bluuebunny/crossref_metadata_2025_split huggingface dataset:
Transport of 2‐Keto‐3‐deoxy‐D‐gluconate in Isolated Membrane Vesicles of Escherichia coli K12
Authors: Lagarde Alain E. , Stoeber François R. 
Published: March 2005

Escherichia coli membrane vesicles isolated from strains derepressed for 2‐keto‐3‐deoxy‐d‐gluconate transport system were found to be able to take up 2‐keto‐3‐deoxy‐d‐gluconate and d‐glucuronate when an energy source was added. The artificial pair ascorbate plus phenazine methosulfate and the physiological electron donors d‐(—)‐lactate and dl‐glycerol 3‐phosphate specifically stimulated transport activity.
Vesicular uptake was lacking in kdgT– mutants and was restored in membranes from a revertant strain. Km values for 2‐keto‐3‐deoxy‐d‐gluconate and for d‐glucuronate closely agree with those previously determined in whole cells. From the additive fact that no binding activity was detected in the periplasm, it was concluded that all 2‐keto‐3‐deoxy‐d‐gluconate transport activity coded by the kdgT gene was restricted to membrane.
By recording difference spectra between oxidized and reduced membrane vesicles, l‐glycerol 3–phosphate oxidation was shown to follow the terminal cytochrome chain common to d(–)‐lactate and other substrates. d‐(–)‐Lactate and d‐glycerol 3‐phosphate oxidation primarily occurs via two membrane‐bound, flavin‐linked specific dehydrogenases and this step was found to be rate‐limiting for 2‐keto‐3‐deoxy‐d‐gluconate uptake in vesicles.
2‐Keto‐3‐deoxy‐d‐gluconate uptake is inhibited by classical electrontransfer inhibitors, by proton‐conducting compounds and thiol reagents. Except for oxamate and rotenone, all inhibitors induced rapid and complete efflux of previously accumulated 2‐keto‐3–deoxy‐d‐gluconate. Considering the site of action of these inhibitors along the respiratory chain it was concluded that (a) the site coupling electron flux to substrate uptake was lying between the primary dehydrogenase and cytochrome b1; and that (b) the carrier coupling site or a coupling component, yet speculative, rather than the recognition site for 2‐keto‐deoxygluconate had active sulfhydryl groups.
The Biochemical Requirements for Bacterial Division
Authors: Pardee Arthur B. 
Published: March 2005

We are searching for biochemical events related to the process of bacterial division. Data obtained with intact bacteria, using inhibitors primarily, suggest that protein synthesis must occur through most of the division cycle in order that a bacterium can divide at the end of the cycle. Hence, one approach is to look for a protein that is made throughout the division cycle and whose production is related to ability of the bacteria to divide. Dr Masayori Inouye modifled the ability of the bacterium Escherichia coli to divide, using mutants, inhibitors or nutritional deficiencies, and then looked for alterations in the pattern formed by electrophoresis of membrane proteins on polyacrylamide gels. One protein peak on the gels was strongly increased whenever the cells were prevented from dividing. We named this protein “X”; it has a molecular weight of about 50000.A role was sought for the protein made throughout the cycle but showing its effect only at the end. Earlier observations by Dr Helene Smith showed that brief heating of the bacteria (16 min at 45 °C) produced synchronous cell division; heating delayed division in a way suggesting that a protein needed for division had been damaged and must be resynthesized before division occurs. Damage to this protein causes the cells to swell; this swelling appears to be the prime basis for the delay of cell division, as shown recently by Dr Po Chi Wu. The swelling caused by an osmotic shift similarly delayed cell division; a result that is support for this hypothesis. A model is proposed for the role of the X protein as a sort of “girdle” that must be built up during the cycle, and that constricts the cells at the time of division.
Distribution of Newly Synthesized Histones during DNA Replication
Authors: Tsanev Roumen , Russev Georgi 
Published: March 2005

A procedure has been developed which allows the separation of chromatin into two fractions highly enriched in preexisting and in newly synthesized DNA, respectively. The procedure includes the following steps. (a) Partial substitution of thymine with 5–bromouracil in the newly synthesized DNA strand. (b) Labelling of proteins with 14C‐labelled amino acids and DNA with [3H]thymidine during the period of DNA replication. (c) Introduction of breaks in the new DNA strand at the points containing 5‐bromouracil by irradiation at 313 nm. (d) Denaturation of chromatin at pH 12.2 and separation of the intact from the fragmented material by centrifugation in alkaline sucrose density gradient. (e) Purification of the two fractions by a recentrifugation under the same conditions.The results from such experiments have shown that during DNA replication the preexisting histones remain bound to the parental DNA strand while the newly synthesized histones join the new DNA strand.
Studies on Seed‐Oil Triglycerides
Authors: Gurr Michael I. , Blades Jon , Appleby Robert S. , Smith Colin G. , Robinson Malcolm P. , Nichols Bryan W. 
Published: March 2005

Crambe seeds harvested 6—30 days after flowering actively incorporated radioactive precursors into lipids. The distribution of label among the different lipid classes depended on the precursor, the age of the seed, and the duration of the incubation.
Labelled acetate was incorporated preferentially into phospholipids of whole seeds but the proportion of the radioactivity located in triglycerides increased as the age of the seed increased.
Uniformly labelled glucose was incorporated readily into the glycerol moiety of glycerolipids but not at all into the fatty acid moiety. The largest proportion was found in the phospholipid fraction and this proportion increased as the age of the seed increased, in contrast to labelled acetate.
Uniformly labelled glycerol was actively incorporated into glycerolipids of whole seeds. Phospholipids were the first fraction to be labelled, and in this fraction labelled phosphatidic acid could be detected at early time periods. Subsequently, diglycerides became labelled and finally triglycerides. Labelled glycerol was quickly converted into a number of water‐soluble metabolites, one of the most rapidly labelled of which was glycerol phosphate. Taken together these data are consistent with the presence of the glycerol phosphate pathway for triglyceride synthesis in maturing Crambe seeds.
A fat fraction has been isolated from mature and maturing Crambe seeds by homogenisation of the seeds followed by flotation in the centrifuge. Electron microscopic examination indicated that the fraction contains the oil bodies that are the storage form of the oil in the seed in vivo.
The isolated oil bodies contain 75% by weight of lipid and 19% of protein. About 90% of the lipid is triglyceride, the remainder is mostly phospholipid. No glycolipid is present.
The isolated fat fraction catalysed the conversion of different substrates, malonyl‐CoA, long chain acyl‐CoA and glycerol 3‐phosphate into lipids. Evidence is presented that this is not due to contamination from other cell fractions.
Mößbauer Effect and Electron Spin Resonance of the (Iron)4‐Sulfur Clusters of Ferredoxin from Clostriclium pasteuriunum
Authors: Gersonde Klaus , Schlaak Heinz‐Eberhard , Breitenbach Michael , Park Fritz , Eicher Hermann , Zgorzalla Werner , Kalvius Michael G. , Mayer Aldabert 
Published: March 2005

Ferredoxin from Clostridiurn pasteurianum which is characterized by two clusters containing four sulfur‐linked iron atoms each, was studied by MöBbaner and elect)ron spin resonance spectroscopy.The iron of oxidized ferredoxin is high‐spin Fe(III) (S=5/2) coupled antiparallel magnetically as conaludcd from the magnitude of the quadrupole splitting and its slight temperature dependence. Thus the net spin of the cluster is 8=0. This explains why no magnetic hyperfine splitting was observed in the Mößbauer absorption spectrum of oxidized ferredoxin at 4.2 K. The observation of at least two quadrupole doublets with a ratio of intensities deviating from unity points to a slight inequivalence of the ligand field symmetry of the individual iron atoms.Reduction of oxidized ferredoxin leads to an uptake of one electron per cluster. The net electron spin of each cluster of reduced ferredoxin is thus S=1/2. Spin‐spin coupling of both the clusters prevents the appearance of a magnetic hyperfine splitting at low temperature. In an external magnetic field of 20 kG the coupling is removed and a hyperfine structure is observed.Under the conditions of X‐band electron spin resonance spectroscopy, i.e. in a magnetic field of 3.5 kG, the spin‐spin coupling is not totally removed. Thedore, an anisotropic 7‐line spectrum originating from a system with S = 1, is observed. Under chemical conditions producing partly reduced ferrcdoxin a 2‐line spectrum from the uncoupled S=1/2 systems is also found. It indicates axial symmetry of the innermolecular electric field. Möβhauer spectroscopy enabled the detection of various amounts of non‐cluster iron in samples of oxidized and reduced ferredoxin.
The Reactivation of C55‐Isoprenoid‐Alcohol‐Phosphokinase Apoprotein by Lipids
Authors: Sandermann Heinrich 
Published: March 2005

The reactivation of C55‐isoprenoid‐alcohol phosphokinase apoprotein by a variety of lipids has been investigated.
A considerable lack of specificity for the chemical structure of the activating lipid is demonstrated.
Reactivation appears to occur by some physical mechanism. An electrostatic interaction of protein and lipid does not appear to be important.
All active lipids found are able to swell and become hydrated in aqueous buffer. Some of these lipids were active only after sonication or in the presence of detergent.
The hydrated lipid phase is concluded to activate by solubilization, as a liquid‐crystalline or micellar solvent for the extremely hydrophobic enzyme apoprotein and for the non‐hydrating C55‐isoprenoid alcohol.
Lateral Mobility and Surface Density of Lipopolysaccharide in the Outer Membrane of Salmonella typhimurium
Authors: Mühlradt Peter F. , Menzel Johannes , Golecki Jochen R. , Speth Volker 
Published: March 2005

Salmonella typhimurium 1195 mutant bacteria were allowed to produce clusters of lipopolysaccharide with new, wild‐type specificity over the export sites. Ferritin‐labeling experiments showed that the lipopolysaccharide diffuses away from the export sites at 37 °C and is found dispersed on the cell surface, whereas at 0 °C it remains immobile.The density of lipopolysaccharide molecules on the bacterial surface, measured as number of lipopolysaccharide molecules per μm2, or as percentage of the bacterial surface covered by lipopolysaccharide was determined in two wild‐type S. typhimurium strains and one mutant strain, which was grown under conditions either permissive or nonpermissive for the formation of wild‐type lipopolysaccharide. There are 0.7—1.0 × 105 lipopolysaccharide molecules per μm2 surface area. About 15—25% of the bacterial surface was found to be covered by lipopolysaccharide. The mean intermolecular distance at hypothetical uniform distribution of lipopolysaccharide was calculated to be in the range of 3—4 nm. It was of no consequence to the lipopolysaccharide surface density whether the mutant strain produced wildtype or mutant lipopolysaccharide.An estimate of the lateral diffusion constant D (D∼ 3 × 10−13 cm2/s) of lipopolysaccharide in the outer membrane was made from earlier published data [2]. Possible reasons for the low order of magnitude of D, based on the observed surface density, are discussed.
Purification and Properties of Ovalbumin Messenger RNA
Authors: Haines Michael E. , Carey Norman H. , Palmiter Richard D. 
Published: March 2005

Ovalbumin messenger RNA was purified 67‐fold from hen oviduct polysomal RNA by sucrose gradient centrifugation and oligo(dT)‐cellulose hybridization. Ovalbumin messenger RNA activity was coincident with the main peak of absorbance after acrylamide gel electrophoresis of the purified RNA. Each purified messenger was translated over 20 times in a rabbit reticulocyte lysate, protein‐synthesizing system. An apparent molecular‐weight of approximately 875 000 was obtained by electrophoresis under both native and denaturing (formamide) conditions. In contrast, on sucrose gradients this messenger sedimented at approximately 15 S, corresponding to a molecular weight of about 550 000. Under certain conditions, ovalbumin messenger RNA activity was observed on both gels and gradients at a position approximately double these molecular weights. The larger messenger RNA species could be dissociated to the smaller one by either heating at 65 °C or formamide treatment, thus demonstrating messenger RNA aggregation.We conclude that ovalbumin messenger RNA contains between 1670 and 2640 nucleotides; a value significantly greater than the number (1161) that are translated into ovalbumin.
l‐Phenylalanyl‐tRNA Synthetase of Escherichia coli K‐10
Authors: Hanke Till , Bartmann Peter , Hennecke Hauke , Kosakowski Heinz M. , Jaenicke Rainer , Holler Eggehard , Böck August 
Published: March 2005

The molecular weight of l‐phenylalanyl‐tRNA synthetase was redetermined. A new value of 267 000 was obtained by high‐speed analytical ultracentrifugation and Sephadex G‐200 gel chromatography. The value is in contrast to the molecular weight of 180000 determined previouslyFrom the molecular weight 267000 and the sedimentation coefficient s20,w= 8.6 S the ratio f/f0= 1.67 was calculated. The magnitude of f/f0 is in accord with the molecular weight 183000 obtained from sucrose gradient centrifugation. The results indicate that the enzyme is highly hydrated and/or assumes the shape of a rod or a disc.The subunit composition was reinvestigated by sodium dodecylsulfate gel electrophoresis, guanidinium chloride gel filtration and by reactivation experiments of pH‐2 inactivated enzyme. The results are consistent with a subunit structure of the α2β2 type. Molecular weights of the subunits are 39000 and 94000, respectively.
Glycoproteins of Chick Embryo Fibroblasts in Cultures with a Finite Life Span
Authors: Courtois Yves , Hughes R. Colin 
Published: March 2005

Chick embryo fibroblasts, that cease to grow after a finite number of passages in culture, have been cultured in the presence of radioactive d‐glucosamine. Incorporation of radioactivity into cellular macromolecules is very similar for cells regardless of their position in the finite life span.
The labelled substances removed by trypsin from the surface of non‐confluent cells growing in different parts of the life span were analysed. Smooth membrane fractions isolated from trypsinised cells were also compared.
The labelled substances were very similar, in particular the extent of sialylation of the total glycoprotein fraction of early phase or late phase cells. Minor differences were found, however.
It was found that mucopolysaccharides constitute a larger proportion of the labelled material from the surface of late phase cells than from the surface of early phase cells. Conversely early phase cells were relatively rich in several glycopeptides compared to late phase cells.
The Rate‐Limiting Step of Adrenal Steroidogenesis and Adenosine 3′:5′‐Monophosphate
Authors: Kahnt Friedrich W. , Milani Alfred , Steffen Hans , Neher Robert 
Published: March 2005

The rate‐limiting step of adrenal steroidogenesis is first defined in detail. The kinetics of the conversion in vitro of double‐labeled exogenous cholesterol as potential precursor by intact and ruptured bovine adrenocortical mitochondria are compared under identical conditions. The results strongly suggest that the transport of cholesterol from the outside to the inside of the mitochondrial membrane is the proper rate‐limiting process and not the subsequent enzymatic conversion of cholesterol to steroids.The involvement of a transport process is supported by findings such as increase of conversion rates in intact mitochondria by (a) preincubation of exogenous precursor with mitochondria; (b) addition of carrier proteins such as non‐defatted bovine serum albumin as against proteins strongly binding cholesterol; (c) addition of Ca2+ concentrations sufficient to increase membrane permeability.The immediate stimulation by adrenocorticotrophic hormone of adrenocortical steroidogenesis seems to be mediated by cyclic AMP formed intracellularly. Such a messenger may be considered to act directly or indirectly upon the rate‐limiting step. It was found that the presence of cyclic AMP (1–5 mM) or cyclic monobutyryl AMP (0.04–1 mM) under optimal and various suboptimal conditions of cholesterol utilization did in no instance increase the conversion rate nor did it influence the utilization of endogenous precursor in intact mitochondria.It is concluded that cyclic AMP has no direct effect on (a) the transport process for exogenous cholesterol; (b) the intramitochondrial enzymatic conversion; (c) the utilization of endogenous steroid precursor. If cyclic AMP is involved, its action seems to have a premitochondrial localization and to be related indirectly only to a transport mechanism or a precursor supply process.
Hexokinase of Human Erythrocytes
Authors: Gerber Gerhard , Preissler Heidemarie , Heinrich Reinhart , Rapoport Samuel M. 
Published: March 2005

Hexokinase was purified 680‐fold from erythrocytes of man. The preparation was not contaminated by other glycolytic enzymes.Initial reaction rate measurements were performed at pH 7.2, ionic strength 0.15 and 37°C by use of coupled optical assays. The experimental data were used to construct a kinetic model of the enzyme, the kinetic parameters of which were estimated by means of a computer‐fit program.The hexokinase of erythrocytes operates by a rapid‐equilibrium random mechanism. The dissociation constant of the E · glucose complex is 0.038 to 0.054 mM, of the E · MgATP complex 1.0 to 2.1 mM. Uncomplexed Mg2+ up to 4 mM is capable of activating the enzyme velocity twofold (KMg2+= 1.0 mM). An inhibition was observed at higher Mg2+ concentrations. α‐d‐Glucose 6‐phosphate and α‐d‐glucose 1,6‐bisphosphate inhibit the hexokinase competitive to MgATP with dissociation constants of 0.069 mM. The enzyme is also inhibited by 2,3‐bisphosphoglycerate competitive with respect to MgATP with a KP2G= 2.7 mM. An inhibition by uncomplexed ATP was not detected.The parameters of the kinetic model of the enzyme in conjunction with the free intracellular Concentrations of substrates and effectors were used to calculate the actual intracellular activity of the hexokinase in dependence on variations of pH, temperature and the state of haemoglobin oxygenation. The calculated hexokinase activities correspond to the glycolytic flux in all conditions considered, except at pH 8.
Interaction of Biguanides with Mitochondrial and Synthetic Membranes
Authors: Schäfer Günter 
Published: March 2005

Biguanides inhibit a large scale of metabolic processes which in most cases are membrane‐linked. Oxidative phosphorylation is strongly inhibited in that part of the respiratory chain located on the substrate site of cytochrome c.It is assumed that inhibition is due to physical changes within the membrane phospholipids, rather than a result of direct interaction with functional intermediates of energy conservation. The following observations have been made.

Binding affinity to mitochondrial, submitochondrial and other membranes is identical with that to pure phospholipids.
Synthetic phospholipid vesicles exactly resemble the binding properties and kinetics of mitochondrial membranes.
Gradual lipid extraction of mitochondrial membranes causes a proportional decrease of binding capacity but no change of affinity constants.
The stoichiometry shows that the capacity for inhibitor binding is indeed in the range of mitochondrial phospholipid content.
Binding of the inhibitors occurs to the aqueous lipid interface of the membrane. Possibilities are discussed for the mode of action of the inhibitors representing firmly fixed positive charges.
Physical and Functional Homology between Ribosomal Protein S1 and Interference Factor i
Authors: Inouye Hiroshi , Pollack Yaakov , Petre Jean 
Published: March 2005

The homology between the 30‐S ribosomal protein S1 and interference factor i has been investigated.The two proteins are indistinguishable by gel electrophoresis under various conditions. Similar peptides are obtained by cleavage with cyanogen bromide. After labelling with N‐ethylmaleimide, enzymatic digestion yields homologous labelled peptides. This homology is confirmed by immunological data.Characteristic activities of the two proteins have been compared. Both bind polyuridylic acid and stimulate polypeptide synthesis on this template by S1‐depleted ribosomes. Furthermore, S1 inhibits the translation of the coat protein cistron on MS2 RNA, while it has little effect on the overall translation of T4 mRNA, two characteristic properties of interference factor i.S1 and factor i are thus physically and functionally indistinguishable, which suggests that they have identical polypeptide chains. Implications are discussed in terms of function.
Effect of Calcium Ions on Steroid‐Binding Spectra and Pregnenolone Formation in Rat–Adrenal Mitochondria
Authors: Simpson Evan R. , Jefcoate Colin R. , Mccarthy John L. , Boyd George S. 
Published: March 2005

The effect of calcium ions on steroid‐induced difference spectra of cytochrome P‐450, and on pregnenolone formation, has been studied in rat adrenal mitochondria. In the presence of phosphate, addition of calcium chloride to suspensions of these mitochondria resulted in an increase in the inverted type‐I difference spectrum induced by pregnenolone or methylandrostenediol, and a disappearance of the type I spectrum induced by 25‐hydroxycholesterol. The difference in the inverted type I spectrum between mitochondria from ether‐stressed animals and those given cycloheximide was abolished. Cholesterol side‐chain cleavage was increased, providing an NADPH generator was used as source of reducing equivalents. The difference in side‐chain cleavage activity between mitochondria from ether‐stressed animals and those given cycloheximide, was maintained.In the absence of phosphate, addition of calcium chloride again resulted in a loss of the type I difference spectrum induced by 25‐hydroxycholesterol, but this time there was almost no change in the inverted type I spectrum induced by pregnenolone in mitochondria from rats subjected to ether anaesthesia, and the change in cycloheximide‐treated rats was only about 50%, of the change in the 25‐hydroxycholesterol‐induced difference spectrum. These observations are interpreted in terms of interconversions of the various forms of cytochrome P‐450 associated with side‐chain cleavage proposed to exist in intact adrenal mitochondria.Factors such as bovine serum albumin and EDTA, which increase respiratory control in these mitochondria, also stimulate cholesterol side‐chain cleavage activity, as do magnesium ions, but without altering the magnitude of the steroid‐binding spectra.