bluuebunny/crossref_metadata_2025_split huggingface dataset:
7‐S Collagen: Characterization of an Unusual Basement Membrane Structure
Authors: RISTELI Juha , TIMPL Rupert , BÄCHINGER Hans Peter , ENGEL Jürgen , FURTHMAYR Heinz 
Published: March 2005

A new type of collageneous structure, tentatively named 7‐S collagen, was isolated from a mouse tumor basement membrane, mouse and human placenta, bovine lens capsule and human kidney. The protein was solubilized from the tissues by limited digestion with pepsin or trypsin and could easily be separated from other collageneous protein because of its resistence towards further degradation by bacterial collagenase at 20°C. 7‐S collagen showed an amino acid composition typical of basement membrane collagen and contained 22% carbohydrate mainly as glucosyl‐galactosyl bound to hydroxylysine but also some mannose and glucosamine. Ultracentrifugal analysis demonstrated that the proteins were homogeneous with a sedimentation coefficient of about 7.2 S and with a molecular weight of about 360000 both in phosphate buffer pH 7 and 6 M guanidine. The peptide was triple helical as shown by circular dichroism and exhibited a biphasic melting profile indicating two conformationally distinct domains with tm= 48°C and 70°C. The more stable domain could be isolated as an homogeneous fragment (Mr= 225000) after a second digestion with collagenase at 37°C. This fragment contained all the disulfide bonds (42 Cys/1000 residues) of the original molecule. Electron microscopy showed a rod‐like structure in agreement with the hydrodynamic properties of 7‐S collagen. The dimensions of these peptides were 3 × 95 nm (long form) and 3.4 × 40–50 nm (short form).Complete reduction of 7‐S collagen under denaturing conditions produced several polypeptide chains in the molecular weight range of 27000–153000 which differ from each other by Mr increments 25000–27000. Separation of the chains on agarose did not reveal any simple stoichiometric relationship indicating that some chains are either cross‐linked or represent fragments produced during proteolytic treatments. Complete reduction of 7‐S collagen under non‐denaturing conditions lowered the thermal transition of the triple helix to 48°C but did not change its molecular weight except when exposed to dissociating solvents.7‐S collagens were potent immunogens and could be characterized by radioimmunoassays. Antigenicity was slightly reduced by reduction and denaturation while collagenase at 37°C produced a larger decrease. Proteins obtained from various sources showed distinct immunological relationships although interspecies differences in affinity exist. No or only little cross‐reaction was observed with type IV and V collagens and some further fragments of basement membrane collagen. The data indicate that 7‐S collagen is a unique component of basement membranes which shows a more compact and stable structure than other collageneous proteins.
Purification and Characterisation of an Unusually Heat‐Stable and Acid/Base‐Stable Class I Fructose‐1,6‐bisphosphate Aldolase from Staphylococcus aureus
Authors: GÖTZ Friedrich , FISCHER Stephan , SCHLEIFER Karl‐Heinz 
Published: March 2005

The fructose‐1,6‐biphosphate aldolase (EC 4.1.2.13) from Staphylococcus aureus ATCC 12600 was purified and biochemically investigated. It was found that this aldolase belongs to the class I type of aldolases since the fructose‐1,6‐bisphosphate cleavage activity was insensitive to high levels of EDTA. Like class I aldolases of higher organisms, the S. aureus aldolase activity is inhibited on incubation with the substrate dihydroxyacetone‐phosphate in the presence of NaBH4. Furthermore, the aldolase activity is not stimulated by monovalent or divalent cations.This enzyme exhibits an extreme stability to high temperature, acid and base. The purified enzyme is not inactivated after heating at 97°C for 1.6 h. An incubation at 130°C for 10 min is necessary to destroy irreversibly the activity of the aldolase. The optimal temperature for activity, however, is 37°C. It is a monomer with a molecular weight of about 33000 and exhibits a relatively broad pH optimum ranging over pH 7.5–9.0. Apart from fructose 1,6–bisphosphate as substrate (Km= 0.045 mM), this aldolase also revealed activity with fructose 1‐phosphate (Km= 25 mM). The pH of the isoelectric point lies between 3.95 and 4.25.
Xylan‐Degrading Enzymes of the Yeast Cryptococcus albidus
Authors: BIELY Peter , VRŠANSKÁ Mária , KRÁTKÝ Zdeněk 
Published: March 2005

During growth on wood β‐1,4‐xylans the yeast Cryptococcus albidus produced at least two enzymes which convert the polysaccharide to xylose catabolized by the cells. The enzyme almost completely secreted into culture fluid was identified as an endo‐1,4‐β‐xylanase.The function of the extracellular β‐xylanase is to hydrolyze xylan to oligosaccharides, mainly to xylobiose and xylotriose, which enter the cell where they are split by the second identified enzyme, a cell‐bound β‐xylosidase (xylobiase). Aryl β‐xylosidase activity detected in the culture fluid was shown to be due to low affinity of β‐xylanase for p‐nitrophenyl β‐D‐xylopyranoside. This property of β‐xylanase was preserved after purification of the enzyme by chromatography on DEAE‐cellulose, CM‐Sephadex and Biogel A 1.5 m or Biogel P 100. Purified β‐xylanase exhibited certain microheterogeneity after polyacrylamide gel electrophoresis.Both extracellular β‐xylanase and intracellular β‐xylosidase were produced in much lower amounts by the cells grown on glucose than by the cells grown on xylan. This suggested that they are not produced constitutively.The investigated strain was not able to grow on cellulose and the crude and purified β‐xylanase were unable to hydrolyze cellulose or its soluble derivatives.
Deoxyribonucleotide Biosynthesis in Synchronous Algae Cells
Authors: FELLER Wolfgang , SCHIMPFF‐WEILAND Gabriele , FOLLMANN Hartmut 
Published: March 2005

Synchronous cells of the green alga, Scenedesmus obliquus, cultured in a 14‐h/10‐h light/dark regime, contain a peak of ribonucleoside‐diphosphate reductase activity and maximum deoxyribonucleoside 5′‐triphosphate concentrations at the 12th hour of the cell cycle, coinciding with DNA synthesis and preceding the formation of eight daughter cells. The intracellular dTTP pool reaches 4.5 pmol and the other pools 2–3 pmol/106 cells. Algal reductase activity is sensitive to cycloheximide, but not to lincomycin. These correlations demonstrate the functioning of the NDP → dNDP → dNTP pathway of DNA precursor biosynthesis in plant cells.In the presence of 20 μg 5‐fluorodeoxyuridine/ml, an inhibitor of thymidylate synthesis, the dTTP pool is rapidly depleted and DNA synthesis ceases. 5‐Fluorouracil and methotrexate produce similar effects. At the same time the ribonucleotide reductase activity and also the dATP pool are greatly increased, especially when fluorodeoxyuridine treatment is combined with continued illumination of the algae. In contrast, arabinosylcytosine, an inhibitor of DNA replication, has no effect on ribonucleotide reduction. The control of de novo enzyme synthesis in the eucaryotic algae therefore appears to depend on the presence of dTTP (or a related nucleotide), but not directly coupled to DNA synthesis. This interdependence resembles the situation observed in HeLa cells, while it may differ in detail from control mechanisms of ribonucleotide reductase studied in bacteria.
Autoxidation of Native Oxymyoglobin
Authors: SUGAWARA Yoshiaki , SHIKAMA Keiji 
Published: March 2005

A complete kinetic description has been made on the pH profile for the autoxidation rate in terms of displacement of superoxide anion, O–2, from MbO2 by the entering water molecule or hydroxyl ion.Using the equation, the effect of temperature on the autoxidation rate has been studied over the pH range 4.8–12.6 in 0.1 M buffer at 15°, 25° and 35°C. The resulting thermodynamic parameters characterize the dissociation groups involved in the reaction as histidyl and tyrosyl residues.Despite the fact that each elementary process of the reaction is primarily protected against autoxidation by the high energy barrier of approximately 85–150 kJ · mol−1, the catalytic proton participates not only in decreasing the value of ΔH°≠ but also in increasing the value of ΔS°≠ to facilitate the formation of the activated complex, thereby promoting most of the autoxidation reaction of MbO2. The proton‐catalyzed process is therefore of primary importance and a mechanistic detail of the reaction is discussed.
Separation of the Proteins of Bovine Milk‐Fat‐Globule Membrane by Electrofocusing with Retention of Enzymatic and Immunological Activity
Authors: MATHER Ian H. , TAMPLIN Catherine B. , IRVING Michael G. 
Published: March 2005

1. Proteins of fat‐globule membrane from bovine milk were solubilized with the non‐ionic detergent Triton X‐100 in the presence of protease inhibitors. Approximately 25% of the total membrane protein was solubilized and the extracts were shown to contain a sample of most of the major membrane proteins and glycoproteins.2. The solubilized proteins were separated in flat‐beds of Ultrodex by electrofocusing and the pI values for the major proteins, glycoproteins and certain enzymes determined. Several of the proteins displayed marked heterogeneity indicating the existence of protein variants and isoenzymes. Principal pI values for the enzymes assayed were as follows: xanthine oxidase, 7.35–7.55; NADH2: iodonitrotetrazolium reductase, less than 4.5; 5′‐nucleotidase, 7.15–7.4; alkaline phosphatase, 5.4–5.7; phosphodiesterase, 4.6–4.8; γ‐glutamyl transpeptidase, 4.4–4.55.3. Fractions after electrofocusing were analyzed by ‘fused rocket’ immunoelectrophoresis and crossed immunoelectrophoresis after separation in polyacrylamide gels containing sodium dodecyl sulphate. Major antigens of the membrane include xanthine oxidase and glycoproteins of apparent molecular weights 67000, 49500 and 46000. The latter two components share common antigenic determinants and could not be separated by gel filtration, ion‐exchange chromatography, lectin‐affinity chromatography or preparative electrofocusing.
Effects of Tertiary Amine Local Anesthetics on the Assembly and Disassembly of Brain Microtubules in vitro
Authors: GENNA Jean‐Marie , COFFE Gérard , PUDLES Julio 
Published: March 2005

From kinetic and electron microscopy studies on the effects of procaine, tetracaine and dibucaine on the polymerization and depolymerization of the microtubules isolated from pig and rat brains the following results were obtained.1. Procaine or tetracaine, at the concentration range of 0.5–20 mM and of 0.5–5 mM respectively, increases the rate of tubulin polymerization (24°C or 37°C) and of microtubule depolymerization (4°C) as a linear function of the concentration of the anesthetics, while identical amounts of microtubules are formed. In the absence of microtubule‐associated proteins the polymerization of tubulin is not induced by 10 mM procaine, furthermore, the critical concentration of microtubule proteins necessary for assembly into microtubules is not affected at this concentration level of the anesthetic. This suggests that procaine affects not the nucleation, but rather the elongation process.2. Dibucaine, from 0.5 mM to 3 mM increases the lag time of the polymerization reaction, while from 0.5 mM to 2 mM it linearly decreases both tubulin polymerization (24°C) and microtubule depolymerization (4°C) rates. Dibucaine, up to 2 mM concentration, does not affect the extent of tubulin polymerization; however, above this concentration it induces the formation of amorphous aggregates.3. Procaine or tetracaine enhances the depolymerizing effect of calcium on microtubules. The half‐maximal values for the depolymerizing effect of calcium were 0.96, 0.71 and 0.51 mM for the control, in the presence of 10 mM procaine and 5 mM tetracaine respectively.
Isolation of Highly Active Photosystem II Particles from a Mutant of Chlamydomonas reinhardtii
Authors: DINER Bruce A. , WOLLMAN Francis‐André 
Published: March 2005

Highly active photosystem‐II particles were rapidly isolated using detergents and obtained in good yield from a mutant of the green alga Chlamydomonas reinhardtii. The particles are completely devoid of reaction centers of photosystem I, and of the secondary electron acceptor to photosystem II. They show: (a) a specific activity (ΔA of C550/unit chlorophyll) 4–7‐times that of the starting material and of spinach chloroplasts; (b) an antenna size of 40 to 50 chlorophyll molecules containing little light‐harvesting chlorophyll a/b complex (chlorophyll a/chlorophyll b= 4–6.4); (c) a ratio of variable to dark‐adapted fluorescence yield of up to 3.Further treatment of these particles by ion‐exchange chromatography largely removes five proteins and further decreases the antenna size with little loss in primary photoactivity.
Antagonistic Action between Antibodies Directed against 7‐Methylguanosine and Polyamines on Translation in vitro of RNA Induced by Measles Virus
Authors: DANTE Robert , NIVELEAU Alain 
Published: March 2005

Antibodies directed against N7‐methylguanosine (m7Guo) were prepared and added to a wheat germ cell‐free protein‐synthesizing system programmed with RNA extracted from monkey cells persistently infected with measles virus.A dose‐dependent inhibition of [35S]methionine incorporation was observed when RNA was preincubated with anti‐m7Guo immunoglobulins.Antibodies preincubated with m7Guo did not show any inhibiting activity. The inhibitory effect of antibodies was abolished when RNA was preincubated with immunoglobulins in the presence of spermine and spermidine. When polyamines were added to the assay programmed with the IgG‐RNA complex, no inhibition was observed.
Quantitative Analysis of the Proton and Charge Stoichiometry of CytochromecOxidase from Beef Heart Reconstituted into Phospholipid Vesicles
Authors: SIGEL Erwin , CARAFOLI Ernesto 
Published: March 2005

The proton and charge stoichiometry of cytochromecoxidase reconstituted into phospholipid vesicles has been analysed using a fast responding oxygen electrode and a system for the simultaneous measurement of H+, K+or lipophilic cations, and O2.From initial rate measurements after addition of reductant (ascorbate/N,N,N′,N′‐tetramethyl‐p‐phenylenediamine) the following stoichiometries could be extrapolated: K+/e−, between 0.85 and 2.05, H+/e−, between 0.58 and 0.75.Lipophilic cations can replace valinomycin/potassium as the charge · compensating system.Net H+extrusion was observed for up to 11 turnovers of the enzyme (11 O2/cytochromeaa3). The variation of the internal pH buffering capacity of the vesicles has an influence on the number of turnovers during which net proton translocation can be observed, but has, no influence on the determined stoichiometries.
31P NMR Studies of Vegetative and Encysted Cells of Acanthamoeba castellanii
Authors: DESLAURIERS Roxanne , BYRD R. Andrew , JARRELL Harold C. , SMITH Ian C. P. 
Published: March 2005

31P NMR studies have been performed on vegetative and encysted Acanthamoeba castellanii. A prominent feature of the spectra is the presence of resonances characteristic of P‐C linkages. Vegetative cells show sharp phosphonate resonances whereas encysted cells show broad phosphonate resonances and narrow phosphate resonances. Studies of model compounds using 31P NMR have shown the narrow resonances to arise from 2‐aminoethylphosphonic acid; the broad components from encysted cells arise from molecules with long rotational correlation times such as aggregates of lipophosplionoglycan or phosphonoprotein. Vegetative cells also show the presence of an electronegatively β‐substituted phosphonic acid, possibly 1‐hydroxy‐2‐aminoethylphosphonic acid, a component identified in the cell membrane lipophosphoglycan. pH titration studies of a number of phosphonic acid model compounds show a chemical shift behaviour quite distinct from that of biological phosphates. Upon acidification from pH 12, resonances shift to high field followed by return, to lower field at pH 1.0. These resonances can be ambiguous as monitors of cellular pH.The pH of both vegetative and encysted cells is 6.0–6.5. Encysted cells under anaerobic conditions show no spectral changes over 24 h. Vegetative cell, under similar conditions show timedependent changes characteristic of variation in cellular pH. There appears to be little correlation between pH and the variously differentiated states (vegetative or encysted) in Acanthamoeba.
Synthesis of Threonine Dehydratase in Neonatal Rat Liver
Authors: YEUNG Yee Guide , YEUNG Desmond Chak Yew 
Published: March 2005

Antiserum against rat threonine dehydratase has been raised in rabbits. The rate of incorporation of [H]leucine into threonine dehydratase was found to increase dramatically after birth. Actinomycin D suppressed the rate of the synthesis of the enzyme in the postnatal rat liver, although the antibiotic was without effect on the incorporation of leucine into total cytosolic protein.The administration of dibutyryl‐adenosine 3′, 5′‐monophosphate to foetal rats in utero or postnatal rats resulted in a marked enhancement of the rates of synthesis of threonine dehydratase.
2‐Deoxy‐d‐galactose Impairs the Fucosylation of Glycoproteins of Rat Liver and Morris Hepatoma
Authors: BÜCHSEL Reinhard , HASSELS‐VISCHER Barbara , TAUBER Rudolf , REUTTER Werner 
Published: March 2005

The consequences of injected 2‐deoxy‐d‐galactose on the biosynthesis of glycoproteins have been studied in rat liver and Morris hepatoma 9121. The incorporation of 2‐deoxy‐d‐galactose into glycoprotein (a), the Subsequent glycosylation with l‐fucose and N‐acetylneuraminic acid (b), and the impairment of the rat uracil nucleotide levels (c) have been determined.a) The rates of uptake and incorporation of 2‐deoxy‐d‐galactose into glycoprotein were evaluated in various tissues of the rat. When compared to d‐galactose they were found to be decreased in most tissues, especially in liver and hepatoma. After labeling with 2‐deoxy‐d‐galactose in vivo the identification of the label in glycoprotein fractions mainly yielded 2‐deoxy‐d‐galactose and no 2‐deoxy‐d‐glucose. Autoradiographic analysis of plasma membranes and serum, using polyacrylamide gel electrophoresis for separation, indicates the presence of the same bands with either 2‐deoxy‐d‐galactose or with d‐galactose as the label.b) 1 h after a single dose of 200 mg 2‐deoxy‐d‐galactose/kg body weight a 25–50% decrease of the subsequent l‐fucose incorporation into the acid‐precipitable fraction of serum, liver and hepatoma was observed compared to untreated controls. Time‐courses and dose‐dependences of the inhibitory effect were obtained in these tissues including the hepatoma plasma membrane fraction. A simultaneous increase in the acid‐soluble l‐fucose concentration was found in both liver and serum. The specificity of this effect was shown by an unchanged incorporation of N‐acetyl‐d‐mannosamine as the precursor of N‐acetylneuraminic acid. Protein synthesis, as measured by l‐leucine incorporation, was also not impaired after 2‐deoxy‐d‐galactose treatment. Injection of 2‐dcoxy‐d‐galactose as followed by labeling with l‐[14C] fucose in vivo and autoradiographic analysis of plasma membrane, cytosol and serum lead to shifts of bands. In contrast, only minimal changes were seen in hepatoma tissue.c) Uracil 5′‐nucleotide concentrations were determined in livers and tumors of ACI rats bearing the Morris hepatoma 9121. 1 h after the injection of 200 mg 2‐deoxy‐d‐galactose/kg body weight the levels of UDP‐glucose and UDP‐galactose in host liver were depressed by 50%. However, no fall was found in hepatoma tissue. Supplementation experiments with uridine showed that the decreased concentrations of uracil nucleotides are not responsible for the reduced l‐fucose incorporation into glycoprotein.From the data presented it can be concluded that the incorporation of 2‐deoxy‐d‐galactose specifically modifies the of oligosaccharide moiety of glycoprotein in vivo. By blocking the C‐2 for the attachment of l‐fucose this d‐galactose analog may serve as an experimental tool for studying the significance of terminal l‐fucose in secreted and membrane‐bound glycoproteins.
Identification of Amino‐Acid Substitutions in the Proteolipid Subunit of the ATP Synthase from Dicyclohexylcarbodiimide‐Resistant Mutants of Escherichia coli
Authors: HOPPE Jürgen , SCHAIRER Hans Ulrich , SEBALD Walter 
Published: March 2005

The amino acid sequence of the proteolipid subunit of the ATP synthase was analyzed in six mutant strains from Escherichia coli K12, selected for their increased resistance towards the inhibitor N,N′‐dicyclohexylcarbodiimide. All six inhibitor‐resistant mutants were found to be altered at the same position of the proteolipid, namely at the isoleucine at residue 28. Two substitutions could be identified. In type I this residue was substituted by a valine resulting in a moderate decrease in sensitivity to dicyclohexylcarbodiimide. Type II contained a threonine residue at this position. Here a strong resistance was observed.These two amino acid substitutions did not influence functional properties of the ATPase complex. ATPase as well as ATP‐dependent proton‐translocating activities of mutant membranes were indistinguishable from the wild type. At elevated concentrations, dicyclohexyicarbodiimide still bound specifically to the aspartic acid at residue 61 of the mutant proteolipid as in the wild type, and thereby inhibited the activity of the ATPase complex.It is suggested that the residue 28 substituted in the resistant mutants interacts with dicyclohexylcarbodiimide during the reactions leading to the covalent attachment of the inhibitor to the aspartic acid at residue 61. This could indicate that these two residues are in close vicinity and would thus provide a first hint on the functional conformation of the proteolipid. Its polypeptide chain would have to fold back to bring together these two residues separated by a segment of 32 residues.
Inhibition of Lipid‐Linked Mannose and Mannoprotein Synthesis in Yeast by Diumycin in vitro
Authors: BABCZINSKI Peter 
Published: March 2005

Diumycin, a phosphoglycolipid antibiotic, inhibits different mannosyl transfer reactions in yeast. Using membrane preparations, the drug effectively inhibited the formation of dolichyl phosphate mannose (DolP‐Man); 50% inhibition was observed at approximately 10 μg/ml. To a lesser extent also mannosyl transfer from DolP‐Man to protein decreased in presence of diumycin. Both mannosyl transfer to protein‐serine/threonine acceptor sites as well as into positions within the asparagine‐linked polymannose part of the yeast mannoprotein are inhibited to about 60% under conditions where DolP‐Man formation is blocked. DolP‐Man synthesis as well as mannosyl transfer from DolP‐Man to protein are also inhibited by diumycin using solubilized enzymes and exogenous acceptor substrates.Glycosyltransfer reactions from GDP‐mannose either to protein‐serine/threonine‐linked mannose (formation of short manno‐oligosaccharides) or to dolichyl‐diphosphate‐linked chitobiose (formation of lipid‐linked trisaccharide) are not inhibited by diumycin under conditions where DolP‐Man synthesis is blocked by the antibiotic.The inhibitory action of diumycin on DolP‐Man formation does not seem to be competitive with respect to dolichyl phosphate, since it cannot be overcome by higher concentrations of dolichyl phosphate.