bluuebunny/crossref_metadata_2025_split huggingface dataset:
Turnover of Polyadenylated Messenger RNA in Fission Yeast
Authors: FRASER Ronald S. S. 
Published: March 2005

Polyadenylated RNA was isolated from fission yeast (Schizosaccharomyces pombe) total RNA using oligo(dT)‐cellulose, and was studied as a model for messenger RNA. The half‐life of polyadenylated RNA was measured by two independent methods. (a) The rate of labelling of polyadenylated RNA during incubation of cells with [5 3H]uridine was measured. A half‐life of 40‐45 min was found by comparing the experimental data with theoretical curves calculated for labelling of RNAs with various half‐lives. The influence of precursor‐pool specific activity on RNA labelling kinetics is considered. (b) Cells were labelled with [5‐3H]uridine then further RNA synthesis was inhibited by addition of 8‐hydroxyquinoline. The rate of loss of radioactivity from polyadenylated RNA indicated a half‐life of 50 min. The half‐life found by these two methods is about one‐third of the cell doubling time, and is much longer than previous estimates by indirect methods of yeast messenger RNA half‐life. Both experimental methods provided evidence for the existence of two populations of polyadenylated RNA. The larger population has a half‐life of 40‐50 min; a much smaller population is probably turning over more rapidly.After inhibition of RNA synthesis by 8‐hydroxyquinoline, the rate of total protein synthesis declined much more rapidly than the polyadenylated RNA content of the cells. However, 60 min after inhibition of RNA synthesis there was a small rise in the rate of protein synthesis. These data are interpreted as evidence for mechanisms controlling protein synthesis which operate at the level of messenger RNA translation.
The Tryptophan Synthase from Escherichia coli
Authors: KIRSCHNER Kasper , WISKOCIL Robert L. , FOEHN Martha , REZEAU Laurel 
Published: March 2005

An improved method is described for the purification of the α‐subunit of tryptophan synthase from Escherichia coli. The standard manganese chloride and acid‐precipitation steps have been replaced by rapid and efficient chromatographic procedures.Indoleethanol phosphate, indolepropanol phosphate and indolebutanol phosphate have been synthesized. They are not cleaved by tryptophan synthase and are strictly competitivee inhibitors versus indoleglycerol phosphate. The inhibition constant decreases as the number of methylene groups in the side chain increases. This may reflect an improved accommodation of the indole and phosphate moieties to their respective subsites. The difference spectra generated by binding indole, indoleglycerol phosphate and indolepropanol phosphate to the α‐subunit are very similar. This reflects the transfer of the indole moiety to an hydrophobic environment within the active center. The binding of indolepropanol phosphate to the α2β2‐complex perturbs the spectrum of pyridoxal 5′‐phosphate located in the β2‐subunit. This demonstrates direct or indirect interactions between the component active sites.Binding studies by spectrophotometric titration and equilibrium dialysis with indolepropanol [32P]phosphate show that there is only one binding site per equivalent of α‐subunit. Complex formation with the β2‐subunit increases the affinity of the α‐subunit for indolepropanol phosphate. It is a general consequence of protein‐protein interaction in this system.
Heterogeneity of Human Milk β(1 – 4)‐d‐Galactosyltransferase
Authors: PRIEELS Jean‐Paul , MAES Eddy , DOLMANS Marcel , LEONIS José 
Published: March 2005

β(1 – 4)‐Galactosyltransferase from human milk (the A protein of lactose synthase) has been found to be heterogeneous when fractionated by affinity chromatography against insolubilized a‐lactalbumin, using a linear gradient of decreasing N‐acetylglucosamine concentration. Three forms were isolated. Molecular weights of the different species, as determined by sodium dodecylsulphate gel electrophoresis, were found to be 38000, 43000 and 50000. The 38000 and 50000 species were studied for their catalytic ability to synthesize either lactose in the presence of α‐lactalbumin, or N‐acetyllactosamine in the presence and absence of the ‘specifier’ protein.Appreciable difference was observed between the two enzyme forms with respect to their catalysis of lactose synthesis with a‐lactalbumins from various sources. Differences in the rate of production of N‐acetyllactosamine in the presence of α‐lactalbumin were also observed. For the lowest‐molecular‐weight species it was found that the inhibitory effect of a‐lactalbumin upon N‐acetyllactosamine syn‐ thesis becomes an activating effect at higher α‐lactalbumin concentrations, while no such inversion was observed for the other species.The results suggest that the conformation at the site of association of the enzyme with the acceptor saccharide or α‐lactalbumin has been changed, presumably by a partial enzymic hydrolysis.
Nitration of Tyrosyl Residues in Human α‐Lactalbumin
Authors: PRIEELS Jean‐Paul , DOLMANS Marcel , LEONIS José , BREW Keith 
Published: March 2005

α‐Lactalbumin isolated from human milk was reacted with tetranitromethane in molar excess of 8–32 mol/mol of tyrosine. After gel filtration on Sephadex G‐75, followed by chromatographic fractionation using DEAE‐Sephadex A‐25, three main components were separated, which differed from one another in the extent of nitration.These protein fractions were found to contain, respectively, one and two nitrotyrosine residues, or two nitrotyrosine residues together with one nitrotryptophan. The lactose synthase specifier activity of each of these components was measured and compared with that of unsubstituted cr‐lactalbumin.Comparison of kinetic parameters showed the chemically modified proteins to be only slightly less active when tyrosines were the sole residues modified. In sharp contrast the additional nitration of a single tryptophan residue totally abolished the specifying activity of α‐lactalbumin.Circular dichroism spectra of the tryptophan derivative revealed some structural alteration when compared with the other two and with the native protein. The conclusion could also be confirmed by using a double‐immunodiffusion technique. After hydrolysis of the derivatives with thermolysin, it was possible to localize the substituted residues in the known sequence of human a‐lactalbumin. Tyrosine‐103 was found to be more easily nitrated than tyrosine‐18. These two residues seem, there‐fore, to be on the outer surface of the molecule and more exposed than tyrosine‐36 and tyrosine‐50.Some precautions are indicated in the use of tetranitromethane as a nitrating agent on the basis of complex products observed in the nitration of the free amino acids tyrosine and tryptophan and their derivatives.
Some Actions of Purified Toxins from Bungarus fasciatus Venom on Isolated Pig‐Heart Mitochondria
Authors: LEBLANC Pierre , LIEVREMONT Maurice 
Published: March 2005

In the presence of succinate as an oxidation substrate, neurotoxins α, β and γ induce the following. Firstly, an increasing stimulation of oxygen uptake, which is potentiated by 25 μM Ca2+. Mg2+ 1.3 mM completely inhibits the effect of toxin α but not of toxins β and γ. Secondly, a depletion of the Mg2+, Ca2+ and K+ content of the water‐soluble and membrane‐bound mitochondrial compartments, following complex kinetics, which suggest a multistep interaction mechanism of the toxins with the mitochondria. 25 μM Ca2+ also potentiates the effect of the toxins on these ionic flows. Thirdly, no decrease of turbidity with toxin α, and a limited decrease with toxins β and γ.
In the absence of respiration, the neurotoxins induce a cationic depletion, the kinetics of which are different than with succinate, suggesting an instantaneous maximal effect on the inner mem‐brane. Toxins β and γ (but not α) induce, under these conditions, a turbidity decrease of large amplitude, which is proportional to the amount of toxin added and tends to reach a maximum. With γ toxin this turbidity decrease is faster than the rate of water uptake (which never exceeds 18%) indicating that it is due rather to structural modifications than to swelling. The same is observed with β toxin, provided the mitochondria1 protein concentration to be lower than 0.7 mg/ml. For higher concentrations, a continuous decrease of turbidity together with a considerable uptake of water probably reflects the onset of phospholipasic activities.
It is postulated that structural modifications of the mitochondrial membranes are initiated which lead to the loss of their selective impermeability. The simultaneous loss of respiratory control with succinate may be due to the direct (though Ca2+‐potentiated) displacement of the fraction of the membrane‐bound Mg2+ ions which controls its energy‐transducing properties.
In addition, correlations between the effects of the toxins on mitochondria and their neuro‐toxicity in vivo are discussed.
Biosynthesis of the 09 Antigen of Escherichia coli
Authors: KOPMANN Heinz J. , JANN Klaus 
Published: March 2005

The O9 antigen of Escherichia coli O9 consists of a mannan, bound to the core oligosaccharide and lipid A. The mannan contains α‐1,2‐ and α‐1,3‐linked mannose in an unknown sequence. The serological specificity of the O9‐antigen is not expressed through the mannan but through an immuno‐dominant group in which isopropylidene ribose seems to play a role.From a non‐capsulated (K−) form of E. coli O9:K29(A):H− a mutant lacking phosphomannose isomerase pmi−) was selected. Membrane preparations were obtained from this mutant using plasmolysis, spheroplasting and sonic rupture of the spheroplasts. Cytoplasmic and outer membranes were separated by sucrose gradient centrifugation.Incorporation in vivo of [14C]mannose into late log phase bacteria resulted in the formation of complete lipopolysaccharide (O9 antigen) which accumulated to a large extent on the cytoplasmic membrane before being exported to the outer membrane. The product was extracted with 45% aqueous phenol and characterized by chemical analysis, dodecylsulfate polyacrylamide gel electro‐ phoresis, gel permeation chromatography and antigen binding using anti‐O9 antiserum.Incorporation in vitro from GDP‐[14C]mannose into membrane mixtures or isolated cytoplasmic membrane resulted in the formation of the mannan part of the lipopolysaccharide which was not substituted by the determinant group and was not transferred to the core‐lipid A part. The product, isolated with 45% aqueous phenol, was characterized as was the product in vivo. Furthermore, it was subjected to methylation analysis with the same results as obtained with authentic O9 (lipo)poly‐saccharide. By monitoring the formation of radioactive polymer the over‐all reaction was found to have an apparent Km= 5 × 10−5 moles per 1, based on GDP‐mannose. It was found that the preferred activated form of mannose in the 09 antigen synthesis is GDP‐mannose, other purine or pyrimidine bases being much less effective; it was also found that the reaction is not dependent on bivalent cations such as Mg2+ or Mn2+, these ions even inhibiting the reaction partially at concentrations above 5 mM; it was further found that bacitracin as well as EDTA are not inhibitory. All attempts to extract an intermediate into organic solvents were unsuccessful. Oligosaccharides could not be isolated at any stage of the synthesis, starting 5 s after the addition of GDP‐[14C]mannose.In pulse‐chase experiments in vitro it was found that an intermediate was formed which can be detected by dodecylsulfate polyacrylamide gel electrophoresis. With increasing incubation time this material shows decreasing charge to mass ratios. Treatment of the incubation mixture with mild acid or mild alkali prior to dodecylsulfate polyacrylamide gel electrophoresis showed that the radio‐ active material (mannan) was bound to a carrier through an alkali‐stable but acid‐labile linkage. The nature of the carrier is discussed.From the results it is concluded that the O9 mannan is synthesized in a single chain mechanism which does not utilize lipid‐bound oligosaccharides.
Purification and Properties of an Esterase B4 from Human Liver
Authors: COATES Paul M. , EDWARDS Yvonne H. , HOPKINSON David A. 
Published: March 2005

A butyryl esterase, designated B4, has been purified from human liver and some of its properties described. The activity of this enzyme comprises 0.48 of the total butyryl esterase activity found in human liver.Esterase B4 has been distinguished from other butyryl esterases by its preference for the esters of the fluorogenic compounds 4‐methyl umbelliferone and fiuorescein over naphthyl esters as substrates. Other distinguishing features of this esterase include a relatively high pI (pH 8.7) a monomeric structure of low molecular weight (20000) and high solubility in solutions of ammonium sulphate.
Preparation and Characterization of the Active Derivative of Bovine Trypsin‐Kallikrein Inhibitor (Kunitz) with the Reactive Site Lysine‐15 – Alanine‐16 Hydrolyzed
Authors: JERING Helmut , TSCHESCHE Harald 
Published: March 2005

The derivative of the trypsin‐kallikrein® inhibitor (Kunitz), TKI+, was prepared with the reactivesite peptide bond Lys‐15‐Ala‐16 hydrolyzed. This was achieved by selective borohydride reduction of the Cys‐14‐Cys‐38 disulfide bond, followed by tryptic cleavage of the reactive‐site peptide bond, air reoxidation of the half‐cystine residues, and purification by ion‐exchange chromatography. The derivative corresponds to the hypothetical ‘modified’ inhibitor TKI+, which so far could not be obtained from virgin inhibitor by a direct modification reaction (partial proteolysis).The derivative isolated was homogeneous as revealed by amino acid analysis, disc electrophoresis, inactivation by carboxypeptidase B, and inactivation by sodium borohydride reduction. The inhibitory activity of the sodium‐borohydride‐reduced inhibitor was fully recovered after air reoxidation. The site of cleavage in the inhibitor was confirmed by performic acid oxidation and subsequent isolation of the two corresponding peptides containing residues 1–15 and 16–58 of the entire polypeptide chain. From several aminopeptidases tested only aminopeptidase K rapidly cleaved Ala‐16 and Arg‐17 from the modified inhibitor and at a reduced rate Ile‐18. Des‐(Ala16,Arg17)‐inhibitor and des‐Ala16‐inhibitor are both lacking a strong inhibitory activity against bovine trypsin. This indicates a decrease in the association constant by factor of at least 108 ‐1010. The reactive‐site‐modified inhibitor is not subject to further enzymic breakdown and therefore is a permanent inhibitor of trypsin. However, the modified inhibitor forms the inactive complex much slower than virgin inhibitor.In the modified inhibitor the hydrolyzed peptide bond was resynthesized to yield virgin inhibitor by forming the complex with trypsin and subjecting the complex to kinetic control dissociation. This proves that the bond Lys‐15‐Ala‐16 is at the reactive site of this inhibitor. Preparation of a modified and still active inhibitor (Kunitz) is in agreement with the general model proposed for the interaction of proteinase‐inhibitor‐proteinase interactions. This presents new evidence that this model is generally applicable.
A Mutant Strain of Chlamydomonas reinhardi Exhibiting Altered Ribulosebisphosphate Carboxylase
Authors: NELSON Patricia E. , SURZYCKI Stefan J. 
Published: March 2005

A mutant, ac i72, of Chlamydomonas reinhardi possessing an altered ribulosebisphosphate carboxylase and unable to grow on minimal medium has been isolated and characterized. Comparison of ribulosebisphosphate carboxylase purified from both wild type and ac i72 strains is given. The enzyme from ac i72 shows alterations in several characteristics : (a) the specific activity is reduced to 35% that of wild type, (b) the V for both substrates is reduced 3–6‐fold, (c) the Mg2+ requirement for maximal activity is 3 times greater, (d) the inhibitory effect of Cl− is greater, and (e) the isoelectric point is changed (6.0 for wild type and 5.8 for ac i72). However, the ribulosebisphosphate carboxylase from ac i72 is identical to that from wild type with respect to pH requirement, temperature sensitivity, subunit structure, and sedimentation characteristic.Other photosynthetic properties of wild type and ac i72 cells were also compared. CO2 fixation in ac i72 in vivo is reduced proportionally to the reduction in activity of the enzyme, but the level of O2 evolution is the. same as in wild‐type cells. Photosynthetic electron transport, 70‐S ribosome content, and chlorophyll content are unaltered in ac i72. The chloroplast ultrastructure of ac i72 cells is distinctly different from that of wild‐type cells. The inheritance of the mutation is Mendelian.
Purification and Characterization of Dopamine β ‐Hydroxylase from Bovine Adrenal Medulla
Authors: LJONES Torbjørn , SKOTLAND Tore , FLATMARK Torgeir 
Published: March 2005

A new purification procedure that permits large‐scale purification of dopamine β‐hydroxylase from bovine adrenal medulla was developed. Whole adrenal medullas were extracted with 0.1 Triton X‐ 100, and the enzyme was purified by precipitation with polyethylene glycol, chromatography on DEAE‐cellulose, and adsorption to concanavalin A linked to agarose. The yield of protein and the specific activity were high compared with previously published methods. The enzyme appeared essentially homogenous by the criteria of polyacrylamide gel electrophoresis in the presence or absence of dodecylsulfate, and sedimentation velocity analysis.The purified protein was subjected to amino acid and carbohydrate analyses, and the results were compared with previously published data. We found about 3 mol of copper per mol of protein (tetramer of 290000 daltons). No free sulfhydryl groups could be found. Analysis for NH2‐terminal amino acids with [14C]dansyl chloride revealed 2 residues of alanine and 2 residues of serine per tetramer. We found the NH2‐terminal amino acid of chromogranin A to be leucine. The results of our analysis for amino acid composition and NH2‐terminal amino acids do not support the suggestion that dopamine β‐hydroxylase and chromogranin A contain identical peptide chains.
Influence of Unsaturated Fatty Acids in Chloroplasts
Authors: SIEGENTHALER Paul‐André , DEPÉRY Françoise 
Published: March 2005

Linolenic acid (C18:3) is the main endogenous unsaturated fatty acid of thylakoid membrane lipids, and seems in its free form to exert significant effects on the structure and function of photosynthetic membranes. In this investigation the effect of linolenic acid was studied at various pH values on the electron flow rate in isolated spinach chloroplasts and related to ΔpH, the proton pump and the pH of the inner thylakoid space (pHi). The ΔpH and pHi were estimated from the extent of the fluorescence quenching of 9‐aminoacridine.Linolenic acid caused a shift (approximately one unit) of the pH optimum for electron flow toward acidity in the following systems: (a) photosystems II + I (from H2O to NADP+ or to 2,6‐dichlorophenolindophenol) coupled or non‐coupled; (b) photosystem II (from H20 to 2,6‐dichloro‐phenolindophenol in the presence of dibromothymoquinone).In photosystem I conditions (phenazine methosulphate), the ΔpH of the control increased as a function of external pHo with a maximum around pH 8.8. When linolenic acid was added, the ΔpH dropped, but its optimum was shifted toward more acidic pHo. The same phenomena were also observed in photosystems II + I (from H2O to ferricyanide) and in photosystem II conditions (from H2O to ferricyanide in the presence of dibromothymoquinone). However, the ΔpH was smaller and the sensitivity of the proton gradient toward linolenic acid was eventually higher than for photosystem I electron flow activity. The proton pump which might be considered as a measure of the internal buffering capacity of thylakoids was optimum at pHo 6.7 in the controls. An addition of linolenic acid diminished the proton pump and shifted its optimum toward higher pHo.As a consequence, pHi increased when pHo was raised. At the optimal pHo 8.6 to 9, pHi were 5 to 5.5. Additions of increasing concentrations of linolenic acid displaced the curves toward higher pHi. A decrease of pHo was therefore required to maintain the pHi in the range of 5 ‐ 5.5 for maximum electron flow.In conclusion, the electron flow activity seems to be delicately controlled by the proton pump (buffer capacity), ΔpH, pHi and pHo. Fatty acids damage the membrane integrity in such a way that the subtle equilibrium between the factors is disturbed.
Protein Degradation and Proteinases during Yeast Sporulation
Authors: BETZ Heinrich , WEISER Ulrike 
Published: March 2005

During ascospore formation in Saccharomyces cerevisiae, at least 60–70% of the pre‐existing vegetative protein was broken down at a rather constant rate until the time mature asci appeared. Under the same conditions in a non‐sporulating haploid derived from the same strain the rate of protein degradation, although initially comparable to that of sporulating cells, decreased much more rapidly. Proteins synthesized at different times during sporulation had approximately the same degradation rates as the vegetative proteins. Similar rates of degradation were observed for the vegetative proteins in all fractions obtained from cell homogenates by differential centrifugation.Protein breakdown after transfer to sporulation medium was blocked by uncouplers and inhibitors of energy metabolism, and was partially inhibited by cycloheximide.Polyacrylamide gel electrophoresis, in the presence of sodium dodecylsulfate, of the proteins extracted from vegetative cells and from isolated asci and ascospores revealed that ascus formation was accompanied by a shift of the cellular proteins to a lower molecular weight.From several proteinase inhibitors tested, only tosyl‐p‐lysine chloromethylketone slightly reduced the rate of ascus formation. During sporulation the total activity of proteinase A increased more than twofold with a maximum at 18 h after transfer to sporulation medium. Total proteinase B activity showed a striking increase in the first hours after transfer to sporulation medium and after that remained constant throughout sporulation. The levels of carboxypeptidase Y and of the proteinase B inhibitor were not significantly altered during sporulation. The proteinases and the proteinase B inhibitor were present within the mature ascospore. The proteinases, from both vegetative and sporulating cells were eluted with the same ionic strength from DEAÉ‐Sephadex, and they were undistinguishable in their sensitivity to different proteinase inhibitors. No additional proteolytic activities could be detected in sporulating cells using 3H‐labelled denatured yeast protein as a substrate.
Regulation of Cyanide‐Insensitive Respiration in Neurospora
Authors: EDWARDS David L. , ROSENBERG Emiko 
Published: March 2005

Inhibition of either mitochondrial transcription or translation in Neurospora crassa results in the rapid production of the cyanide‐insensitive pathway of mitochondrial respiration. Protein synthesis an cytoplasmic ribosomes is required for the appearance of cyanide‐insensitive respiration in the culture.
Removal of the inhibition of transcription or translation results in a rapid return to cyanide‐sensitive respiration. Additional protein synthesis in the cytoplasm is required for the loss of cyanide‐insensitive respiratory activity while additional mitochondrial protein synthesis has no effect.
These studies indicate that a mitochondrial gene product is involved in a negative manner in the regulation of cyanide‐insensitive respiration. When the mitochondrial product is present, the pathway is not expressed, when the mitochondrial product is absent, the pathway is expressed.
Studies with forced heterokaryons formed from respiratory‐deficient mutants having cyanideinsensitive respiration and respiratory‐competent auxotrophs indicate that the site of action of the mitochondrial gene product is external to the mitochondrion.