bluuebunny/crossref_metadata_2025_split huggingface dataset:
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.
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.