bluuebunny/crossref_metadata_2025 huggingface dataset:
Kinetics of Factor VIII Light‐Chain Cleavage by Thrombin and Factor Xa
Authors: Donath Marie‐José S. H. , Lenting Peter J. , Van Mourik Jan A. , Mertens Koen
Published: July 2004

Activation and limited proteolysis of factor VIII have been investigated with respect to the role of the heavy‐chain region Lys713‐Arg740. The kinetics of factor VIII activation have been analyzed in a system consisting of human factor VIII, factor IXa, factor X, phospholipids, and thrombin or factor Xa. Plasma‐derived factor VIII is activated by thrombin with a second‐order rate constant of 3.3±0.3×106M‐1S‐l, which proved to be slightly higher than for activation by factor Xa. The second‐order rate constant of activation by thrombin of plasma‐derived factor VIII in the presence of a monoclonal antibody against the sequence Lys713‐Arg740 is markedly reduced. The same result was obtained for activation by thrombin and factor Xa of factor VIII with a deletion including the sequence Lys713‐Arg740, des‐(713–1637)‐factor VIII. This suggests that the region Lys713‐Arg740 promotes factor VIII activation by both thrombin and factor Xa. Since factor VIII activation is associated with proteolysis, cleavage of factor VIII heavy and light chains was analyzed quantitatively. These studies indicated that heavy‐chain cleavage of des‐(713–1637)‐factor VIII is similar to that of plasma‐derived factor VIII. In contrast, cleavage of the light chain of des‐(713–1637)‐factor VIII is clearly reduced. Furthermore, the second‐order rate constant (0.2±0.1×106 M‐1 S‐1) of des‐(713–1637)‐factor VIII light‐chain cleavage by thrombin was reduced tenfold compared with that of plasma‐derived factor VIII. Proteolysis by factor Xa yielded similar results. The rate of des‐(713–1637)‐factor VIII light‐chain cleavage by thrombin is similar to that of isolated light‐chain, but isolated light‐chain is cleaved by factor Xa 20‐fold more efficiently than the light chain in des‐(713–1637)‐factor VIII. We conclude that activation of factor VIII by both thrombin and factor Xa is closely associated with light‐chain cleavage. Furthermore, within the factor VIII heterodimer, the heavy‐chain sequence Lys713–Arg740 promotes both activation and light‐chain proteolysis.
Intrinsic Conformational Preferences of the Hoechst Dye Family and their Influence on DNA Binding
Authors: Vega M. Cristina , Coll Miquel , Alemán Carlos
Published: July 2004

Quantum mechanical calculations have been used to investigate the molecular conformation of the Hoechst family of DNA‐binding dyestuffs. Compounds in which the phenolic substituent adopts either a meta or para position were studied. Two different environments have been considered, which are the gas phase and aqueous solution; the conformation in aqueous solution has been modeled through a self‐consistent reaction field strategy. The results clearly indicate that Hoechst dyes do not adopt a planar conformation and that the degree of planarity is controlled by the external environment. A comparison with experimental data reveals that the conformation of Hoechst dyes in the gas phase is similar to that observed in DNA complexes by X‐ray crystallography. In aqueous solution, the conformation deviates from planarity more than in the gas phase, since non‐bonded interactions with the solvent offset the loss of conjugative interactions. The role of the drug conformation in the binding mechanism with DNA is discussed.
A Functional Heme‐Binding Site of Soluble Guanylyl Cyclase Requires Intact N‐Termini of α1 and β1 Subunits
Authors: Foerster John , Harteneck Christian , Malkewitz Jürgen , Schultz Günter , Koesling Doris
Published: July 2004

Soluble guanylyl cyclase, a heterodimeric enzyme, is the most important intracellular target for the signalling molecule nitric oxide (NO). NO stimulates the enzyme by binding to a prosthetic heme group. The identity of the axial heme ligand, however, is still unknown. Here we show that guanylyl cyclase mutated at the residue His105 on the β1 subunit, a mutant that we have shown before to contain no heme after purification [Wedel, B., Humbert, P., Harteneck, C., Foerster, J., Malkewitz, J., Böhme, E., Schultz, G. & Koesling, D. (1994) Proc. Natl Acad. Sci. USA 91, 2592–2596] can be reconstituted with heme. The reconstituted mutant remains NO‐insensitive and displays an ultraviolet absorption spectrum consistent with an altered axial coordination. Thus, this residue is a strong candidate for the axial heme‐ligating residue and appears to be necessary for NO stimulation. Apart from the axial heme ligand, the role of the enzyme's two subunits, α1 and β1, in heme binding has not been clarified to date. To address this question, we purified mutant heterodimers in which the non‐conserved amino termini of either α1 (131 residues deleted), or β1 (64 residues) have been deleted. These deletion mutants had previously been found to be marginally (α1 truncated) or not at all NO sensitive (β1 truncated) in cytosolic fractions [Wedel, B., Harteneck, C., Foerster, J., Friebe, A., Schultz, G. & Koesling, D. (1995) J. Biol. Chem. 270, 24871–24875]. Here, we show that the purified enzyme truncated on α1 has a significantly reduced capacity to bind heme which explains the reduced NO sensitivity. By contrast, the β1‐truncated enzyme binds an amount of heme comparable to the wild type but is only marginally NO‐responsive and displays a shift in the heme ultraviolet absorption maximum indicative of altered heme coordination. In conclusion, the heme binding site of soluble guanylyl cyclase requires the presence of both subunits in full length to be able to bind wild‐type quantities of heme and to be capable of mediating the NO‐heme‐induced stimulation. Despite some structural similarity, both subunits appear to participate differently in NO‐heme‐mediated enzyme regulation.
Photomodification of Mitochondrial Proteins by Azido Fatty Acids and Its Effect on Mitochondrial Energetics
Authors: Schönfeld Peter , Ježek Petr , Belyaeva Elena A. , Borecký Jiří , Slyshenkov Vyacheslav S. , Więckowski Mariusz R. , Wojtczak Lech
Published: July 2004

Azido derivatives of long‐chain fatty acids, 12‐(4‐azido‐2‐nitrophenylamino)dodecanoic acid (N3‐NpNH‐Lau) and 16‐(4‐azido‐2‐nitrophenylamino)hexadecanoic acid (N3‐NpNH‐Pam), were used to study the mechanism of the protonophoric function of long‐chain fatty acids in mitochondrial membranes. N3‐NpNH‐Lau was found to increase resting‐state respiration and decrease the membrane potential in a dose‐dependent way in a manner similar to that of the natural fatty acid, myristate. Both effects of N3‐NpNH‐Lau as well as of the myristate were reversed or prevented by the inhibitor of the mitochondrial ADP/ATP carrier (AAC), carboxyatractyloside. This protective effect of carboxyatractyloside was well expressed in rat heart mitochondria and less so in mitochondria within digitonin‐permeabilized Ehrlich ascites tumour cells. Photomodification of Ehrlich ascites tumour mitochondria by ultraviolet irradiation in the presence of N3‐NpNH‐Lau made them more resistant to the uncoupling effect of myristate, and photomodification of rat heart mitochondria resulted in a strong inhibition of AAC which could not be reversed by serum albumin.

Photolabelling of rat heart mitochondria with tritiated N3‐NpNH‐Pam revealed around 10 labelled bands on SDS/polyacrylamide gel electrophoresis. Based on immunodetection with a specific antibody, one of them, corresponding to 30 kDa, was identified as AAC. Specific interaction of AAC with azido fatty acids was confirmed by a high radiolabelling of this band.

The role of fatty acids in fine control of the efficiency of oxidative phosphorylation is discussed.
Rationalization of the Strength of Metal Binding to Human Serum Transferrin
Authors: Li Hongyan , Sadler Peter J. , Sun Hongzhe
Published: July 2004

A wide range of metal ions of natural, therapeutic, diagnostic and toxic interest are transported by serum transferrin (80 kDa). It is therefore important to understand the factors that control the strength of metal binding. We show here that even though Sc3+ has only slightly larger ionic radius than Fe3+ (0.075 nm versus 0.065 nm), it binds to the C‐lobe and N‐lobe sites much more weakly: logK*1 (bicarbonate‐independent binding constant) 14.6 ± 20.2, logK*1 13.3 ± 0.3, respectively (10 mM Hepes, 5 mM bicarbonate, 310 K). Preferential binding to the C‐lobe was established by 1H‐NMR spectroscopy. We show that the strength of binding of divalent and trivalent metal ions to human serum transferrin correlates with metal ion acidity [and therefore with the strength of binding to hydroxide, K1(OH)]. The correlations are of predictive value for a range of other metal ions. The plot of logK*1 (human serum transferrin) versus logK1(OH) has a negative intercept consistent with unfavorable entropy effects due to lobe closure of apotransferrin on binding of metal ions. This interpretation was tested by comparison with similar correlations of the strength of metal binding to the enzymes carbonic anhydrase and carboxypeptidase with that for the low‐Mr ligand imidazole. These plots have positive intercepts consistent with the preorganized (entatic) state of these metalloenzymes (favorable entropy effects on metal binding).
Dimethylsulfide:Acceptor Oxidoreductase from Rhodobacter sulfidophilus
Authors: Hanlon Steven P. , Toh Tze‐Hsien , Solomon Peter S. , Holt Robert A. , Mcewan Alastair G.
Published: July 2004

Dimethylsulfide:acceptor oxidoreductase was purified from the purple non‐sulfur phototrophic bacterium Rhodobacter sulfidophilus. The native form of the enzyme had a molecular mass of 152 kDa and was composed of three distinct subunits of 94, 38 and 32 kDa. Dimethylsulfide:acceptor oxidoreductase did not oxidise other thioethers which were tested. The enzyme was able to reduce a variety of N‐oxides using reduced methylviologen as electron donor but it reduced dimethylsulfoxide at a very low rate. The resting form of dimethylsulfide:acceptor oxidoreductase exhibited a spectrum which was characteristic of a reduced cytochrome with absorbance maxima at 562 nm, 533 nm and 428 nm. Pyridine haemochrome analysis established that the cytochrome contained a b ‐type haem and a content of 0.65 mol protohaem/mol enzyme was determined. After oxidation of the haem with ferricyanide, the absorbance spectrum of the reduced cytochrome was restored by reduction with dimethylsulfide. Metal analysis revealed that dimethylsulfide:acceptor oxidoreductase contained 0.5 mol Mo and 3.5 mol Fe/mol enzyme. Heat treatment of the enzyme released material with fluorescence excitation and emission spectra which were characteristic of form B of the pterin component of the pterin molybdenum cofactor. From this analysis it is concluded that dimethylsulfide: acceptor oxidoreductase is a molybdenum oxotransferase which may also contain a iron‐sulfur cluster. It is suggested that the haem and pterin molybdenum cofactor are associated with the 94‐kDa subunit.
The Lyn‐Catalyzed Tyr Phosphorylation of the Transmembrane Band‐3 Protein of Human Erythrocytes
Authors: Brunati Anna Maria , Bordin Luciana , Clari Giulio , Moret Vittorio
Published: July 2004

Band‐3 protein (approximately 95 kDa), the major and multifunctional transmembrane protein of human erythrocytes, has been shown to be phosphorylated by endogenous Tyr‐protein kinases on different Tyr residues at its N and C cytoplasmic domains.

Both the added p36syk (catalytic domain of 72syk) and Lyn kinases are able to phosphorylate the isolated cytoplasmic domain of band 3 (cdb3), yielded by chymotryptic digestion of band 3 in the isolated membranes (ghosts).

However, the two Tyr‐protein kinases exhibited different phosphorylation behaviours when added to the isolated erythrocyte membranes. More precisely, the added p36syk markedly Tyr phosphorylates the band‐3 protein, whereas the added Lyn phosphorylates it very poorly.

It is of interest that Lyn can associate with membranes and markedly phosphorylate band 3 when this latter protein has been previously phosphorylated by p36syk, i.e. the p36syk ‐catalyzed phosphorylation is proposed to be a prerequisite for the association of Lyn with the membrane (likely to band 3) and for the Lyn‐catalyzed phosphorylation of different band‐3 Tyr sites.
Purification and Characterisation of Haemolymph 3‐Dehydroecdysone 3β‐Reductase in Relation to Ecdysteroid Biosynthesis in the Cotton Leafworm Spodoptera littoralis
Authors: Chen Jian‐Hua , Webb Tracey J. , Powls Roy , Rees Huw H.
Published: July 2004

The in vitro secretion of ecdysteroids from the prothoracic glands of last instar larvae of Spodoptera littoralis was detected and analysed by HPLC‐RIA. The primary product was identified as 3‐dehydroecdysone (≈ 82%), with lesser amounts of ecdysone (≈ 18%). Interconversion of ecdysone and 3‐dehydroecdysone by prothoracic glands was not detectable.

3‐Dehydroecdysone 3β‐reductase activity was demonstrated in the haemolymph. Ecdysone, the end‐product, was characterised by reverse‐phase and adsorption HPLC, chemical transformation into ecdysone 2, 3‐acetonide, and mass spectrometry. The conditions for optimal activity were determined. The enzyme requires NADPH or NADH as cofactor and Km values for NADPH and NADH were determined to be 0.94 μM and 22.8 μM, respectively. Investigation of the kinetic properties of the enzyme, using either NADPH or NADH as cofactor, revealed that it exhibits maximal activity at low 3‐dehydroecdysone substrate concentrations, with a drastic inhibition of activity at higher concentrations (>5 μM). The results suggest that the 3‐dehydroecdysone 3β‐reductase has a high‐affinity (low Km) binding site for 3‐dehydroecdysone substrate, together with a lower‐affinity inhibition site.

The 3β‐reductase enzyme was purified to homogeneity using a combination of poly(ethylene glycol) 6000 precipitation and successive FPLC fractionation on Mono‐Q, phenyl Superose (twice), and hydroxylapatite columns. The native enzyme was shown to be a monomer with molecular mass of 36 kDa by SDS/PAGE and gel‐filtration chromatography. Furthermore, the activity of the enzyme during the last larval instar was found to reach a peak prior to that of the haemolymph ecdysteroid titre, supporting a role for the enzyme in development.
Cloning and Characterization of the 5′‐Upstream Regulatory Region of the Ca2+‐Release Channel Gene of Cardiac Sarcoplasmic Reticulum
Authors: Ntshida Kazuhiko , Otsu Kinya , Hori Masatsugu , Kuzuya Tsunehiko , Tada Michihiko
Published: July 2004

To elucidate the transcriptional regulation mechanism for the Ca2+‐release channel gene of the cardiac sarcoplasmic reticulum (RYR2), we isolated and analyzed the 5′‐upstream flanking region of the gene. Sequence analysis indicated that the core promoter region lacks canonical TATA and CAAT boxes, but contains three overlapping GC boxes. A gel shift assay indicated that Sp1 binds to the region containing the GC boxes. Different 5′‐deletion constructs in the 5′‐flanking region of the RYR2 gene were fused to the luciferase gene, and their promoter activity in rat neonatal cardiac myocytes was subsequently determined. The results revealed the presence of a region containing positive regulatory elements in the 5′‐flanking region. Analyses of substitutional mutations introduced into the GC boxes and the regulatory region indicated that in addition to the GC box located at –56 to –51, two regulatory elements (RYR2P1 and RYR2P2) are essential for the promoter activity. These results indicated that Sp1 and transcription factors that bind to RYR2Pl and RYR2P2 cooperatively enhance the expression of the RYR2 gene. In a transient transfection experiment involving promoter‐luciferase gene constructs in skeletal muscle cells, we identified a negative regulatory region between positions–209 and –90 that represses the expression of the RYR2 gene in skeletal muscle cells.
Polyamine Spider Toxins and Mammalian N‐Methyl‐d‐Aspartate Receptors
Authors: Raditsch Martin , Geyer Matthias , Kalbitzer Hans Robert , Jahn Werner , Ruppersberg J. Peter , Witzemann Veit
Published: July 2004

Recombinant N‐methyl‐d‐aspartate receptors composed of NR1/NR2A subunits were expressed in Xenopus oocytes to analyse the voltage‐dependent and use‐dependent channel blocking activity of argiotoxin636. Functional assays demonstrate that the toxin competes with other open channel blockers such as Mg2+ and MK‐801. Direct binding or competition assays using radiolabeled ligands and isolated rat brain membranes, in contrast, reveal no specific binding or yield binding constants which differ by orders of magnitude from the IC50 values of the functional assays. One explanation is that argiotoxin636 does not bind with high affinity to the inhibitory site in the N‐methyl‐d‐aspartate‐receptor channel under in vitro conditions when membranes are depolarised. The structure of argiotoxin636 was investigated by NMR spectroscopy. In solution the positively charged argiotoxin636 acquires an extended conformation and its dimensions might allow permeation deep into the channel. In the absence of direct structural information on the channel protein, the detailed analysis of blockade in conjunction with structural information, as provided here, may be of aid in the deduction of structural features of glutamate‐receptor channel ion pores.
L‐Aspartate Oxidase from Escherichia coli
Authors: Mortarino Michele , Negri Armando , Tedeschi Gabriella , Simonic Tatjana , Duga Stefano , Gassen Hans Gunther , Ronchi Severino
Published: July 2004

This paper reports the biochemical characterization of the flavoprotein l‐aspartate oxidase from Escherichia coli. Modification of a previously published procedure allowed overexpression of the holo‐enzyme in an unproteolysed form. l‐Aspartate oxidase is a monomer of 60 kDa containing 1 mol of non‐covalently bound FAD/mol protein. A polarographic and two spectrophotometric coupled assays have been set up to monitor the enzymatic activity continuously. l‐Aspartate oxidase was subjected to product inhibition since iminoaspartate, which results from the oxidation of l‐aspartate, binds to the enzyme with a dissociation constant (Kd) equal to 1.4 μM. The enzyme binds FAD by a simple second‐order process with Kd 0.67 μM. Site‐directed mutagenesis of the residues E43, G44, S45, F47 and Y48 located in the putative binding site of the isoallossazinic portion of FAD reduces the affinity for the coenzyme.
Different Susceptibilities of Fibulin‐1 and Fibulin‐2 to Cleavage by Matrix Metalloproteinases and Other Tissue Proteases
Authors: Sasaki Takako , Mann Karlheinz , Murphy Gillian , Chu Mon‐Li , Timpl Rupert
Published: July 2004

Fibulin‐1 and fibulin‐2 are two novel rod‐like proteins which occur either in basement membranes or in interstitial fibrils in close association with fibronectin. They were examined for their sensitivity to proteolysis by matrix metalloproteinases (stromelysin, matrilysin), circulating proteases (thrombin, plasmin, kallikrein), leucocyte elastase and mast cell chymase. Fibulin‐1 (95 kDa) was readily cleaved by leucocyte elastase, weakly by matrilysin and not by the other proteases. Cleavage occurred in a domain‐connecting link region close to the N‐terminus, giving rise to fragments of 70 kDa and 26 kDa. A much more extensive cleavage by all seven proteases was observed for fibulin‐2 (195 ma), giving rise to many fragments in the range 15–150 kDa. Vulnerable sites included two central link regions, the cysteine‐free part of the large N‐terminal globular domain but also several regions of epidermal‐growth‐factor(EGF)‐like repeats which are a major part of the rod‐like domain. The latter domain became much more sensitive to proteolysis in the presence of EDTA, demonstrating that calcium is required for stabilization. Edman degradation demonstrated cleavage of peptide bonds corresponding to the known specificities of these proteases. A similar proteolysis was also observed for fibulin‐2 deposited by cultured fibroblasts into a dense fibrillar network. Since fibulin‐2 is an abundant component of small and large blood vessels it could be a major target for proteolysis during vascular injuries.
Immunological Demonstration of Protein Kinase Cμ in Murine Tissues and Various Cell Lines
Authors: Rennecke Jörg , Johannes Franz‐Josef , Richter Karl Hartmut , Kittstein Walter , Marks Friedrich , Gschwendt Michael
Published: July 2004

13 murine tissues and 12 cell lines were tested for the expression of the novel protein kinase C (PKC) isoenzyme μ. Using two different PKCμ antibodies (sc‐639 and P26720), PKCμ was detected in all tissues and cells and thus proved to be an ubiquitous PKC isotype. However, in some tissues, PKCμ was recognized only by the antibody P26720 and not by sc‐639. Thymus, lung and peripheral blood mononuclear cells expressed the greatest amount of PKCμ.

Recognition of PKCμ by the antibody sc‐639 was drastically impaired when treating keratinocytes or mouse skin in vivo with the phorbol ester 12‐O‐tetradecanoylphorbol‐13‐acetate (TPA), thus mimicking down‐regulation of PKCμ. The lack of a decrease in the PKCμ amount and, thus, the lack of down‐regulation could be proved using the antibody P26720. This antibody was able to recognize PKCμ in extracts of untreated as well as TPA‐treated tissues or cells. Phosphorylation of proteins in a cell‐free system (cell or tissue extracts) in the presence and absence of TPA or other PKC activators and various protein kinase inhibitors indicated that phosphorylation of activated PKCμ caused its reduced interaction with the antibody sc‐639. Therefore, this antibody might present a well suited tool for the detection of activated PKCμin vivo. Moreover, our results clearly show that some antibodies, such as sc‐639, might be able to selectively detect non‐phosphorylated or phosphorylated forms of a protein, and that such properties of an antibody have to be studied carefully before the latter can be used for reliable quantitative determination of this protein. We consider this information important to avoid misinterpretation of data concerning the immunological quantification of proteins such as PKCμ.
The O‐Specific Polysaccharide Chain of Campylobacter fetus Serotype B Lipopolysaccharide is a d‐Rhamnan Terminated with 3‐O ‐Methyl‐d‐Rhamnose (d‐Acofriose)
Authors: Senchenkova Sof'ya N. , Shashkov Alexander S. , Knirel Yuriy A. , Mcgovern Joseph J. , Moran Anthony P.
Published: July 2004

An O‐specific polysaccharide was liberated from Campylobacter fetus subsp. fetus serotype B lipopolysaccharide by mild acid hydrolysis followed by gel chromatography. This polysaccharide was found to contain d‐rhamnose and 3‐O ‐methyl‐d‐rhamnose (d‐Rha3Me, d‐acofriose) in a ratio of approximately 24:1, as well as lipopolysaccharide core constituents. The structure of the polysaccharide was studied by 1H‐NMR and 13C‐NMR spectroscopy, which included two‐dimensional COSY, rotating‐frame NOE spectroscopy (ROESY), and computer‐assisted analysis of the 13C‐NMR spectrum. Methylation analysis using [2H3]methyl iodide and Smith degradation followed by GLC/MS of the derived acetylated oligosaccharide‐alditols was used to determine the location of d‐acofriose. The O‐specific polysaccharide is linear, consists on average of 12 disaccharide repeating units, and is terminated by a residue of d‐acofriose. The following structure of the d‐rhamnan chain was established:

image
Reversible Denaturation of Myo ‐Inositol Monophosphatase
Authors: Moreno Fermin , Corrales Susana , Blanco Francisco Garcia , Gore Michael G. , Rees‐Milton Karen , Churchich Jorge E.
Published: July 2004

The unfolding of bovine brain myo ‐inositol monophosphatase by guanidine · HCl (Gdn · HCl) has been investigated. The recovery of circular dichroism, emission spectra, and catalytic activity after dilution of Gdn · HCl‐treated samples indicate that the overall process is reversible. The steepness of the spectroscopic changes between 3 M and 5 M Gdn · HCl, and the lack of any discernible plateau suggest that unfolding of the protein is a cooperative process.

The sensitized luminescence of bound Tb(III) was used as a probe of conformational changes of the metal‐binding loop. Denaturation of the enzyme by Gdn · HCl does not abolish sensitized luminescence. A 50% decrease in sensitized luminescence was observed in 5 M Gdn · HCl. Under this set of experimental conditions, the protein binds terbium with an association constant of 1×106M‐1. It is suggested that a residual structure of denatured myo ‐inositol monophosphatase is responsible for the binding of terbium ions.

The kinetics of unfolding and refolding as a function of Gdn · HCl concentration were monitored by protein fluorescence in a stopped‐flow instrument. The monophosphatase unfolded in a single kinetic phase with rate constants in the range 80–65 s−1 at 25°C. The refolding kinetics fit monoexponential functions with rate constants in the range 120–65 s−1 depending on the Gdn · HCl concentration. Substantial refolding of the protein occurs within the dead time of mixing.
Identification of Substrate Proteins for Cathepsin L that are Selectively Hydrolyzed During the Differentiation of Imaginal Discs of Sarcophaga peregrina
Authors: Homma Ko‐ichi , Natori Shunji
Published: July 2004

Previously, we showed that cathepsin L is essential for the differentiation of imaginal discs of the flesh fly [Homma, K. & Natori, S. (1994) J. Biol. Chem. 269, 15258–15264]. We have now identified imaginal disc proteins that are susceptible to digestion by cathepsin L and showed that they are selectively hydrolyzed during imaginal disc differentiation. Two of these proteins, with molecular masses of 210 and 200 kDa, were further characterized. Immunofluorescence studies suggested that they were intrinsic components of the basement membranes of various tissues. They were selectively hydrolyzed at the elongation stage of imaginal leg disc differentiation. Western blotting of embryos at various developmental stages showed that these proteins were only detected at the end of embryogenesis.
Activation of the Uncoupling Protein by Fatty Acids is Modulated by Mutations in the C‐Terminal Region of the Protein
Authors: González‐Barroso M. Mar , Fleury Christophe , Arechaga Ignacio , Zaragoza Pilar , Levi‐Meyrueis Corinne , Raimbault Serge , Ricquier Daniel , Bouillaud Frédéric , Rial Eduardo
Published: July 2004

The transport properties of the uncoupling protein (UCP) from brown adipose tissue have been studied in mutants where Cys304 has been replaced by either Gly, Ala, Ser, Thr, Ile or Trp. This position is only two residues away from the C‐terminus of the protein, a region that faces the cytosolic side of the mitochondrial inner membrane. Mutant proteins have been expressed in Saccharomyces cerevisiae and their activity determined in situ by comparing yeast growth rates in the presence and absence of 2‐bromopalmitate. Their bioenergetic properties have been studied in isolated mitochondria by determining the effects of fatty acids and nucleotides on the proton permeability and NADH oxidation rate. It is revealed that substitution of Cys304 by non‐charged residues alters the response of UCP to fatty acids. The most effective substitution is Cys for Gly since it greatly enhances the sensitivity to palmitate, decreasing threefold the concentration required for half‐maximal stimulation of respiration. The opposite extreme is the substitution by Ala which increases twofold the half‐maximal concentration. We conclude that the C‐terminal region participates in the fatty acid regulation of UCP activity. The observed correlation between yeast growth rates in the presence of bromoplamitate and the calculated activation constants for respiration in isolated mitochondria validates growth analysis as a method to screen the in situ activity of UCP mutants.
Structural Characterization of the N‐Linked Carbohydrate Chains of the Zona Pellucida Glycoproteins from Bovine Ovarian and Fertilized Eggs
Authors: Katsumata Toshiyuki , Noguchi Satoru , Yonezawa Naoto , Tanokura Masaru , Nakano Minoru
Published: July 2004

The N‐linked oligosaccharides that were released by hydrazinolysis from glycoproteins of zonae pellucidae of bovine ovarian eggs, were composed of neutral (23%) and acidic (77%) carbohydrate chains; almost all the acidic chains were neutralized by sialidase digestion. Sugar mapping analysis of pyridylaminated N‐linked chains by reverse‐phase and normal‐phase HPLC and 500‐MHz 1H‐NMR spectroscopy revealed that the major neutral chain is a high‐mannose‐type oligosaccharide and the acidic chains are di‐, tri‐, and tetra‐antennary, fucosylated complex‐type chains that have N‐acetyllactosamine repeats in the non‐reducing regions. The structures of the neutral chain and the core regions of the acidic chains of N‐linked oligosaccharides from the zona proteins of fertilized eggs, which were obtained by the in vitro fertilization method, were essentially the same as those of the ovarian egg zonae. The amount, however, of the acidic chains decreased to 32 mol/100 mol in the fertilized egg zonae, which suggests that a sialidase released from the oocyte during fertilization operates on the zona glycoproteins.
Transcriptional Induction of Rat Liver Apolipoprotein A‐I Gene Expression by Glucocorticoids Requires the Glucocorticoid Receptor and a Labile Cell‐Specific Protein
Authors: Saladin Régis , Vu‐Dac Ngoc , Fruchart Jean‐Charles , Auwerx Johan , Staels Bart
Published: July 2004

Treatment with glucocorticoids increases the concentration of plasma high‐density lipoprotein (HDL), which is inversely correlated to the development of atherosclerosis. Previously, we demonstrated that repeated administration of glucocorticoids increases apolipoprotein (apo) A‐I gene expression and decreases apoA‐II gene expression in rat liver. In the present study, the mechanism of glucocorticoid action on hepatic apoA‐I and apoA‐II expression was studied. A single injection of rats with dexamethasone increased hepatic apoA‐I mRNA levels within 6 h and further increases were observed after 12 h and 24 h. In contrast, liver apoA‐II mRNA levels gradually decreased after dexamethasone treatment to less than 25% control levels after 24 h. In rat primary hepatocytes and McARH8994 hepatoma cells, addition of dexamethasone increased apoA‐I mRNA levels in a time‐dependent and dose‐dependent manner, whereas apoA‐II mRNA levels were unchanged. Simultaneous addition of the glucocorticoid antagonist RU486 prevented the increase in apoA‐I mRNA levels after dexamethasone treatment, which suggests that the effects of dexamethasone are mediated through the glucocorticoid receptor. Inhibition of transcription by actinomycin D and nuclear‐run‐on experiments in McARH8994 cells and primary hepatocytes showed that dexamethasone induced apoA‐I, but not apoA‐II, gene transcription. Transient‐transfection assays in McARH8994 cells with a chloramphenicol acetyl transferase vector driven by the rat‐apoA‐I‐gene promoter demonstrated that the proximal apoA‐I promoter could be induced by dexamethasone, and this effect could be abolished by simultaneous treatment with RU486. However, in COS‐1 cells, apoA‐I promoter transcription was not induced by dexamethasone or cotransfected glucocorticoid receptor. In addition, the induction of apoA‐I gene transcription by dexamethasone was blocked by the protein‐synthesis inhibitor cycloheximide, which suggests the presence of a labile protein involved in apoA‐I gene activation by dexamethasone. In conclusion, our results demonstrate that dexamethasone regulates rat apoA‐I, but not apoA‐II, gene expression through direct action on the hepatocyte. The induction of apoA‐I gene transcription by dexamethasone requires the glucocorticoid receptor and a labile cell‐specific protein.
The Effects of Calcium and Other Polyvalent Cations on Channel Formation by Escherichia coliα‐Hemolysin in Red Blood Cells and Lipid Bilayer Membranes
Authors: Döbereiner Andreas , Schmid Angela , Ludwig Albrecht , Goebel Werner , Benz Roland
Published: July 2004

Channel formation by Escherichia coliα‐hemolysin (HlyA) was studied in lipid bilayer membranes and in erythrocytes as a function of the concentration of divalent and trivalent cations. Hemolysin showed full channel‐forming activity in artificial lipid bilayers, even in the presence of 5 mM EDTA and when the E. coli cells were grown in calcium‐depleted media (< 1 μM Ca2+). The addition of divalent cations decreased the single‐channel conductance by about 50% with half‐saturation constants of S mM and less, while the mean lifetime of the HlyA channel was not affected. The addition of trivalent cations, such as Fe3+ or La3+, had a similar effect on the channel conductance, but the half‐saturation constant was 1 μM or below. These effects may be caused by the binding of the cations to negatively charged groups at the channel mouth and have probably nothing to do with the possible binding of these cations to the repeat domain of the toxin, which is essential for target cell recognition.

When cells were grown in calcium‐depleted media, the supernatants showed absolutely no hemolytic activity. Addition of small amounts of Ca2+ to the supernatant led to toxin‐mediated hemolysis. Its half‐saturation constant was 120 μM. Of the other earth alkaline cations only strontium (Sr2+), which has an ion radius similar to Ca2+, led to full activation of HlyA with a Km of 1.5 mM. Ba2+ induced only weak hemolytic activity, while Mg2+ and several heavy metal cations had no effect. These results led to the conclusion that the target cell recognition sites formed by the repeat region of HlyA have defined sizes and bind only ions with defined radii.
Isolation and Expression of a cDNA Clone Encoding Human Kynureninase
Authors: Alberati‐Giani Dsniela , Buchli Rico , Malherbe Pari , Broger Clemens , Lang Gabrielle , Köhler Christer , Lahm Hans‐Werner , Cesura Andrea M.
Published: July 2004

Kynureninase (L‐kynurenine hydrolase), a pyridoxal‐5′‐phosphate‐(pyridoxal‐P)‐dependent enzyme, catalyses the cleavage of L‐kynurenine and L‐3‐hydroxykynurenine into anthranilic and 3‐hydroxyanthranilic acids, respectively. In this report, we describe the isolation of a cDNA clone encoding human kynureninase. Degenerate oligonucleotides designed from the amino acid sequences of peptides from rat liver kynureninase, were used as primers for reverse‐transcription PCR of rat kidney RNA. The resulting rat cDNA product was then used to screen a human hepatoma cell line (Hep G2) cDNA library. Analysis of a positive cDNA clone showed the presence of an insert of 1651 nucleotides containing an open reading frame coding for a protein of 456 amino acids (theoretical molecular mass = 52357 Da). The predicted amino acid sequence of human kynureninase displayed high similarity to that reported for the rat enzyme and to a Saccharomyces cerevisiae gene product putatively ascribed to kynureninase. Profile analysis of kynureninase primary structure indicated the presence of a pyridoxal‐P ‐binding site consensus sequence assigned to class‐V aminotransferases, with Lys276 being the residue binding the cofactor. RNA blot analysis of human tissues, including brain, showed the presence of an ≈2.0‐kb mRNA species in all tissues tested. A second mRNA species (≈2.6 kb) was also detected in some tissues. After transfection of HEK‐293 cells with the cDNA coding for kynureninase, the Km, values of l‐kynurenine and dl‐3‐hydroxykynurenine for the recombinant enzyme were 671±37 μM and 13.2±2.0 μM, respectively.
Chromosomal Localization, Genomic Structure and Characterization of the Human Gene and a Retropseudogene for 6‐Pyruvoyltetrahydropterin Synthase
Authors: Kluge Claudia , Brecevic Lukrecija , Heizmann Claus W. , Blau Nenad , Thöny Beat
Published: July 2004

Autosomal recessive mutations in the 6‐pyruvoyltetrahydropterin synthase (PTPS) gene are the most common reason for hyperphenylalaninemia due to tetrahydrobiopterin deficiency. We used the previously isolated PTPS cDNA as a probe and identified the human gene, PTS, located on chromosome 11q22.3‐q23.3, and a retropseudogene, PTS‐P1, assigned to 9p12‐p13 (symbols approved by the human genome nomenclature committee). PTS‐P1 has 74% similarity to the 3′ portion of PTPS cDNA. The PTS gene spans about 8 kb and consists of 6 exons, as revealed by DNA‐sequence analysis. This gene structure differs from that published previously which was reported to contain only two exons [Ashida, A., Owada, M. & Hatakeyama, K. (1994) Genomics 24, 408–410]. By means of intron‐specific primers, we amplified exon 3 from genomic DNA of a PTPS‐deficient patient and found a mutation in the 3′ acceptor splice site, which is responsible for skipping of exon 3.
An Immobilised Peptide Array Identifies Antibodies to a Discontinuous Epitope in the Extracellular Domain of the Bovine Growth Hormone Receptor
Authors: Beattie James , Shand John H. , Flint David J.
Published: July 2004

Using an array of overlapping decapeptides representing the extracellular domain of the bovine (b) growth‐hormone receptor (GHR) we have mapped the continuous, dominant epitopes defined by five rabbit and one guinea pig polyclonal antisera to recombinant bovine growth‐hormone‐binding protein (rbGHBP). We report that six major epitopes are identified by these antisera and that these largely occur in areas of non‐ordered secondary structure, although there is some contribution from the extensive β‐sheet structure of GHBP. Similar to our previously described studies for growth hormone (GH), we have again found slight differences between animals in the exact location of these epitopes. Using peptide‐affinity chromatography we have isolated a population of antibodies reactive with epitope 1 (the N‐terminal epitope:GHBP residues 21–38). Analysis of these antibodies by further peptide affinity chromatography and competitive radioimmunoassay experiments indicated cross‐reactivity of epitope‐1‐specific antibodies with epitope 4 (in the interdomain hinge region of the GHBP:residues 111–126). We suggest that, although separate in the primary structure of the molecule, the tertiary fold exhibited by GHBP may bring into close proximity areas of sequence representing epitope 1 and epitope 4 such that they represent a conformational epitope. Under these conditions our experiments indicate that peptides 1 and 4 may represent partial functional epitopes for this antibody population and consequently demonstrate that this approach may be useful in describing discontinuous epitopes.
Enzymatic Properties and Inhibition by Single‐Stranded Autonomously Replicating Sequences of Adenylosuccinate Synthase from Saccharomyces cerevisiae
Authors: Gallert Karl‐Christian , Ohanjan Tanja , Daignan‐Fornier Bertrand , Lottspeich Friedrich , Krauss Gerhard
Published: July 2004

Adenylosuccinate synthase (ASS) from Saccharomyces cerevisiae has been shown to bind specifically to the T‐rich side of the autonomously replicating sequence (ARS) core consensus sequence [Zeidler, R., Hobert, O., Johannes, L., Faulhammer, H. & Krauss, G. (1993) J. Biol. Chem. 268, 20191–20197]. We have cloned and sequenced the gene for ASS and have studied in detail the enzymatic properties and DNA‐binding activity of ASS. The deduced amino acid sequence of the yeast ASS is highly similar to the same enzymes from other sources from which it is however distinguished by its more basic nature.

We show that the enzymatic activity of ASS is inhibited in a highly specific manner by the binding of a 44‐base DNA oligonucleotide carrying the ARS core consensus sequence. Other nucleic acids, rNTP and dNTP are not able to mimic the specific inhibitory effect. Single‐base substitutions in the ARS core sequence lead to a tenfold reduction in inhibition. The inhibition data corroborate the earlier report on the DNA‐binding specificity of this enzyme. The homologous enzymes from Escherichia coli and Dictyostelium discoideum do not show specific binding to single‐stranded ARS sequences and their enzymatic activity is not influenced by the presence of a 44‐base DNA oligonucleotide carrying the ARS core consensus sequence.

Treatment of ASS with alkaline phosphatase leads to a loss of DNA binding and to a loss of the inhibition by DNA of the enzymatic activity which suggests that the DNA‐binding activity but not the enzymatic activity may be regulated by the phosphorylation status of the protein.
The Regulation of Human Immunodeficiency Virus Type‐1 Gene Expression
Authors: Kingsman Susan M. , Kingsman Alan J.
Published: July 2004

Despite 15 years of intensive research we still do not have an effective treatment for AIDS, the disease caused by human immunodeficiency virus (HIV). Recent research is, however, revealing some of the secrets of the replication cycle of this complex retrovirus, and this may lead to the development of novel antiviral compounds. In particular the virus uses strategies for gene expression that seem to be unique in the eukaryotic world. These involve the use of virally encoded regulatory proteins that mediate their effects through interactions with specific viral target sequences present in the messenger RNA rather than in the proviral DNA. If there are no cellular counterparts of these RNA‐dependent gene‐regulation pathways then they offer excellent targets for the development of antiviral compounds. The viral promoter is also subject to complex regulation by combinations of cellular factors that may be functional in different cell types and at different cell states. Selective interference of specific cellular factors may also provide a route to inhibiting viral replication without disrupting normal cellular functions. The aim of this review is to discuss the regulation of HIV‐1 gene expression and, as far as it is possible, to relate the observations to viral pathogenesis. Some areas of research into the regulation of HIV‐1 replication have generated controversy and rather than rehearsing this controversy we have imposed our own bias on the field. To redress the balance and to give a broader view of HIV‐1 replication and pathogenesis we refer you to a number of excellent reviews [Cullen, B. R. (1992) Microbiol. Rev. 56, 375–394; Levy, J. A. (1993) Microbiol. Rev. 57, 183–394; Antoni, B. A., Stein, S. & Rabson, A. B. (1994) Adv. Virus Res. 43, 53–145; Rosen, C. A. & Fenyoe, E. M. (1995) AIDS (Phila.) 9, S1–S3].
Asparaginyl‐tRNA Synthetase from Thermus thermophilus HB8
Authors: Seignovert Laurence , Härtlein Michael , Leberman Reuben
Published: July 2004

The gene for the asparaginyl‐tRNA synthetase, a class IIb enzyme, from the extreme thermophile Thermus thermophilus HB8 has been cloned and sequenced. Sequence analysis revealed an open reading frame that codes for a protein of 438 amino acid residues (50875 Da). Codon usage in the asparaginyl‐tRNA synthetase gene (asnS) is similar to the characteristic usage in the genes for proteins from bacteria of the genus Thermus, and the G+C content in the third position of the codons is as high as 94%. The amino acid sequence of asparaginyl‐tRNA synthetase from T. thermophilus shows high similarity with other bacterial asparaginyl‐tRNA synthetase sequences (30–55% identity). By expression of the T. thermophilus asnS gene in Escherichia coli, the thermostable enzyme was overproduced and purified to homogeneity by heat treatment and two chromatography steps. The protein obtained is remarkably thermostable and retains 50% of its initial tRNA aminoacylation activity after 1 h of incubation at 90°C or 21 h at 85°C. Crystals of the enzyme were obtained from polyethylene glycol 6000 solutions by vapour diffusion techniques. The crystals diffract X‐rays beyond 2.8 Å.
Cyclosporin A Inhibits the Degradation of Signal Sequences after Processing of Presecretory Proteins by Signal Peptidase
Authors: Klappa Peter , Dierks Thomas , Zimmermann Richard
Published: July 2004

Targeting of presecretory proteins to, and insertion into, the microsomal membrane are mediated by signal sequences. These signal sequences are removed from presecretory proteins by signal peptidase. We demonstrate that the signal sequence of preprolactin, after translocation into microsomes and cleavage by signal peptidase, is converted to an intermediate form. This intermediate was found outside the microsomes, where it was degraded in the presence of cytosol. Degradation of the signal sequence of another presecretory protein, preprocecropinA, occurred even in the absence of cytosol. The immunosuppressant cyclosporin A inhibited trimming of the preprolactin signal sequence and degradation of the preprocecropinA signal sequence. We observed by cross‐linking studies that cleaved signal sequences are bound to two microsomal proteins prior to degradation.
Expression of Aryl Hydrocarbon Receptor (AhR) and Aryl Hydrocarbon Receptor Nuclear Translocator (Arnt) in Adult Rabbits Known to be Non‐Responsive to Cytochrome P ‐450 1A1 (CYP1A1) Inducers
Authors: Takahashi Yoshiki , Nakayama Kazuo , Shimojima Tsukasa , Itoh Susumu , Kamataki Tetsuya
Published: July 2004

Induction of aryl hydrocarbon hydroxylase by aryl hydrocarbons occurs only in neonatal rabbits and not in adult rabbits [Kahl, G. F., Friederich, D. E., Bigelow, S. W., Okey, A. B. & Nebert, D. W. (1980) Dev. Pharmacol. Ther 1, 137–162], In the present study, we isolated cDNA clones encoding aryl hydrocarbon receptor (AhR) and aryl hydrocarbon receptor nuclear translocator (Arnt) from adult rabbits. The deduced amino acid sequences of rabbit AhR and Arnt showed 80% and 94% identities with those of human AhR and Arnt, respectively. Rabbit AhR mRNA was predominantly expressed in the lung and liver. In contrast, rabbit Arnt mRNA was expressed at almost the same level in all tissues except for the heart, liver, and small intestine. Gel shift analysis showed that the AhR · Arnt complex could bind to the consensus xenobiotic‐responsive element, which indicates that AhR expressed in adult rabbit livers possessed binding activity to the consensus xenobiotic‐responsive element in vitro, although aryl hydrocarbons did not induce the activity of AHH in adult rabbits. We propose that the incapability of adult rabbits to induce cytochrome P ‐450 1A1 (CYP1A1) is caused by factors other than AhR and Arnt.
Cloning and Biochemical Characterisation of an Aspergillus Niger Glucokinase
Authors: Panneman Henk , Ruijter George J. G. , Van Den Broeck Hetty C. , Driever Eugenie T. M. , Visser Jaap
Published: July 2004

The Aspergillus niger glucokinase gene glkA has been cloned using a probe generated by polymerase chain reaction with degenerate oligonucleotides. The DNA sequence of the gene was determined, and the deduced amino acid sequence shows significant similarity to other eukaryotic hexokinase and glucokinase proteins, in particular to the Saccharomyces cerevisiae glucokinase protein. The encoded protein was purified from a multicopy glkA transformant, and extensively characterised. The protein has a molecular mass of 54536 Da and a PI of 5.2. The enzyme has high affinity for glucose (Km, 0.063 mM at pH 7.5) and a relatively low affinity for fructose (Km, 120 mM at pH 7.5), and in vivo fructose phosphorylation by glucokinase is consequently negligible. The configurations at C1 and C4 of the substrate appear to be essential for substrate specificity. The A. niger glucokinase shows non‐competitive inhibition by ADP towards ATP and uncompetitive inhibition by ADP towards glucose. The kcat, (turnover number) decreases rapidly below pH 7.5 (56% at pH 7.0 and 17% at pH 6.5) and this may have important implications for the in vivo regulation of activity. In addition, proof is provided for the presence of a second hexose‐phosphorylating enzyme in A. niger. This enzyme is probably a hexokinase, since unlike glucokinase, this activity is inhibited by trehalose 6‐phosphate.