bluuebunny/crossref_metadata_2025 huggingface dataset:
Fructose Transport in Bacillus subtilis
Authors: GAY Philippe , DELOBBE André
Published: March 2005

The transport of fructose in Bacillus subtilis was studied in various mutant strains lacking the following activities: ATP‐dependent fructokinase (fruC), the fructose 1‐phosphate kinase (fruB) the phosphofructokinase (pfk), the enzyme I of the phosphoenolpyruvate phosphotransferase system (the thermosensitive mutation ptsIl), and a transport activity (fruA). Combinations of these mutations indicated that the transport of fructose in Bacillus subtilis is tightly coupled to its phosphorylation either in fructose 1‐phosphate, identified in vivo and in vitro or in fructose 6‐phosphate identified by indirect lines of evidence. These steps of fructose metabolism were shown to depend on the activity of the enzyme I of the phosphoenolpyruvate phosphotransferase systems.

The fruA mutations affect the transport of fructose when the bacteria are submitted to catabolite repression. The mutations were localized on the chromosome of Bacillus subtilis in a cluster including the fruB gene. When grown in a medium supplemented by a mixture of potassium glutamate and succinate the fruA mutants are able to carry on the two vectorial metabolisms generating fructose 6‐phosphate as well as fructose 1‐phosphate. A negative search of strictly negative transport mutants in fruA strains indicated that more than two structural genes are involved in the transport of fructose.
Lipopolysaccharides d'une Souche à Virulence Atténuée de Yersinia pestis
Authors: BORDET Claudette , BRUNETEAU Maud , MICHEL Georges
Published: March 2005

Lipopolysaccharides from a Slightly virulent strain of Yersinia pestis were investigated. Two types of lipopolysaccharides were obtained from the bacteria: lipopolysaccharide I was extracted with aqueous phenol/chloroform/hexane while lipopolysaccharide II was extracted with phenol/water. Chemical analysis of these lipopolysaccharides shows the presence of D‐glucose, galactose, glucosamine, L‐glycero, ‐D‐manno‐heptose, D‐glycero‐D‐manno‐heptose, 3‐deoxyoctulosonic acid, phosphate, 3‐hydroxymyristic acid, and small quantities of palmitic acid and palmitoleic acid. In addition, lipopolysaccharide II contains arabinose.

Lipopolysaccharides I and II were methylated; analysis by gas‐liquid chromatography and by mass spectrometry indicated the presence of 2,3,4,6‐tetra‐O‐methyl glucose, 2,3,4,6‐tetra‐O‐methyl galactose and of tri‐, tetra‐ and penta‐methylated heptose derivatives. Arabinose in lipopolysaccharide II was not methylated and is therefore entirely substituted, while glucose and galactose are not substituted. Sodium metaperiodate nearly entirely oxidized glucose and galactose. Part of the heptose was not degraded by periodate oxidation, while part was converted into mannose.

Lipopolysaccharides I and II could be the R and S forms of the lipopolysaccharide respectively, the S form containing an O‐specific chain with arabinose.

The lipopolysaccharides were hydrolysed with acetic acid and degraded polysaccharides I and II were obtained. A large part of the arabinose was lost during the hydrolysis of lipopolysaccharide II into polysaccharide II, thus indicating that the arabinose might be in the furanose form.

The absence of phosphate groups in polysaccharides I and II suggests the absence of bridges between the chains. However, if such bridges exist in the lipopolysaccharides, they are either on 3‐deoxyoctulosonic acid or on lipid A.
The Involvement of Protein L 16 on Ribosomal Peptidyl Transferase Activity
Authors: BERNABEU Carmelo , VÁZQUEZ David , BALLESTA Juan P. G.
Published: March 2005

Radioactive ribosomes from Escherichia coli were treated with increasing concentrations of NH4Cl in the presence of 50 % ethanol. The resulting particles were tested for peptidyl transferase activity as well as for the binding of (U)C‐A‐C‐C‐A‐Leu‐Ac, (U)C‐A‐C‐C‐A‐Leu, chloramphenicol, lincomycin and erythromycin. At the same time the proteins present in the particles were quantitatively estimated and the amount of each related to the residual activity displayed by the treated ribosomes. It was found that the loss of protein L 16 closely paralleled the inactivation of the particles implying an important role for this protein in the structure of the peptidyl transferase center.
Biochemical Markers of the Progress of Differentiation in Cloned Teratocarcinoma Cell Lines
Authors: ADAMSON Eileen D. , EVANS Martin J. , MAGRANE Gregg G.
Published: March 2005

The progeny of single teratocarcinoma cells will give rise to several different cell types in vitro, and the latter were shown to be functionally differentiated by biochemical criteria. In these studies, cloned lines of mouse teratocarcinoma cells were assayed during the course of differentiation for some biochemical products characteristic of the tissues formed. The carcinoembryonic protein, α‐foetoprotein was not synthesized by undifferentiated embryonal carcinoma (EC) cells, but was synthesized in increasing amounts during their differentiation to endoderm‐type cells in suspension culture. α‐Foetoprotein was shown to be a product of endoderm cells, but not all endoderm cells synthesized this protein.

During the course of further differentiation when EC cells or aggregates were grown in tissue‐culture dishes, other biochemical products appeared. In cultures containing predominantly nerve‐type cells, there was a 30‐fold increase in the specific activity of acetylcholinesterase, with concomitant appearance of the aldolase isoenzyme characteristic of mouse brain. In some cultures, a small amount of muscle‐type cell formation was marked by the appearance of the MB isoenzyme of creating phosphokinase. Generally, biochemical differentiation was immature.
The Low‐Spin ⇌ High‐Spin Transition of Camphor‐Bound Cytochrome P‐450
Authors: LANGE Reinhard , BONFILS Claude , DEBEY Pascale
Published: March 2005

The spin state of camphor‐bound cytochrome P‐450 is shown to depend largely on medium and temperature in aqueous as well as in mixed organic buffer. At sub‐zero temperatures a variation of paH, ionic strength or camphor concentration modifies the spin equilibrium from nearly pure high‐spin form to nearly pure low‐spin form. Since the apparent pKa of transition is a linear function of log I, the spin state seems to be controlled by the electrostatic potential in the heme proximity. K+ is found to have a specific effect on the spin state.

The change of enthalpy, ΔH values, of the spin transition depends on the same parameters as the equilibrium constant, in the organic cosolvent as well as in aqueous buffer. As the cosolvent effect is reflected by higher ΔH values, and KCI and pH tend to lower ΔH, the cosolvent effect can easily be compensated. Therefore kinetic studies of the spin conversion might well be undertaken at sub‐zero temperature in this solvent.
Endonucleolytic Cleavage of Parental DNA and T4 Late‐Gene Expression: Distribution Analysis of Single‐Strand and Double‐Strand Breaks
Authors: GROSSI Gianfranco , MACCHIATO Maria , GIALANELLA Giancarlo , CASCINO Antonino
Published: March 2005

In order to investigate the dependency of late transcription on concurrent DNA replication during bacteriophage T4 development, we analyzed the endonucleolytic cleavage kinetics of the DNA of a T4 mutant lacking DNA polymerase, DNA ligase and exonuclease by using the sucrose gradient sedimentation technique. Our results can be summarized as follows.

1. The single‐strand endonucleolytic cleavage of the T4 mutant DNA is not a random process.

2. The number of single‐strand nicks reaches a plateau level of 10–12 nicks/molecule.

3. The occurrence of a double‐strand break is delayed and their number is at any time lower than the number of single‐strand nicks.

4. The circular permutation of T4 genome, as computer‐simulated by the Monte Carlo method, produces a smoothing of the discrete distribution which would be expected if nicks were localized in the promoter sites of late transcription units.

We conclude that our findings support the model which relates single‐strand DNA nicks to the late transcription initiation sites.
Synthesis of Transfer RNA during the Synchronous Nuclear Division Cycle in Physarum polycephalum
Authors: FINK Kirsten , TURNOCK Geoffrey
Published: March 2005

1. The synthesis of tRNA during the synchronous nuclear division cycle in plasmodia of Physarum polycephalum has been investigated using an isotope dilution procedure and compared to the pattern of synthesis of rRNA.

2. The synthesis of both tRNA and rRNA stops during mitosis. As the genes for the two types of RNA are not linked, this finding suggests that cessation of synthesis during nuclear division is a common characteristic of all types of transcription, supporting earlier work using pulse labelling and autoradiography to investigate the same problem.
Energy Requirements for Maltose Transport in Yeast
Authors: SERRANO Ramón
Published: March 2005

Maltose transport in yeast (Saccharomyces cerevisiae) is inhibited by uncouplers under conditions where the intracellular concentration of the sugar is lower than in the medium. The uncouplers did not deplete the ATP content of the yeast cells and a 50–100‐fold reduction in ATP caused by antimycin and 2‐deoxyglucose had no effect on maltose transport. In ATP‐depleted cells, the maltose transported is partially hydrolyzed to glucose but not further metabolized and therefore a mechanism of transport involving phosphorylation can be discarded. One proton is cotransported with every maltose molecule. The fact that maltose transport is inhibited by KCl but not by NaCl, Tris · Cl or KSCN suggest that the electroneutrality during maltose and proton uptake can be maintained by the exit of K+ from the cells or by the entry of a permeable anion as SCN−. These results indicate that the translocation of maltose across the yeast plasma membrane is not dependent on ATP and is coupled to the electrochemical gradient of protons in this membrane. When this gradient is abolished by uncouplers, the transport system is not able to function even in favour of a concentration gradient of the sugar.
Transcription of Ribosomal 5‐S RNA by RNA Polymerase C in Isolated Chromatin from HeLa Cells
Authors: YAMAMOTO Mikio , JONAS Dagmar , SEIFART Klaus
Published: March 2005

The synthesis of ribosomal 5‐S RNA in vitro in isolated chromatin from HeLa cells was studied by hybridization of the product to a plasmid DNA probe which contained the 5‐S RNA genes from Xenopus mulleri. It was found that endogenous RNA polymerase C, contained in the chromatin, actively synthesizes RNA which hybridizes to the DNA for 5‐S RNA. The molecular weight of this RNA, judged by gel filtration and electrophoresis in formamide‐containing polyacrylamide gels, is of distinct 5‐S size. Hybridisation of the product synthesized in vitro to the DNA for 5‐S RNA can be competed quantitatively by the presence of unlabelled cytoplasmic 5‐S RNA. Presaturation of the DNA‐containing filters with cytoplasmic 5‐S RNA prevents hybridisation, indicating that antisense transcripts are not synthesized to a measurable extent in this system. The addition of Escherichia coli RNA polymerase to chromatin stimulates the synthesis of bulk RNA, and beyond certain concentrations also leads to enhanced formation of hybridizable sequences. These are, however, predominantly contained in molecules of high molecular weight, indicating abberent initiations and/or terminations of the bacterial polymerase at random sites. These data re‐emphasize the necessity of concurrent hybridization and size analyses for the assay of specific transcription. Homologous RNA polymerase C, added to chromatin either as a crude preparation in the presence of polymerase A or as purified enzyme, resulted in increased production of hybridizable 5‐S RNA sequences, the formation of which was suppressible by high concentrations of α‐aminitin. The RNA products synthesized under these conditions were predominantly asymmetric and hybridized in a discrete peak at the 5‐S position. This is in contrast to results found for the bacterial enzyme which synthesized a high proportion of antisense transcripts on isolated chromatin. These findings indicate strongly that a particular RNA polymerase species of homologous source should be used when analyzing the transcription of specific genes in vitro.
Immunological Studies on Beef‐Heart Ubiquinol — Cytochrome c Reductase (Complex III)
Authors: NELSON B. Dean , MENDEL‐HARTVIG Ib
Published: March 2005

Antibodies against isolated beef‐heart ubiquinol – cytochrome c reductase (complex III) have been characterized. Antibodies to complex III react strongly with isolated beef heart complex III and intact beef heart mitochondria, as shown by immunodiffusion and rocket electrophoresis experiments. The complex III content of intact mitochondria can be quantitated with rocket electrophoresis using isolated complex III as a standard. Antibodies to complex III also react with beef liver mitochondria and with both heart and liver mitochondria from rats. The latter are very weak antigens compared to beef heart material. Antibodies to complex III do not react with respiratory chain complexes I and IV, or F1‐ATPase from beef heart mitochondria, but gives a slight, but variable, reaction with complex II and the membrane fraction isolated from complex V (oligomycin‐sensitive ATPase). Antigenic sites are located on at least five of the seven peptides of complex III. These peptides are presumably lacking in respiratory chain complexes which do not react with antibodies to complex III, and are assumed to be uniquely located in complex III.

Antiserum against complex III inhibits duroquinol–cytochrome c reductase activity in isolated complex III and in complex III incorporated into phospholipid vesicles. Oxidation of NADH and succinate is not affected in submitochondrial particles treated with 6‐times more antibody than required for complete inhibition of enzyme activity in free complex III or in complex III‐phospholipid vesicles.
The Protein Data Bank
Authors: BERNSTEIN Frances C. , KOETZLE Thomas F. , WILLIAMS Graheme J. B. , MEYER Edgar F. , BRICE Michael D. , RODGERS John R. , KENNARD Olga , SHIMANOUCHI Takehiko , TASUMI Mitsuo
Published: March 2005

The Protein Data Bank is a computer‐based archival file for macromolecular structures. The Bank stores in a uniform format atomic co‐ordinates and partial bond connectivities, as derived from crystallographic studies. Text included in each data entry gives pertinent information for the structure at hand (e.g. species from which the molecule has been obtained, resolution of diffraction data, literature citations and specifications of secondary structure). In addition to atomic co‐ordinates and connectivities, the Protein Data Bank stores structure factors and phases, although these latter data are not placed in any uniform format. Input of data to the Bank and general maintenance functions are carried out at Brookhaven National Laboratory. All data stored in the Bank are available on magnetic tape for public distribution, from Brookhaven (to laboratories in the Americas), Tokyo (Japan), and Cambridge (Europe and worldwide). A master file is maintained at Brookhaven and duplicate copies are stored in Cambridge and Tokyo. In the future, it is hoped to expand the scope of the Protein Data Bank to make available co‐ordinates for standard structural types (e.g.α‐helix, RNA double‐stranded helix) and representative computer programs of utility in the study and interpretation of macromolecular structures.
Mechanism of Activation of the Protein Kinase I from Rabbit Skeletal Muscle
Authors: HOPPE Jürgen , MARUTZKY Rainer , FREIST Wolfgang , WAGNER Karl G.
Published: March 2005

The high‐affinity binding site for ATP of the holoenzyme of cAMP‐dependent protein kinase (type I) from rabbit skeletal muscle has been investigated. Binding affinity of a series of ATP derivatives substituted at different sites in the molecule was determined by competition with tritiated ATP. The results were compared with data available from cAMP derivatives with the same substituents, in order to analyse the electronic and steric features of these two sites on the protein kinase. The comparison revealed significant differences of the effect of substituents towards the two sites. In particular the N6‐derivatives of ATP and substituents affecting the γ‐phosphate indicate that the high‐affinity ATP site of the protein kinase has similar properties as those found for phosphotransferase sites. The present results are consistent with the supposition that the high‐affinity site for ATP on the holoenzyme is congruent with the phosphotransferase site of the catalytic subunit. Upon combination of catalytic and regulatory subunits this site would be transformed into a high‐affinity site for ATP with simultaneous blocking of the phosphotransferase activity.
The Purification, Characterization and Localization of a Parvalbumin‐Like Protein from Chicken‐Leg Muscle
Authors: HEIZMANN Claus W. , HÄUPTLE Marie‐Theresè , EPPENBERGER Hans M.
Published: March 2005

A parvalbumin‐like protein was purified to homogeneity from chicken leg muscle, 40 mg of protein was isolated from 1 kg muscle which is 20% of the amount obtained for parvalbumin isolated from rabbit muscle by a similar procedure. The protein contained 1–2 residues of tyrosine and 1 residue of tryptophan, three arginine residues and a rather small number (4–5) of phenylalanine residues. The differences in the aromatic amino acid content account for the less distinct vibronic structure in the ultraviolet region below 275 nm compared to most other parvalbumins. The protein contained <0.04 residue of γ‐carboxyglutamic acid per molecule. However, the molecular weight of 12000, high content of glutamic acid and aspartic acid, the low content of proline and histidine, and the presence of two calcium atoms per molecule were properties common to parvalbumins. Immunprecipitation experiments in agar using anti‐chicken parvalbumin‐like protein indicated that an immunologically indistinguishable form of this protein is present in heart, white and red back muscles, breast, stomach, brain, pancreas, small intestine and kidney, but not in spleen, liver and blood.

The distribution is therefore as ubiquitous as G‐actin or β‐actinin. Parvalbumin‐like protein was found to accumulate in differentiating primary muscle cell cultures after 120 h. This approximates to the time on which myosin and actin start to accumulate, proteins necessary for myofibril assembly.

Experiments to investigate the localization of parvalbumin‐like protein, using the indirect immunofluorescence technique showed regular cross‐striation patterns within the I band of isolated myofibrils.

The results suggest that parvalbumin‐like protein may interact with one or more of the protein components of the thin filament thus being involved in the contractile process. This component could be β‐actinin which copurified with parvalbumin‐like protein through the DEAE‐cellulose step (see following paper).
The Purification, Characterization and Localization of β‐Actinin from Chicken Muscle
Authors: HEIZMANN Claus W. , HÄUPTLE Marie‐Therese
Published: March 2005

β‐Actinin was purified to homogeneity from water extracts of chicken leg muscle by chromatography on DEAE‐cellulose and gel filtration on Sephadex G‐200. 60–100 mg of protein was obtained from 1 kg of muscle. The molecular weight estimated from gel electrophoresis in the presence of sodium dodecylsulfate was 65000, from gel filtration on Sephadex G‐200 66000, and from sedimentation equilibrium experiments 67000. The results show that β‐actinin is a monomeric protein.

The amino acid composition was determined and differed from β‐actinin of rabbit muscle.

Double‐immunodiffusion experiments in Ouchterlony plates using anti‐chicken β‐actinin indicated that an immunologically indistinguishable form of this protein is present in muscle and non‐muscle tissues of the chicken. The distribution of β‐actinin is therefore similar to G‐actin and parvalbumin‐like protein.

β‐Actinin was found to accumulate in differentiating primary muscle cell cultures after 120 h approximately together with myosin and actin, proteins necessary for myofibril assembly and functioning.

Experiments to investigate the localization of β‐actinin, using the indirect immunofluorescence technique, showed a regular, intensive cross‐striation pattern within the I band of isolated myofibrils. An electronmicroscopic investigation demonstrated that β‐actinin does not polymerize to filamentous structures under conditions where G‐actin is transformed to F‐actin. It is able to inhibit the polymerization of G‐actin and promotes the depolymerization of preformed F‐actin filaments in vitro.

The immumological, physico‐chemical and electronmicroscopic data show that β‐actinin is a quite distinct protein from actin.
Isolation and Function of the Subunits of Citramalate Lyase and Formation of Hybrids with the Subunits of Citrate Lyase
Authors: DIMROTH Peter , BUCKEL Wolfgang , LOYAL Rosemarie , EGGERER Hermann
Published: March 2005

1. The enzyme complex citramalate lyase from Clostridium tetanomorphum, composed of six copies of three different proteins, has been dissociated into its protein components and these were isolated in an enzymically active state. Citramalate lyase has been reconstituted from the isolated subunits.

2. The enzyme complex consists of an acyl carrier protein and two different enzymes, the α and β subunits. The isolated enzymes were active if acetyl‐thio‐acyl carrier protein and (3S)‐citramalyl‐thio‐acyl carrier protein were substituted for the corresponding acyl‐CoA derivatives.

3. The α subunit in the presence of (3S)‐citramalyl‐CoA and acetate catalyzed the formation of acetyl‐CoA and citramalate. The β subunit catalyzed the cleavage of (3S)‐citramalyl‐CoA to acetyl‐CoA and pyruvate. This reaction was dependent on Mg2+ and was abolished if EDTA was added in excess. It was concluded from these results that the α subunit represents an acetyl‐thio‐acyl carrier protein: citramalate acyl carrier protein transferase within the native complex, and the β subunit a (3S)‐citramalyl‐thio‐acyl carrier protein lyase.

4. Results similar to those given above have previously been obtained with the enzyme complex citrate lyase from Klebsiella aerogenes, but with citrate, (3S)‐citryl‐thio‐acyl carrier protein or (3S)‐citryl‐CoA instead of citramalate and its corresponding derivatives. Thus, not only molecular weight, subunit composition and chemical events during substrate turnover are nearly identical in the two different enzyme complexes but also the functions of the individual subunits.

5. The close relationship between the two different enzymes from the two different microorganisms was confirmed by cross reactions between the isolated subunits of each enzyme complex producing a hybrid enzyme complex.
Enzyme Memory
Authors: RICARD Jacques , BUC Jean , MEUNIER Jean‐Claude
Published: March 2005

When wheat germ hexokinase LI is rapidly mixed with its substrates in a stopped‐flow apparatus, the reaction proceeds and exhibits a ‘slow’ burst. The length of the negative induction time, τ, is independent of enzyme concentration but depends on glucose and magnesium‐ATP concentration. At low and high glucose concentrations, the burst disappears but becomes maximum at intermediate hexose concentrations.

The approach to steady state can be considered and treated as a relaxation spectrum. Two relaxations can be detected experimentally: a ‘fast’ (0.2 s) and a ‘slow’ process (100 s). The ‘slow’ relaxation, studied at low glucose concentration, is very similar to the free enzyme relaxation which takes part in the mnemonical transition. This relaxation has been studied in further detail. When the reciprocal of the ‘slow’ relaxation time, λ2, is plotted against glucose concentration, one obtains a curve concave down, that reaches a plateau for high glucose concentrations. This result is taken to mean that glucose binding and enzyme transconformation occur as two distinct steps and that the binding step is faster than the transconformation process.

The burst obtained at the start of the reaction is interpreted as indicative of a progressive shift from an enzyme species able to bind glucose easily and to undergo the proper conformation change, to an other form less able to bind glucose in a productive way. This shift would not be spontaneous but would be forced by the reaction process itself until the dynamic equilibrium that occurs under steady state is reached. Evidence in favor of enzyme memory has been obtained from pre‐mixing experiments with glucose 6‐phosphate. If the enzyme, already premixed with glucose 6‐phosphate in one of the drive syringes of the stopped‐flow apparatus, is mixed with the others reactants, the reaction exhibits a ‘slow’ lag instead of a burst. This transient would correspond to the shift from a weakly ‘reactive’ enzyme form to a strongly ‘reactive’ one. Thus, as expected with the concept of mnemonical transition, the free enzyme must occur under two forms having different reactivities for glucose and glucose 6‐phosphate.
Enzyme Memory
Authors: BUC Jean , RICARD Jacques , MEUNIER Jean‐Claude
Published: March 2005

When a temperature‐jump from 2 to 35°C is applied to wheat‐germ hexokinase LI in solution, the enzyme undergoes an extremely slow conformation change which can be followed by monitoring the fluorescence emission of tryptophanyl residues of the protein. This process occurs in one detectable step and is not associated with any inactivation of the enzyme, although it involves a limited unfolding of the polypeptide chain.

The reciprocal of the relaxation time of this slow conformation change (λ2) slightly increases with glucose concentration. A plot of λ2 against glucose concentration exhibits a downward curvature. If gucose 6‐phosphate is present in the reaction mixture, the reciprocal of the relaxation time, λ2, decreases when the ligand concentration is increased. The decay of λ2 with respect to glucose 6‐phosphate is hyperbolic. This kinetic behavior is identical to that expected for the conformational transition involved in enzyme memory.

From this kinetic study one gets the values of the rate constants governing the conformational transition of the enzyme. The forward rate constant k5′ of the interconversion of the ‘rhombu’ conformation (the conformation state that binds glucose 6‐phosphate) to the ‘circle’ conformation (the conformation state that binds glucose prefenetially) has a value of 10−3 s−1, while the backward rate constant k−5′ has a value of about 5 × 10−6 s−1. The conformation change is thus almost irreversible; at 35°C and in the absence of any ligand, 99.5% of the free enzyme is under the ‘circle’ conformation.

A thermodynamic study of the conversion of the ‘rhombus’ to the ‘circle’ conformation has shown that the entropy of activation ΔS≠ of this process is highly negative (−42 cal or −175 J K−1 mol−1). This is taken to mean that the transition state which has to be reached during the conformation change must have a highly folded structure. This situation clearly distinguish the conformational transition of enzyme memory from a thermal denaturation process.
Correlation between Hypomethylation of DNA and Expression of Globin Genes in Friend Erythroleukemia Cells
Authors: CHRISTMAN Judith K. , PRICE Peter , PEDRINAN Lauri , ACS George
Published: March 2005

This report identifies l‐ethionine as an inducer of differentiation in murine erythroleukemia cells. When Friend erythroleukemia cells are grown in the presence of 4 mM l‐ethionine, globin mRNA accumulates and in 4–5 days, 25–30% of the cells in the culture contain hemoglobin. Incubation of the cells with bromodeoxyuridine prevents both ethionine‐induced accumulation of globin mRNA and erythroid differentiation.

At the concentration where l‐ethionine acts as an inducer of FL cell differentiation it inhibits methylation of DNA and tRNA in vivo but does not prevent macromolecular synthesis or cell division.

To establish whether a link existed between inhibition of a specific methyltransferase and activation of globin synthesis in FL cells, we examined the degree of hypomethylation of DNA and tRNA from FL cells induced to differentiate with dimethylsulfoxide and butyrate.

In contrast to the tRNA from ethionine‐treated cells, tRNA from cells induced by butyrate or Me2SO cannot be methylated in vitro using homologous enzymes.

DNA isolated from cells exposed to any of the three inducers, however, was significantly hypomethylated when compared with DNA from uninduced cells. These data suggest that methylation of DNA may play a role in the regulation of gene expression.
Reconstitution of Model Membranes from Phospholipid and Outer Membrane Proteins of Proteus mirabilis
Authors: NIXDORFF Kathryn , FITZER Hildegard , GMEINER Jobst , MARTIN Hans Herbert
Published: March 2005

Outer membrane proteins extracted from isolated cell walls of Proteus mirabilis were able to combine with cell wall phospholipids in a model membrane system. The presence of outer membrane proteins in vesicular model membranes mediated the release of previously entrapped [14C]sucrose while [3H]inulin was retained. Incorporation of lipopolysaccharide from the same cell walls was not required for the formation of such selectively permeable membranes.

Three major outer membrane proteins of apparent molecular weights 39000, 36000 and 17000 were isolated using acetic acid and sodium deoxycholate solution as solvents and avoiding the strongly denaturing sodium dodecyl sulfate. The isolated proteins were assayed for their ability to form hydrophilic pores in reconstituted membranes. The trypsin‐sensitive 39000‐Mr protein and the peptidoglycan‐associated 36000‐Mr protein were equally effective in this function whereas the 17000‐Mr protein mediated little penetration of low molecular weight solute. The 39000‐Mr and 36000‐Mr proteins also protected reconstituted membrane vesicles from disruption by detergent while 17000‐Mr protein was ineffective in this regard.
The Hysteretic Properties of Glycogen Synthase I
Authors: SØLLING Henrik , ESMANN Viggo
Published: March 2005

Glycogen‐free synthase I from human polymorphonuclear leukocytes is activated by its own substrate, glycogen, in a slow, time‐dependent proces (hysteretic activation). This lag in response to addition of glycogen depends on the concentration of glycogen, pH and temperature. At pH 7.4 and at a temperature of 30 °C, the half‐time of activation, t1/2, decreases from 89 min at 0.004 mg/ml glycogen to 6 min at 25 mg/ml. The activation is accelerated by increasing temperature and pH, but is not influenced by enzyme concentration, glucose 6‐phosphate, UDP, high ionic strength, EDTA, mercaptoethanol, glucose, sucrose or amylase limit dextrin. The Km for UDP‐glucose (0.024 mM) and the activity ratio were unchanged during the activation process.

The activation can be described by vt=vf+ (vo−vf) e−kt where vt, vf and vo are velocities at times t, 0 and ∞ and k is a complex rate constant. Evidence from ultracentrifugation and kinetic studies is presented to substantiate the hypothesis that the underlying mechanism is a simple bimolecular process: enzyme + glycogen ⇌ enzyme‐glycogen complex, with the dissociation constant Ks= 0.003 mg/ml. The hysteretic activation may become rate‐limiting during experiments in vitro with synthase. The possibility of a physiological role in glycogen metabolism, perhaps in the form of a concerted hysteresis with H+ is discussed.
Inhibition of the Lysosomal Pathway of Protein Degradation in Isolated Rat Hepatocytes by Ammonia, Methylamine, Chloroquine and Leupeptin
Authors: SEGLEN Per O. , GRINDE Bjørn , SOLHEIM Anne E.
Published: March 2005

Protein degradation in isolated rat hepatocytes was measured as the release of [14C]valine from pre‐labelled protein. To reduce background radioactivity, the intracellular [14C]valine pool was depleted by serial extraction at 37 °C, effecting equilibration between the intracellular pool and the valine‐free extracellular medium. After extraction, a small, non‐equilibrating intracellular [14C]valine pool remained; this pool could only be labelled in the presence of ongoing protein synthesis, and might represent valine and valine‐containing oligopeptides derived from protein degradation.

The [14C]valine released from degraded protein was not significantly re‐utilized for protein synthesis intracellularly (no effect of cycloheximide or high concentrations of unlabelled valine), reflecting the low rate of protein synthesis and the rapid transport of valine into the extracellular medium, both characteristic of isolated hepatocytes.

From cells pre‐labelled for 24 h in vivo, [14C]valine was released linearly at a rate of 5%/h, probably representing the true over‐all protein degradation rate. The lysosomotropic inhibitor ammonia (10 mM NH4Cl) inhibited 70% of the degradation, presumably the contribution by the lysosomal pathway.

From 1‐h pre‐labelled cells, [14C]valine was released at a declining rate, and ammonia inhibited degradation only by 45%, consistent with the view that the majority of short‐lived proteins are degraded by the non‐lysosomal pathway(s).

Chloroquine and methylamine, which accumulate in lysosomes by virtue of their weak base properties, inhibited hepatocytic protein degradation to the same extent as ammonia, with no additivity. These compounds therefore seem to block the lysosomal pathway of protein degradation selectively and completely. Leupeptin, which binds to and inhibits the activity of certain lysosomal proteases, also inhibited protein degradation almost to the same extent as ammonia, but with a small part of the effect (< 20%) being additive to the NH3 effect and thus probably reflecting a slight inhibition of non‐lysosomal protein degradation as well.

Of the four inhibitors tested, only the effect of ammonia was rapidly reversible within the experimental period (2 h). Leupeptin, on the other hand, was the only degradation inhibitor which did not also affect protein synthesis. Chloroquine caused significant cell death at concentrations above 0.2 mmol/l in this protein‐free medium, i.e. in the concentration range needed for maximal inhibition of protein degradation.

Incubation of hepatocytes under anoxic conditions resulted in an inhibition of protein degradation which was greater than, and partially additive to, the effect of ammonia, i.e. most of the degradation by the lysosomal pathway and more than one‐half of the degradation by the non‐lysosomal pathways appears to be energy‐dependent.
Hepatocyte Chromosomal Non‐histone Proteins in Developing Rats
Authors: GUGUEN‐GUILLOUZO Christiane , TICHONICKY Lydie , KRUH Jacques
Published: March 2005

Rat hepatocytes taken at different stages of the perinatal period were partially purified. On sodium dodecylsulphate acrylamide electrophoresis chromosomal non‐histone proteins showed important variations in complexity during development. Chromosomal phosvitin kinase strongly increased during the last days of fetal life; it strongly decreased just after birth and increased again for a short time, while the cytosol phosvitin kinase increased more significantly after birth.

Chromosomal non‐histone proteins prepared at various stages were incubated with [γ‐32P]ATP and resolved on polyacrylamide gel. The incorporation was very low in samples taken at the 15th say of the fetal life. A dramatic increase was observed at the 17th day. This incorporation strongly decreased in the samples taken thereafter and it was negligible in proteins from adult rats. The variations in protein kinase and in 32P incorporation into non‐histone proteins were correlated with the pattern of appearance of enzymes in this period of life, with cell growth and with the hormone‐induced maturation.
Conformational Studies by 1H Nuclear Magnetic Resonance of the Basic Pancreatic Trypsin Inhibitor after Reduction of the Disulfide Bond between Cys‐14 and Cys‐38
Authors: WAGNER Gerhard , KALB (GILBOA) A. Joseph , WÜTHRICH Kurt
Published: March 2005

The disulfide bond Cys‐14–Cys‐38 in the basic pancreatic trypsin inhibitor was reduced and the cysteinyl side chains were protected with iodoacetamide, iodoacetic acid or ethyleneimine. At pH values near 5, the first of these protecting groups is neutral, the second negatively charged and the third positively charged. High‐resolution 1H nuclear magnetic resonance showed that the average spatial structures of the three modified proteins are essentially identical. The influence of the different electrostatic charges on the stability and the internal flexibility of the proteins could thus be studied in a fixed spatial arrangement of the polypeptide chain. In 2H2O solution at p2H 5.3, the denaturation temperatures are 79°C for the carboxymethylated, 76°C for the carboxyamidomethylated and 70°C for the aminoethylated reduced inhibitor. Comparison of the three species indicates that stabilizing and destabilizing effects of the charged groups are mainly due to local interactions with nearby charged amino acid side chains, rather than to the variations of the overall charge of the proteins. The stabilizing or destabilizing effects per charged group were estimated to be of the order of 1.0–2.0 kJ mol‐1. The different stabilities of the three proteins are also manifested in the amide proton exchange rates, whereas the internal mobilities of corresponding aromatic rings were found to be identical in the three species.
Demonstration by Membrane Reconstitution of a Butanol‐Soluble Intermediate in the Biosynthesis of the O9 Antigen of Escherichia coli
Authors: KANEGASAKI Shiro , JANN Klaus
Published: March 2005

The activity in vitro of the mannan‐synthesizing system of Escherichia coli O9 depends on the presence of glucose in the growth medium of the bacteria. Inactive membranes of E. coli strain F988 grown without glucose gain mannan‐synthesizing activity by reconstitution with a butanol extract obtained from the same bacteria grown with glucose. Inactive membranes could also be restored to biosynthetic activity by incubation with UDP‐glucose in the presence of magnesium chloride. In this magnesium‐ion‐dependent reaction, a glucolipid was formed which was extractable with butanol. It could be used for the reconstitution of inactive membranes.

The products of incubations with GDP‐mannose of reconstituted and active membranes were analyzed for electrophoretic mobility in sodium dodecylsulfate/polyacrylamide gel electrophoresis, molecular weight and composition. In all cases they proved to be the mannan attached to a hydrophobic mannose carrier, presumably a glucolipid.

These results suggest that a glucolipid is the intermediary mannose acceptor in the biosynthesis of the O9 antigen.
Cooperative Binding of α Subunits to the Apo‐β2 Subunit of Tryptophan Synthase from Escherichia coli
Authors: BARTHOLMES Peter , TEUSCHER Brigitte
Published: March 2005

The binding parameters for the interaction of α subunits with the dimeric apo‐β2 subunit of tryptophan synthase from Escherichia coli were determined by gel chromatography, equilibrium dialysis and sedimentation velocity experiments. In 0.1 M pyrophosphate buffer pH 7.5 the binding of two α subunits to the stable apo‐β2 dimer is cooperative. Fitting the data to the Adair equation yielded the apparent microscopic dissociation constants for the complexes with one and two bound α chains. Since they differ significantly, the apo‐β2 dimer seems to exist in distinct conformational states depending on the saturation with α subunits.

This finding is in agreement with a recently published scheme summarizing the interactions between the apo‐β2 subunit and its ligands, α subunit and pyridoxal 5′‐phosphate. The binding between the subunits shows a moderate temperature‐dependence, suggesting that hydrophobic effects may be involved in the subunit association to form the apo‐multienzyme complex.
A Kinetic Analysis of the Uptake of Cytosine‐β‐d‐Arabinoside by Rat‐B77 Cells
Authors: KOREN Ruth , SHOHAMI Esther , YEROUSHALMI Sarit
Published: March 2005

We present here a differentiation by kinetic methods of the tandem processes of transport and metabolic trapping during uptake of cytosine‐β‐d‐arabinoside by intact rat fibroblasts. Transport across the cell membrane occurs by a carrier‐mediated mechanism displaying a Km of ∼ 500 μM and a V of ∼ 300 pmol × min‐1× (106 cells)‐1. The subsequent metabolic trapping (phosphorylation) has a Km of ∼ 15 μM and V of ∼ 0.25 pmol × min‐1× (106 cells)‐1.

In this system, transport is rate‐limiting for the first phase of the uptake process whereas phosphorylation becomes rate‐limiting when internal concentration of radioactive labeled substrate exceeds that in the extracellular medium. The duration of the first phase depends on the substrate concentration.
Biochemical and Genetic Evidence for Two Extracellular Adenosine 3′:5′‐Monophosphate Phosphodiesterases in Dictyostelium purpureum
Authors: TSANG Adrian S. , COUKELL M. Barrie
Published: March 2005

Two extracellular adenosine 3′:5′‐monophosphate phosphodiesterases (designated phosphodiesterase 1 and 2) were purified from growth media of the cellular slime mold, Dictyostelium purpureum. Both enzymes have a broad pH optimum of 7.5–8.6 but they differ in molecular weight, stability, kinetic behaviour and sensitivity to inactivation by the phosphodiesterase inhibitor of D. discoideum.

The polypeptides associated with the two enzyme activities were identified on two‐dimensional gels. Under these conditions, the polypeptide associated with the phosphodiesterase‐1 activity has a molecular weight of 60000 and an apparent isoelectric point of pH 8.5. Two polypeptides (Mr 50000 and Mr 48000) were associated with the phosphodiesterase‐2 activity. These two polypeptides have the same isoelectric point of about pH 7.5 and very similar peptide maps. The ratio of the 50000‐Mr polypeptide to the 48000‐Mr polypeptide, when stained with Coomassie blue was 3:1, while the same polypeptides appeared to be present in a 6:1 ratio when stained with periodate‐Schiff's reagent. These results suggest that the major difference between the 50000‐Mr and 48000‐Mr polypeptides is in the size of their carbohydrate moieties.

Several developmental mutants were isolated which synthesize the phosphodiesterases abnormally. Results obtained from an examination of enzyme production in two of these mutants provided further evidence for two extracellular adenosine 3′: 5′‐monophosphate phosphodiesterases in D. purpureum. In addition, these results revealed that phosphodiesterase‐1 is synthesized during vegetative growth whereas phosphodiesterase‐2 is synthesized towards the end of the growth phase and during early development.
Direct Evidence for Extracellular Adenosine 3′:5′‐Monophosphate Phosphodiesterase Induction and Phosphodiesterase Inhibitor Repression by Exogenous Adenosine 3′:5′‐Monophosphate in Dictyostelium purpureum
Authors: TSANG Adrian S. , COUKELL M. Barrie
Published: March 2005

The phosphodiesterase inhibitor of Dictyostelium purpureum was purified from buffered suspensions of starved amoebae and identified on two‐dimensional electrophoretic gels. The polypeptide associated with the inhibitor activity has an apparent molecular weight of 50000 and an isoelectric point of about pH 4.0.

To determine whether or not exogenous adenosine 3′: 5′‐monophosphate (cAMP) stimulates the synthesis de novo of one or both of the extracellular phosphodiesterases of D. purpureum [1], pre‐aggregative cells were incubated with 14C‐labelled protein hydrolysate in the presence and in the absence of 1 mM cAMP. The extracellular proteins of the labelled suspensions were then separated by two‐dimensional gel electrophoresis and the relative amount of label incorporated into the polypeptides was analyzed by autoradiography. It was found that the polypeptides associated with the phosphodiesterase‐2 activity were labelled in the cAMP‐treated suspension, but not in the control. Moreover, the polypeptide associated with the phosphodiesterase‐1 activity was not labelled under these conditions. These results suggest that exogenous cAMP induces the synthesis de novo of phosphodiesterase‐2, but not phosphodiesterase‐1, during the early development of D. purpureum.

Under the same conditions, there was little or no incorporation of labelled amino acids into the polypeptide associated with the phosphodiesterase inhibitor activity either in the presence or in the absence of cAMP. However, when the extracellular proteins, which accumulated in the cell suspensions during the first 10 h of starvation, were separated on two‐dimensional gels and then stained with Coomassie blue, the inhibitor polypeptide was found to be missing from the cAMP‐treated suspension. These results suggest that exogenous cAMP either represses the synthesis of the phosphodiesterase inhibitor, or, perhaps, interfers with the processing of precursor inhibitor molecules.
Synthesis of Albumin and Malic Enzyme in Wheat‐Germ Lysates and Xenopus laevis Oocytes Programmed with Chicken‐Liver Messenger RNA
Authors: GOODRIDGE Alan , CIVELLI Olivier , SCHERRER Klaus , YIP Cecil
Published: March 2005

Undegraded, biologically‐active, polyadenylated RNA was isolated from chicken liver by a rapid, simple procedure. Liver cells were dispersed mechanically and then broken gently by controlled Dounce homogenization in the absence of detergent or ribonuclease inhibitors. After removing lysosomes and mitochondria by centrifugation, RNA was precipitated at pH 5.2. Polyadenylated mRNA was isolated directly from the detergent‐solubilized precipitate by oligo(dT)‐cellulose chromatography. The resulting RNA was translated into liver‐specific peptides in both the wheat germ lysate and Xenopus laevis oocytes. Translatable albumin mRNA was detected in the liver cytoplasm of both fed 3‐week‐old chicks and unfed, day‐old chicks. Translatable malic enzyme mRNA was only detected in the livers from the fed chicks.
A Novel SH‐Type Carboxypeptidase in the Inner Membrane of Rat‐Liver Mitochondria
Authors: HAAS Rainer , HEINRICH Peter C.
Published: March 2005

A carboxypeptidase from rat liver mitochondria was partially purified by discontinuous sucrose gradient centrifugation, washes with NaCl/KBr/Tris buffer, and solubilization with 2 M NaCl in the presence of soybean trypsin inhibitor bound to CM‐cellulose. By means of dodecylsulfate/polyacrylamide gel electrophoresis a molecular weight of 34500 was determined; a value of 38000 was estimated by Sephadex G‐100 gel filtration. The carboxypeptidase was completely inhibited by 3 mM Hg2+. In the presence of 3 mM Cu2+ 50% of the catalytic activity was inhibited. Among several peptides tested Cbz‐Ala‐Phe, Cbz‐Leu‐Phe, Cbz‐Phe‐Leu, and Cbz‐Phe‐Phe, were good substrates. The enzyme activity exhibited a pH optimum of around 9 with Cbz‐Ala‐Phe as a substrate. After submitochondrial fractionation it was found that the carboxypeptidase is located in the inner mitochondrial membrane.