bluuebunny/crossref_metadata_2025 huggingface dataset:
Photoelectric and Spectral Responses of Bacteriorhodopsin Modified by Carbodiimide and Amine Derivatives
Authors: OVCHINNIKOV Yuri A. , ABDULAEV Nazhmutdin G. , DERGACHEV Alexander E. , DRACHEV Alexander L. , DRACHEV Lei A. , KAULEN Andrey D. , KHITRINA Lyubov V. , LAZAROVA Zvetana P. , SKULACHEV Vladimir P.
Published: March 2005

Photoelectric activity and photocycle kinetics of bacteriorhodopsin successively treated with a carbodiimide and several amine derivatives have been studied. Both spectral and electrical responses were initiated by a 15‐ns laser flash inducing a single turnover of bacteriorhodopsin. Photocycle kinetics were measured in a suspension of purple sheets. Photoelectric potential generation was monitored by a voltmeter, using a phospholipid‐impregnated collodion film covered on one side with purple sheets or proteoliposomes.

It is shown that in bacteriorhodopsin treated with a carbodiimide derivative and ethylenediamine or methyl‐o‐arginine, significant acceleration of the millisecond phase of the photoelectric response is observed, whereas the kinetics of the microsecond phase remain unchanged. This effect is accompanied by an increase in the fast (τ 2 ms) component of the 412‐nm intermediate decay. Methylamine‐modified bacteriorhodopsin shows no such changes in electric and spectral kinetics.

Treatment with ethylenediamine or arginine, rather than methylamine, desensibilizes, completely or partially, photoelectric response to inhibition by cationic effectors such as La3+ or by increase in the H+ concentration. At the same time modification sensibilizes this response to the inhibiting effect of an anion, citrate.

Dark adaptation of bacteriorhodopsin modified by ethylenediamine or arginine slows down both microsecond and millisecond electrogeniac phases, whereas that of untreated or methylamine modified bacteriorhodopsin does not.

Proteoliposomes of ethylenediamine‐modified bacteriorhodopsin generate a photoelectric potential of opposite direction (interior negative) as compared with those of unmodified or methylamine‐modified bacteriorhodopsin.

It is concluded that the modifications by ethylenediamine and arginine change the decomposition of the 412‐nm intermediate in such a fashion that a portion of this intermediate decays faster. This results in accelerating those electrogenic phases which are associated with the 412‐nm intermediate decay. Such an effect may be due to replacement of negatively charged groups in the bacteriorhodopsin polypeptide chain by positively charged ones.

Different methods for measurement of changes in the bacteriorhodopsin activity are discussed. The conclusion is made that the analysis of the kinetics of photoelectric potential generation in the purple sheet‐collodion film system is a sensitive probe for changes in bacteriorhodopsin activity.
The Intracellular Concentration of Pyrophosphate in the Batch Culture of Escherichia coli
Authors: KUKKO Eila , HEINONEN Jukka
Published: March 2005

We applied our colorimetric PPi determination method [Heinonen, J., Honkasalo, S. and Kukko, E. (1981) Anal. Biochem. 117, 293–300] to bacterial cultures and measured the intracellular concentration of PPi in the batch culture of Escherichia coli. The cells growing in minimal medium contained about 2.5 nmol PPi/mg protein (0.5 mM). There was no extracellular PPi. A similar concentration of PPi was found also in cells growing in minimal medium enriched by amino acid mixture.
Relationship between Membrane Integrity and the Inhibition of Host Translation in Virus‐Infected Mammalian Cells
Authors: LACAL Juan Carlos , CARRASCO Luis
Published: March 2005

The inhibition of host protein synthesis after picornavirus infection, and its relationship to the modification of membrane permeability has been studied. In encephalomyocarditis (EMC)‐virus‐infected L cells, cellular protein synthesis is reduced from the third hour after infection, at which time an inhibition in 86Rb+ uptake by the infected cell was observed. The bulk of viral protein synthesis takes place in a cell in which a profound modification in the ionic concentration has occurred. This is in agreement with the idea that optimal viral translation requires a higher concentration of monovalent ions as compared to cellular protein synthesis.

A parallel measurement of (a) protein synthesis, (b) 86Rb+ content, (c) membrane potential and (d) modification of permeability to hygromycin B in EMC‐virus‐infected and poliovirus‐infected HeLa cells, indicates that in EMC‐virus‐infected cells a correlation exists between membrane alterations and the inhibition of host protein synthesis. This correlation was not apparent in poliovirus‐infected cells, suggesting that in this system other factors, different from monovalent ion alterations, are involved in the shut‐off. However, in this system, the synthesis of viral proteins also takes place when the cell membrane has been modified and permeability to ions is altered.

The analysis of the shut‐off of protein synthesis and permeability modification in different cell lines infected with EMC virus or poliovirus indicates that the kinetic pattern of the shut‐off is variable and depends on the cell line used and the multiplicity of infection used in each experiment.

The ATP content of picornavirus‐infected cells was examined throughout infection. A decrease of this nucleotide in the infected cells was apparent from the third hour post‐infection and this factor could perhaps contribute to the inhibition of viral protein synthesis that occurs late in infection. Another factor that could influence this inhibition is the transport of amino acids. For instance, the transport of glycine decreased from the beginning of the infection in EMC‐virus‐infected cells and a leakage of methionine was also observed late in infection. These modifications in membrane permeability could contribute to the development of the cytopathic effect observed in virus‐infected cells.
The Binding of NADH and NADPH to Bovine‐Liver Glutamate Dehydrogenase
Authors: DELABAR Jean M. , MARTIN Stephen R. , BAYLEY Peter M.
Published: March 2005

The binding of NADH and NADPH to bovine liver glutamate dehydrogenase has been studied using circular dichroism (CD) and fluorescence spectroscopy, under comparable conditions of enzyme concentration. Spectroscopic titrations have been analysed using non‐linear least‐squares techniques to determine dissociation constants and parameters for the different chromophoric portions of the bound coenzymes, according to different models for the binding equilibria.

For NADPH, enhancement of dihydronicotinamide fluorescence and positive CD at 340 nm reflect binding of the coenzyme at the active site (site I) via the dihydronicotinamide moiety with a dissociation constant KI of 50 μM; negative CD at 260 nm reflects the binding of a second molecule of coenzyme at the non‐active site (site II) via the adenine moiety with a KII of 650 μM.

For NADH, the same spectroscopic assignment is made; however, site II affinity is much greater (KII 20 μM) and exceeds the affinity at the active site (KI 50 μM).

In no case was spectroscopic evidence obtained with either coenzyme to support the concept of non‐identical active site binding as three sites IA and three sites IB per hexamer.

Coenzyme affinities are approximately twofold higher in triethanolamine buffer (0.05 + 0.1 M KCl, pH 7.6) compared with phosphate buffer (0.1 M at pH 7.0).

ADP enhances the CD of bound NADPH at 340 nm by up to twofold; phosphate ion also enhances the CD and binds with a Kd of 60–80 mM. GTP causes strong negative CD at 340 nm but only for the NADPH bound at site II. These results are consistent with independent binding sites for ADP (site II) and GTP (site III); phosphate ion is apparently able to affect certain properties of all three sites.
(1,3)‐β‐d‐Glucan Synthase from Budding and Filamentous Cultures of the Dimorphic Fungus Candida albicans
Authors: ORLEAN Peter A. B.
Published: March 2005

UDPglucose: (1,3)‐β‐d‐glucan 3‐β‐d‐glucosyltransferase (EC 2.4.1.34) was obtained as a particulate fraction from cell‐free extracts prepared after mechanical breakage of cells of a strain of the dimorphic fungus Candida albicans. The properties of this glucosyltransferase were investigated. Budding and filamentous cultures of C. albicans were grown after dilution of a stationary phase inoculum of yeast cells into fresh medium at 30°C and 40°C respectively and the specific activities of (1,3)‐β‐d‐glucan synthase, obtained from budding and filamentous cultures harvested during the first 3 h of their growth, were compared.

UDPglucose was the only glucosyl donor in the reaction (assayed by following the incorporation of radioactivity from UDP[14C]glucose into polymer) and the radioactive product was exclusively β‐(1,3)‐glucan.

Glycogen synthase activity was not detected.

(1,3)‐β‐d‐Glucan synthase activity was enhanced by ATP and GTP.

A threefold increase in the specific activity of the glucosyltransferase was obtained when cell breakage, and subsequent steps in the preparation of the enzyme, were carried out in the presence of 25 μM GTP. A Km value for UDPglucose of 1.5–1.9 mM was obtained for the glucosyltransferase prepared in the presence or absence of GTP.

The glucosyltransferase reaction was not affected by ADPglucose, CDPglucose, GDPglucose or glucose 6‐phosphate, but was competitively inhibited by TDPglucose. GDPglucose was not a glucosyl donor under the present conditions.

There was no evidence that the product was N‐glycosidically linked to protein, since the reaction was neither enhanced in the presence of UDP‐N‐acetylglucosamine, nor inhibited by tunicamycin.

The specific activity of the glucosyltransferase from 3‐h‐old filamentous cultures was about 1.5‐times higher than that from 3‐h‐old budding cultures.

The specific activities of (1,3)‐β‐d‐glucan synthase prepared from budding and filamentous cultures of C. albicans during their first 90 min of growth were similar.
Purification and Properties of Diamine Oxidase from Euphorbia Latex
Authors: RINALDI Augusto , FLORIS Giovanni , FINAZZI‐AGRÒ Alessandro
Published: March 2005

A diamine oxidase has been purified to homogeneity from the latex of an herbaceous shrub, Euphorbia characias. This enzyme has a relative molecular mass of 144000 and is composed of two identical subunits. It contain two Cu(II) and two carbonyl‐like groups per dimer. The purified enzyme is pink and shows a broad absorption in the visible region centered at 480 nm, which is modified by the addition of phenylhydrazine or semicarbazide. The electron paramagnetic resonance spectrum is typical of copper(II) in a tetragonal symmetry. This enzyme oxidizes putrescine and cadaverine at fairly high rate and, less efficiently a few related compounds, but not histamine, spermine or spermidine.
Fingerprint Analysis of Bovine Hypothalamic Preprosomatostatin
Authors: IVELL Richard , RICHTER Dietmar
Published: March 2005

Messenger RNA from bovine hypothalami was used to direct the synthesis in vitro of a precursor to somatostatin (SRIF) of Mr 15500. Specific antibodies, raised against the chemically synthesized tetradecapeptide SRIF‐14, were used for the preliminary characterzation. The radioactively labelled preprosomatostatin was then cleaved by trypsin or cyanogen bromide and the products were assayed by two‐dimensional fingerprinting techniques. The results conclusively demonstrated the presence of the tetradecapeptide SRIF‐14 sequence and its naturally occurring N‐terminally extended form, SRIF‐28. This 28‐amino‐acid sequence was shown to occupy the C terminus of the 15 500‐dalton precursor and is probably preceded by basic amino acid(s).
A 13C Nuclear Magnetic Resonance Study of CO2/HCO−3 Exchange Catalyzed by Human Carbonic Anhydrase I
Authors: SIMONSSON Ingvar , JONSSON Bengt‐Harald , LINDSKOG Sven
Published: March 2005

Rates of CO2/HCO−3 exchange, catalyzed by human carbonic anhydrase I (or B) at chemical equilibrium, were estimated from the nuclear magnetic resonance linewidths of 13C‐labeled substrates. The results show that the maximal exchange rate constant is independent of pH in the range 5.7–8.0, whereas the apparent substrate dissociation constant depends on pH. Exchange proceeds rapidly in the absence of added buffers, and the addition of buffers has negligible effects on exchange rates. Exchange is equally rapid with 1H2O or 2H2O as solvents. Chloride ions inhibit CO2/HCO−3 exchange competitively.

The maximal exchange rates obtained with human carbonic anhydrase I are 50 times slower than those obtained with human isoenzyme II (or C). From a comparison of the exchange kinetics with the steady‐state kinetics of CO2 hydration and HCO−3 dehydration it is tentatively concluded that the transfer of H+ between active site and medium proceeds with rates of similar magnitudes in the two isoenzymes, whereas the central catalytic step, the interconversion of enzyme‐bound CO2 and HCO−3, is much slower in isoenzyme I than in isoenzyme II.
The Effect of Phospholipase A2. on Intact Cells and Isolated Membranes of Mycoplasma gallisepticum
Authors: MARKOWITZ Ora , GROSS Zvi , ROTTEM Shlomo
Published: March 2005

When isolated Mycoplasma gallisepticum membranes were treated with phospholipase A2 approximately 95% of the phospholipids were rapidly degraded. During the phospholipase A2 treatment unesterified fatty acids but not lysophospholipids accumulated. The enzyme had little effect on phospholipids of viable M. gallisepticum cells. The susceptibility of these cells to phospholipase A2 was, however, gradually increased upon preincubation of the cells at 37°C with a maximal susceptibility obtained after 2 h of preincubation. The preincubation did not affect the phospholipid and protein composition of the membrane and was not required with iodoacetatepoisoned cells. Extensive phospholipase A2 treatment of preincubated viable cells resulted in an increase in cell leakiness and a rapid decrease in cell viability, except when bovine serum albumin was added to the reaction mixture. In the presence of 1% albumin, cell viability was fully preserved, even after prolonged (up to 3 h) incubation periods with an excess (50 μg/ml) of phospholipase A2. Yet, with either intact cells or isolated membranes, the presence of albumin resulted in a selective hydrolysis of membrane phospholipids. Phosphatidylglycerol was readily hydrolyzed whereas the hydrolysis of the disaturated phosphatidylcholine was almost completely inhibited. In liposomes prepared from total lipid extracts of M. gallisepticum, phospholipase A2 hydrolyzed the phosphatidylglycerol and phosphatidylcholine equally well. Following the rate and extent of hydrolysis of phosphatidylglycerol in intact cells and isolated membranes by excess phospholipase A2 in the presence of 1% albumin revealed that at 37°C 54% of the phosphatidylglycerol in intact cells were hydrolyzed without affecting cell intactness and viability compared with 83% hydrolysis in isolated membranes. At lower temperatures (15°C and 22°C) the rate and extent of hydrolysis was lower in both cells and membranes. The phosphatidylglycerol pool size susceptible to phospholipase A2 in intact cells was found to decrease from 54% at 37°C to 11% at 15°C with an increase in the size of the phosphatidylglycerol pool not susceptible to the enzyme from 17% to 59% respectively. The results are discussed in relation to the possible use of this enzyme in studies of transbilayer distribution of phosphatidylglycerol in M. gallisepticum.
Properties of Purified Orange II Azoreductase, the Enzyme Initiating Azo Dye Degradation by Pseudomonas KF46
Authors: ZIMMERMANN Thomas , KULLA Hans G. , LEISINGER Thomas
Published: March 2005

Orange II azoreductase [NAD(P)H: 1‐(4′‐sulfophenylazo)‐2‐naphthol oxidoreductase], an enzyme catalyzing the reductive cleavage of the azo bridge of Orange II and related dyes, was purified to electrophoretic homogeneity from Pseudomonas species, strain KF46. This organism utilized carboxy‐Orange II [1‐(4′‐carboxyphenylazo)‐2‐naphthol] but not Orange II as the sole source of carbon, energy, and nitrogen. Orange II azoreductase was induced 80‐fold by both Orange II and carboxy‐Orange II. With two successive runs of affinity chromatography using two chromatographic media with different triazinyl dyes as ligands, the enzyme was purified 120‐fold with 43% yield. The purified enzyme is a monomer with a molecular weight of 30000. Its Km values were 1.5 μM for both Orange II and carboxy‐Orange II. 5 μM for NADPH, and 180 μM for NADH. A survey of the efficiency of various Orange dyes as substrates for Orange II azoreductase showed that: (a) a hydroxy group in the 2‐position of the naphthol ring is required: (b) charged groups in proximity to the azo group hinder the reaction; (c) a second polar substituent on the dye molecule impedes the reaction; (d) electron‐withdrawing groups on the phenyl ring accelerate the reaction.
Control of Protein Synthesis in Cuboidal Rat Mammary Epithelial Cells in Culture
Authors: BARRACLOUGH Roger , DAWSON Karin J. , RUDLAND Philip S.
Published: March 2005

The cuboidal rat mammary tumour cell line, Rama 25, spontaneously converts to elongated cells during culture. The polypeptides synthesised by the two morphological forms are compared by two‐dimensional polyacrylamide gel electrophoresis. In addition to major changes in the intermediate filament proteins, there are two abundant acidic polypeptides of molecular weights 9000 and 15 500, which are synthesised by the elongated cells but which are not detectable in the cuboidal cells.

When mRNA preparations isolated from the cuboidal and elongated cells are translated in the reticulocyte lysate, the patterns of radioactive products resemble the patterns of polypeptides synthesised in the intact cells. Translation products corresponding to the 9000‐Mr and 15 500‐Mr polypeptides are synthesised when mRNA from elongated cells is translated, but are not synthesised when mRNA from cuboidal cells is translated. Evidence is presented that the appearance of these two proteins represents a change in gene expression between the two cell types and that control is exerted prior to mRNA translation.
Kinetics of Phosphatidylglycerol Synthesis in Isolated Membrane Vesicles from Escherichia coli Containing Different Amounts of Membrane‐Bound Phosphatidic Acid
Authors: DEMANT Erland J. F.
Published: March 2005

Cytoplasmic membrane vesicles of Escherichia coli containing various amounts of phosphatidic acid ranging from 0.2% to more than 50% (mol/mol) of the total lipid has been prepared by de novo synthesis of phosphatidic acid in the isolated membranes from acyl‐CoA esters and sn‐glycerol 3‐phosphate. The kinetics of CTP‐inituted phosphatidylglycerol synthesis in the phosphatidic‐acid‐enriched membranes has been studied.

Phosphatidic acid pools with high and low reactivity as substrate for phosphatidylglycerol synthesis were present in the membranes. The two pools were found identical with respect to fatty acid composition and content of molecular species. The rate of phosphatidylglycerol synthesis from the highly reactive phosphatidic acid pool was independent of the phosphatidic acid concentration in membranes containing from 0.2–30 (mol/mol) phosphatidic acid. Ca2+‐ions were found to inhibit the synthesis of phosphatidylglycerol.

On the basis of the findings presented it is suggested that phosphatidic acid probably plays a minor role as a feed‐back modulator of sn‐glycerol 3‐phosphate acylation in E. coli, and that phosphatidylglycerol synthesis can occur at near maximal rate in growing cells.
DNA Polymerases a and y during Pre‐emergence and Early Larval Development of Artemia
Authors: SLATER John M. , McLENNAN Alexander G.
Published: March 2005

DNA polymerases a and γ have been studied in cryptobiotic cysts and developing embryos and larvae of the brine shrimp Anemia. The two enzymes readily separate on Cibacron blue 3‐GA Matrex gel. Assay requireaments with activated DNA as primer‐template are pH 8.0, 1 mM Mg2+, 50 mM K+ for DNA polymerase α and pH 8.4, 10 mM Mg2+, 80 mM K+ for DNA polymerase γ. DNA polymerase α is inhibited by N‐ethylmaleimide (94% and 100% at 1 mM and 10 mM respectively) and aphidicolin (96% at 60 μM). DNA polymerase γ is also sensitive to N‐ethylmaleimide (83% and 100% inhibition at 1 mM and 10 mM respectively) but is resistant to aphidicolin, 2′,3′‐Dideoxythymidine 5′‐triphosphate (ddTTP) inhibits the γ polymerase by 88% when in fivefold excess over dTTP whereas the α polymerase is unaffected by this compound. DNA polymerase α has a sedimentation coefficient of 7.6 S which is reduced to 6.2 S by a phenylmethylsulphonyl fluoride‐sensitive proleinase. The γ polymerase sediments at 8.3 S. No DNA polymerase β activity could be detected. After the reinitiation of development both activities increased twofold up to 8 h (γ polymerase) and 16 h (α polymerase), then declined before the onset of nuclear DNA replication after hatching. Thymidine kinase activity increased over 200‐fold up to the time of replication. Analysis on Percoll density gradients of the intracellular distribution of both polymerases during development suggests that the changes in their activities may be due to migration from storage sites to replication complexes in the nuclei and mitochondria. The content of the mitochondrial DNA polymerase γ in different batches of cysts may reflect the relative viabilities of the cysts.
Purification and Characterization of tRNA (Adenine‐1‐)‐Methyltransferase from Thermus flavus Strain 71
Authors: MOROZOV Ivan A. , GAMBARYAN Alexandra S. , LVOVA Tatjana N. , NEDOSPASOV Andrey A. , VENKSTERN Tatjana V.
Published: March 2005

tRNA (adenine‐1‐)‐methyltransferase was isolated from the extreme thermophile Thermus flavus, strain 71. It was purified about 2000‐fold by ammonium sulfate fractionationand affinity chromatography on tRNA bound to aminohydroxybutylcellulose via its oxidized 3′ end. The purified protein preparation is free of nucleasc and aminoacyl‐tRNA synthetase activity and contains no more than 40% of tRNA (guanine‐7‐)‐methyltransferase activity. The only activity of the enzyme is to methylate A58 in the TΨ loop of tRNA. Out of the eight purified tRNAs examined, only yeast tRNATrp was not utilized as substrate. The enzyme highly thermostable. It is most active at 75°C. tRNA (adenine‐1‐)‐methyltransferase has a Km of 0.4–0.5 μM for tRNAGln2 from Escherichia coli and a Km of 6 μM for S‐adenosyl‐L‐methionine.
Heterogeneity of Microtubule‐Associated Proteins and Brain Development
Authors: FRANCON Jacques , LENNON Ana Maria , FELLOUS Arlette , MARECK Alain , PIERRE Michel , NUNEZ Jacques
Published: March 2005

Developmental changes in the composition of brain microtubule‐associated proteins have been studied in three species the rat and the mouse, which are characterized by post‐natal brain development, and the guinea‐pig, whose Drain is mature at birth.

At an adult stage, and whatever the species, two major microtubule‐associated proteins, which have been referred to MAP2 and τ, have been identified by polyacrylamide gel electrophoresis Rat τ is composed of four closely spaced bands; mouse τ contains only three components with one of them being present in higher proportion than the others; adult guinea‐pig τ is essentially present as a single band.

Microtubule‐associated proteins were also prepared at different stages of brain development. In the three species only two bands were seen in the τ region at immature stages of development (fast τ and slow τ). However adult τ factors progressively replace the young entities. In contrast, only small changes were seen in the proportion of MAP2.

Peptide mapping analysis of the purified T entities confirmed that the four adult rat proteins are very similar. In contrast, peptide mapping of the two young rat T proteins were very different from each other and from those of the adult ones. Peptide mappings of young and adult MAP2 were only slightly different.

The activities of young T proteins and young MAP2 in promoting pure tubulin assembly were much lower than those of the adult ones. Young fast T and young slow T were purified and both show to be active in promoting pure tubulin polymerization.

These data demonstrate the existence of two types of heterogeneity of microtubule‐associated proteins: plurality of protein species at every stage of brain development and changes in composition ana activity dependent on development.
Structure‐Function Relations between Fatty Acid Oxidation and the Mitochondrial Inner‐Membrane — Matrix Region
Authors: OTTO David A. , ONTKO Joseph A.
Published: March 2005

Decreasing the osmolarity of the incubation media for rat liver mitochondria, by lowering the KCl or sucrose content, greatly increased the oxidation of palmitate, palmitoyl‐CoA, palmitoyl‐(–)‐carnitine, octanoate and butyrate, but only slightly affected the oxidation of succinate, glutamate plus malate, and 3‐hydroxybutyrate. Kinetic studies indicated that the increased rate offatty acid oxidation commenced at the onset of incubation in the hypotonic media and remained relatively constant. This was accompanied by an increase in the 3‐hydroxy‐butyrate: acetoacetate ratio, a higher rate of oxygen consumption, an increase in the extramitochondrial adenine‐nucleotide phosphorylation‐state ratio, and a greater state of reduction of the respiratory chain cytochromes b. c, and oxidase. Incubation of mitochondria in the hypotonic media caused a rapid decrease in the intermembrane and intracristal spaces and concurrent expansion of the matrix, without changing the total volume of the mitochondria.

These observations indicate that expansion of the inner‐membrane–matrix compartment of mitochondria in vitro specifically activates fatty acid oxidation at the site of β‐oxidation or at the site of its interaction with the respiratory chain, with a consequent increase in the states of reduction and phosphorylation of the mitochondria, Evidence is also presented which suggests that the activation of mitochondrial fatty acid oxidation by Ca2‐ is initiated by a Ca2+ ‐induced alteration of the structure of volume of the inner‐membrane–matrix region.
Transferrin Uptake by Cultured Rat Embryo Fibroblasts
Authors: OCTAVE Jean‐Noël , SCHNEIDER Yves‐Jacques , HOFFMANN Pierre , TROUET André , CRICHTON Robert R.
Published: March 2005

The effect of different drugs which affect lysosomal function (chloroquine, methylamine) and endocytosis (colchicine) or of the iron chelators desferrioxamine B, rhodotorulic acid, bipyridyl, 2,3‐dihydroxybenzoic acid, isoniazid pyridoxal hydrazone on the uptake of 59Fe‐loaded 3H‐labelled transferrin by cultured rat embryo fibroblasts has been studied. All these substances, except isoniazid pyridoxal hydrazone reduce the uptake of iron by fibroblasts, without any significant effect on the uptake of [3H]transferrin, nor of its specific binding to the cells. Cell fractionation experiments indicate that in the presence of methylamine and chloroquine, most of the cell associated 3H label is no longer transferred into lysosomes and remains associated with subcellular compartments which have a 5'‐nucleotidase activity (a marker enzyme of plasma membrane and related structures such as endocytic vesicles).

Short‐term kinetic experiments with fibroblasts incubated for 1 min at 37 °C with doubly labelled transferrin show that on reincubation in fresh medium almost all of the 59Fe initially bound remains associated with the cells whereas the 3H‐labelled transferrin is released with a t1/2 of about 1.5 min. In the presence of desferrioxamine B both 59Fe and transferrin, the former in part as ferrioxamine, are released from the cells with the same kinetics as is observed for transferrin in the control experiments. Similar studies with cells preincubated for 16 h with chloroquine or methylamine indicate that the release of transferrin from the cells is slowed down considerably. Chloroquine‐treated cells also release large amounts of 59Fe.

These results are consistent with the model for iron release from transferrin proposed previously [1] involving receptor mediated endocytosis of transferrin followed by release of iron during a rapid passage of the transferrinreceptor complex in lysosomes and recycling of the complex to the plasma membrane where the iron‐depleted transferrin is released. The inhibition of iron uptake by iron chelators such as desferrioxamine is consistent with complexation of iron by the chelator subsequent to its release from transferrin inside the cells and transfer of the iron‐chelate into the extracellular medium.
Binding of Disaccharides by Peanut Agglutinin as Studied by Ultraviolet Difference Spectroscopy
Authors: NEUROHR Klaus J. , BUNDLE David R. , YOUNG N. Martin , MANTSCH Henry H.
Published: March 2005

The binding of the disaccharides methyl β‐d‐lactoside and 2‐acetamido‐2‐deoxy‐3‐O‐(β‐d‐galactopyranosyl)‐ β‐dD‐galactopyranose [β‐d‐Gal‐(l→3)‐d‐GalNAc] to peanut agglutinin was studied by ultraviolet difference spectroscopy. The magnitude of the difference spectra varied with the concentration of the carbohydrates; association constants and thermodynamic parameters were determined from titration experiments at different temperatures. The enthalpy and entropy changes for binding of methyl β‐d‐lactoside were found to be ΔH°= ‐65 ± 4 kJ mol−1, ΔSdeg; =−156 ± 14 J mol−1 K−1. For β‐dD‐Gal‐ (1→3)‐d‐Gal NAc the observed thermodynamic parameters were ΔH°=−78 ± kJ mol−1, ΔS°=−177 ± 16 J mol−1 K−1. For both disaccharides, the enthalpy change upon binding to the lectin is much larger than that found for the binding of methyl α‐ and β‐d‐galactopyranosides, strongly suggesting the existence of an extended binding site on peanut agglutinin. The observed parameters are compared with those found for the binding of monosaccharides and oligosaccharides to other lectins and to lysozymc. Molecular models of the minimum energy conformers of β‐GaI‐(1→3)‐d‐GalNAc and methyl β‐d‐Iactoside are used to interpret the interaction of these, and structurally related ligands, with the peanut agglutinin binding site.
Enhanced Breakdown of Arylsulfatase A in Multiple Sulfatase Deficiency
Authors: WAHEED Abdul , HASILIK Andrej , VON FIGURA Kurt
Published: March 2005

Multiple sulfatase deficiency (mucosulfatidosis) is a lysosomal storage disorder characterized by the decrease in activities of all known sulfatases. To measure the apparent rate of synthesis and the half‐life of arylsulfatase A in multiple sulfatase deficiency, fibroblasts from patients with the disease and from controls were subjected to pulse‐chase labelling with radioactive amino acids. Arylsulfatase A and cathepsin D, a lysosomal enzyme that is not affected in multiple sulfatase deficiency, were isolated from cells and media by immunoprecipitation. The labelled polypeptides were separated by polyacrylamide gel electrophoresis, visualized by fluorography and quantified by liquid scintillation counting. Using single and double isotope techniques it was found that, as compared to cathepsin D, the apparent rate of synthesis of arylsulfatase A was 2–5‐times lower and the half‐ life 4–9‐times shorter in multiple sulfatase deficiency than in control fibroblasts. In multiple sulfatase deficiency fibroblasts the rates of endocytosis and the stabilities of endocytosed arylsulfatases A isolated from human urine and bovine testes were equal to those in metachromatic leucodystrophy fibroblasts.

We postulate that in normal cells a gene product exists that affects the stability of sulfatases and that multiple sulfatase deficiency is due to a mutation in this gene.
Enzymic Synthesis of Li:gnin Precursors
Authors: SCHMID Günter , GRISEBACH Hans
Published: March 2005

UDPglucose :coniferyl‐alcohol glucosyltransferase was isolated from cambial sap of spruce (Picea abies). An apparently homogeneous enzyme was obtained by a seven‐step procedure including dye‐ligand chromatography. The enzyme has an Mr, of about 50000 and consists of one polypeptide chain. Transferase activity is not influenced by metal ions.

The enzyme shows a pronounced substrate specificity towards UDPglucose and coniferyl alcohol with Km values of respectively 220 μM and 250 μM. The only reaction product is coniferin (coniferyl alcohol 7‐O‐β‐d‐glucopyranoside). No formation of ‘isoconiferin’ (coniferyl alcohol 1 ‐O‐β‐d‐glucoside) was detected. The reversibility of the reaction was proved by formation of [3H]UDPglucose from [3H]UDP and coniferin in the presence of the transferase. The products UDP and coniferin inhibit the reaction noncompetitively. Product inhibition patterns are consistent with a mono‐iso‐ordered bibi mechanism involving two isomeric enzyme forms.
Local Anaesthetics Including Benzyl Alcohol Activate the Adenylate Cyclase in Trypanosoma brucei by a Calcium‐Dependent Mechanism
Authors: VOORHEIS H. Paul , MARTIN B. Richard
Published: March 2005

The adenylate cyclase of Trypanosoma brucei is activated by local anaesthetics.

Activation of adenylate cyclase by the local anaesthetic, benzyl alcohol, requires Ca2+.

The kinetics of the presteady state reveal that the activation step occurs prior to and is separate from the catalytic step.

The activation step in the presence of Ca2+ can be stimulated some 200‐fold and is highly co‐operative with respect to benzyl alcohol concentration.

The mechanism by which local anaesthetics activate may be by exposing the Ca2+‐binding site of the catalytic step. Ca2+ receptor which is oriented towards the hydrophobic regions of the plasma membrane in the basal state.
The Electrochemical Proton Gradient of Saccharomyces
Authors: DE LA PEÑA Pilar , BARROS Francisco , GASCóN Santiago , RAMOS Sofia , LAZO Pedro S.
Published: March 2005

The electrochemical gradient of protons (ΔμΔH+) across the plasma membrane of Saccharomyces× 17/17 has been measured as a function of external pH and the external potassium concentration. The proton gradient (ΔpH) has been determined from the distribution of the weak permeable [14C]propionic acid across the plasma membrane. The accumulation of the lipophilic cation [3H]tetraphenylphosphonium has been used to calculate the membrane potential (ΔΨ). It has been observed that yeast cells are able to maintain a ΔμH+ whose values vary from −150 mV at pH 4.5 to −90 mV at pH 7.0. When considered independently ΔpH and ΔΨ have values which vary reciprocally depending on the external pH. At pH 4.5 δpH is the main component of ΔμH+ having a value of −105 mV. Then its value decreases as the external pH increases reaching a value of −10 mV at pH 7.0. At this pH ΔΨ is the main cornponcnt of δμ,H++ having a value of −80 mV, while at pH 4.5 its value is −45 mV. As the external concentration of potassium increases the value of ΔΨ becomes smaller. This decrease in ΔΨ is accompanied by an increase in the value of ΔpH. For any given concentration of potassium, the decrease in ΔΨ is larger than the increase in δpH the net result being, therefore, a decrease in the protonmotive force. In cells treated with the protonophores carbonylcyanide M‐chlorophenylhydrazone or dinitrophenol, δpH is drastically reduced at every pH while ΔΨ is not significantly changed. Upon treatment with dicyclohexylcarbodiimide ΔpH is reduced at any pH tested. This decrease is accompanied by an increase in the value of ΔΨ so that the total protonmotive force remains essentially constant. If the treatment with dicyclohexylcarbodiiniide is carried out in the presence of 200 mM potassium carbodiimide effect on ΔpH and ΔΨ is abolished. The results show that the movements of H+ and K+ are closely connected and play a central role in the establishment and maintenance of an energized state of the cell. They further suggest that δμH+ regulates the fluxes of protons while ΔμH+ regulates the establishment of a ΔμK+.
A Study of Some Molecular and Kinetic Properties of Two tRNA Methyltransferases from Mouse Plasmocytoma
Authors: NAU François , PHAM‐CœUR‐JOLY Ghislaine , DUBERT Jean‐Marie
Published: March 2005

A tRNA(adenine‐1)methyltransferase and a tRNA(cytosine‐5)methyltransferase have been partially purified from mouse plasmocytoma MOPC 173. Their apparent Mr are 200000—230000 and 110000—140000, respectively, as determined by gel filtration and density gradient centrifugation. Both enzymes exhibit maximum activity in the presence of high concentrations of monovalent cations (0.175 M and 0.25 M KCl, respectively) and in the absence of magnesium. Their kinetic constants have been determined at various KCl concentrations, with several tRNA species as substrates. These constants may differ by more than one order of magnitude, depending upon the substrate used, and they are strongly dependent upon the ionic concentration as well. The possibility that the tRNA(adenine‐1)methyltransferase from mouse plasmocytoma is different from the homolgous enzyme purified from a normal rat tissue [Glick, J. M. and Leboy, P. S. (1977) J. Biol. Chem. 252, 4790–4795] is discussed.
Intramitochondrial ATP and Cell Functions: Yeast Cells Depleted of Intramitochondrial ATP Lose the Ability to Grow and Multiply
Authors: GBELSKÁ Yvetta , ŠUBÍK Július , SVOBODA Augustin , GOFFEAU André , KOVÁČ Ladislav
Published: March 2005

Cells of the yeast Saccharomyces cerevisiae could be depleted of their intramitochondrial ATP by culturing on glucose in the presence of antimycin A, which prevents production of ATP in mitochondria, along with bongkrekic acid, which prevents transport of ATP from the cytosol into mitochondria. Alternatively, the depletion could be achieved by culturing respiration‐deficient mutants in the presence of bongkrekic acid. The depleted cells of the respiration‐deficient mutant did not grow on glucose in a synthetic medium and growth for a few generations was made possible by adding peptone, yeast extract or some amino acids into the medium. The depleted cells did not differ from control cells in their content of amino acids, proteins, nucleic acids and major phospholipids and had preserved the ability to carry on protein and nucleic acid syntheses and to mate to other cells. No conspicuous cytological differences were found between the control and depleted cells. After culturing in a semi‐synthetic medium in the presence of bongkrekic acid the cells of the respiration‐deficient mutant exhibited almost no cytochrome c in their spectra and their azide‐sensitive ATPase activity was drastically reduced. The results suggest that intramitochondrial syntheses of some low‐molecular compounds as well as import and/or assembly of some cytoplasmically synthesized mitochondrial proteins into mitochondria may be impaired in cells lacking intramitochondrial ATP and this may be responsible for their inability to grow and multiply.
The Distribution of Redox Equivalents in the Anaerobic Respiratory Chain of Paracoccus denitrificans
Authors: KUČERA Igor , DADÁK Vladimir , DOBRÝ Roman
Published: March 2005

The branching of the electron flow to individual teminal acceptors NO3−, NO2− and N2O was investigated in denitrifying cells of Paracoccus denitrificans. It was found that the extent of electron flow to nitrate depends on the activity of electron flow through the terminal part of the respiratory chain comprising cytochrome c.

NO2− and N2O either endogenously formed or exogenously added decreased the rate of nitrate reduction by oxidizing the respiratory chain via nitrite reductase and nitrous oxide reductase. The inhibitory effect of both intermediates of denitrification was released by antimycin. The rate of the first reaction of the sequence NO3−→ NO2−→ N2O → N2 is in the type of cells used under feedback redox control exerted by concentration of intermediates. This regulation can be designated as ‘inhibition by product via respiratory chain’.

The reaction rate of nitrate denitrification could also be influenced by changing the portion of electron flow from substrate to cytochrome c by artificial electron acceptors and donors. By varying the concentration of N,N,N′,N′‐tetramethyl‐p‐phenylenediamine in the presence of ascorbate, the desired redox state of cytochrome c and consequently various rates of nitrate reductase activity could be established. The basis of the enhancing effect of dithionite on the rate of nitrate reduction could also be explained by its functioning as an electron donor for the terminal part of respiratory chain.

The quantitative evalution of the extent of electron flow revealed that both branches of the respiratory chain (to nitrate reductase and to cytochrome c) compete for a constant limited flow of redox equivalents supplied from dehydrogenases. A strong evidence exists that the same mechanism is implied in the inhibition of nitrate reduction brought about by oxygen.
Catalytically Active Monomer Forms of Immobilized Arginase
Authors: AGUIRRE Raúl , KASCHE Volker
Published: March 2005

Native rat liver arginase was covalently coupled to Sepharose beads and the resulting matrix‐bound tetramer was subsequently dissociated by acid or EDTA treatment. The immobilized derivatives that remain in the matrix were renatured in the presence of the cofactor Mn2+ and analyzed in terms of recoverable activity before and after incubation with soluble enzyme. The activity of the immobilized enzyme was determined under conditions where it is directly proportional to the enzyme content. Both the acid‐treated and EDTA‐treated and renatured immobilized rat liver arginase have about one‐fourth the activity of the untreated immobilized enzyme. The activity of the renatured immobilized enzyme after reassociation with free enzyme was 85%(acid‐dissociated oligomer) and 60% (EDTA‐dissociated oligomer) of the activity of the initially immobilized oligomer. The acid‐dissociated and renatured immobilized arginase had a Km value (18 mM) that is approximately four times the value of the immobilized tetrameric enzyme (3.4 mM). After renaturation and reassociation with free enzyme the Km value of the immobilized enzyme was found to be equal to the value of the immobilized oligomer before dissociation. This indicates that the dissociated and renatured immobilized enzyme is monomeric. The Mn2+‐free buffer. The immobilized tetramer, on the other hand, does not lose Mn2+ after this procedure, and this ion seems to be bound even more tightly than in the case of the free tetramer. The reconstituted oligomer had a Km value approximating the value of the initially bound oligomer.

From these results and binding curves we conclude that rat liver arginase oligomer can be dissociated into monomers, which are catalytically active after renaturation with cofactor (Mn2+) at neutral pH. Thus, for arginase the tertiary structure is sufficient for the expression of the function of this enzyme.

The immobilized monomeric arginase has been used to purify crude arginase by subunit exchange (affinity) chromatography.
The Role of EF‐Tu in the Expression of tufA and tufB Genes
Authors: van der MEIDE Peter H. , VIJGENBOOM Erik , TALENS Anneke , BOSCH Leendert
Published: March 2005

We have studied the regulation of the expression of tufA and tufB, the two genes encoding EF‐Tu in Escherichia coli. To this aim we have ditermined the intracellular concentrations of EF‐TuA and EF‐TuB under varying growth conditions by an immunological assay in mutants of E. coli constructed for this purpose. The data show that in wild‐type cells the expression of tufA and tufB is regulated coordinately. This coordination is not restricted to steady‐state growth conditions but is maintained throughout the life cycle of the cells up till the stationary phase. The ratio in which the two genes are expressed, however, may vary among cells with different genetic constitutions. Neither complete elimination of EF‐TuB from the cell (by insertion of bacteriophage Mu DNA into tufB) nor elevation of the intracellular EF‐TuB concentration (by transformation with plasmids harbouring tufB) has any effect on the expression of tufA. A specific single‐site mutation of tufA, however, rendering EF‐TuA resistant to the antibiotic kirromycin, disturbs the coordinate expression oftufA and tufB, enhancing tufB expression exclusively. These results have been interpreted by assuming that in wild‐type cells the EF‐Tu protein itself is involved in the regulation of the expression of tufB and that the mutant species of EF‐Tu has lost this capacity either partially or completely. In agreement with this hypothesis are experiments performed in vitro with a coupled transcription/translation system programmed with DNA from a plasmid harbouring the entire tRNA‐tufB transcriptional unit as a template. They show that addition to this system of EF‐Tu in concentrations 2–5% of the endogenous amount results in strong inhibition of EF‐Tu synthesis.

We hypothesize that EF‐Tu acts as an autogenous repressor, inhibiting tufB expression post‐transcriptionally.
Hyperproduction of Phosphate‐Binding Protein, phoS, and pre‐phoS Proteins in Escherichia coli Carrying a Cloned phoS Gene
Authors: MORITA Takashi , AMEMURA Mitsuko , MAKINO Kozo , SHINAGAWA Hideo , MAGOTA Koji , OTSUJI Nozomu , NAKATA Atsuo
Published: March 2005

A large amount of phosphate‐binding protein, the phoS gene product, accumulated in the periplasmic space of the cells when an Escherichia coli strain carrying a multicopy plasmid containing a chromosomal fragment of the phoS‐phoT region (pSN507) was grown in a low‐phosphate medium. When the same strain carrying a plasmid containing only the phoS gene (pSN518 or pSN5182) was grown in low‐phosphate medium, phosphate‐binding protein accumulated in the periplasm, and in addition a larger protein accumulated in the non‐periplasmic fraction. The apparent Mr of this protein and the phosphate‐binding protein were 39000 and 35000 respectively, as judged by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. This larger protein showed immunological cross‐reaction with the phosphate‐binding protein. The 39000‐Mr protein was also detected in cells carrying pSN507 when the proteins were pulse‐labeled with radioactive amino acids. From these findings, together with the fact that this protein is recovered from the membrane fraction, we conclude that this protein is an unsecreted precursor protein of the phosphate‐binding protein.

Kinetics and regulation of accumulation of these proteins were studied. This system will be useful for preparation and purification of the precursor protein for biochemical studies in relation to the mechanism of protein secretion.