bluuebunny/crossref_metadata_2025 huggingface dataset:
Botulinum ADP‐ribosyltransferase C3
Authors: AKTORIES Klaus , RÖSENER Sigrid , BLASCHKE Ute , CHHATWAL Gursharan S.
Published: March 2005

A novel ADP‐ribosyltransferase C3 was purified to homogeneity from filtrates of certain strains of Clostridium botulinum type C by ammonium sulfate precipitation, gel filtration, ion‐exchange chromatography and heat treatment. The molecular mass of botulinum ADP‐ribosyltransferase C3 was found to be 25 kDa. In the presence of [32P]NAD but not with [carbonyl‐14C]NAD, C3 labelled 21–24‐kDa protein(s) in membranes of human platelets and other tissues. The Km value of the ADP‐ribosylation reaction for NAD was about 2 μM. Labelling of the 21–24‐kDa protein(s) by C3 was largely reduced by addition of nicotinamide. Snake venom phosphodiesterase cleaved the ADP‐ribose attached to the 21–24‐kDa protein(s) by C3 and released 5'AMP. C3 catalyzed hydrolysis of [carbonyl‐14C]NAD and released [carbonyl‐14C]nicotinamide. ADP‐ribosylation of 21–24‐kDa platelet membrane protein(s) was biphasically regulated by Mg2+, Mn2+ and Ca2+. In the absence of free divalent cations GTP, GTP [γS] and GDP but not GDP[βS], GMP, ATP or ATP[γS] increased labelling by C3. In the presence of Mg2+, GTP[γS] was inhibitory. Guanine nucleotides prevented heat inactivation of the substrate protein(s) with the rank order GTP[γS] = GTP = GDP > GDP[βSL] > GMP ≫ ATP = ATP[γS]. The data support the view that the novel ADP‐ribosyltransferase C3 modifies eukaryotic 21–24‐kDa GTP‐binding protein(s).
Control by pH of urea synthesis in isolated rat hepatocytes
Authors: BOON Louis , MEIJER Alfred J.
Published: March 2005

Control by pH of urea synthesis has been studied in isolated rat hepatocytes incubated with a physiological mixture of amino acids.

Inhibition of urea synthesis by decreasing the pH of the medium was caused by diminished production of ammonia and not, as suggested in the literature, by inhibition of entry of ammonia into the ornithine cycle. The decrease by low pH of the rate of degradation of the added amino acids, that of alanine being quantitatively the most important, was accompanied by a decrease in their intracellular concentration.

It is concluded that inhibited transport of amino acids across the plasma membrane of the hepatocyte is responsible, at least in part, for the fall in urea synthesis with decreasing pH.

It is proposed that inhibition by low pH of other steps in the ureogenic pathway, distal to the production of ammonia, does not affect flux through the ornithine cycleper se, but rather contributes to the buffering of the intrahepatic concentration of ammonia.
Inorganic pyrophosphate synthesis during methanogenesis from methylcoenzyme M by cell‐free extracts of Methanobacterium thermoautotrophicum (strain ΔH)
Authors: KELTJENS Jan T. , van ERP René , MOOIJAART Rob J. , van der DRIFT Chris , VOGELS Godfried D.
Published: March 2005

Cell‐free extracts of Methanobacterium thermoautotrophicum (strain ΔH) were found to contain high concentrations of inorganic pyrophosphate (up to 40 mM). The compound was accumulated by the organism despite high activity of inorganic pyrophosphatase which was found to be present in the cell extracts (1–2 μmol min−1mg protein−1). This activity was strongly inhibited at [PP1]> 1.0 mM. It was demonstrated that PPi synthesis occurred during methylcoenzyme M reduction under hydrogen atmosphere: in the first stage of the reaction for each mole of methane formed one mole of PPi was produced. Inhibition of the methylcoenzyme M reduction by 2‐bromoethanesulfonic acid or by high concentrations (> 3 μM) of tetrachlorosalicylanilide also inhibited PPi synthesis. In contrast, low concentrations (1.3 μM) of tetrachlorosalicylanilide only inhibited PPi synthesis to the same extent as the methylcoenzyme M reduction was affected. In a later stage of the methylcoenzyme M reduction, PPi synthesis dropped and a second, as yet unidentified, unstable compound was formed. Synthesis of this compound also paralleled methane formation in a stoichiometric way and was affected by the inhibiting substances in a similar way as PPi synthesis.
Atrial natriuretic peptide induces breakdown of phosphatidylinositol phosphates in cultured vascular smooth‐muscle cells
Authors: RESINK Thérèse J. , SCOTT‐BURDEN Timothy , BAUR Ursula , JONES C. Richard , BÜHLER Fritz R.
Published: March 2005

Discrepancies exist between extent of guanylate cyclase activation by atrial natriuretic peptide (ANP) in cell‐free systems and ANP‐stimulated levels of cyclic GMP in whole cells, and also between receptor affinity and dose effectiveness of ANP. Therefore, we have investigated whether, in addition to receptor‐coupled guanylate cyclase activation, other second‐messenger cascade systems may be involved in mediating both an increase in cyclic GMP and the physiological response to ANP. Equilibrium 125I‐ANP binding studies on cultured thoracic aorta smooth muscle cells revealed the existence of low‐affinity (∼ 10−8 M, 84.5 fmol/105 cells) and high‐affinity (∼ 10−10 M, 12.5 fmol/105 cells) binding sites. We confirm that ANP elevates intracellular cyclic GMP (EC50∼ 108 M) and inhibits agonist‐(isoproterenol and forskolin)‐induced increases in intracellular cyclic AMP (IC50∼ 10−9 M). ANP also stimulated breakdown of phosphatidylinositol phosphates and generation of inositol phosphates with a half‐maximally effective concentration of ∼ 10‐10 M. The extent of phosphatidylinositol polyphosphate hydrolysis was small (120%) in comparison to that of phosphatidylinositol (Ptd‐Ins) (200%). Ptd‐Ins hydrolysis was paralleled by the appearance of glycerophosphoinositol, and there was also a close temporal relationship between these processes and the accumulation of intracellular cyclic GMP. Smooth muscle cells released [3H]arachidonic acid label in response to ANP (EC50∼ 10−10 M). Taken together, the data suggest that the vasorelaxant hormone ANP has stimulatory effects on phosphoinositol lipid metabolism via both phospholipase C (generation of inositol phosphates) and phospholipase A2 (generation of releasable [3H]arachidonic acid and indirectly glycerophosphoinositol). In contrast, stimulation of phosphatidylinositol phosphate breakdown by the vasoconstrictive hormone angiotensin II is not associated with glycerophosphoinositol formation, and neither cyclic GMP nor cyclic AMP levels were influenced by this hormone.
Structural studies on the core and lipid A region of a 4‐amino‐L‐arabinose‐lacking Rc‐type mutant of Proteus mirabilis
Authors: RADZIEJEWSKA‐LEBRECHT Joanna , BHAT U. Ramadas , BRADE Helmut , MAYER Hubert
Published: March 2005

The structure of the 4‐amino‐L‐arabinose‐lacking lipopolysaccharide of the Proteus mirabilis Rc‐type mutant R4, derived from wild‐type O28, was elucidated. The lipopolysaccharide core structure has previously been partially characterized. The linkage between heptose and deoxyoctulosonic acid(dOclA) is now reported, as well as the structure of the lipid A moiety of this mutant strain. Besides the tentative identification of an α‐linked glucosamine disaccharide in the lipid A backbone accompanying the usual β1 → 6‐linked glucosamine‐disaccharide, the only significant structural variation to previous studies was the lack of substitution of the C‐4′ phosphate by 4‐amino‐L‐arabinose. In addition, the substitution at C‐8 of one dOclA unit by 4‐amino‐L‐arabinose, previously reported for the R45 mutant of P. mirabilis 1959, is lacking in this R mutant. Also in addition to previous findings, the terminal unit of heptose was found to be substituted at C‐7 with phosphorylethanolamine (PEtN) and not only with phosphate, although this substitution is not complete as demonstrated by the relevant signals in 31P‐NMR. Additional studies with the wild‐type strain P. mirabilis O28 revealed the presence of 4‐amino‐L‐arabinose in both the core and the lipid A regions suggesting that the R4 mutant is defective in the biosynthesis of this amino sugar rather than in its transfer. Otherwise the lipid A regions of the mutant and the wild‐type strain show no structural differences.

The following formula is proposed for the lipopolysaccharide of 4‐amino‐L‐arabinose‐lacking mutant R4/O28 P. mirabilis:
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The Paracoccus denitrificans cytochrome aa3 has a third subunit
Authors: HALTIA Tuomas , PUUSTINEN Anne , FINEL Moshe
Published: March 2005

The presence of a third polypeptide subunit in Paracoccus cytochrome c oxidase is demonstrated. This protein (apparent molecular mass 23 kDa) binds dicyclohexylcarbodiimide in membranes of aerobically grown bacteria and in the purified enzyme.

The N‐terminal amino‐acid sequence of this dicyclohexylcarbodiimide‐binding protein is identical to the deduced sequence of the COIII gene product [Raitio et al. (1987)EMBO J. 6, 2825–2833].

We conclude that the aa3‐type oxidase in Paracoccus is composed of at least three subunits, which correspond to the three mitochondrially coded polypeptides in the eukaryotic enzyme.
Polyphthienoyl trehalose, glycolipids specific for virulent strains of the tubercle bacillus
Authors: DAFFÉ Mamadou , LACAVE Charlotte , LANÉELLE Marie‐Antoinette , GILLOIS Monique , LANÉELLE Gilbert
Published: March 2005

Phthienoic acids constitute a family of dextro‐rotary odd‐numbered unsaturated fatty acids isolated exclusively from virulent strains of human and bovine tubercle bacilli. In the bacterial cell they are not free and a search for their linked form in complex wall lipids of Mycobacterium tuberculosis (strain Canetti) showed that they esterified trehalose.

Structural elucidation of the major phthienoyl trehalose showed the occurrence of five acyl residues located at 2, 2′, 3′, 4 and 6′ positions of trehalose. The acyl substituents were mainly 2,4, 6‐trimethyl tetracos‐2‐enoic acid (C27 phthienoic acid) accompanied by its homologs. In addition to these branched fatty acids, straight‐chain C16 and C18 acyls composed about 20% of the substituents. The proposed structure is a new one, both for the mycobacterial‐specific glycolipid and for the substituted positions on trehalose.

Other minor acyl trehaloses were detected in M. tuberculosis(strain Canetti), differing from the major component by the occurrence of an additional hydroxy fatty acid (3‐hydroxy‐2,4,6‐trimethyl tetracosanoic acid) or by the number of acyl substituents. The major glycolipid presented a weak activity in vitro on mitochondrial oxidative phosphorylation. These glycolipids and phthienoic acids could serve as virulence indicators.
Ionic‐strength‐dependent substrate inhibition of the lysis of Micrococcus luteus by hen egg‐white lysozyme
Authors: VERHAMME Ingrid M. A. , VAN DEDEM Gijsbert W. K. , LAUWERS Albert R.
Published: March 2005

The kinetics of lysis of Micrococcus luteus by hen egg‐white lysozyme in dilute buffer media is characterized by pronounced substrate inhibition. This effect occurs within the complete pH range where lysozyme activity is detectable. The electrostatic potential of the negatively charged cell‐wall proteoglycan increases with decreasing ionic strength, resulting in an enhanced affinity between proteoglycan and lysozyme and probably favouring multipoint substrate attachment. For the lysozyme‐catalyzed hydrolysis of cell‐wall proteoglycan three plausible mechanisms of substrate inhibition can be postulated. Two out of the three models fit our experimental data, the simplest of the two providing the most rigorous information on the kinetic parameters Km, V and Ki. Three graphical methods consistent with the chosen model were applied for preliminary parameter estimation and the constants obtained were compared to those from nonlinear least‐squares analysis. If substrate inhibition is neglected it is shown that serious bias is imposed upon the parameters.
Nucleotide sequence of cloned cDNA coding for pumpkin 11‐S globulin β subunit
Authors: HAYASHI Makoto , MORI Hitoshi , NISHIMURA Mikio , AKAZAWA Takashi , HARA‐NISHIMURA Ikuko
Published: March 2005

cDNA coding for preproglobulin β, a precursor protein of 11‐S globulin β subunit, was cloned and the nucleotide sequence has been determined. The sequence covers the whole coding region (1440 base pairs) with 5′ and 3′ noncoding region (30 and 214 base pairs, respectively). The deduced amino acid sequence of preproglobulin β consists of a 21‐amino‐acid N‐terminal signal peptide, preceding the acidic γ polypeptide region (275 amino acids) and the subsequent basic δ region (184 amino acids). The site for post‐translational cleavage of the precursor polypeptide to make the γ and δ chains is estimated to be located between the asparagine‐glycine residues. The N‐terminal amino acid of the γ chain of mature 11‐S globulin β subunit was reported to be blocked by 5‐oxoproline (pyroglutamic acid) [Ohmiya et al. (1980) Plant Cell Physiol. 21, 157–1671. It was shown that the blocked N‐terminal amino acid is coded as a glutamine residue. The derived amino acid sequence was also compared with those of precursor proteins of other 11‐S globulins such as soybean glycinin, cotton β globulin, pea legumin and rape 11‐S globulin by dot matrix analysis.
Shape and location of eukaryotic initiation factor eIF‐2 on the 40s ribosomal subunit of rat liver
Authors: BOMMER Ulrich‐Axel , LUTSCH Gudrun , BEHLKE Joachim , STAHL Joachim , NESYTOVA Natalya , HENSKE Annemarie , BIELKA Heinz
Published: March 2005

The location of initiation factor eIF‐2 and of its subunits in quaternary initiation complexes (40s‐ribosomal‐subunit · eIF‐2 · GuoPP[CH2]P· Met‐tRNA1) was investigated by immunoelectron microscopy. Quaternary complexes were fixed with glutaraldehyde and reacted with affinity‐purified polyclonal antibodies against eIF‐2α, eIF‐2β or eIF‐2γ. The dimeric immune complexes obtained by sucrose gradient centrifugation were investigated electron microscopically after negative staining. Antibody‐binding sites were observed on the interface side of the 40s ribosomal subunit in the region between the ‘head’ and the ‘body’ (neck region) of the 40s ribosomal subunit. Within this region, eIF‐2α points to the rear side, whereas eIF‐2β and eIF‐2γ point to the frontal side of the 40s subunit indicating an elongated shape of eIF‐2 about 15 nm long.

By analytical ultracentrifugation of isolated eIF‐2 the sedimentation and diffusion coefficients were determined to be 6.54 S and 4074 × 10−7 cm2/s respectively. From these data, a molar mass of 122.4kg/mol and a dry volume of 147.4nm3 were calculated. For the shape of eIF‐2 a prolate ellipsoid of revolution is assumed with a maximal length of about 15nm and with an axial ratio of about 1:3.5. This conclusion is further confirmed by a calculated frictional ratio of 1.37 and a Stokes radius of about 4.54 nm.
Fluorescence‐quenching‐resolved spectroscopy of proteins
Authors: WASYLEWSKI Zygmunt , KOLOCZEK Henryk , WASNIOWSKA Alicja
Published: March 2005

A new procedure is described for using fluorescence‐quenching data of tryptophan residues in proteins to resolve their fluorescence emission spectra. In this concept the Stern‐Volmer quenching plot is determined at each particular emission wavelength and itterative non‐linear least‐squares fitting procedure allowed to resolve the steady‐state emission spectra into components. The resolved components, attributed to each of tryptophan residue, can be characterized by different accessibility to the quencher. The ability to resolve fluorescence emission spectra can be improved by using different kinds of efficient quenchers, which can selectively quench the emission of exposed or both exposed and buried fluorophores. The method was used to decompose emission fluorescence spectra in two‐tryptophan‐containing proteins; horse liver dehydrogenase, sperm whale apomyoglobin and metalloprotease from Staphylococcus aureus. The resolved spectra of alcohol dehydrogenase and metalloprotease are in excellent agreement with those previously obtained by single‐photon counting or phase methods. The method presented here is technically simple and does not require expensive instrumentation.
Biosynthesis and turnover of a 34‐kDa protein growth factor in human cytotrophoblasts
Authors: ROY‐CHOUDHURY Susanta , SEN‐MAJUMDAR Anis , MURTHY Uma , MISHRA Vishnu S. , KLIMAN Harvey J. , NESTLER John E. , STRAUSS Jerome F. , DAS Manjusri
Published: March 2005

Recently we isolated a new protein growth factor of 34 kDa from synctial membranes of human placenta. In its polypeptide molecular mass, antigenic structure, receptor binding specificity and partial amino acid sequence, it is unlike several known growth factors, hormones and other proteins. Here we report studies on its biosynthesis and turnover in cultured cytotrophoblasts from term human placenta. Expression of the 34‐kDa protein in these cells was studied by immunoprecipitation and Western blot analyses using a highly specific antibody. The experiments have produced the following results.

a) Immunostaining and Western blot analyses have demonstrated the presence of immunoreactive 34‐kDa protein in isolated cytotrophoblasts. The protein is present in both freshly isolated cells and in cells that have fused in culture to form multinuclear syncytiotrophoblasts.

b) Trophoblastic biosynthesis of the protein has been demonstrated by in vitro translation of cellular mRNA and by metabolic labelling experiments with intact cells.

c) Pulse‐chase experiments show that biosynthesis of the protein does not involve any detectable precursors of higher or lower molecular mass.

d) Studies on turnover indicate that the synthesized protein is unusually stable with a half‐life of 50–70 h.
The conformation and stability of recombinant‐derived granulocyte‐macrophage colony stimulating factors
Authors: WINGFIELD Paul , GRABER Pierre , MOONEN Peter , CRAIG Stewart , PAIN Roger H.
Published: March 2005

The conformation and stability of recombinant‐derived human and murine granulocyte‐macrophage colony stimulating factors produced in Escherichia coli have been investigated by analytical ultracentrifugation, urea‐gradient polyacrylamide gel electrophoresis and several spectroscopic methods.

The proteins were demonstrated to be physically homogeneous monomeric proteins with compact globular shapes and shown to have similar secondary structures containing both α‐helix and β‐sheet structure.

The intramolecular disulphide linkages of both proteins were shown to be essential for maintaining native conformation as reduction with dithiothreitol resulted in protein unfolding.

Comparison of the human E. coli‐derived (non‐glycosylated) and mammalian cell culture‐derived (glyco‐sylated) proteins by urea‐gradient electrophoresis indicated that glycosylation had no major effect on the conformational stability and kinetics of urea induced unfolding and refolding.
Kinetics, anion binding and mechanism of Co(II)‐substituted bovine muscle carbonic anhydrase
Authors: REN Xilin , SANDSTRÖM Anders , LINDSKOG Sven
Published: March 2005

The binding of N−3 to Co(II)‐substituted bovine carbonic anhydrase III was measured at various pH values by spectrophotometric titrations. The apparent Ki values were found to increase with pH in the studied range between pH 5.8 and 8.9. The inhibition of CO2 hydration by N−3 was found to be essentially uncompetitive at all investigated pH values (pH 6.3–8.9). The Ki values for the inhibition of kcat are much smaller than those obtained in the spectrophotometric titrations indicating that an enzyme form with a high affinity for N−3, presumably having a metal‐bound H2O, accumulates in the steady state at saturating CO2 concentrations. Assuming that the low pH limit of Ki= 9 μM for the inhibition of kcat represents the affinity of N−3 for the Co(II)‐OH2 form, a pKa value near 5 can be estimated for Co(II)‐bound water from the pH dependence of N− binding in the absence of CO2.

Measurements of time‐resolved absorption spectra during CO2 hydration in the presence of a low N−3 concentration showed the transient appearance of the characteristic spectrum of the enzyme‐N−3 adduct clearly demonstrating the accumulation in the steady state of an enzyme form with a high affinity for N−3. In similar experiments without inhibitor the transient formation of a spectral form corresponding to a Co(II)‐OH2 species has been demonstrated. This spectral form is rather featureless lacking the absorption maxima at 618 nm and 640 nm characteristic of the Co(II)‐OH− species. Our results strongly support the hypothesis that the rate‐limiting step in CO2 hydration catalyzed by carbonic anhydrase III is the protolysis of metal‐bound water.
Expression, renaturation and purification of recombinant human interleukin 4 from Escherichia coli
Authors: van KIMMENADE Anita , BOND Martha W. , SCHUMACHER Jodi H. , LAQUOI Carole , KASTELEIN Rob A.
Published: March 2005

The lymphokine human interleukin 4 (IL‐4) has been expressed from a plasmid in the cytoplasm of Escherichia coli. Advantage has been taken of insolubility of the human IL‐4 in E. coli for rapid purification of this protein in only a few steps. We describe extraction and renaturation procedures which solubilize human IL‐4 yielding biologically active protein. The protein was purified to homogeneity by one passage over a gel‐filtration column. The refolded human IL‐4 was characterized by N‐terminal sequence analysis, amino acid analysis and bioassays. The refolded E. coli‐derived human IL‐4 has biological activity on T and B cells and binds to the human IL‐4 receptor, comparable to mammalian expressed human IL‐4, indicating that the protein is folded correctly.
Ornithine cyclodeaminase from Ti plasmid C58: DNA sequence, enzyme properties and regulation of activity by arginine
Authors: SANS Notker , SCHINDLER Ulrike , SCHRÖDER Joachim
Published: March 2005

Nopaline, an abundant opine in plant cells transformed with nopaline‐type Ti plasmids, is catabolized in Agrobacterium by three Ti‐plasmid‐coded steps via arginine and ornithine to proline. The last enzyme, ornithine cyclodeaminase (OCD), converts ornithine directly into proline with release of ammonia. We describe the DNA sequence of the ocd gene from Ti plasmid C58, antiserum against an OCD fusion protein overexpressed in Escherichia coli, induction and identification of the gene product in Agrobacterium and enzymatic properties of the protein. The DNA sequence suggests a soluble protein with a stretch of some homology with ornithine carbamoyltransferases from other bacteria. OCD activity is subject to substrate inhibition, is stimulated by NAD+ {presumably acting as a catalytic cofactor) and is regulated by l‐arginine which has pronounced effects on the optima for pH and temperature and on the Km, for ornithine. The regulation of OCD activity by l‐arginine is discussed as part of the mechanisms which integrate the pathway of Ti‐plasmid‐coded opine utilization with general metabolism in Agrobacterium.
The structure of melittin
Authors: BAZZO Renzo , TAPPIN Michael J. , PASTORE Annalisa , HARVEY Timothy S. , CARVER John A. , CAMPBELL Iain D.
Published: March 2005

The conformation of the 26‐residue polypeptide melittin has been studied using 1H‐NMR spectroscopy in methanolic solution. The 1H‐NMR spectrum of melittin has been assigned using two‐dimensional NMR techniques and the secondary structure has been calculated from nuclear Overhauser enhancement data using distance geometry and restrained molecular dynamics analyses. The structure is found to be mainly helical, and similar to that found in crystals from diffraction data: residues 2 – 11 and 13 – 26 form regular α‐helices joined by a ‘hinge’ between residues 11 – 12. The structure in this hinge region is shown to be significantly different from that in the crystal structure, leading to a smaller angle between the two helices. The possible significance of the praline residues in this and similar membrane‐spanning peptides is discussed.
Structural variability of the neutral carbohydrate moiety of cow colostrum κ‐casein as a function of time after parturition
Authors: FIAT Anne‐Marie , CHEVAN Jeanine , JOLLÈS Pierre , De WAARD Pieter , VLIEGENTHART Johannes F. G. , PILLER Frederic , CARTRON Jean‐Pierre
Published: March 2005

New neutral oligosaccharides from cow colostrum κ‐casein were identified and characterized by 500‐MHz 1H‐NMR spectroscopy. Their structures are Galβ(1→3)GalNAc‐ol, Galβ(1→3)[GlcNAcβ(1→6)]GalNAc‐ol, Galβ(1→3)[Galβ(1→4)GlcNAcβ(1→6)]GalNAc‐ol, Galβ(1→3){Fucα(1→3)[Galβ(1→4)]GlcNAcβ(1→6)}Gal‐NAc‐ol. The tetrasaccharide and the cow colostrum κ‐caseinoglycopeptide which contains this oligosaccharide inhibit the hemagglutination of blood group I human erythrocytes. In cow mature milk only the disaccharide is characterized. The variability of these neutral oligosaccharides in cow κ‐casein as a function of time after calving is studied.
Regulation of collagen metabolism and cell growth by epidermal growth factor and ascorbate in cultured human skin fibroblasts
Authors: HATA Ryu‐ichiro , SUNADA Hironobu , ARAI Katsuhiko , SATO Toshiaki , NINOMIYA Yoshifumi , NAGAI Yutaka , SENOO Haruki
Published: March 2005

Epidermal growth factor (2–50 ng/ml), prepared from mouse submaxillary glands, stimulated growth and the synthesis of non‐collagenous proteins and hyaluronic acid, but inhibited collagen synthesis in cultured human skin fibroblasts, both stimulation and inhibition being dose‐dependent. All these effects may be intrinsic functions of the epidermal growth factor molecule, because these effects were cancelled by the co‐presence of antiserum specific for epidermal growth factor and because they were also observed following the addition of human epidermal growth factor produced urogastrone cDNA. On the other hand, l‐ascorbate (vitamin C) stimulated growth and collagen synthesis, as well as synthesis of non‐collagenous proteins, with no significant effect on hyaluronic acid synthesis.

Co‐presence of epidermal growth factor and ascorbate gave additive effects on growth and protein synthesis of the cells. These results suggest that the two growth‐promoting factors, epidermal growth factor and l‐ascorbate, modulate metabolism of extracellular matrix components as well as cell growth in a quite different manner in human skin fibroblasts.
Purification and properties of a protein component of messenger ribonucleoprotein particles that shares a common epitope with eucaryotic elongation factor Tu
Authors: SLOBIN Lawrence I. , GREENBERG Jay R.
Published: March 2005

A 62‐kDa polypeptide, which reacts with antibodies directed against a peptide corresponding to a portion of the amino‐terminal structure of eucaryotic elongation factor Tu(eEF‐Tu), was purified from the 0.5 M NaCl wash of rabbit reticulocyte polysomes. Previous work has shown that this polypeptide is a constituent of messenger ribonucleoprotein particles (mRNPs) from a variety of mammalian cell types [Greenberg, J. R. and Carroll, E. C. (1985) Mol. Cell Biol. 5, 342–351]. The purified polypeptide bound mRNA as well as rRNA using a nitrocellulose‐filter assay. The same nitrocellulose‐filter assay failed to detect binding to GTP. Using a competition‐binding assay, it was established that the purified polypeptide interacts with poly(U) and poly(G) but not with poly(A). This preference for synthetic polynucleotides was the same as found for eEF‐Tu [Slobin, L. I. (1983) J. Biol. Chem. 258, 4895–4900]. Furthermore, treatment of the purified RNA‐binding protein with trypsin resulted in a rapid cleavage of two peptide bonds resulting in fragments of 60 kDa and 53 kDa. Trypsin also cleaves eEF‐Tu rapidly at two bonds resulting in two large polypeptide fragments [Slobin, L. I., Clark, R. V. & Olson, M. O. J. (1981) Biochemistry 20, 5761–5767]. The amino acid sequence of the first 39 residues of the purified RNA‐binding protein was determined and found to possess no homology to eEF‐Tu.
Purification and characterization of dihydropyridine receptor from rabbit skeletal muscle
Authors: SOLDATOV Nikolai M.
Published: March 2005

Digitonin and 3‐[(3‐cholamidopropyl)dimethylammonio]‐2‐hydroxy‐1‐propane sulfonate (Chapso) were used to solubilize the receptor of dihydropyridine calcium antagonists from the transverse tubule membranes of rabbit skeletal muscle. The receptor retained the ability for selective adsorption from either detergent extract by dihydropyridine‐Sepharose. Incubation of the affinity resin with nitrendipine resulted in the elution of the receptor protein composed of two main polypeptides with molecular masses of 160 kDa and 53 kDa, as shown by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Only these two subunits were found in the receptor preparation purified to a specific dihydropyridine‐binding activity of 2500–2800 pmol/mg protein (60–70% purity) from digitonin‐solubilized membranes by a combination of wheat‐germ‐agglutinin–Sepharose, anion‐exchange and dihydropyridine‐Sepharose chromatography steps.

The individual subunits were isolated in dodecyl‐sulfate‐denatured form from the preparation of the receptor, enriched by a two‐step large‐scale procedure applied to Chapso‐solubilized membranes. The 160‐kDa subunit slowly changed its apparent molecular mass to 125 kDa upon disulfide bond reduction without formation of novel peptides. This finding implies that 160‐Da subunit is cross‐linked by intramolecular S‐S bridge(s). Chemical deglycosylation with trifluoromethanesulfonic acid showed that the carbohydrate content of large and small subunits accounted for 7.5% and 6.6% by mass, respectively. The dihydropyridine receptor subunits are glycosylated through N‐glycoside bonds only. In their ratio of polar to hydrophobic amino acid residues in the amino acid composition of the receptor subunits, these polypeptides behave rather as peripheral proteins. It is suggested that the main portion of polypeptide chains is located outside the membrane in contact with solvent.
Neurospora mitochondria contain an acyl‐carrier protein
Authors: BRODY Stuart , MIKOLAJCZYK Steve
Published: March 2005

Mitochondria of Neurospora crassa were found to contain a protein which was labelled with [14C]pantothenic acid and which carried an acyl group. This protein, when purified 6000‐fold, closely resembled the bacterial and chloroplast acyl‐carrier protein(s) [ACP(s)] in its physical and chemical properties. The predominant acyl group esterified to the purified protein was 3‐hydroxytetradecanoate, as determined by gas chromatographic mass spectrometry. The amino acid sequence of the tryptic peptide carrying the 4′‐phosphophantetheine moiety showed a high degree of sequence similarity to the analogous bacterial and chloroplast ACP peptide sequences. The possible functions of this ACP in lipid metabolism are discussed in view of the fact that Neurospora has a separate cytoplasmic enzyme complex which carries out the de novo biosynthesis of fatty acids.
Tryptic cleavage inhibits but does not uncouple Ca2+ATPase of sarcoplasmic reticulum
Authors: TÖRÖK Katalin , TRINNAMAN Brian J. , GREEN N. Michael
Published: March 2005

Sarcoplasmic reticulum Ca2+ATPase is cleaved by trypsin at two sites, T1 and T2. Cleavage at T1 is complete, whereas only about 50% of the Ca2+ATPase is digested at the T2 site. In the absence of Ca2+ ionophor, Ca2+‐ATPase activity of the digested enzyme remains virtually unchanged. In the presence of Ca2+ ionophor, however, the calculated specific activity of the doubly cleaved Ca2+ATPase is decreased by about 40%. The decrease in Ca2+ transport activity is much more rapid than cleavage of the T2 site, and could be correlated with an increased leak of Ca2+ from the digested vesicles. We obtained evidence that this leakiness is independent of the digestion of the Ca2+ ATPase itself and is presumably due to the digestion of some other components of the sarcoplasmic reticulum vesicles. Examination of steady‐state phosphoenzyme levels resulting from phosphorylation by ATP and Pi, or dephosphorylation by ADP or ADP/EGTA revealed no difference between the digested and the undigested Ca2+ATPase indicating no change in the equilibria caused by the T2 cleavage. Analysis of the substrate concentration dependence of the Ca2+ATPase activity also led to the conclusion that the digestion at T2 reduced the Vmax of ATP hydrolysis but leaves the Km, unchanged. The above results are consistent with the model that cleavage at the T2 site reduces the turnover rate of the Ca2+ATPase reaction cycle by about 40% by slowing down or altering the rate‐limiting step without affecting the equilibrium constants of the examined steps. We found no evidence of true uncoupling of Ca2+ transport from ATP hydrolysis correlated with cleavage at the T2 site.
Chromomycin A3 binds to left‐handed poly(dG‐m5dC)
Authors: SHAFER Richard H. , ROQUES Bernard P. , LePECQ Jean‐Bernard , DELEPIERRE Muriel
Published: March 2005

The interaction of chromomycin A3 (an antitumor antibiotic) with right‐handed and left‐handed polynucleotides has been studied by absorbance, fluorescence, circular dichroism, 31P‐NMR and 1H‐NMR techniques. Binding to either the B form of poly(dG‐dC) or the Z form of poly(dG‐m5dC) shifts the absorbance maximum to higher wavelength and enhances the fluorescence of the drug. Circular dichroic spectra of solutions containing various concentrations of chromomycin A3 and fixed concentrations of either B or Z polynucleotides show well defined isoelliptic points at similar wavelengths. At the isoelliptic point, the drug complex with B DNA exhibits positive ellipticity while with ZDNA it exhibits negative ellipticity. 31P‐NMR spectra of the chromomycin A3 complex with the Z form of poly(dG‐m5dC) demonstrate that the Z conformation is retained in the drug complex up to one molecule drug/four base pairs. At Mg2+ concentrations lower than that necessary to stabilize the left‐handed conformation of poly(dG‐m5dC) alone, 31P analysis shows that chromomycin A3 can bind simultaneously to both the B and Z conformations of poly(dG‐m5dC), with no effect on the B–Z equilibrium. These data demonstrate that chromomycin A3 binds to left‐handed poly(dG‐m5dC) with retention of the left‐handed conformation up to saturating drug concentrations.
Increase in nucleolytic activity of ribonuclease T1 by substitution of tryptophan 45 for tyrosine 45
Authors: NISHIKAWA Satoshi , MORIOKA Hiroshi , KIMURA Tsuyoshi , UEDA Yukari , TANAKA Toshiki , UESUGI Seiichi , HAKOSHIMA Toshio , TOMITA Ken‐ichi , OHTSUKA Eiko , IKEHARA Morio
Published: March 2005

The preparation and analysis of a mutant ribonuclease (RNase) T1 which possesses higher nucleolytic activity than the wild‐type enzyme are described. The gene for the mutant RNase T1 (Tyr45 → Trp45), in which a single amino acid at the binding site of the guanine base has been changed, was constructed by the cassette mutangenesis method using a chemically synthesized gene [Ikehara, M. et al. (1986) Proc. Natl Acad. Sci. USA 83, 4695–4699]. In order to reduce the nucleolytic activity of the enzyme in vivo, this gene was expressed in Escherichia coli as a fused protein connected through methionine residues to other proteins at both the N‐ and C‐termini. After liberation from the fused protein by cleavage with cyanogen bromide at the methionine junctions, the mutant RNase T1 was purified by column chromatography. The nucleolytic activity toward pGpC increased to 120% of that of wild‐type RNase T1. The kinetic parameters of the mutant enzyme demonstrate that this higher nucleolytic activity is due to a higher affinity for the substrate, probably because of an increased stacking effect in the binding pocket for the guanine base. This mutant enzyme also possessed a higher nucleolytic activity against pApC than wild‐type RNase T1.
Positive supercoiling catalysed in vitro by ATP‐dependent topoisomerase from Desulfurococcus amylolyticus
Authors: SLESAREV Alexey I.
Published: March 2005

A topoisomerase capable of introducing positive supercoils into closed‐circular DNA has been isolated from the extremely thermophilic anaerobic archaebacterium Desulfurococcus amylolyticus. This polypeptide has an Mr of 135000, as determined by electrophoresis under denaturing conditions. The enzyme is active in the temperature range from 65°C to 100°C and catalyzes positive supercoiling both in negatively supercoiled DNA and in relaxed DNA. These reactions require the presence of ATP. The enzyme's action on a single topoisomer has shown the linking number to increase by an integral number upon the relaxation of negative supercoils and the introduction of positive ones. This means that the reverse gyrase from D. amylolyticus is a type I topoisomerase. The presence of an extended AT sequence within the closed‐circular DNA enhances the activity of the Desulfurococcus topoisomerase. Even though the enzyme is isolated from a strictly anaerobic bacterium, it is fully active in the presence of oxygen.
Rapid‐scan stopped‐flow studies of the flavoenzyme mercuric reductase during catalytic turnover
Authors: SANDSTRÖM Anders , LINDSKOG Sven
Published: March 2005

Time‐resolved absorption spectra of the FAD‐containing enzyme mercuric reductase were recorded during the catalytic reaction at 25°C, pH 7.3. With an excess of NADPH over Hg2+ there was a rapid (k= 43 s−1) initial formation of a spectral species similar to that previously assigned to an NADPH complex of two‐electron‐reduced enzyme, EH2‐NADPH. This spectrum persisted during the quasisteady‐state phase of the reaction suggesting that EH2‐NADPH is a true catalytic intermediate and that the rate of catalysis is limited by the oxidation of EH2‐NADPH by Hg2+. Also with an excess of Hg2+ over NADPH a spectrum similar to that of EH2‐NADPH was rapidly formed. As the NADPH was exhausted, the spectrum of oxidized enzyme, E, did not reappear but rather a spectrum similar to that previously assigned to an NADP+ complex of two‐electron‐reduced enzyme, EH2‐NADP+. These results suggest that EH2‐NADP+ cannot rapidly reduce the Hg2+ substrate. However, eventually all reducing equivalents from NADPH added to oxidized, activated enzyme are utilized for the reduction of Hg2+. A mechanism model is proposed that does not involve the free, oxidized enzyme in the catalytic cycle.
Sequence and expression of Tangier apoA‐I gene
Authors: MAKRIDES Savvas C. , RUIZ‐OPAZO Nelson , HAYDEN Michael , NUSSBAUM Alexander L. , BRESLOW Jan L. , ZANNIS Vassilis I.
Published: March 2005

We have isolated and characterized the apoA‐I gene from a λ L47.1 genomic library constructed with DNA obtained from the lymphocytes of a Tangier disease patient. The DNA‐derived protein sequence of Tangier apoA‐I was found to be identical to normal apoA‐I. Transfection of mouse C127 cells with a recombinant vector containing the Tangier apoA‐I gene (pSV2‐gpt apoA‐I) allowed selection of stable clones resistant to aminopterin and mycophenolic acid. Analysis of these clones for apoA‐I synthesis showed that the protein secreted by cells expressing the Tangier apoA‐I gene was indistinguishable from the apoA‐I secreted by HepG2 cells. These experiments establish that the Tangier apoA‐I gene is structurally normal. It appears that the molecular basis of Tangier disease is not related to apoA‐I structure or regulation of expression, but rather to other factors pertinent to apoA‐I and high‐density lipoprotein metabolism.
Characterisation of binding of the methoxyacrylate inhibitors to mitochondrial cytochrome c reductase
Authors: BRANDT Ulrich , SCHÄGGER Hermann , von JAGOW Gebhard
Published: March 2005

The three E‐β‐methoxyacrylate (MOA) inhibitors oudemansin A, strobilurin A and MOA stilbene [3‐methoxy‐2(2‐styrylphenyl)propenic acid‐methylester], which differ by more than one order of magnitude in their binding affinity to the mitochondrial ubihydroquinone: cytochrome c oxidoreductase (bc1 complex), bind to a site that is not identical to the binding site for ubihydroquinone, the substrate of the outer ubiquinone reaction site (Qo centre). Although the ubihydroquinone molecule is still bound in the presence of the MOA inhibitors, its electrons cannot be transferred to the iron‐sulfur centre. A shift of the relative position of the ubihydroquinone molecule in the reaction centre due to a conformational distortion of cytochrome b induced by the binding of the MOA inhibitor seems to be the reason for the blocked electron transfer.

Further analysis shows that ubihydroquinone affects the Kd values of all three MOA inhibitors tested: the values are raised by a constant factor of two, although the inhibitors bind with quite different affinity. The iron‐sulfur protein is not involved in the binding of the MOA inhibitors.

These results have direct implications for the proper use of MOA inhibitors in experiments designed to analyse the structure/mechanism relationship in cytochrome c reductase. In particular, point mutations recently described in MOA‐inhibitor‐resistant mutants can no longer be taken to affect necessarily the ubihydroquinone binding site.
Primary structure of glycolate oxidase from spinach
Authors: CEDERLUND Ella , LINDQVIST Ylva , SÖDERLUND Gustaf , BRÄNDÉN Carl‐Ivar , JÖRNVALL Hans
Published: March 2005

The primary structure of glycolate oxidase from spinach has been determined. Six different types of peptide digest were investigated, utilizing CNBr, proteolytic enzymes, and chemical modifications to change a specificity of cleavage. In total, 90 peptides were purified and analyzed. The studies were aimed at correlation with crystallographic analysis of the same protein carried through in parallel and with cDNA studies which utilized initially determined amino acid sequences for synthesis of oligonucleotide probes. Continuous comparisons with the results from the crystallographic studies helped at an early stage to secure peptide overlaps, at the same time as the peptide data secured residue assignments in the electron density maps. In the end, all data agree and regions from all parts of the molecule have been checked by independent methods of analysis. The primary structure establishes the type of N‐terminal post‐translational processing, and yields information on segments not fully defined in electron density maps. Combined, the chemical, crystallographic, and cDNA data give extensive reliability. The peptide analysis shows that the N‐terminus is blocked by acylation of the initiator methionine, which is in a primary structure typical for non‐removal of the methionine in the processing events of the nascent protein chain. The molecule is comparatively rich in menthionine and some other generally less common residues, but has only one cysteine residue and no extensive hydrophobic segment. An amino acid sequence homology with flavocytochrome b2 from yeast, as expected from known similarities in tertiary structure, is observed (33% residue identities).
Structural changes induced by Triton X‐100 on sonicated phosphatidylcholine liposomes
Authors: URBANEJA María‐Angeles , GOÑI Félix M. , ALONSO Alicia
Published: March 2005

Solubilization of sonicated unilamellar vesicles by Triton X‐100 is a complex process. Solubilization starts at low detergent concentrations, as compared to the case of large vesicles, and is accompanied by the simultaneous rapid formation of large multilamellar liposomes. Measurements of lipid and detergent distribution indicate that, at a 1:1 lipid: detergent mole ratio, about one‐third of the lipid, with most of the detergent, is solubilized in the form of mixed micelles. The remaining two‐thirds are in the form of multilamellar liposomes, virtually free of detergent. Higher detergent concentrations also bring about the solubilization of these liposomes.
Expression of the chemically synthesized gene for ribonuclease T1 in Escherichia coli using a secretion cloning vector
Authors: QUAAS Rainer , McKEOWN Yvonne , STANSSENS Patrick , FRANK Ronald , BLÖCKER Helmut , HAHN Ulrich
Published: March 2005

The gene for ribonuclease T1 from Aspergillus oryzae has been chemically synthesized using the segmental support technique. An Escherichia coli clone producing the ribonuclease at high levels was constructed by linking the gene downstream to the region coding for the signal peptide of the OmpA protein (a major outer membrane protein of E. coli), using the secretion cloning vector pIN‐III‐ompA2. This strategy was employed in order to circumvent a possible toxic effect of the gene product on the host cell. Active ribonuclease containing four additional amino acids at the N‐terminus could be isolated from the periplasmic fraction of the host. The final yield after purification was 20 mg enzyme/l liquid culture. With respect to immunological, catalytic and specific behaviour, no qualitative differences could be detected between the enzyme from the over‐producing E. coli strain and ribonuclease T1 isolated from A. oryzae.
Involvement of various organs in the initial plasma clearance of differently glycosylated rat liver secretory proteins
Authors: GROSS Volker , HEINRICH Peter C. , vom BERG Daniela , STEUBE Klaus , ANDUS Tilo , TRAN‐THI Thuy‐Anh , DECKER Karl , GEROK Wolfgang
Published: March 2005

The initial plasma clearance and organ distribution of α1‐acid glycoprotein and α2‐macroglobulin carrying different types of oligosaccharide, side chains was studied in rats. The differently glycosylated proteins were synthesized by rat hepatocytes in culture in the presence of tunicamycin (unglycosylated form), swainsonine (hybrid type), or 1‐deoxymannojirimycin (high‐mannose type). Deglycosylated glycoproteins (Asn‐GlcNAc) were obtained by endoglucosaminidase H treatment of high‐mannose‐type glycoproteins.

Ten minutes after intravenous injection 3% of complex type, 26% of hybrid type, 84% of high‐mannose type. 64% of unglycosylated and 80% of deglycosylated α1‐acid glycoprotein disappeared from the plasma. The respective values for α2‐macroglobulin were 26%, 42%, 59% and 67%. When the clearance of total hepatic secretory proteins was examined, major differences between glycosylated and unglycosylated (glyco)proteins were found, particularly in the case of low‐molecular‐mass polypeptides.

Whereas complex‐type α1‐acid glycoprotein and α2‐macroglobulin showed no accumulation in various organs, hybrid‐type α1‐acid glycoprotein and α2‐macroglobulin were present in spleen and liver. High‐mannose‐type α1‐acid glycoprotein and α2‐macroglobulin also accumulated mainly in spleen and liver. Spleen had the highest specific activity; liver, due to its larger organ mass, represented the major organ for the uptake of high‐mannose‐type glycoproteins. Competition experiments with mannan and GlcNAc–bovine‐serum‐albumin showed a mannose/GlcNAc receptor‐mediated removal. Whereas unglycosylated α1‐acid glycoprotein was taken up by the kidney, unglycosylated α2‐macroglobulin was found in the spleen. Deglycosylated glycoproteins (Asn‐GlcNAc) were removed from the plasma via two different mechanisms: firstly, clearance by the kidney similar to the unglycosylated glycoproteins; secondly, clearance by a mannose/GlcNAc receptor‐mediated uptake mainly into the spleen.

We conclude that N‐linked oligosaccharide side chains are important for the plasma survival of hepatic secretory glycoproteins and that unphysiologically glycosylated forms are cleared by different mechanisms.
The regulation of the intracellular pH in cells from vertebrates
Authors: FRELIN Christian , VIGNE Paul , LADOUX Annie , LAZDUNSKI Michel
Published: March 2005

Eukaryotic cells control their intracellular pH using ion‐transporting systems that are situated in the plasma membrane. This paper describes the different mechanisms that are involved and how their activity is regulated.