bluuebunny/crossref_metadata_2025 huggingface dataset:
Affinity Labelling of Rat‐Muscle Hexokinase Type II by a Glucose‐Derived Alkylating Agent
Authors: CONNOLLY Bernard A. , TRAYER Ian P.
Published: March 2005

The glucose‐derived alkylating agent N‐bromoacetylglucosamine (GlcNBrAc) is shown to cause a time‐dependent irreversible inactivation of rat muscle hexokinase type II. The kinetics of inactivation are in accord with the reversible formation of an enzyme‐inhibitor complex prior to modification, indicating that the reagent is active‐site‐directed. A Ki of 0.57 mM obtained for this reversible complexing is in agreement with a Ki of 0.65 mM obtained for the inhibition caused by N‐propionylglucosamine, an isosteric analogue of GlcNBrAc and a competitive inhibitor with respect to glucose. Glucose itself protects competitively against inactivation. A KG of 0.26 mM obtained for the formation of enzyme‐glucose complex from these studies is in agreement with the kinetically‐determined Km of 0.2 mM. The substrate‐unrelated but chemically similar alkylatig agents bromoacetic acid and N‐bromoacetylgalactosamine inactivate the enzyme at 20% of the rate caused by GlcNBrAc. The inactivation rate increases rapidly over the pH range 7–9. Analysis of this pH dependence shows that a single residue of pKa 8.9 is reacting with GlcNBrAc with a kmax (pH corrected, pseudo‐first‐order rate constant) of 1.5 × 10−3 s−1. These values are typical of the reaction of model thiols with alkylating agents and suggests the reacting residue is probably a cysteine. Use of radioactively labelled GlcNBrAc indicates that uptake of 1 mol of reagent per mol protein causes complete activity loss. Finally the behaviour of this enzyme with active‐site‐directed alkylating agents is compared with published results of similar experiments carried out with yeast hexokinase and bovine brain hexokinase type I.
Anisotropy Decay of Labelled Actin
Authors: IKKAI Takamitsu , WAHL Philippe , AUCHET Jean‐Claude
Published: March 2005

G actin, labelled presumably on cysteine‐373 with the fluorescent chromophore N‐iodoacetylN′‐(5 sulfo‐1‐naphthyl)‐ethylenediamine and purified by Sephacryl S‐200 gel chromatography, migrated in one band on polyacrylamide gel electrophoresis and had the same polymerizability as unlabelled purified G actin. Anisotropy decays of labelled actin solutions have been studied at different ionic strengths and protein concentrations.

It was found that these anisotropy decays could be fitted by a sum of two exponential functions. Under low ionic strength or below the critical concentrations the longer correlation time (45 ns at 3.5°C) was independent of protein concentration and ionic strength. Above the critical concentration, the longer correlation time increased with ionic strength and protein concentration. In order to take into account that, under these conditions, the solutions contained a mixture of F and G actin at the critical concentration, the anisotropy decays were analysed as a sum of three exponential functions in which the longest correlation time characterized F actin. Since F actin correlation time also depended on actin concentration, an analysis with a sum of four exponential functions was performed, in which two fixed correlation times (100 ns and 900 ns at 3.5°C) were introduced in order to characterize the F actin motions. The longer of these correlation times was attributed to regions where two actin filaments interact side by side, while the shorter one was attributed to filament regions free from intermolecular interactions.

The small value of the free F actin correlation time indicates that the protomer peptide chain is very flexible around its C terminus, probably involving the motion of a molecular lobe. This flexibility might be an important factor in the interaction of actin with myosin during the muscular contraction.
The (2′‐5′)Oligoadenylate (pppA2′‐5′A2′‐5′A) Synthetase and Protein Kinase(s) from Interferon‐Treated Cells
Authors: HOVANESSIAN Ara G. , KERR Ian M.
Published: March 2005

Two enzyme activities have been partially purified from interferon‐treated mouse L cells: the (2′‐5′)An synthetase responsible for the synthesis of pppA2′‐5′A2′‐5′A (and related oligomers) and one or more protein kinases responsible for the phosphorylation of (a) a polypeptide of molecular weight 67000 (67000‐Mr polypeptide kinase), (b) the 35000‐Mr subunit of the protein synthesis initiation factor eIF‐2 and (c) added histones. Chromatography on columns of poly(I) · poly(C)‐Sepharose provided a more than 1000‐fold purification of these enzymes in a single step and in high yield. The partially‐purified poly(I) · poly(C)‐Sepharose‐bound (2′‐5′)An synthetase is stable and on incubation with ATP is active for several days. This provides a convenient means for the synthesis and accumulation of pppA2′‐5′A2′‐5′A. A simple two‐step elution of the bound enzymes from the poly(I) · poly(C)‐Sepharose yielded a kinase fraction free of detectable (2′‐5′)An synthetase. The (2′‐5′)An synthetase from these columns, on the other hand, always contained detectable kinase activity. This latter could be removed on DEAE‐cellulose, but the resultant ‘kinase‐free′ (2′‐5′)An synthetase required, in addition to double‐stranded RNA (dsRNA), a second component for optimum activity. This second component chromatographed with the kinase but no evidence for phosphorylation of the synthetase by it was obtained. It may be, therefore, that the association of the second component with the kinase(s) is fortuitous. The most purified kinase fractions still contained the 67000 ‐Mr polypeptide kinase, eIF‐2 kinase and histone kinase activities. A partial separation of the histone kinase activity was obtained, however, emphasising the possible heterogeneity of the kinase fraction. In contrast to the purified (2′‐5′)An synthetase, the purified kinase activities were no longer dependent upon dsRNA.
χ‐Carrageenase from Pseudomonas carrageenovora
Authors: McLEAN Maitland W. , WILLIAMSON Frank B.
Published: March 2005

A χ‐carrageenase was isolated from the cell‐free medium of cultured Pseudomonas carrageenovora. From dodecylsulphate/polyacrylamide gel electrophoresis, a single protein (identified as the χ‐carrageenase) was detected in the medium. Activity against nominal carrageenan types and inspection of the products indicate the enzyme to be a χ‐carrageenase.

Purification is described here by ammonium sulphate precipitation and subsequent CM‐Sepharose CL‐6B ion‐exchange chromatography. Molecular weight was estimated as 35000 by dodecylsulphate/polyacrylamide gel electrophoresis. Products of degradation were analysed by gel filtration, spectrophotometric assays and 13C nuclear magnetic resonance. These results are consistent with the product of limit digest being neocarrabiose 4‐O‐sulphate.
Detergents Linked to Polysaccharides: Preparation and Effects on Membranes and Cells
Authors: PITHA Josef , KOCIOLEK Karol , CARON Marc G.
Published: March 2005

Residues of Triton X‐100 [C8H17‐C6H4‐(O‐CH2‐CH2)9‐10‐O‐] were bound by ether bonds to inulin, dextran, amylose, and cellulose‐yielding compounds containing 10–30, 5, 19 and 6% (w/w) of Triton X‐100 residue respectively. The water‐soluble inulin derivative was studied in detail. This compound was fractionated on the basis of molecular weight by gel chromatography and on the basis of degree of substitution by adsorption to polystyrene resin. Even the residues of Triton X‐100 which were bound to a single inulin macromolecule were able to form a micelle; in addition to these monomolecular micelles the inulin derivative was able to form polymolecular micelles as well. The inulin derivative was effective in solubilizing proteins and phospholipids from membranes of human erythrocytes and liberated reverse transcriptase activity from the membrane‐enveloped virions of murine leukemia. The Triton X‐100 inulin derivative abolished binding of the 3H‐labelled antagonist dihydroalprenolol to solubilized preparations of β‐adrenergic receptors from frog erythrocytes in a dose‐related manner similar to the inactivation produced by Triton X‐100, while digitonin, a detergent containing a bulkier hydrophobic group, did not cause inactivation. On the basis of its Triton X‐100 content, the inulin derivative was found to be less detrimental to the growth of murine erythroleukemic cells in vitro than Triton X‐100 alone when short (2–4 h) exposures were used, but this difference disappeared at longer (1–3 day) exposures. Results thus suggest that the increase in size of the hydrophilic part of the detergent brings about moderation in those effects of the detergent which are dependent on the rate of diffusion, while the solubilizing and inactivating effects of the detergent were not changed. It is probably the size of the hydrophobic part which is important in protein‐inactivating properties of the detergent.
Conformational Studies of Neurotoxin II from Naja naja oxiana
Authors: TSETLIN Viktor I. , ARSENIEV Aleksandr S. , UTKIN Yuri N. , GUREVICH Aleksandr Z. , SENYAVINA Lyudmila B. , BYSTROV Vladimir F. , IVANOV Vadim T. , OVCHINNIKOV Yuri A.
Published: March 2005

After treatment of neurotoxin II, a component part of the venom of the Middle Asian cobra Naja naja oxiana, with acetoxysuccinimide all five possible ∼‐acetylated‐lysyl derivatives were obtained and the position of the label was established. Trifluoroacetylation of both the derivatives and the parent toxin yielded, respectively, the five acetyl‐penta(trifluoroacetyl)‐neurotoxins II and the hexa(trifluoroacetyl)‐neurotoxin II, which were studied by circular dichroism (CD), 1H and 19F nuclear magnetic resonance (NMR) spectroscopy. The availability of this series of compounds made possible assignment of all six fluorine signals (from the N‐terminal and the five ɛ‐amino groups) in the hexa(trifluoroacetyl)‐neurotoxin II NMR spectra and disclosure of the proximity of the Lys‐26 and Lys‐46 trifluoroacetyl groups. The pH dependence of the 19F NMR signals was determined and the pK values of the groups affecting the signal chemical shifts were calculated by a computer iterative program. In order to ascertain the relative accessibility of the Iysyl side chains, the change in halfwidths of the hexatrifluoroacetylated neurotoxin II 19F signals, with addition of varying amounts of an iminoxyl spin probe, was determined. The data obtained are compared with the X‐ray data on sea snake neurotoxins and the significance of the side chain interactions observed in solution is discussed.
Spin Transition of Camphor‐Bound Cytochrome P‐450
Authors: LANGE Reinhard , DEBEY Pascale
Published: March 2005

Variations of the spin state in camphor‐bound cytochrome P‐450 are interpreted in the light of the polyelectrolyte theory and its implications on the microenvironment of the heme. The ratio of high‐spin to low‐spin iron can serve as a tool to determine the local paH in the microenvironment of a group (pK0,app∼ 5.4) which governs the spin state. The local paH depends on the electrostatic potential created by negatively charged groups (pKa= 5.6), modulated in turn by paH and by the screening effect of ionic strength. A model is given for the proton‐coupled spin state change.
Norepinephrine‐Stimulated Fatty‐Acid Release and Oxygen Consumption in Isolated Hamster Brown‐Fat Cells
Authors: NEDERGAARD Jan , LINDBERG Olov
Published: March 2005

Brown fat cells isolated from adult golden hamsters have earlier been found to respond to addition of the physiological agonist norepinephrine with an increased rate of oxygen consumption and with fatty acid release. Working with these cells, we found the following.

The presence of albumin in the incubation medium (phosphate buffer) increases norepinephrine‐induced fatty acid release and tends to stabilize the rate of oxygen consumption; bubbling of phosphate buffer with 5% CO2 in air has only a slight effect on fatty acid release.

In the presence of albumin, the norepinephrine‐induced rate of oxygen consumption is also stable in bicarbonate buffer; it is higher than in the phosphate + CO2 buffer and the brown fat cells have a higher sensitivity to norepinephrine.

20 mM phosphate (as e.g. present in a phosphate buffer) inhibits both fatty acid release and oxygen consumption.

Insulin inhibits the rate of oxygen consumption, but only at suboptimal concentrations of norepinephrine.

Atractylate inhibits submaximal norepinephrine‐induced respiration, indicating that some oxidative phosphorylation takes place in norepinephrine‐stimulated brown fat cells.

Fatty acid export from brown fat should be regarded as physiologically important.
(Photo)chemistry of 5‐Deazaflavin
Authors: DUCHSTEIN Hans‐Jürgen , FENNER Helmut , HEMMERICH Peter , KNAPPE Wolfgang‐R.
Published: March 2005

The catalytic action of 5‐deazaflavin in the photochemical reduction of flavin and iron proteins [Massey, V. and Hemmerich, P. (1978) Biochemistry, 17, 9–17] is shown to be due to the highly reactive 5‐deazaflavosemiquinone. This radical is generated in a complex sequence of reactions, which involves (a) covalent photoaddition of the substrate residue to the deazaflavin, (b) fast secondary photoreaction of this adduct with starting deazaflavin to yield a covalent radical dimer, accompanied by the liberation of the oxidized substrate, and (c) deazaflavin‐sensitized cleavage of the radical dimer to the monomers. The structure and properties of this radical (redimerisation or dismutation) and the precursor intermediates as well as the mechanism of the photoreaction are described. Deazaflavins and their natural parent compounds are compared with respect to their different redox behavior and radical stability. The syntheses of 5‐deuterated deazaflavins are described and their redox reactions are compared with those of normal deazaflavins.
Formate‐Nitrite Reduction in Escherichia coli K12
Authors: ABOU‐JAOUDÉ Antoine , CHIPPAUX Marc , PASCAL Marie‐Claire
Published: March 2005

Some physiological characteristics of the formate nitrite reductase activity were studied on whole cells.

Highest level of activity was found in cells grown in anaerobiosis on glycerol in the presence of nitrite. Maximal activity was obtained at pH 8 and at 45 °C. Copper ions and cyanide inhibit the activity whereas 0.1 mM azide is without effect.

No reduction of nitrite was detected in the presence of oxygen, even at very low concentrations. Nitrate has a double effect: it competes with nitrite for electrons supplied by formate, and has an inhibitory action on nitrite reductase activity.

The stoichiometry between formate and nitrite is 3:1; the formate nitrite reduction is accompanied by CO2 evolution with the same stoichiometry.
Construction in vitro of Hybrid Plasmids Carrying All the EcoRI Fragments from λrifd18 DNA
Authors: BERNARDI Alberto , BERNARDI Françoise
Published: March 2005

The cloning of all the eleven fragments obtained by degrading the phage λrifd18 by the restriction enzyme EcoRI into the plasmid pSF2124 has been achieved: nine of these fragments have been cloned individually, whereas two others have been cloned jointly in the same plasmid. These fragments harbor, in addition to λ genes, the genes for ribosomal proteins, the elongation Factor Tu, the β and β' subunits of RNA polymerase and the ribosomal RNAs.

The clones carrying the ribosomal RNA genes have been constructed to provide convenient plasmids to determine the primary structure of ribosomal RNAs. Some further genetic manipulations in vitro have been performed on two of them to remove extraneous non‐ribosomal RNA gene sequences; the ribosomal genes purified this way have been subcloned into to the plasmid pBR322.

Other clones of interest have been obtained which carry the genes for the elongation factor Tu, a number of 50‐S ribosomal proteins and the β subunit of RNA polymerase.
Light‐Induced Proton Transport by Chloroplasts Suspended in Fluid Media at Sub‐Zero Temperatures: Kinetics and Stoichiometry
Authors: OLSEN Lars F. , COX Raymond P.
Published: March 2005

Chloroplasts suspended in a medium containing ethanediol and water (1:1, v/v) at –16°C show light‐induced proton uptake and subsequent dark efflux. Proton uptake in continuous light showed biphasic kinetics.

A 1 ms flash caused a single turnover of the photochemical centres at –16 °C. Under the same conditions 3 H+ were taken up from the external medium in the presence of methyl viologen as electron acceptor.

The flash‐induced proton uptake was exponential and monophasic with t1/2= 3 s. The flash‐induced proton release into the thylakoid interior was biphasic, with half‐times of less than 0.1 s and 3 s. The fast phase represented approximately 30% of the total release and may be correlated with the oxidation of water.

The half‐time of reduction of cytochrome f in the dark following illumination in the presence of 2 mM NH4Cl (2.5 s) is similar to the half‐time of the slow phase of proton release, suggesting a correlation between the kinetics of cytochrome f reduction and plastoquinol oxidation.
Cathepsin D of Rat Spleen Affinity Purification and Properties of Two Types of Cathepsin D
Authors: YAMAMOTO Kenji , KATSUDA Nobuo , HIMENO Masaru , KATO Keitaro
Published: March 2005

Two types of cathepsin D were purified from rat spleen by a rapid procedure involving an acid precipitation of tissue extract, affinity chromatography with pepstatin–Sepharose 4B and concanavalin‐A–Sepharose 4B, and chromatography on Sephadex G‐100 and DEAE‐Sephacel. The purified major enzyme (85% of the cathepsin D activity after DEAE‐Sephacel chromatography), termed cathepsin D‐I, represented about a 1000‐fold purification over the homogenate and about a 20% recovery. The purified minor enzyme (15%), termed cathepsin D‐II, represented about a 900‐fold purification and about a 3% recovery. Both enzymes showed four (PI: 4.2, 4.9, 6.1 and 6.5) and three (pI: 4.6, 5.6 and 5.8) multiple forms after isoelectric focusing, respectively. The purified enzymes appeared homogeneous on electrophoresis in polyacrylamide gel and had a molecular weight of about 44000. In sodium dodecylsulfate/polyacrylamide gel electrophoresis both enzymes showed a single protein band corresponding to a molecular weight of 44000. The enzymes had similar amino acid compositions except for serine, proline and methionine. Cathepsin D‐I contained 6.6% carbohydrate, consisting of mannose, glucose, galactose, fucose and glucosamine in a ratio of 8:2:1:1:5 with a trace of sialic acid. The properties of purified enzymes were also compared.
Purification and Partial Characterization of a Factor, a Mating Hormone Produced by Mating‐Type‐a Cells from Saccharomyces cerevisiae
Authors: BETZ Richard , DUNTZE Wolfgang
Published: March 2005

Cells of Saccharomyces cerevisiae exhibiting the a mating type secrete into the culture medium a mating‐type‐specific hormone activity (a factor), which specifically causes a transient arrest of DNA replication and cell division in cells of the opposite mating type, α. Three compounds exhibiting a factor activity have been found in culture filtrates from a cells. The most active compound has been purified more than 105‐fold and appears to be homogeneous on the basis of thin‐layer chromatography and thin‐layer electrophoresis in different systems. We propose that this compound, which exhibits in α cells the biological activities that have been attributed to a factor, represents pure a factor. a factor has been characterized as a very hydrophobic undecapeptide with the following amino acid composition: H2N‐Tyr(Asx1, Gly1, Ala1, Val1, Ile2, Phe1, Lys1, Trp1, Pro1). Although in their respective target cells the biological effects of a factor and of α factor, the corresponding mating hormone of mating‐type‐α cells, are remarkably similar, the primary structures of both hormones appear to be quite different.
Regulation of Glycogen Synthase Activity in Choriocarcinoma Cells Effect of Glucose Starvation on the Conversion of Synthase D to Synthase I and the Kinetic Properties of the Enzyme
Authors: HUANG Kuo‐Ping
Published: March 2005

Choriocarcinoma cell glycogen synthase exists predominantly in the form dependent on glucose‐6‐phosphate during growth. This enzyme can not be converted to the form independent of glucose 6‐phosphate by endogenous phosphoprotein phosphatase, rabbit liver phosphoprotein phosphatase, or human placental alkaline phosphatase. However, the glycogen synthase present in the extract of cells which have been subjected to glucose starvation for 24 h can be converted to the form independent of glucose 6‐phosphate by these phosphatases. The activity which converts the form dependent on glucose 6‐phosphate to the form independent of glucose 6‐phosphate by the endogenous phosphatase is progressively increased during starvation. This increase in activity is blocked by the presence of 20 μM cycloheximide in the medium. Analysis of the kinetic properties of glycogen synthase isolated from cells during starvation shows that the A0.5 value (the activator concentration giving 50% maximal activity) for glucose 6‐phosphate and the S0.5 value (the substrate concentration giving 50% maximum activity) for UDP‐glucose are reduced. This reduction in the kinetic constants of the enzyme is also prevented by the presence of cycloheximide. The starvation‐induced change in the kinetic properties of the enzyme can be reversed by the incubation of the starved‐cell extract with ATP, Mg2+, and cyclic AMP or by refeeding the starved cells with medium containing high glucose. Also, the glycogen synthase which has similar kinetic properties to the enzyme found in the starved cells can be derived from the enzyme present in normal cultures. This involves mixing a small portion of the starved‐cell extract with a large portion of normal cellular extract and incubating under conditions favoring dephosphorylation. It appears that during glucose starvation glycogen synthase is partially dephosphorylated, and the resulting enzyme, still in a form dependent on glucose 6‐phosphate, has a lower A0.5 value for glucose 6‐phosphate and a lower S0.5 value for UDP‐glucose than the enzyme from cells without starvation. The starvation‐induced modification of the form of the enzyme dependent on glucose 6‐phosphate transforms it into a better substrate for phosphoprotein phosphatase.
Modification of Peptidoglycan Structure by Penicillin Action in Cell Walls of Proteus mirabilis
Authors: MARTIN Hans Herbert , GMEINER Jobst
Published: March 2005

Composition of cell wall peptidoglycan was studied in the gram‐negative bacterium Proteus mirabilis and in different wall‐defective growth forms of this organism which survive and multiply in the presence of penicillins. The results show that action of the classical benzylpenicillin does not proceed by a uniform mechanism of inhibition of peptide crosslinkage in peptidoglycan.

Peptidoglycan of the unstable spheroplast L‐form of P. mirabilis grown in medium with 120 μg/ml benzylpenicillin contained monomers, and peptide‐crosslinked dimers and trimers of disaccharide‐peptide building blocks in a similar ratio as peptidoglycan of the normal bacterial form. Moreover, peptide side‐chains in peptidoglycan from penicillin‐grown L‐form spheroplasts were predominantly tetrapeptides l‐alanyl‐d‐γ‐glutamyl‐meso‐diaminopimelyl‐d‐alanine. Thus, specific penicillin‐sensitive enzymes of peptidoglycan synthesis, peptidoglycan transpeptidase and dd‐carboxypeptidase, must have remained uninhibited by the antibiotic in the L‐form. L‐form peptidoglycan, however, differed characteristically from peptidoglycan of normal Proteus bacteria by having a much lower content of O‐acetyl groups linked to muramic acid residues of the polymer.

P. mirabilis grew actively in the form of osmotically stable spheres in medium with up to 100 μg/ml of the amidino penicillin mecillinam. Neither the peptide crosslinkage nor the O‐acetyl content of peptidoglycan were influenced to any great extent by mecillinam.

Only the combined action of benzylpenicillin and mecillinam caused complete inhibition of growth in P. mirabilis, concomitant with deformation of cells to very large spheres. It is hypothesized that benzylpenicillin and mecillinam each inhibit different parts of a multiple, isofunctional enzyme system whose total inhibition requires the presence of both antibiotics.
Fatty‐Acid Synthesis in Lactating‐Goat Mammary Gland 1. Medium‐Chain Fatty‐Acid Synthesis
Authors: GRUNNET Inger , KNUDSEN Jens
Published: March 2005

Tissue slices from lactating goat‐mammary gland synthesized short (C4:0 and C6:0), medium (C8:0 and C10:0) and long‐chain (C12:0 to C16:0) fatty acids in proportions similar to that found in goat milk fat. In contrast, the particle‐free supernatant fraction and the purified fatty acid synthetase from this tissue synthesized predominantly short‐chain and long‐chain fatty acids.

Terminating acyl‐thioesterases of low molecular weight could not be detected in the particle‐free supernatant.

Addition of the microsomal fraction to the particle‐free supernatant induced the synthesis of medium‐chain fatty acids in proportions which were similar to those found in goat milk fat.
β‐Like Globin RNA Sequences in Hemoglobin Lepore Disease
Authors: GIGLIONI Barbara , COMI Paola , TARAMELLI Roberto , POZZOLI Maria , ZANOLLO Adriana , OTTOLENGHI Sergio , GIANNI Alessandro Massimo
Published: March 2005

A Southern Italian patient homozygous for hemoglobin Lepore disease synthesizes approximately 3% Lepore Δβ‐globin chains (relative to α chains) in the reticulocytes. Measurement of β‐like RNA sequences by hybridization to complementary DNA specific for β‐globin demonstrates a low level (1–2% relative to α sequences) of these sequences in cytoplasmic RNA from reticulocytes or spleen cells, suggesting that the Lepore gene is expressed into mRNA at a lower extent than normal α or β genes; the comparison with the level of β‐like sequences found in nuclear RNA (6–8%) further supports this conclusion and indicates, in addition, that Lepore RNA might be degraded at a faster rate than normal. 2–3%β‐like sequences are found in nuclear RNA in three cases of homozygous β0‐thalassemia, setting the highest possible estimate for the Δ‐RNA level; this figure suggests that the ‘Δ‐promoter’‐dependent Lepore Δβ gene is somehow more actively expressed than the Δ gene.
Localization of Phospholipids in the Membrane of Bacillus megaterium
Authors: DEMANT Erland J. F. , OP DEN KAMP Jos A. F. , VAN DEENEN Laurens L. M.
Published: March 2005

Phospholipase C treatment of Bacillus megaterium protoplasts at 37°C removed up to 63% of the total phospholipid without disrupting the membrane. More than 95% of the total phosphatidylethanolamine and 3′‐glucosaminylphosphatidylglycerol, 80% of the phosphatidylglycerol and about 20% of the cardiolipin and 2′‐glucosaminylphosphatidylglycerol were hydrolyzed.

Similar results were obtained with isolated membranes and dispersions of lipids extracted from the membranes, demonstrating that these data do reflect, at least in part, the substrate specificity of phospholipase C and not necessarily the distribution of phospholipids over the two membrane layers.

When protoplasts and isolated membranes were incubated with excess phospholipase C at temperatures in between 5°C and 25°C a different hydrolysis pattern was obtained and it was concluded that 50% of the phosphatidylethanolamine and 70% of the 3′‐glucosaminylphosphatidylglycerol are located in the outer membrane layer, the residual being in the cytoplasmic layer. At least 40% of the phosphatidylglycerol was found to be present also in the outer layer. The data do not allow conclusions about the localization of cardiolipin and 2′‐glucosaminylphosphatidylglycero1. Trinitroben‐zenesulfonic acid reacted with 50% of the phosphatidylethanolamine in protoplasts and with nearly 100% of this lipid in isolated membranes at various temperatures and concentrations, thus confirming the symmetric localization of this lipid.

The data furthermore indicate that a temperature‐dependent transbilayer movement of phospholipids is induced by phospholipase C treatment of the Bacillus megaterium protoplasts.
The Phosphoenolpyruvate‐Dependent Phosphotransferase System of Staphylococcus aureus
Authors: SCHMIDT‐ADERJAN Ulrike , RÖSCH Paul , FRANK Rainer , HENGSTENBERG Wolfgang
Published: March 2005

Upon nitration of the phosphocarrier protein HPr three nitrated derivatives of the protein were isolated: mononitrated HPr, dinitrated HPr and trinitrated HPr. Tryptic digestion of the derivatives leads to nitrotyrosine‐containing peptides which were isolated and characterized by amino acid analysis. This resulted in the determination of the positions of the nitrated tyrosyl residues in the amino acid sequence. In mononitrated HPr only Tyr‐56 was modified, in dinitrated HPr both Tyr‐56 and Tyr‐37 had reacted with the nitrating agent; modification of all three tyrosyl residues in trinitrated HPr required more drastic reaction conditions.

The nuclear magnetic resonance spectra of the three derivatives allowed the assignments of the tyrosine resonances as follows: Tyr‐A and Tyr‐B with pK values of 10.5 and 11.5 were designated Tyr‐56 and Tyr‐37 whereas Tyr‐C, whose protons are not titratable before denaturation of the protein, was assigned to Tyr‐6 in the amino acid sequence.

The nitration studies, together with the titration behaviour of the three tyrosines, indicate the topology of the tyrosyl residues to be as follows: Tyr‐56 is located at the surface, Tyr‐37 is slightly buried, Tyr‐6 is deeply buried.

The nitrotyrosyl derivatives retain their biological activity.
Procaine, a Local Anesthetic Interacting with the Cell Membrane, Inhibits the Processing of Precursor Forms of Periplasmic Proteins in Escherichia coli
Authors: LAZDUNSKI Claude , BATY Daniel , PAGÈS Jean‐Marie
Published: March 2005

Treatment of Escherichia coli cells with procaine (0.55%, w/v) results in the accumulation of precursor in addition to mature forms of two periplasmic proteins, alkaline phosphatase and glutamine‐binding protein. The precursor form of alkaline phosphatase has a higher molecular weight than the mature form by about 2600. An experimental technique is described to isolate and purify precursor forms of any presumably exported protein. After the membrane solubilization step in the presence of nonionic detergent, a peptidase is stimulated, resulting in partial cleavage of the precursors. The products of this cleavage have been identified as the mature protein and presumably the signal peptide in the case of alkaline phosphatase. The amino acid composition of this peptide, which is comprised of 25 residues, has been determined.

Procaine (0.55%, w/v) causes an increase in molecular packing of lipid molecules in the membrane which might result in an alteration of membrane fluidity sufficient for selective inhibition of processing of precursors of exported proteins.
Kinetic Complexity of Nuclear Poly(A)‐Containing RNA in Normal and Regenerating Rat Liver
Authors: KRIEG Liselotte , ALONSO Angel , VOLM Manfred
Published: March 2005

Poly(A)‐containing RNA isolated from liver nuclei of untreated rats and 3 h or 12 h after partial hepatectomy or sham operation was hybridized to the complementary DNAs (cDNAs). In the homologous reactions two major components could be seen. When compared to normal liver, the complexity of the least abundant class was lower in nuclei from livers 3 h after partial hepatectomy and was higher in those isolated 12 h after operation. The heterologous reactions revealed an increase of some abundant poly(A)‐containing sequences and a loss or dilution of rare sequences 3 h after operation. The latter effect was not specific to the regeneration process but occurred after laparotomy as well. 12 h after partial hepatectomy, however, about 10% new poly(A)‐containing sequences were detected, corresponding to about 5000 molecules of 4500 nucleotides length, which are unique to regenerating nuclei.
Infrared Spectroscopic Investigations of Tuftsin and Its Analogs
Authors: SUCHARDA‐SOBCZYK Anna , SIEMION Ignacy Z. , KONOPIŃSKA Danuta
Published: March 2005

The ability of tuftsin and its analogs to form folded intramolecularly hydrogen‐bonded structures has been investigated by infrared spectroscopy. The appearance of β‐turns is documented for peptides containing proline in position 3 of the chain. Correlation of spectroscopic data with biological activity in the investigated series is attempted.
Physarum Thymidine Kinase A Step or a Peak Enzyme Depending upon Temperature of Growth
Authors: WRIGHT Michel , TOLLON Yvette
Published: March 2005

The variations of thymidine kinase or ATP: thymidine 5′‐phosphotransferase (EC 2.7.1.21) during the cell cycle of Physarum polycephalum plasmodia have been studied at two extreme physiological temperatures: 22°C and 32°C. At 22°C the enzyme activity increases near mitosis and stays constant during late S and G2 phases, exhibiting the typical pattern of a ‘step enzyme’. But at 32°C thymidine kinase activity goes through a maximum 1 h 30 min after mitosis and decreases during the subsequent phases as expected for a ‘peak enzyme’. The rate of enzyme degradation and/or inactivation, measured in the presence of metabolic poisons (cycloheximide or dinitrophenol), appears to follow a simple exponential function with a half‐life of approximately 3 h and 1 h at 22°C and 32°C respectively. The effect of growth temperature on the decrease of thymidine kinase activity can account entirely for the differences in the pattern of enzyme activity at the two extreme temperatures. Tentative calculations indicate that the rate of enzyme synthesis is nearly constant during the cell cycle except near mitosis, where it is temporarily increased. The results suggest the existence of a regulatory mechanism able to modulate the rate of synthesis of thymidine kinase during the cell cycle.
Properties of the Methane Mono‐oxygenase from Extracts of Methylosinus trichosporium OB3b and Evidence for Its Similarity to the Enzyme from Methylococcus capsulatus (Bath)
Authors: STIRLING David I. , DALTON Howard
Published: March 2005

The methane mono‐oxygenase from Methylosinus trichosporium OB3b was soluble. The only suitable electron donor was NAD(P)H, neither sodium l‐ascorbate nor electrons derived from the oxidation of methanol could substitute for NAD(P)H. Evidence is presented for the existence of an NAD+‐linked formaldehyde dehydrogenase.

Mono‐oxygenase activity was not inhibited by a range of potential inhibitors including potassium cyanide, amytal, carbon monoxide or various metal‐chelating agents, although 8‐hydroxyquinoline and ethyne were effective in this respect.

Although the enzyme preparations were unstable on storage, the crude extract could be resolved into two components by ion‐exchange chromatography. Activity could be restored to one of the components on addition of purified components from Methylococcus capsulatus (Bath).

Cross‐reactivity of mono‐oxygenase components and other similarities between the enzymes from M. trichosporium and M. capsulatus are discussed. The properties of the M. trichosporium methane mono‐oxygenase reported here are contrasted with the properties of the same enzyme reported by others.
Permissive Effect of Dexamethasone on Glucagon Induction of Urea‐Cycle Enzymes in Perifused Primary Monolayer Cultures of Rat Hepatocytes
Authors: GEBHARDT Rolf , MECKE Dieter
Published: March 2005

Parenchymal cells from adult rat liver, cultured in perifused monolayers, increased the levels of urea‐cycle enzymes between 15% and 60% in response to glucagon within 24 h. This stimulation was drastically enhanced by the simultaneous presence of dexamethasone, especially in the case of argininosuccinate synthetase and argininosuccinate lyase, which increased nearly threefold. Dexamethasone itself produced only negligible stimulation, but exerted a similar effect on the stimulatory action of glucagon, if it was exclusively present during 6 h prior to the glucagon treatment, suggesting a permissive action of this hormone.

The effect of glucagon, particularly in the presence of dexamethasone, was mimicked by dibutyryl adenosine 3′:5′‐monophosphate, whereas epinephrine was ineffective. All stimulations induced by hormones or dibutyryl adenosine 3′: 5′‐monophosphate were abolished by cycloheximide, suggesting the involvement of protein synthesis in the induction process.

Using the usual culture technique with a discontinuous supply of medium no significant effect of glucagon and dexamethasone could be measured. This striking difference between both culture systems indicates that perifusion is the more adequate in vitro system for studies of the regulation of enzyme levels. Possible reasons for the failure of hormonal stimulation of urea‐cycle enzymes in normal monolayer culture are discussed.
Phytochrome‐Induced Appearance of mRNA Activity for the Apoprotein of the Light‐Harvesting Chlorophyll a/b Protein of Barley (Hordeum vulgare)
Authors: APEL Klaus
Published: March 2005

Illumination of dark‐grown barley plants induces a massive insertion of the light‐harvesting chlorophyll u/b protein into the developing thylakoid membrane. Light induces specifically the appearance of mRNA activity for the apoprotein of this chlorophyll‐binding protein [Apel, K. and Kloppstech, K. (1978) Eur. J. Biochem. 85, 581–588]. This change in mRNA activity can also be induced by a 15‐s red light pulse followed by 4 h darkness. The red light effect is reversed by a subsequent far‐red light treatment. It is concluded that the change in mRNA activity is controlled by phytochrome. In continuous white light the light‐harvesting chlorophyll a/b protein appears within the membrane after a lag period of 4–5 h. This lag period could be partially eliminated by a red light pulse given prior to the onset of continuous illumination. The possible role of the phyto‐ chrome‐induced appearance of mRNA activity as a rate‐limiting step of the greening process is discussed.

The light‐harvesting chlorophyll a/b protein could not be detected among the plastid membrane proteins of plants which had been treated with a red light pulse alone. The assembly of the light‐harvesting chlorophyll a/b protein takes place only under continuous illumination which allows chlorophyll synthesis. Thus, the synthesis and ultimate assembly of this chlorophyll‐binding protein depends on the cooperation of two distinct light reactions.
Endogenous Role of Epoxide Hydratase
Authors: BINDEL Ulla , SPARROW Alan , SCHMASSMANN Hansueli , GOLAN Mario , BENTLEY Philip , OESCH Franz
Published: March 2005

A highly sensitive and rapid radiometric assay for the determination of specific epoxide hydratase activity with a steroid epoxide (16α, 17α‐epoxy‐1,3,5(10)‐estratrien‐3‐ol, ‘estroxide’) has been developed. The unreacted substrate was separated from the product 1,3,5(10)‐estratrien‐3,16β,17α‐triol by extraction into light petroleum. The product was then extracted into ethyl acetate and measured by scintillation spectrometry. Radiochromatography established that after subtraction of the blank the entire radioactivity measured in the ethyl acetate phase resulted from the product 1,3,5(10)‐estratrien‐3,16,17‐triol, whilst high performance liquid chromatography with the four possible isomers of 1,3,5(10)‐estratrien‐3,16,17‐triols as standards showed that the product was the 3,16β,17α‐triol. Limits of linearity with time and protein were established with microsomal fractions from liver, testes and ovaries.

The apparent Km and V values were determined with liver and testes microsomes. The apparent Km for estroxide was very similar in both organs (liver 10 μM; testes 6.3 μM) which was similar to the value obtained for the series of epoxides derived from polycyclic hydrocarbons but substantially lower than that for styrene oxide. The specific activity of epoxide hydratase with estroxide as substrate was about twice as high as with benzo(a)pyrene 4,5‐oxide. The ratio of the specific epoxide hydratase activity with the two substrates was similar (1.6–2.2) in all investigated organs [liver, kidney, lung, testes (ovaries) and adrenals] of either sex of rats. Immunoprecipitation of solubilized liver microsomes by antibodies raised against epoxide hydratase purified to homogeneity was measured with estroxide, benzo(a)pyrene 4,5‐oxide and styrene oxide as substrates. The extent of precipitation of activity towards all three substrates with increasing amounts of added antibody was the same which suggests that all three reactions are catalysed by the same microsomal enzyme.
Subcellular Distribution of Membrane‐Bound Glycosyltransferases from Pea Stems
Authors: DÜRR Mathias , BAILEY David S. , MACLACHLAN Gordon
Published: March 2005

Particulate and subcellular membrane preparations from growing regions of etiolated pea stems catalyse the transfer of sugars from UDP‐[14C]glucose and GDP‐[14C]mannose to a variety of endogenous lipid, glycoprotein and polysaccharide acceptors. Glycolipids were fractionated and identified by chromatography on DEAE‐cellulose and silica gel. They included neutral components, e.g. sterylglycosides, and polar lipids, comprising polyprenylmonophospho‐monosaccharides and polyprenyldiphospho‐oligosaccharides. High‐molecular‐weight material was partially hydrolyzed with pronase or acidic cyanogen bromide in order to solubilize glycoproteins and products bound to proteins, and to separate these from insoluble polysaccharide.

Pea membranes with densities at which endoplasmic reticulum equilibrates in linear sucrose gradients contained most of the recovered capacity for glycosylation of endogenous polyprenyl monophosphate. When dolichyl phosphate was added to the membrane preparations, it was readily glycosylated in the presence of lysophosphatidylcholine but there was no indication that dolichylphospho‐monosaccharide served as an intermediate for synthesis of other products. In contrast, glycosyltransfer to endogenous neutral lipids, polyprenyl diphosphate and polymeric products occurred to a limited extent in the endoplasmic reticulum region and was most extensive in membranes which equilibrate at higher densities, i.e. in regions containing Golgi and possibly plasma membrane vesicles. The data are consistent with the conclusion that glycosylation of polymeric products occurs throughout the pea endomembrane system, with polyprenylmonophospho‐monosaccharide available to act as an intermediate (precursor) primarily in the endoplasmic reticulum.
The Ternary 5‐S RNA Complex of Proteins L18 and L25
Authors: ÖSTERBERG Ragnar
Published: March 2005

The 5‐S RNA (A) and the proteins L18 (B) and L25 (C) from Escherichia coli ribosomes form a ternary complex of the type ABC with a stepwise stability constant, log K111∼6.5. This is indicated from X‐ray scattering titrations recorded at 21°C in ribosomal reconstitutional buffer. When the ternary ABC complex forms there is only a limited change in the scattering curve compared to that of 5‐S RNA, indicating that 5‐S RNA does not undergo a major conformational change during the complex formation. The increase in the radius of gyration from 3.61 nm (5‐S RNA) to 3.95 nm (ABC complex) as well as the experimental scattering curve can be explained by models where it is assumed that the elongated L18 and L25 models are quite far from the electron density centre and where the protein molecules interact mainly with the minor arms of the supposed Y‐shaped 5‐S RNA molecule.
Cytochrome c Oxidase Subunits in Nuclear and Extranuclear Cytochrome‐aa3‐Deficient Mutants of Neurospora crassa
Authors: BERTRAND Helmut , WERNER Sigurd
Published: March 2005

The mitochondria of cytochrome‐aa3‐deficient Neurospora crassa mutants were screened for the seven polypeptide constituents of cytochrome c oxidase. The polypeptides of the holoenzyme and the unassembled or partially assembled subunits were detected by sodium dodecyl sulfate/acrylamide gel electrophoresis of immunoprecipitates obtained with antiserum to the holoenzyme as well as to several individual subunits. With respect to the mitochondrially synthesized polypeptides of the oxidase, subunits 1 to 3, the results obtained from the analysis of immunoprecipitates were confirmed through the direct electrophoretic analysis of mitochondrial translation products. The results were as follows.

The mitochondria of the cya‐2‐8 and cya‐3‐16 nuclear mutants and the [exn‐5] cytoplasmic mutant contained a protein complex immunoprecipitated by anti‐holoenzyme antibody and composed of the complete set of the seven cytochrome oxidase polypeptides. Only the oxidase sub‐units 5 and 6 were immunoprecipitated by anti‐holoenzyme antibody from the mitochondria of the cyt‐2‐1 and 299‐1 nuclear mutants, even though at least some of the mitochondrially synthesized polypeptides were detected in both mutants by subunit specific immunoprecipitation.

A ‘subunit 1’ polypeptide larger than the authentic subunit‐1 polypeptide of wild‐type cytochrome oxidase was found in the mitochondria from two nuclear mutants, cyt‐2‐1, and 299‐1 and the [mi‐3] cytoplasmic mutant. This larger polypeptide may be an unprocessed precursor of the ‘mature’ subunit 1 protein of the holoenzyme. No changes in the apparent molecular weights were found for the polypeptide subunits of cytochrome oxidase in mitochondria of the [exn‐5] cytoplasmic mutant and the cya‐2‐8 and cya‐4‐23 nuclear mutants.

A nuclear mutant, 299‐1, lacks the mitochondrially synthesized subunit‐2 polypeptide of cytochrome oxidase. When cells were labelled in the presence of cycloheximide, the subunit 2 content of mitochondria from mutants [exn‐5], cya‐2‐8, cya‐3‐16 and cya‐4‐23 was lower than in mitochondria from wild‐type. This deficiency, however, does not appear to be sufficiently severe to fully account for the lack of cytochrome aa3 in these mutants. The cya‐4‐23 nuclear mutant either is severely deficient in or lacks cytochrome oxidase subunits 5 and 6.

On the basis of these and previously reported observations, it is proposed that the cytochrome oxidase deficiencies of as many as seven of the eight N. crassa cytochrome‐aa3‐deficient mutants could be caused by genetically imposed alterations in regulatory systems controlling the production of different components of the enzyme.
Synthesis of Cytochrome f by Isolated Pea Chloroplasts
Authors: DOHERTY Andrew , GRAY John C.
Published: March 2005

Chloroplasts, isolated from the leaves of 7‐day‐old pea seedlings, were incubated in the light with [35S]methionine or [3H]leucine. After extraction from the washed chloroplast membranes using a mixture of ethyl acetate, ethanol and ammonia, cytochrome f was precipitated with a monospecific antiserum and resolved by gradient polyacrylamide gel electrophoresis in sodium dodecylsulphate. The cytochrome f band was identified by its intrinsic fluorescence in ultraviolet light and was shown to be radioactive by autoradiography or fluorography of dried polyacrylamide gels. One‐dimensional peptide mapping of the products of papain hydrolysis confirmed that the radioactivity was an integral part of cytochrome f. The incorporation of [35S]methionine into cytochrome f was inhibited by d(–)threo‐chloramphenicol but not by cycloheximide and did not occur in the dark. The synthesis was resistant to ribonuclease. It is concluded that cytochrome f is synthesised in intact isolated pea chloroplasts.