bluuebunny/crossref_metadata_2025 huggingface dataset:
Further characterization of an early expressed glycoprotein of the rabbit small intestinal brush border
Authors: GORVEL Jean‐Pierre , RIGAL Alain , BERNADAC Alain , MAROUX Suzanne
Published: March 2005

Mouse 23 B 921 monoclonal antibody recognized a rabbit brush‐border antigen with an apparent molecular mass 140 kDa (140‐kDa Ag) which, unlike most hydrolases, is expressed in the poorly differentiated crypt cells of the small intestine. Immunoelectron microscopy of brush‐border vesicles showed that the 23 B 921 bound to an epitope localized on the outside of the membrane. As is the case with hydrolases the external domain of the 140‐kDa Ag constitutes the main part of the molecule, which can be released by papain treatment of brush‐border vesicles. The presence of a small hydrophobic domain, anchoring the molecule into the membrane and responsible for its amphipatic character, was shown by its affinity for Triton‐X114 micelles. The topological organization in the membrane of 140‐kDa Ag seemed to be identical to that of hydrolases. Unlike hydrolases, however, the native structure of the antigen was found from its sensitivity to proteolysis to be very dependent on its integration into the lipid bilayer. Nevertheless, detergent‐extracted 140‐kDa Ag can be purified by immunoaffinity chromatography although it cannot be stocked for more than 48 h even in the presence of protease inhibitors. The carbohydrate moiety of 140‐kDa Ag, bearing the human blood group A determinant, amounts to 20% of the total molecular mass.

The existence of some privileged relationship was established between 140‐kDa Ag and hydrolases: (a) in the membrane, hydrolases protect the 140‐kDa Ag from papain action; (b) after detergent extraction, 140‐kDa Ag is strongly associated with several hydrolases particularly disaccharidases.
Isolation of human‐p53‐specific monoclonal antibodies and their use in the studies of human p53 expression
Authors: BANKS Lawrence , MATLASHEWSKI Greg , CRAWFORD Lionel
Published: March 2005

The isolation and construction of a complete human p53 cDNA and subsequent expression in monkey cells is described. A set of new anti‐(human p53) monoclonal antibodies has also been obtained and used to show the expression of the human p53 cDNA in cos‐1 cells. These antibodies enable the specific detection of human p53, which is synthesised in the presence of p53 from other species. Fusion proteins of p53 with β‐galactosidase were used firstly as antigen and secondly, in conjunction with competition assays, to localise the determinants recognised by the antibodies. At least two previously unrecognised epitopes are involved and two of the antibodies are human‐p53‐specific. The epitopes are denaturation‐resistant and the antibodies are, therefore, valuable for immunoblotting as well as immunoprecipitation and enzyme‐linked immunoassay. Transfection of plasmids containing complete human p53 cDNA into monkey (cos‐1) cells cause expression of human p53 recognised by the monoclonal antibodies. Control plasmids did not induce immunoreactive protein.
Effect of tropomyosin on the interactions of actin with actin‐binding proteins isolated from pig platelets
Authors: PRULIÈRE Gérard , d'ALBIS Anne , DER TERROSSIAN Elisabeth
Published: March 2005

Several non‐muscle tropomyosins have been reported to lack the ability to polymerize in a head‐to‐tail manner [Dabrowska, R. et al. (1983) J. Muscle Res. Cell Motil. 1, 83–92; Côté, G. P. (1983) Mol. Cell. Biochem. 57, 127–146]. Unlike rabbit skeletal muscle tropomyosin, these proteins could therefore not protect the F‐actin microfilaments neither from disassembly or from cross‐linking by the other actin‐associating factors. However, we have provided evidence that, in vitro, pig platelet tropomyosin, although shorter in molecular length, exhibits the same properties as the muscle protein: it self‐associates and forms a 1:6 complex with platelet filamentous actin under physiological conditions [Prulière et al. (1984) J. Muscle Res. Cell Motil. 6, 126]. In this paper, we examine the effects of several other actin‐binding proteins on the microfilaments saturated with this non‐muscle tropomyosin. Since contractile proteins often vary with the cell type and may require different conditions for their interactions, we have developed a procedure which allows the parallel purification of actin‐binding protein (ABP), vinculin, α‐actinin, gelsolin as well as actin and tropomyosin from the same batch of cells. Thus, using an homogeneous system, we show by viscometry, sedimentation and densitometry, and by electron microscopy, that pig platelet tropomyosin can protect the structure of the microfilaments from the action of the modulating factors to the same extent as rabbit skeletal muscle α‐tropomyosin. Our data suggest that interaction of ABP, vinculin or α‐actinin can occur only with the ends of the filaments when F‐actin is saturated with tropomyosin, while cross‐linking takes place by interactions with sites localized along the entire length of F‐actin in the absence of tropomyosin. Moreover, the presence of tropomyosin on F‐actin leads to the total inhibition of gelsolin severing activity, although it did not prevent the binding of gelsolin to the F‐actin – tropomyosin complex. This suggests that pig platelet as well as skeletal muscle tropomyosins have the ability to increase the strength of the interaction between actin monomers within the filament. This also suggests that the binding sites of gelsolin along the filaments are not localized in the groove of the F‐actin helix.
Structural differences around hormonogenic sites on thyroglobulins from different species detected by monoclonal antibodies
Authors: CHAN Chi‐Tsung J. , BYFIELD Peter G. H. , CALLUS Marian , CHAMPION Brian , HIMSWORTH Richard L.
Published: March 2005

Thyroxine remains attached to its synthetic site in thyroglobulin until it is released by proteolysis. Strong homology in the primary sequence surrounding thyroxine‐forming residues in thyroglobulins from various species suggests a unique three‐dimensional structure at hormonogenic sites. To examine this, two thyroxine‐binding mouse anti‐(chicken thyroglobulin) monoclonal antibodies, 1A10 and 5F6, were used as probes for this region in an enzyme‐linked immunosorbent inhibition assay. The thyroxine content of thyroglobulins had a marked positive influence on the monoclonal antibody binding: when the thyroxine content of human thyroglobulin rose by 6.6‐fold, cross‐reactivities rose 25‐fold for the 1A10 monoclonal antibody and 17.6‐fold for the 5F6 monoclonal antibody. However, interspecies comparison of thyroglobulin preparations with similar thyroxine content showed lower than expected cross‐reactivities for human, pig and sheep thyroglobulins when compared with chicken thyroglobulin. Only when the thyroxine content of heterologous thyroglobulin preparations was two or three times higher did the cross‐reactivities equal or surpass that of chicken thyroglobulin. It is concluded that in thyroglobulin there are structural differences in the different animal species near the thyroxine‐forming sites bound by these monoclonal antibodies. The known primary sequence similarity does not seem to result, therefore, in identical three‐dimensional structures about this site. These differences may reflect species‐specific variations in distant regions brought close as a result of chain folding to form the hormonogenic site, such as those around the donor diiodotyrosine residue or in polysaccharide structures. These monoclonal antibodies provide information about the structure of thyroglobulin, which cannot be obtained from knowledge of the amino acid sequence alone.
The amino‐acid sequence of the 2S sulphur‐rich proteins from seeds of Brazil nut (Bertholletia excelsa H.B.K.)
Authors: AMPE Christophe , VAN DAMME Jozef , de CASTRO Luiz A. B. , SAMPAIO Maria José A. M. , VAN MONTAGU Marc , VANDEKERCKHOVE Joël
Published: March 2005

Storage proteins of the albumin solubility fraction from seeds of Bertholletia excelsa H.B.K. were separated by reversed‐phase high‐performance liquid chromatography and their primary structures were determined by gas‐phase sequencing on intact polypeptides and on the overlapping tryptic and thermolysin peptides. The 2S storage proteins consist of two subunits linked by disulphide bridges. The large subunit (8.5 kDa) is expressed in at least six different isoforms while the small subunit (3.6 kDa) consists of only one form. These proteins are extremely rich in glutamine, glutamic acid, arginine and the sulphur‐containing amino acids cysteine and methionine. One of the variants even contains a sequence of six methionine residues in a row. Comparison with known sequences of 2S proteins of other dicotyledonous plants shows limited but distinct sequence homology. In particular, the positions of the cysteine residues relative to each other appear to be completely conserved, suggesting that tertiary structure constraints imposed by disulphide bridges dominate sequence conservation. It has been proposed that the two subunits of a related protein (the Brassica napus storage protein) is cleaved from a precursor polypeptide [Crouch, M. L., Tenbarge, K. M., Simon, A. E. & Ferl, R. (1983) J. Mol. Appl. Genet. 2, 273–283]. The amino acid sequence homology of the Brazil nut protein with the former suggests that a similar protein processing event could occur.
Stereoselective effects in the interactions of pterin cofactors with rat‐liver phenylalanine 4‐monooxygenase
Authors: HAAVIK Jan , DØSKELAND Anne P. , FLATMARK Torgeir
Published: March 2005

The (6R) and (6S) epimers of l‐erythro‐tetrahydrobiopterin (BH4) and some of its structural analogs, were tested as cofactors and non‐covalent effectors in the phenylalanine 4‐monooxygenase (phenylalanine hydroxylase, EC 1.14.16.1) reaction. The oxidation‐reduction potentials (Em,7) of the free (not enzyme‐bound) form of the (6R) and (6S) epimers were rather similar (range 174–184 mV) for the oxidation of tetrahydropterins to quinonoid dihydropterins. Rapid‐mixing kinetic experiments were performed at 20°C under conditions which allow only a few turnover reactions of the enzyme. Three main oxidation products were identified spectroscopically at pH 6.8 for all three tetrahydropterins tested: (a) the C(4a)‐hydroxy derivatives, (b) the quinonoid dihydropterins, and (c) the stable 7,8‐dihydropterins (in that sequence). The formation of the C(4a)‐hydroxy forms closely paralleled that of tyrosine, and supports the proposal that this covalent adduct is formed as an immediate product on completion of the catalytic cycle. Assay of the initial rate of C(4a)‐hydroxy derivative formation represents a new approach in kinetic studies of this enzyme, and the kinetic parameters obtained for the phenylalanine‐activated enzyme are presented.

The affinity of binding of (6R)‐BH4 and (6S)‐BH4 to phenylalanine hydroxylase was also estimated on the basis of their quenching of the intrinsic tryptophan fluorescence of the enzyme. The apparent affinities were found to correspond well to the Km values estimated in kinetic studies of the hydroxylation reaction with the phenylalanine activated enzyme, i.e. higher for (6R)‐BH4 than for (6S)‐BH4. The lower V value observed for the native enzyme with the (6R) epimer in steady‐state kinetics is explained by its higher potency as a negative effector, since the oxidation‐reduction potentials of the two diastereomers were similar.

Dihydrobiopterin (BH2) was found to inhibit the hydroxylation reaction and quenched the intrinsic tryptophan fluorescence of the enzyme with the same concentration dependence as that observed with (6S)‐BH4.
Transport and metabolism of 5′‐nucleotidase in a rat hepatoma cell line
Authors: van den BOSCH Renee , GEUZE Hans J. , du MAINE Atala P. M. , STROUS Ger J.
Published: March 2005

The biosynthesis of the ectoenzyme 5′‐nucleotidase in the rat hepatoma cell line H4S has been studied by pulse‐labeling with [35S]methionine and subsequent immunoprecipitation of the cell lysate. 5′‐Nucleotidase is a membrane glycoprotein with an apparent molecular mass on SDS‐gels of 72 kDa. The enzyme is initially synthesized as a 68‐kDa precursor which is converted to the mature 72‐kDa form in 15–60 min (t1/2= 25 min). The molecular mass of the unglycosylated enzyme is approximately 58 kDa. Culturing the cells in the presence of varying concentrations of tunicamycin, an inhibitor of N‐glycosylation, revealed six species of 5′‐nucleotidase after sodium dodecyl sulfate/polyacrylamide electrophoresis. This indicates the presence of five N‐linked oligosaccharide chains accounting for the difference between the 58‐kDa polypeptide backbone and the 68‐kDa species. The 68‐kDa precursor is susceptible to cleavage by endo‐β‐N‐acetylglycosaminidase H; the 72‐kDa mature protein is converted to several bands upon this treatment. This result indicates that part of 5′‐nucleotidase keeps one or two high‐mannose or hybrid chains in the mature form, even after prolonged pulse‐chase labeling. The newly synthesized mature enzyme reaches the cell surface after 20–30 min. The half‐life of 5′‐nucleotidase is about 30 h in H4S cells. No immunoprecipitable 5′‐nucleosidase is released into the culture medium.
Molecular cloning and expression of cDNA for human antileukoprotease from cervix uterus
Authors: HEINZEL Regina , APPELHANS Heribert , GASSEN Günter , SEEMÜLLER Ursula , MACHLEIDT Werner , FRITZ Hans , STEFFENS Gerd
Published: March 2005

We have isolated cDNA clones for the human antileukoprotease HUSI‐I, an elastase inhibitor, from a library, containing cDNA inserts made from human cervix uterus. A library of 10000 recombinants was screened using a mixture of 16 different oligodeoxyribonucleotides which correspond to amino acids 79–84 and one 20mer oligodeoxyribonucleotide corresponding to amino acids 19–26. Two overlapping cDNA clones, containing the entire coding sequence and part of the 5′‐and 3′‐untranslated region, were isolated. DNA sequence data showed that our clone corresponds with the available protein sequence data. For expression, the cDNA fragment was inserted in a derivative of plasmid pPLc236 and expressed under the control of λPL promoter. Expression of antileukoprotease was proven by Western blot analysis and inhibition of chymotrypsin.
Flavin‐dependent alcohol oxidase from yeast
Authors: GEISSLER Johanna , GHISLA Sandro , KRONECK Peter M. H.
Published: March 2005

The kinetic course of the reaction of methanol and deutero‐methanol with FAD‐dependent alcohol oxidase was investigated under single‐turnover conditions [ked∼ 15000 min−1 (1H3COH) and ∼4300 min−1 (2H3COH)] and multiple‐turnover conditions [TNmax∼6000 min−1 (1H3COH) and ∼3100 min−1 (2H3COH)]. A kinetic scheme for the overall catalytic mechanism is proposed, which is characterized by (1) formation of a Michaelis complex between enzyme and substrate, (2) the reductive step involving partly rate‐limiting scission of the substrate C‐H bond, (3) reaction of the complex of reduced enzyme and aldehyde with dioxygen, and (4) a significant contribution of the dissociation rate of product from its complex with reoxidized enzyme to the overall rate.

Prolonged turnover of various alcohols, including methanol, results in progressive inactivation of the enzyme by two processes. In the absence of catalase the inactivation rate increases with time due to accumulation of hydrogen peroxide, which is a potent inactivator (Kd∼ 1.6 mM; kinact∼ 0.55 min−1). In the presence of catalase inactivation during turnover is much slower, the process showing pseudo‐first‐order kinetics (Kinact∼ 0.6 mM; kinact∼ 0.005 min−1 with methanol). The ratio kcat/kinact varies with different alcohols but is always > 105. Propargyl alcohol and methylenecyclopropyl alcohol cannot be considered as suicide substrates, as compared to analogous substrates of other flavin oxidases.
Conformational interactions between α and β subunits in the F1 ATPase of Escherichia coli as shown by chemical modification of uncA401 and uncD412 mutant enzymes
Authors: STAN‐LOTTER Helga , BRAGG Philip D.
Published: March 2005

In contrast to wild‐type F1 adenosine triphosphatase, the β subunits of soluble ATPases from Escherichia coli mutant strains AN120 (uncA401) and AN939 (uncD412) were not labeled by the fluorescent thiol‐specific reagents 5‐iodoacetamidofluorescein, 2‐(4′‐iodoacetamidoanilino)naphthalene‐6‐sulfonic acid or 4‐[N‐(iodoacetoxy)ethyl‐N‐methyl]amino‐7‐nitrobenzo‐2‐oxa‐1,3‐ diazole. The mutation in the α subunit (uncA401) of F1 ATPase thus influences the accessibility of the single cysteinyl residue in the β subunit. Following reaction of ATPase with 4‐chloro‐7‐nitrobenzo‐2‐oxa‐1,3‐diazole or N,N′‐dicyclohexylcarbodiimide, the α and β subunits of the uncA401, but not of the uncD412 mutant F1 ATPase were intensely labeled by a fluorescent thiol reagent. The mutation in the β subunit (uncD412) thus influences the accessibility of the cysteinyl residues in the α subunit. In other work [Stan‐Lotter, H. and Bragg, P. D. (1986) Arch. Biochem. Biophys. 248] we have shown that 4‐chloro‐7‐nitrobenzo‐2‐oxa‐1,3‐diazole and 2‐(4′‐iodoacetamidoanilino)naphthalene‐6‐sulfonic acid react with a different β subunit from that labeled by N,N′‐dicyclohexylcarbodiimide. This asymmetry with respect to modification by 4‐chloro‐7‐nitrobenzo‐2‐oxa‐1,3‐diazole and N,N′‐dicyclohexylcarbodiimide was seen in both mutant enzymes. In addition, the modification of one β subunit of the uncA401 F1 ATPase induced the previously unreactive sulfhydryl group of another β subunit to react with 2‐(4′‐iodoacetamidoanilino‐naphthalene‐6‐sulfonic acid. These results provide evidence for at least three types of conformational interactions of the major subunits of F1 ATPase: from α to β, from β to α, and from β to β. As in wild‐type ATPase, labeling of membrane‐bound unc mutant ATPase by a fluorescent thiol reagent modified the α subunits. This suggests that a conformational change of yet a different type occurs when the enzyme binds to the membrane.
Structure and expression of the chicken epidermal growth factor receptor gene locus
Authors: OLOFSSON Birgitta , PIZON Véronique , ZAHRAOUI Ahmed , TAVITIAN Armand , THERWATH Amu
Published: March 2005

Similarity between the carboxyl‐terminal portion of the human epidermal growth factor (EGF) receptor and the deduced protein sequence of the chicken‐derived oncogene v‐erbB, of avian erythroblastosis virus strain H, has suggested that the chicken cellular erbB locus, c‐erbB, might be part of a longer EGF‐receptor gene in the chicken, whose entire coding capacity remained to be defined. The c‐erbB locus spans more than 20 × 103 base pairs (20 kbp) of DNA and contains at least 1.8 kbp homologous to the v‐erbB oncogene. We show here that human EGF receptor cDNA and chicken genomic DNA share homology not only within the c‐erbB locus but also within a 25.1‐kbp DNA region situated 5′ to this locus. The 3′ region of the EGF receptor overlaps, in sequence homology, the c‐erbB locus. The EGF receptor/c‐erbB locus in chicken generates six related but distinctly different mRNAs of sizes 12, 9, 5, 3.6, 3.2 and 2.6 kb. The transcripts of 12, 9, and 3.6 kb contain sequences coding for both the extracellular EGF‐binding domain of the receptor and the intracellular tyrosine kinase domain. The 12‐kb and 9‐kb transcripts, which have already been shown to contain the sequences coding for the v‐erbB, were found to possess, in addition, sequences that encode the entire chicken EGF receptor. The 3.2‐kb and 2.6‐kb mRNAs are homologous only to the 5′ portion of the EGF receptor gene.

These results therefore indicate that the c‐erbB locus, initially defined by homology to the viral transforming gene, corresponds to the 3′ region of the EGF receptor gene in the chicken genome. The multiple, related, chicken EGF receptor RNA transcripts reported here are reminiscent of the various human EGF receptor RNA transcripts observed in normal and transformed cells.
Multiple crosslinks of proteins S7 and S9 to domains 3 and 4 of 16S ribosomal RNA in the Escherichia coli 30S particle
Authors: CHIARUTTINI Claude , MILET Michèle , HAYES Donal H. , EXPERT‐BEZANCON Alain
Published: March 2005

RNA‐protein cross‐links were introduced into Escherichia coli 30S subunits by treatment with 1‐ethyl‐3(3‐dimethylaminopropyl)carbodiimide. 16S rRNA, cross‐linked to 30S ribosomal proteins, was isolated and hybridized with seven single‐stranded bacteriophage M13‐DNA probes. These probes, each carrying an inserted rDNA fragment, were used to select contiguous RNA sections covering domains 3 and 4 (starting at nucleotide 868 and ending at the 3′OH terminus) of the 16S rRNA. The proteins covalently linked to each selected RNA section were identified by two‐dimensional polyacrylamide gel electrophoresis. Proteins S7 and S9 were shown to be efficiently cross‐linked to multiple sites belonging to both domains.
Rate constants and equilibrium constants for binding of the gelsolin‐actin complex to the barbed ends of actin filaments in the presence and absence of calcium
Authors: SELVE Norma , WEGNER Albrecht
Published: March 2005

The equilibrium constant for binding of the gelsolin‐actin complex to the barbed ends of actin filaments was measured by the depolymerizing effect of the gelsolin‐actin complex on actin filaments. When the gelsolin‐actin complex blocks monomer consumption at the lengthening barbed ends of treadmilling actin filaments, monomers continue to be produced at the shortening pointed ends until a new steady state is reached in which monomer production at the pointed ends is balanced by monomer consumption at the uncapped barbed ends. By using this effect the equilibrium constant for binding was determined to be about 1.5 × 1010 M−1 in excess EGTA over total calcium (experimental conditions: 1 mM MgCl2, 100 mM KCl, pH 7.5, 37°C). In the presence of Ca2+ the equilibrium constant was found to be in the range of or above 1011 M−1. The rate constant of binding of the gelsolin‐actin complex to the barbed ends was measured by inhibition of elongation of actin filaments. Nucleation of new filaments by the gelsolin‐actin complex towards the pointed ends was prevented by keeping the monomer concentration below the critical monomer concentration of the pointed ends where the barbed ends of treadmilling actin filaments elongate and the pointed ends shorten. The gelsolin‐actin complex was found to bind fourfold faster to the barbed ends in the presence of Ca2+ (10 × 106 M−1 s−1) than in excess EGTA (2.5 × 106 M−1 s−1). Dissociation of the gelsolin‐actin complex from the barbed ends can be calculated to be rather slow. In excess EGTA the rate constant of dissociation is about 1.7 × 10−4 s−1. In the presence of Ca2+ this dissociation rate constant is in the range of or below 10−4 s−1.
New thiol inhibitor of Achromobacter iophagus collagenase
Authors: YIOTAKIS Athanasios , DIVE Vincent
Published: March 2005

New synthetic mercaptotripeptides (HS‐CH2‐CH2‐CO‐Pro‐Yaa) which inhibit Achromobacter iophagus collagenase were produced in order to obtain more powerful bacterial collagenase inhibitors than currently available, and to investigate the specificity of the S3′ subsite of the enzyme.

Since similar binding constants were found for inhibitors carrying uncharged residues of various sizes in the P3′ position (Yaa = Ala, Leu, Phe, Pro, Hyp) steric hindrance at the collagenase S3′ appears relatively limited.

The compound (HS‐CH2‐CH2‐CO‐Pro‐Arg), which carries an arginine residue in the position P3′ and had the highest inhibition constant of the series tested (Ki= 0.5 μM), proved to be the strongest inhibitor so far reported in the literature. The weakest in the present series was the compound (HS‐CH2‐CH2‐CO‐Pro‐Asp) which carries an aspartic residue in position P3′ and had a Ki= 70 μM.

The present work revealed that the charged groups in the P3′ position play a key role in the interaction of the inhibitors with the enzyme.
Gene distribution and nucleotide sequence organization in the mouse genome
Authors: SALINAS Julio , ZERIAL Marino , FILIPSKI Jan , BERNARDI Giorgio
Published: March 2005

Mouse DNA was fractionated by preparative centrifugation in density gradients of Cs2SO4 containing 3,6‐bis(acetatomercurimethyl)dioxane (BAMD). The effects of temperature, BAMD/nucleotide molar ratio and solvent on the fractionation were explored. The fractions so obtained were investigated by analytical centrifugation in CsCl density gradient and by hybridization with a number of gene probes. These approaches led (a) to the definition of satisfactory conditions for the rapid fractionation of mouse DNA; (b) to the localization of a number of genes in mouse DNA fractions; and (c) to a better understanding of the mosaic organization of the mouse genome and, more specifically, to a better estimate of both the intermolecular and intramolecular compositional heterogeneity of mouse DNA in the (75–150) × 103‐base size range.
Gene distribution and nucleotide sequence organization in the human genome
Authors: ZERIAL Marino , SALINAS Julio , FILIPSKI Jan , BERNARDI Giorgio
Published: March 2005

Human DNA was fractionated by centrifugation in Cs2SO4 density gradients containing 3,6‐bis(acetatomercurimethyl)dioxane (BAMD). Fractions were investigated in their analytical CsCl profiles and a number of specific sequences were localized in them. The results so obtained led to an improved understanding of the organization of nucleotide sequences in the human genome, as well as to the discovery that a class of DNA having a very high G + C content and not represented in the mouse genome, is particularly rich in genes and interspersed repetitive sequences.
Purification and characterization of human interleukin‐1β expressed in recombinant Escherichia coli
Authors: WINGFIELD Paul , PAYTON Mark , TAVERNIER Jean , BARNES Marjory , SHAW Alan , ROSE Keith , SIMONA Marco G. , DEMCZUK Stephen , WILLIAMSON Karen , DAYER Jean‐Michel
Published: March 2005

The high‐level expression of human interleukin‐1β in Escherichia coli is described. The protein contributes about 12% of the total cell protein and is associated with the soluble cytoplasmic fraction of the cell. A method for the purification of the protein is given which is based on anion‐ and cation‐exchange chromatographies. The isolated protein, shown to be homogeneous by several analytical methods, has been characterized by amino acid analysis, N‐ and C‐terminal sequence analysis and analytical centrifugation. The protein is biologically active as demonstrated by two different in vitro assays, namely, the mononuclear cell factor (IL‐1/MCF) assay and lymphocyte activating factor (IL‐1/LAF) assay. The specific activities determined with the IL‐1/MCF and IL‐1/LAF assays, are 2 × 107 and 4 × 107 units mg−1, respectively.
Glutamate decarboxylase side reactions catalyzed by the enzyme
Authors: CHOI Soo Young , CHURCHICH Jorge E.
Published: March 2005

A homogeneous glutamate decarboxylase isolated from pig brain contains 0.8 mol of tightly bound pyridoxal 5‐phosphate/enzyme dimer. Upon addition of exogenous pyridoxal 5‐phosphate (pyridoxal‐5‐P), the enzyme acquires maximum catalytic activity.

Preincubation of the enzyme with l‐glutamate (10 mM) brings about changes in the absorption spectrum of bound pyridoxal‐5‐P with the concomitant formation of succinic semialdehyde. However, the rate of this slow secondary reaction, i.e. decarboxylative transamination, is 10−4 times the rate of normal decarboxylation. It is postulated that under physiological conditions enzymatically inactive species of glutamate decarboxylase, generated by the process of decarboxylative transamination, are reconstituted by pyridoxal‐5‐P produced by the cytosolic enzymes pyridoxal kinase and pyridoxine‐5‐P oxidase.

The catalytic activity of resolved glutamate decarboxylase is recovered by preincubation with phospho‐pyridoxyl‐ethanolamine phosphate. The experimental evidence is consistent with the interpretation that the resolved enzyme binds the P‐pyridoxyl analog, reduces the stability of the covalent bond of the phospho‐pyridoxyl moiety, and catalyzes the formation of pyridoxal‐5‐P.
DNA synthesis in vitro with an endoplasmic‐reticulum‐DNA‐polymerase complex from unfertilized sea urchin eggs
Authors: SHIODA Masaki
Published: March 2005

An endoplasmic‐reticulum — DNA‐polymerase complex was prepared from unfertilized sea urchin eggs and its DNA‐synthesizing activity was examined using single‐stranded DNA of bacteriophage fd as a template. The complex catalyzed the ribonucleotide‐dependent DNA synthesis which required dNTPs, NTPs, Mg2+ and single‐stranded DNA. The DNA synthesis was sensitive to aphidicolin and N‐ethylmaleimide but was resistant to 2′,3′‐dideoxyribosylthymine 5′‐triphosphate (ddTTP) and α‐amanitin, suggesting the involvement of DNA polymerase α. In parallel with the DNA synthesis, a small amount of RNA was synthesized in the presence of 100 μg/ml α‐amanitin. The Km value of ribonucleotides for the RNA synthesis coincided with that for the DNA synthesis, suggesting a correlation between the DNA and RNA syntheses. Labelling of the products with [γ‐32P]ATP followed by DNA digestion with pancreatic DNase I revealed the attachment of an oligoribonucleotide (7–11 bases in length) at the 5′ ends of the DNA products. These observations suggest that in DNA synthesis, primer RNA synthesis occurs first, followed by DNA chain elongation. During 1–90‐min incubation, the amount of the DNA synthesized increased but the length was not significantly increased. Over 80% of the number of synthesized DNA molecules comprised a single population of short DNA fragments (60–200 bases, on average 120 bases in length) and the number of fragments increased, depending on the incubation time. However, DNA fragments of various sizes (about 100–6000 bases) were synthesized with DNA polymerase α solubilized from the endoplasmic‐reticulum — DNA‐polymerase complex. All this evidence suggests that in vitro, the complex preferentially synthesizes a particular size of short DNA fragments. The significance of the fragments is discussed.
DNA‐supercoiling is affected in vitro by the peptide antibiotics tyrocidine and gramicidin
Authors: BOHG Anne , RISTOW Hansjürgen
Published: March 2005

Tyrocidine, a peptide antibiotic produced by Bacillus brevis (ATCC 8185), relaxes superhelical DNA in a biphasic manner and induces ‘packaging’ of the DNA at higher concentrations. This was concluded from studies using the sensitive 4,5′,8‐trimethylpsoralen photobinding technique [Sinden, R. R., Carlson, J. O. & Pettijohn, D.‐E. (1980) Cell 21, 773–783]. Relaxed DNA is not affected by tyrocidine wheras linearized molecules become packaged. The linear gramicidin synthesized by the same strain reverses the tyrocidine‐induced relaxation as well as the packaging, an observation which might be of biological relevance.
Dynamic interactions of enzymes involved in triosephosphate metabolism
Authors: OROSZ Ferenc , OVÁDI Judit
Published: March 2005

A steady‐state kinetic analysis of the coupled reactions catalysed by the three‐enzyme system, aldolase, glyceraldehyde‐3‐phosphate dehydrogenase and triosephosphate isomerase, was performed. The kinetic parameters of the progress curves of end‐product formation calculated for noninteracting enzymes were compared with those measured in the two‐enzyme and three‐enzyme systems. Changes in the fluorescence anisotrophy of labelled dehydrogenase upon addition of aldolase and/or isomerase were also measured.

Glyceraldehyde‐3‐phosphate oxidation catalysed by glyceraldehyde‐3‐phosphate dehydrogenase in the presence of isomerase (which ensures rapid equilibration of the triosephosphates) follows single first‐order kinetics. The rate constant depends simply on the concentration of the dehydrogenase, indicating no kinetically significant isomerase‐dehydrogenase interaction. Fluorescence anisotropy measurements also fail to reveal complex formation between the two enzymes.

The steady‐state velocity of 3‐phosphoglycerate formation from fructose 1,6‐bisphosphate in the reactions catalysed by aldolase and dehydrogenase is not increased twofold on addition of the isomerase, even though a 1:2 stoichiometry of fructose 1,6‐bisphosphate/glyceraldehyde 3‐phosphate is expected. In fact, by increasing the concentration of the isomerase, the steady‐state velocity actually decreases. This effect of the isomerase may be a kinetic consequence of an aldolase‐isomerase interaction, which results in a decrease of aldolase activity. Furthermore, the fluorescence anisotropy of labelled dehydrogenase, measured at different aldolase concentrations, is significantly lower when the sample contains isomerase.

The decrease in the steady‐state velocity of the consecutive reactions caused by the elevation of isomerase concentration could be negated by increasing the dehydrogenase concentration in the three‐enzyme system. All of these observations fit the assumption that the amount of aldolase‐dehydrogenase complex is reduced due to competition of isomerase with dehydrogenase. The alternate binding of dehydrogenase and isomerase to aldolase may regulate the flux rate of glycolysis.
Effects of long‐chain fatty acids on the inhibition by antimycin of respiration in hepatocytes and isolated mitochondria from rat liver
Authors: GREGORY Roland B. , BERRY Michael N.
Published: March 2005

A previous study [Berry, M. N., Gregory, R. B., Grivell, A. R. & Wallace, P. G. (1983) Eur. J. Biochem. 131, 215–222] suggested that long‐chain fatty acid (palmitate) oxidation by hepatocytes was less sensitive than short‐chain fatty acid (hexanoate) oxidation to inhibition by a given concentration of antimycin.

Re‐examination of this phenomenon showed that palmitate oxidation by hepatocytes could be depressed by antimycin to the same degree as other NAD+‐linked substrates, only if the concentration of the inhibitor was raised 2–4‐fold. The presence of palmitate also reduced the sensitivity to antimycin of hepatocytes metabolizing lactate or pyruvate.

Over the range of fatty acids tested, butyrate (C4) to stearate (C18), only long‐chain (> C10) fatty acids endowed cells with decreased sensitivity towards antimycin.

2‐Bromopalmitate, a non‐metabolizable fatty acid, and inhibitor of fatty acid oxidation, also decreased the inhibitory effect of antimycin in cells, suggesting that long‐chain fatty acids per se rather than their metabolites, reverse the inhibition by antimycin. Moreover, another inhibitor of fatty acid oxidation, 2‐tetradecylglycidic acid, did not diminish the effects of palmitate.

Succinate oxidation in isolated mitochondria that had been inhibited by a low concentration of antimycin could be restored by subsequent addition of palmitate or other long‐chain fatty acids such as dodecanoate, tetradecanoate and oleate under conditions where fatty acid oxidation was prevented. 2‐Bromopalmitate, likewise partially restored antimycin‐depressed succinate oxidation. This amelioration of antimycin inhibition was counteracted by the addition of more antimycin and was not seen upon addition of defatted bovine serum albumin, palmitoylcarnitine or octanoate.

The total amount of antimycin bound to mitochondria was not affected by the presence of palmitate.

The data suggest that long‐chain fatty acids are able to interact with the mitochondrial inner membrane in a manner which can relieve the inhibitory effect of antimycin, whether the antimycin is added to the cell or mitochondrial suspension before or after fatty acid addition.
The interaction of phosphatidylcholine bilayers with Triton X‐100
Authors: GOÑI Félix M. , URBANEJA Maria‐Angeles , ARRONDO José Luis R. , ALONSO Alicia , DURRANI Aziz A. , CHAPMAN Dennis
Published: March 2005

The interaction of multilamellar phosphatidylcholine vesicles with the non‐ionic detergent Triton X‐100 has been studied under equilibrium conditions, specially in the sub‐lytic range of surfactant concentrations. Equilibrium was achieved in less than 24 h. Estimations of detergent binding to bilayers, using [3H]Triton X‐100, indicate that the amphiphile is incorporated even at very low concentrations (below its critical micellar concentration); a dramatic increase in the amount of bound Triton X‐100 occurs at detergent concentrations just below those producing membrane solubilization. Solubilization occurs at phospholipid/detergent molar ratios near 0.65 irrespective of lipid concentration.

The perturbation produced by the surfactant in the phospholipid bilayer has been studied by differential scanning calorimetry, NMR and Fourier‐transform infrared spectroscopy. At low detergent concentration (lipid/detergent molar ratios above 3), a reduction in 2H‐NMR quadrupolar splitting occurs, suggesting a decrease in the static order of the acyl chains; the same effect is detected by Fourier‐transform infrared spectroscopy in the form of blue shifts of the methylene stretching vibration bands. Simultaneously, the enthalpy variation of the main phospholipid phase transition is decreased by about a third with respect to its value in the pure lipid/water system. For phospholipid/detergent molar ratios between 3 and 1, the decrease in lipid static order does not proceed any further; rather an increase in fluidity is observed, characterized by a marked decrease in the midpoint transition temperature of the gel‐to‐fluid phospholipid transition. At the same time an isotropic component is apparent in both 31P‐NMR and 2H‐NMR spectra, and a new low‐temperature endotherm is detected in differential scanning calorimetric traces. When phospholipid and Triton X‐100 are present at equimolar ratios some bilayer structure persists, as judged from calorimetric observations, but NMR reveals only one‐component isotropic signals. At lipid/detergent molar ratios below unity, the NMR lines become narrower, the main (lamellar) calorimetric endotherm tends to vanish and solubilization occurs.
Molecular cloning of the cDNA for androgen‐dependent sperm‐coating glycoproteins secreted by the rat epididymis
Authors: BROOKS David E. , MEANS Anthony R. , WRIGHT Elizabeth J. , SINGH Surinder P. , TIVER Kristen K.
Published: March 2005

cDNA clones coding for two closely related androgen‐dependent sperm‐coating glycoproteins secreted by the rat epididymis were selected by screening an epididymal cDNA library constructed in λgt 11 with affinity‐purified antibody directed against the glycoproteins. The largest clone of 956 nucleotides provided coding information for a protein of 246 amino acids of which the first 19 residues comprise a putative signal peptide sequence which when cleaved would produce a mature protein of 227 residues and a molecular mass of 26 kDa. Confirmation of the identity of the clone was provided by a match between the amino acid sequence predicted from the cDNA sequence and the actual amino acid sequence determined for a tryptic peptide fragment of one of the pure glycoproteins. It is probable that the primary amino acid sequence of the two glycoproteins is identical. Northern blot and slot‐blot analysis revealed that the mRNA for the glycoproteins is approximately 1250 nucleotides long and that the concentration of the mRNA in the epididymis is androgen‐dependent. The glycoproteins and their mRNAs were unique to the epididymis as determined by Western and Northern blots, respectively, since signals were absent from skin, brain, liver, kidney, heart, skeletal muscle and testis. Cross‐reacting proteins of slightly smaller apparent molecular mass were detected in extracts of mouse and guinea‐pig epididymis, but not rabbit or bull epididymis. Comparison with existing protein data bases revealed that the epididymal glycoproteins display significant sequence homology with yeast carboxypeptidase Y.
Localisation of light chain and actin binding sites on myosin
Authors: MITCHELL E. Jane , JAKES Ross , KENDRICK‐JONES John
Published: March 2005

A gel overlay technique has been used to identify a region of the myosin S‐1 heavy chain that binds myosin light chains (regulatory and essential) and actin. The 125I‐labelled myosine light chains and actin bound to intact vertebrate skeletal or smooth muscle myosin, S‐1 prepared from these myosins and the C‐terminal tryptic fragments from them (i.e. the 20‐kDa or 24‐kDa fragments of skeletal muscle myosin chymotryptic or Mg2+/papain S‐1 respectively). MgATP abolished actin binding to myosin and to S‐1 but had no effect on binding to the C‐terminal tryptic fragments of S‐1. The light chains and actin appeared to bind to specific and distinct regions on the S‐1 heavy chain, as there was no marked competition in gel overlay experiments in the presence of 50–100 molar excess of unlabelled competing protein. The skeletal muscle C‐terminal 24‐kDa fragment was isolated from a tryptic digest of Mg2+/papain S‐1 by CM‐cellulose chromatography, in the presence of 8 M urea. This fragment was characterised by retention of the specific label (1,5‐I‐AEDANS) on the SH1 thiol residue, by its amino acid composition, and by N‐terminal and C‐terminal sequence analyses. Electron microscopical examination of this S‐1 C‐terminal fragment revealed that: (a) it had a strong tendency to form aggregates with itself, appearing as small ‘segment‐like’ structures that formed larger aggregates, and (b) it bound actin, apparently bundling and severing actin filaments. Further digestion of this 24‐kDa fragment with Staphylococcus aureus V‐8 protease produced a 10–12‐kDa peptide, which retained the ability to bind light chains and actin in gel overlay experiments. This 10–12‐kDa peptide was derived from the region between the SH1 thiol residue and the C‐terminus of S‐1. It was further shown that the C‐terminal portion, but not the N‐terminal portion, of the DTNB regulatory light chain bound this heavy chain region. Although at present nothing can be said about the three‐dimensional arrangement of the binding sites for the two kinds of light chain (regulatory and essential) and actin in S‐1, it appears that these sites are all located within a length of the S‐1 heavy chain of about 100 amino acid residues.
Biosynthesis of the wall neutral polysaccharide in Bacillus cereus AHU 1356
Authors: MURAZUMI Naoyuki , ARAKI Yoshio , ITO Eiji
Published: March 2005

The pathway for the biosynthesis of a cell wall polysaccharide, composed of glucosamine, mannosamine, galactosamine and glucose in a molar ratio of 4:1:1:1, was studied with a membrane system from Bacillus cereus AHU 1356. In this system a glycolipid characterized as GalNAc(α1→4)ManNAc(β1→4)GlcNAc‐PP‐undecaprenol was formed from GlcNAc‐PP‐undecaprenol by sequential transfer of N‐acetylmannosamine and N‐acetylgalactosamine residues from UDP‐ManNAc and UDP‐GalNAc respectively. An additional N‐acetylglucosamine residue and a glucose residue were individually transferred from their UDP derivatives to the trisaccharide‐linked lipid with the formaiton of tetrasaccharide‐linked lipids, which seem to serve as intermediates in the polysaccharide synthesis. Incubation of membranes with the trisaccharide‐linked lipid even in the absence of sugar‐linked nucleotides led to the formation of polysaccharide. These results, together with the data on Smith degradation of the synthesized polysaccharide, indicate that the repeating trisaccharide units of the main chain of the polysaccharide arise from the GalNAc‐ManNAc‐GlcNAc moiety of the glycolipid intermediates and that the sugar residues in the lateral branches of the polymer are at least partly introduced through oligosaccharidelinked lipid intermediates. In addition, the structure of native polysaccharide was re‐examined, and the presence of the disaccharide sequence ManNAc(β1→4)GlcNAc in the polysaccharide chain was confirmed.
Interactions of pig plasma gelsolin with G‐actin
Authors: WEEDS Alan G. , HARRIS Harriet , GRATZER Walter , GOOCH John
Published: March 2005

Pig plasma gelsolin forms a ternary complex with monomeric actin in 0.1 mM CaCl2 and a binary complex in EGTA (<0.01 μM calcium), as shown by gel filtration and fluorescence changes when actin which had been treated with N‐ethylmaleimide and 7‐chloro‐4‐nitrobenzeno‐2‐oxa‐1,3‐diazole (NBD‐actin) or with N‐(1‐pyrenyl)iodoacetamide (PI‐actin) binds to gelsolin. The fluorescence enhancement per actin molecule bound is similar in the binary and ternary complexes, but the affinity of gelsolin for labelled actin is very much greater in the presence of calcium. Furthermore, the formation of ternary complex exhibits strong positive cooperativity.
Characterization of the thioredoxin system in the facultative phototroph Rhodobacter sphaeroides Y
Authors: CLEMENT‐METRAL Jenny D. , HÖÖG Jan‐Olov , HOLMGREN Arne
Published: March 2005

This paper reports the purification and characterization of a thioredoxin system (thioredoxin, thioredoxin reductase, NADPH) from the facultative phototroph Rhodobacter sphaeroides Y. Rhodobacter sph. Y thioredoxin was purified to homogeneity with an assay based on the reduction of 5,5′‐dithiobis(2‐nitrobenzoic acid) by NADPH and Escherichia coli thioredoxin reductase. Rhodobacter sph. Y thioredoxin reductase was purified with the same assay using NADPH and E. coli thioredoxin.

Rhodobacter sph. Y thioredoxin contained 102 amino acid residues and had a single intrachain disulfide bond. The two half‐cystine residues are part of the active site made up of the sequence ‐Ala‐Glu‐Trp‐Cys‐Gly‐Pro‐Cys‐Arg‐ which is identical to that of E. coli thioredoxin except for the presence of an Arg instead of a Lys. Rhodobacter sph. Y thioredoxin contains two tryptophan residues. The fluorescence intensity of the tryptophan residues is quenched in oxidized thioredoxin; on reduction, a much smaller increase is observed with Rhodobacter sph. Y thioredoxin than with the E. coli protein. However, the presence of 5 M guanidine · HCl results in the complete exposure of the two tryptophan residues.

Rhodobacter sph. Y thioredoxin reductase has structural and functional similarities to E. coli thioredoxin reductase: it has a molecular mass of 68 kDa, and consists of two, probably identical, subunits. Each subunit has one bound FAD molecule. The enzyme is highly specific for NADPH; it is also highly specific for Rhodobacter sph. Y thioredoxin with a Km value of 3.3±0.6 μM. A kinetic study of the two thioredoxin systems shows that they have a high degree of cross‐reactivity.
Covalent labelling of histones with aurothiomalate
Authors: GREYLING H. Johann , HAPGOOD Janet P. , SEWELL B. Trevor , VON HOLT Claus
Published: March 2005

Histone octamers were covalently labelled with aurothiomalate at amino groups by the method of carbodiimide activation. The labelling procedure was demonstrated to result in the specific covalent coupling through a single bond of the heavy metal atom label to protein amino groups. Such octamers were dissociated to yield soluble H2A‐H2B dimers containing three gold atoms per dimer. The dimers were reconstituted with native H3‐H4 tetramers to form labelled octamers, which were crystallized to form helical tubes. This strongly suggests that this procedure resulted in minimal changes of protein conformation.