentiation of the leukemic cells.
The Ets family of transcription factors
Authors: WASYLYK Bohdan , HAHN Soonjung L. , GIOVANE Antoine
Published: March 2005

Interest in the Ets proteins has grown enormously over the last decade. The v‐ets oncogene was originally discovered as part of a fusion protein expressed by a transforming retrovirus (avian E26), and later shown to be transduced from a ceilular gene. About 30 related proteins have now been found in species ranging from flies to humans, that resemble the vEts protein in the so‐called ‘ets domain’. The ets domain has been shown to be a DNA‐binding domain, that specifically interacts with sequences containing the common core trinucleotide GGA. Furthermore, it is involved in protein–protein interactions with co‐factors that help determine its biological activity. Many of the Ets‐related proteins have been shown to be transcription activators, like other nuclear oncoproteins and anti‐oncoproteins (Jun, Fos, Myb, Myc, Rel, p53, etc.). However, Ets‐like proteins may have other functions, such as in DNA replication and a general role in transcription activation.

Ets proteins have been implicated in regulation of gene expression during a variety of biological processes, including growth control, transformation, T‐cell activation, and developmental programs in many organisms. Signals regulating cell growth are transmitted from outside the cell to the nucleus by growth factors and their receptors, G‐proteins, kinases and transcription factors. We will discuss how several Ets‐related proteins fit into this scheme, and how their activity is regulated both post‐and pre‐translationally. Loss of normal control is often associated with conversion to an onco‐protein. vEts has been shown to have different properties from its progenitor, which might explain how it has become oncogenic. Oncogene‐related products have been implicated in the control of various developmental processes. Evidence is accumulating for a role for Ets family members in Drosophila development, Xenopus oocyte maturation, lymphocyte differentiation, and viral infectious cycles. An ultimate hope in studying transformation by oncoproteins is to understand how cells become cancerous in humans, which would lead to more effective treatments. vEts induces erythroblastosis in chicken. Cellular Ets‐family proteins can be activated by proviral insertion in mice and, most interestingly, by chromosome translocation in humans. We are at the beginning of understanding the multiple facets of regulation of Ets activity. Future work on the Ets family promises to provide important insights into both normal control of growth and differentiation, and deregulation in illness.
Epitope mapping and immunoinactivation of human gastric lipase using five monoclonal antibodies
Authors: AOUBALA Mustapha , DANIEL Cécile , DE CARO Alain , IVANOVA Margarita G. , HIRN Michel , SARDA Louis , VERGER Robert
Published: March 2005

Five monoclonal antibodies (mAb) directed against human gastric lipase (HGL) have been produced by hybridization of myeloma cells with spleen cells of BALB/c immunized mice. All these mAb belong to the IgG1 class with a k light chain. The effects of these mAb on the enzymic activity of HGL were studied and used to define three classes of antibodies, depending upon their immunoinactivation properties. As determined by ELISA and immunoinactivation studies, four overlapping epitopes were found to be part of the functional sites of the enzyme. The mAb appear to be suitable probes for studying the lipid binding and catalytic domains of HGL. The results of the ELISA additivity test were used to describe tentatively the epitopes of HGL in terms of a schematic spatial map.
Purification and characterization of an aminopeptidase A fromStaphylococcus chromogenesand its use for the synthesis of amino‐acid derivatives and dipeptides
Authors: YOSHPE‐BESANCON Iris , AURIOL Daniel , PAUL Francois , MONSAN Pierre , GRIPON Jean‐Claude , RIBADEAU‐DUMAS Bruno
Published: March 2005

An aminopeptidase with original speciticity was purified 3800‐fold to homogeneity from a cellular extract ofStaphylococcus chromogenes.The enzyme was specific for acidic amino acids (Asp and Giu) at the N‐terminus of peptides and thus can be classified as an aminopeptidase A. However, its specificity was not restricted to acidic amino acids: α‐hydroxy acids such as L‐malic and L‐lactic acids were also accepted in position P1. The enzyme had a broad specificity for the residue at position P′1, accepting all types of amino acids, including Pro, in this position. The optimal conditions for the hydrolysis of Asp‐Phe‐NH2were pH 9.5 and 60°C. The enzyme was inhibited by chelating agents and serine‐protease inhibitors. The activity lost by treatment with chelating agents could be restored by Mn2+or Zn2+which also stimulated the native enzyme. This suggests that it is a metalloprotease with a serine residue essential for the activity. The native enzyme had an apparent molecular mass of 430 kDa on gradient‐gel electrophoresis and subunits of 43 kDa as determined by SDS/PAGE. The enzyme catalyzed the synthesis of peptide and amino acid derivatives such as Asp‐Phe‐OMe (Aspartame) and malyl‐Tyr‐OEt from L‐Asp and L‐malic acid as acyl donors and L‐Phe‐OMe and L‐Tyr‐OEt as nucleophiles, respectively. The use of the enzyme as a reagent in protease‐catalyzed peptide synthesis, N‐terminal protection and subsequent deprotection, is described.
Transcriptional induction of β‐galactoside α‐2,6‐sialyltransferase in rat fibroblast by dexamethasone
Authors: VANDAMME Valérie , PIERCE Annick , VERBERT André , DELANNOY Philippe
Published: March 2005

The β‐galactoside α‐2,6‐sialyltransferase activity of Fisher rat fibroblasts is enhanced by dexamethasone while the activity of the β‐galactoside α‐2,3‐sialyltransferase remains unchanged. This glucocorticoid‐dependent activation can be inhibited by the antagonist RU 38.486 and results from an elevated transcription rate of the 4.7‐kb mRNA previously characterized in rat fibroblasts, distinct from the 4.3‐kb liver‐restricted mRNA. As shown by the binding of radiolabelled Sambucus nigra agglutinin, this activation leads to an increase of NeuNAc(α2–6)Gal sequences on glycoproteins isolated from the dexamethasone‐treated cells.
The use of lac‐type promoters in control analysis
Authors: JENSEN Peter Ruhdal , WESTERHOFF Hans V. , MICHELSEN Ole
Published: March 2005

For control analysis, it is necessary to modulate the activity of an enzyme around its normal level and measure the changes in steady‐state fluxes or concentrations. We describe an improved method for effecting the modulation, as elaborated for Escherichia coli. The chromosomal gene, encoding the enzyme of interest, is put under the control of a lacUV5 or a tacI promoter. The alternative use of the two promoters leads to an expression range which should make it suitable for the use in control analysis of many enzymes. The lacUV5 promoter should be used when the wild‐type expression level is low, the tacI promoter when the latter is high. The endogenous lac operon is placed under the control of a second copy of the lacUV5 promoter and a lac Yam mutation (eliminating lactose permease, the transport system for the inducer isopropyl‐thio‐β‐D‐galactoside) is introduced.

The method was demonstrated experimentally by constructing E. coli strains, in which the chromosomal atp operon is transcribed from the lacUV5 and the tacI promoter. We measured the concentration of the c subunit of H+‐ATPase, and found that the expression of this enzyme could be modulated between non‐detectable levels and up to five times the wild‐type level. Thus, in the absence of inducer, no expression of atp genes could be detected when the atp operon was controlled by the lacUV5 promoter, and we estimate that the expression was less than 0.0025 times the wild‐type level. We show that the introduction of a lac Y mutation facilitated the attainment of steady induction levels of partially induced cells. The mutation also reduced positive cooperativity in the dependence of expression on the concentration of isopropyl‐thio‐β‐D‐galactoside (the inducer) and shifted the concentration of inducer needed for half maximum induction to higher values. These properties should facilitate the experimental modulation of the enzyme activity by varying the concentration of the inducer.
Human and rodent Alzheimer β‐amyloid peptides acquire distinct conformations in membrane‐mimicking solvents
Authors: OTVOS Laszlo , SZENDREI Gyorgyi I. , LEE Virginia M.‐Y. , MANTSCH Henry H.
Published: March 2005

The major constituent of senile plaques (one of the hallmark lesions of Alzheimer's disease) is a 42(43)‐amino‐acid polypeptide, termed the A4 or β‐amyloid peptide. The β‐amyloid peptide or A4 is derived from one or more larger β‐amyloid precursor proteins. The precursor protein from whence the A4 peptide is derived is highly conserved throughout evolution, and humans, monkeys, dogs, and bears develop brain deposits of A4 peptide in amyloid fibrils. However, similar accumulations of A4 amyloid are negligible in the brains of rats and mice for reasons that remain unexplored. Notably, the A4 sequence of rodents, deduced from the cDNA clones, differs only in three amino acids from the A4 isolated from the brain of humans. Hence, these differences could account for the inability of rodents to develop Alzheimer‐like A4 amyloid plaques. To test this hypothesis directly, using physical and chemical model systems, we synthesized, purified, and characterized A4 peptides corresponding to the human and rodent sequences. Circular dichroic and Fourier‐transform infrared spectroscopy were used with various membrane‐mimicking solvents, different peptide concentrations, and variable pH to identify those environmental conditions that promoted β‐pleated sheet formation of the human versus rodent A4. At an intermediate alkaline pH (≤ 10), the rodent peptide has more β‐pleated sheet structure than the human sequence. The β‐pleated sheets for both peptides could be eliminated at very high pH (≥ 12). The amount of the β‐structure increased in an octyl glucoside solution, compared to that found in SDS, as well as in several of the other solutions tested here. This suggests that particles originated from prior membrane damage may play a role in the stabilization of β‐pleated sheets with subsequent formation of amyloid deposits. Finally, we found that higher β‐pleated sheet content was observed for the rodent sequences in acetonitrile/water mixtures. In contrast, more β‐pleated sheets were detected with the human A4 in trifluoroethanol/water mixtures at neutral pH. Remarkably, at relatively low peptide concentrations, only the human sequence assumed an extended secondary structure. These data suggest that subtle inter‐species amino‐acid differences may account for the inability of the rodent peptide to form amyloid fibrils in situ.
Purification, complete amino acid sequence and structural characterization of the heat‐stable sweet protein, mabinlin II
Authors: LIU Xiaozhu , MAEDA Shoji , HU Zhong , AIUCHI Toshihiro , NAKAYA Kazuyasu , KURIHARA Yoshie
Published: March 2005

A new sweet protein, named mabinlin II, was extracted with 0.5 M NaCl solution from the seeds of Capparis masaikai Lévl. and purified by ammonium sulfate fractionation, carboxymethylcellulose‐Sepharose ion‐exchange chromatography and gel filtration. The sweetness of mabinlin II was unchanged by at least 48 h incubation at nearly boiling temperature. Purified mabinlin II thus obtained gave a single band having a molecular mass of 14 kDa on SDS/PAGE. In the presence of dithiothreitol, mabinlin II gave two bands having molecular masses of 4.6 kDa and 5.2 kDa on SDS/PAGE. Two peptides (A chain and B chain) were separated from reduced and S‐carboxamidomethylated mabinlin II by HPLC. The amino acid sequences of the A chain and B chain were determined by the automatic Edman‐degradation method. The A chain and B chain consist of 33‐amino‐acid and 72‐amino‐acid residues, respectively. The A chain is mostly composed of hydrophilic amino acid residues and the B chain also contains many hydrophilic residues. High similarity was found between the amino acid sequences of mabinlin II and 2S seed storage proteins, especially 2S albumin AT2S3 in Arabidopsis thaliana (mouse‐ear cress).
Three‐dimensional structure of the ternary complex between ribonuclease T1, guanosine 3′,5′‐bisphosphate and inorganic phosphate at 0.19 nm resolution
Authors: LENZ Andrea , CHOE Hui‐Woog , GRANZIN Joachim , HEINEMANN Udo , SAENGER Wolfram
Published: March 2005

The ternary complex formed between RNase T1, guanosine 3′,5′‐bisphosphate (3′,5′‐pGp) and Pi crystallizes in the cubic space group I23 with a= 8.706(1) nm. In a previous publication [Lenz, A., Heinemann, U., Maslowska, M. & Saenger, W. (1991) Acta Crystallogr. B47, 521–527], the structure of the complex (in which Pi was not located) was described at a resolution of 0.32 nm. This is now extended to 0.19 nm with newly grown, larger crystals. Refinement with restrained least‐squares converged at R= 17.8% for 8027 reflections with |F0|≥ 1σ(|F0|); the final model comprises 120 water molecules. 3′,5′‐pGp is bound to RNase T1 in the anti form, with guanine in the specific recognition site; the 3′‐phosphate protrudes into the solvent, and the 5′‐phosphate hydrogen bonds with Lys41 O and Asn43 N4. A tetrahedral anion assigned as Pi occupies the catalytic site and hydrogen bonds to the side chains of Tyr38, Glu58, Arg77 and His92. The overall polypeptide fold of RNase T1 in the cubic space group does not differ significantly from that in the orthorhombic space group P212121 except for changes ≤ 0.2 nm in loop regions 69–72 and 95–98.
Phosphorylation of Focal Adhesion Vasodilator‐Stimulated Phosphoprotein at Ser157 in Intact Human Platelets Correlates with Fibrinogen Receptor Inhibition
Authors: Horstrup Karen , Jablonka Bernd , Hönig‐Liedl Petra , Just Melitta , Kochsiek Kurt , Walter Ulrich
Published: March 2005

Integrins and other adhesion receptors are essential components for outside‐in and inside‐out signaling through the cell membrane. The platelet glycoprotein IIb‐IIIa (also known as fibrinogen receptor or integrin αIIbβ3) is activated by platelet agonists, inhibited by cyclic‐nucleotide‐elevating agents, and is involved in the activation of protein tyrosine kinases including the 125‐kDa focal adhesion kinase (pp125FAK). However, the molecular details of glycoprotein IIb‐IIIa regulation are not well understood. Here we report that in ADP‐activated human platelets cAMP‐ and cGMP‐dependent protein‐kinase‐mediated phosphorylation of the focal adhesion vasodilator‐stimulated phosphoprotein (VASP) at Ser157 correlates well with glycoprotein IIb‐IIIa inhibition. Human platelets contain similar concentrations of glycoprotein IIb‐IIIa complexes (fibrinogen binding sites) and VASP. Using gel‐filtered platelets, cAMP‐elevating agents [e.g. prostaglandin E1 and the forskolin analog 6‐(3‐dimethylaminopropionyl)forskolin (NKH 477)] caused VASP Ser157 phosphorylation and inhibited glycoprotein IIb‐IIIa activation up to 70–100%. NO‐generating, cGMP‐elevating agents [e.g. 3‐morpholinosydnonimine hydrochloride (SIN1) and sodium nitroprusside] stimulated VASP Ser157 phosphorylation and inhibited glycoprotein IIb‐IIIa activation up to a maximal extent of 30–50%. The effects of cAMP‐ and cGMP‐elevating agents on VASP phosphorylation and fibrinogen binding were reversible and could be mimicked by membrane‐permeant selective activators of platelet cAMP‐ or cGMP‐dependent protein kinase, respectively. Using threshold concentrations, the nitrovasodilator SIN 1 potentiated the effects of the forskolin analog NKH 477 with respect to inhibition of platelet aggregation, VASP phosphorylation and glycoprotein IIb‐IIIa inhibition. It is proposed that the inhibition of glycoprotein IIb‐IIIa induced by cyclic nucleotide involves cAMP‐and cGMP‐dependent protein‐kinase‐mediated VASP phosphorylation at Ser157.
Serine 16 of Oncoprotein 18 is a Major Cytosolic Target for the Ca2+/Calmodulin‐Dependent Kinase‐Gr
Authors: Marklund Ulrica , Larsson Niklas , Brattsand Göran , Osterman Örjan , Chatila Talal A. , Gullberg Martin
Published: March 2005

Oncoprotein 18 (Op18) is a cytosolic protein that was initially identified due to its up‐regulated expression in acute leukemia and its complex pattern of phosphorylation in response to diverse extracellular signals. We have previously identified in vivo phosphorylation sites and some of the protein kinase systems involved. Two distinct proline‐directed kinase families phosphorylate Ser25 and Ser38 of Op18 with overlapping but distinct site preference. These two kinase families, mitogen‐activated protein (MAP) kinases and cyclin‐dependent cdc2 kinases, are involved in receptor‐regulated and cell‐cycle‐regulated phosphorylation events, respectively. During analysis of Op18 phosphorylation in the Jurkat T‐cell line, we also found that Ser16 of Op18 is phosphorylated in response to a Ca2+ signal generated by T‐cell receptor stimulation or the Ca2+ ionophore ionomycin. As suggested by a previous study, T‐cell‐receptor‐induced phosphorylation events may be mediated by the Ca2+/CaM‐dependent protein kinase type Gr (CaM kinase‐Gr). The present study shows that activation of this protein kinase correlates with phosphorylation of Ser16 of Op18, and in vitro experiments reveal efficient and selective phosphorylation of this residue. The CaM kinase‐Gr is only expressed in certain lymphoid cell lines, and the present study shows that ionomycin‐induced phosphorylation of Op18 Ser16 is restricted to cells expressing this protein kinase. Finally, CaM kinase‐Gr‐dependent in vitro phosphorylation of a crude cellular extract reveals a striking preference of this protein kinase for Op18 compared to other cellular substrates. In conclusion, the results suggest that Ser16 of Op18 is a major cytosolic target for activated CaM kinase‐Gr.
Association of the Regulatory Subunit of a Type II cAMP‐dependent Protein Kinase and its Binding Proteins with the Fibrous Sheath of Rat Sperm Flagellum
Authors: Macleod Janet , Mei Xiaohua , Erlichman Jack , Orr George A.
Published: March 2005

Demembranated rat sperm flagellar polypeptides capable of binding the regulatory subunit (RII) of a type II cAMP‐dependent protein kinase, having apparent subunit molecular masses of 120, 80 and 57 kDa were identified by an RII overlay procedure [Horowitz, J. A., Wasco, W., Leiser, M. & Orr, G. A. (1988) J. Biol. Chem. 263, 2098–2104]. In this study it is shown that all three polypeptides capable of binding RII on a solid‐phase blot are tightly associated with the fibrous sheath. Purified fibrous sheath preparations were capable of binding (a) [3H]cAMP and (b) purified catalytic subunits of cAMP‐dependent protein kinase forming a functional holoenzyme. The 57‐kDa protein was identified as RII by photoaffinity labeling with 8‐azido[32P]cAMP. This peptide was phosphorylated by the catalytic subunit of cAMP‐dependent protein kinase. RIIα was also shown to form tight, specific complexes with the fibrous sheath demonstrating the presence of functional RIIα‐binding sites. Truncated RIIβ fusion proteins were used to identify the N‐terminal amino acids at positions 1–50 as a primary determinant for RII‐binding protein interaction. Differential extraction of adult testis with buffers containing Triton X‐100, urea and sodium dodecyl sulfate revealed the presence of 80‐kDa (major) and 120‐kDa (minor) RII‐binding proteins in particulate extracts. The 80‐kDa polypeptide is only expressed at late stages of spermatogenesis, i.e. during spermiogenesis, suggesting a developmental role for RII anchoring to the sperm flagellar fibrous sheath.
ATP Synthesis Catalyzed by the ATP Synthase of Escherichia coli Reconstituted into Liposomes
Authors: Fischer Susanne , Etzold Carsten , Turina Paola , Deckers‐Hebestreit Gabriele , Altendorf Karlheinz , Gräber Peter
Published: March 2005

The H+‐translocating F0F1‐ATPase from Escherichia coli (EF0F1) was purified and reconstituted into preformed reverse‐phase liposomes prepared from egg yolk phosphatidylcholine/phosphatidic acid. The EF0F1 liposomes were energized by an acid/base transition (pHout= 8.3; pHin= 5.0) and a superimposed K+/valinomycin diffusion potential ([K+]out= 100 mM; [K+]in= 0.6 mM) yielding a maximum rate (turnover number) of ATP synthesis of 27±8 mol ATP · molEF0F1−1· s−1), i.e. 27±8 s−1 This reaction was inhibited by NH4Cl or by addition of the F0F1, inhibitor N,N′‐dicyclohexylcarbodiimide. The rate of ATP synthesis measured as a function of the phosphate and ADP concentrations, can be described by Michaelis‐Menten kinetics with a Km of 0.7±0.2 mM for phosphate ([ADP] = 200 μM) and a Km, of 27±7 μM for ADP ([phosphate] = 5 mM), respectively.
Control by Enzymes, Coenzymes and Conserved Moieties
Authors: Kholodenko Boris N. , Sauro Herbert M. , Westerhoff Hans V.
Published: March 2005

The control and regulation of metabolic systems are determined by their responses to changes in the internal metabolites (the internal state) and parameters of the system. In many cases, the concentrations of the intermediates are constrained by moiety conservations, for example those requiring that all intermediate forms of any enzyme sum to the conserved total concentration of that enzyme. In this study, we show how responses to changes in the internal state are related to responses to changes in the total amounts of conserved moieties. The relationship between these two different measures of control leads to a generalisation of the connectivity theorems. The results have important implications for the study of a variety of phenomena such as metabolite (coenzyme) sequestration, group‐transfer and channelling. The relationships we derive make it possible to determine the control features of these pathways. As an illustration, two examples are chosen. The first shows the effect of sequestration of substrate moiety while the second deals with the sequestration of the enzyme moieties and enzyme/enzyme interactions.
Further Characterisation of Forms of Haemosiderin in Iron‐Overloaded Tissues
Authors: Ward Roberta J. , Ramsey Michael , Dickson Dominic P. E. , Hunt Collette , Douglas Trevor , Mann Stephen , Aouad Fouad , Peters Timothy J. , Crichton Robert R.
Published: March 2005

The biochemical and biophysical properties of isolated haemosiderins have been compared to that of another iron‐containing protein, termed prehaemosiderin, which sediments through chaotropic potassium iodide only after 20 h of ultracentrifugation, in contrast to that of haemosiderin which is recovered after 2 h of ultracentrifugation. The irodprotein ratio and irodphosphate ratio were less that that of the corresponding haemosiderin, while the elemental composition was also reduced in many of the prehaemosiderin samples. Mossbauer spectroscopy and electron diffraction identified the predominant presence of ferrihydrite in prehaemosiderin species even though the secondary haemochromatosis haemosiderin iron cores were essentially goethite‐like. The majority of the pre‐haemosiderins isolated showed the presence of an additional peptide band at 17 kDa in addition to that at 21 kDa.

Further Mossbauer studies of haemosiderin isolated from untreated secondary haemochromatosis patients showed that goethite was the predominant form of iron present, thereby indicating that the presence of this form of ferrihydrite was not wholly attributable to chelation therapy.
Analysis of the Phasing of Four Spectrin‐like Repeats in α‐actinin
Authors: Gilmore Andrew P. , Parr Timothy , Patel Bipin , Gratzer Walter B. , Critchley David R.
Published: March 2005

Selected fragments of the central rod of chicken gizzard α‐actinin were expressed as fusion proteins in Escherichia coli, with the aim of determining the positions in the sequence of the four successive spectrin‐like repeats that make up this domain. The criteria for an independently folding unit were resistance to proteolysis and the high α helicity characteristic of the native protein. Sequences containing repeats 1–4, 2–4, 3–4 and 4 all generated stable fragments on digestion with trypsin and/or thermolysin and N‐terminal sequencing gave the most probable starting position of each repeat. The sequences of all four inferred repeats and the sequences of the entire rod, were separately expressed and were shown to assume a stable, protease‐resistant fold in solution. The repeat boundaries established in this way differed from those originally deduced from sequence alignments; the N‐terminal boundaries of the repeats were 14–24 residues nearer the C‐terminus than predicted. The ability to express individual repeats should facilitate identification of the binding sites for the cytoplasmic domains of βl integrins and intercellular cell adhesion molecule‐1 which have been localised to the rod domain of α‐actinin.
Purification and Characterization of a Ribonuclease from Aspergillus giganteus IFO 5818, the Gigantin
Authors: Salvarelli Stephanie , Muñoz Saturnino , Conde Francisco Pedro
Published: March 2005

A protein has been isolated from a culture medium of Aspergillus giganteus IFO 5818 (gigantin) and purified by ion‐exchange chromatography successively on DEAE‐cellulose and carboxymethyl‐cellulose, and gel‐filtration chromatography on Biogel P10. With a high purity, gigantin was found to be a non‐glycosylated basic protein with a relative molecular mass of 17000±200 determined in PAGE/SDS. Gigantin was able to digest the synthetic homopolymers of nucleic acids poly(A), poly(I), poly(C) and poly(U). The catalytic action has an optimal pH around 7.0, an optimal temperature at 45–55°C and can be inhibited by cations. Gigantin activity, analyzed as its capacity to hydrolyze RNA from yeast, was comparable to that of alphasarcin, a similar biochemical protein produced by the strain A. giganteus MDH 18894. A study of the substrate specificity for alphasarcin indicated a preference for poly(A) and poly(I), while gigantin had greater activity on poly(C) and poly(U). The cross reaction of gigantin with a rabbit antiserum to alphasarcin suggests a high sequence similarity between both proteins. However, gigantin is immunologically distinguishable from alphasarcin as alphasarcin antiserum detects epitopes in alphasarcin that are not present in gigantin.