bluuebunny/crossref_metadata_2025_split huggingface dataset:
ΔpH Induced Calcium Fluxes in Rat Liver Mitochondria
Authors: BERNARDI Paolo , AZZONE Giovanni Felice , VERONESE Paolo 
Published: March 2005

The paper reports an investigation on ΔpH‐driven Ca2+ fluxes in rotenone‐treated rat liver mitochondria. The H+ concentration gradient (acidic inside) required to drive Ca2+ influx is obtained from the K+ concentration gradient through a H+/K+ exchange. The rate of H+/K+ exchange is very low in native mitochondria and is markedly enhanced by the electroneutral ionophore nigericin. The rate of Ca2+ influx depends on the rate of generation of ΔpH; it is negligible in native mitochondria and fast after nigericin.
The process of ΔpH‐driven Ca2+ influx shows three basic features. First, the rate of Ca2+ influx is markedly increased by the addition of uncouplers. Second, the rate of Ca2+ influx is inhibited by ruthenium red with the same sensitivity as the processes driven by the H+ pump or K+ diffusion. The electroneutral H+/Ca2+ ionophore A23187 restores Ca2+ influx in mitochondria inhibited by ruthenium red. Third, both rate and extent of ΔpH‐driven Ca2+ influx are inhibited by the addition of weak bases but not of weak acids.
The process of ΔpH‐driven Ca2+ influx is accompanied by H+ efflux. A correlation also exists between generation of ΔpH (acidic inside) due to H+/K+ exchange and utilization of ΔpH due to H+/Ca2+ exchange. The process of ΔpH‐driven, A23187‐catalyzed, H+/Ca2+ exchange is not preceeded by rapid H+/K+ exchange in the absence of nigericin.
After termination of the phase of Ca2+ influx a phase of Ca2+ efflux ensues. Ca2+ efflux is not coupled to H+ or K+ reentry and is completely inhibited by ruthenium red. Ca2+ efflux is not observed when Ca2+ transport is catalyzed by A23187 in mitochondria inhibited by ruthenium red. Furthermore A23187 abolishes Ca2+ efflux also when Ca2+ influx occurs in the absence of ruthenium red.
The data suggest that the ΔpH‐driven Ca2+ influx and the subsequent Ca2+ efflux take place through the native carrier as electrical uniport.
Specific Labeling of a Phosphate‐Transporting Protein from Rat‐Liver Mitochondria by [203Hg]Mersalyl
Authors: HOFMANN Hans‐Dieter , KADENBACH Bernhard 
Published: March 2005

A highly specific labeling of a phosphate‐transporting protein from rat‐liver mitochondria was obtained with [203Hg]mersalyl under the following conditions: (a) labeling of mitoplasts, isolated by the French‐Press procedure; (b) washing of the isolated inner membranes with NaCl/octylglucoside.
The concentration‐dependent labeling of the protein with mersalyl showed a biphasic saturation which correlated well with the protection and inhibition of phosphate transport.
The molecular weight (Mr 31000) and the amount of labeled carrier protein (24 nmol/g mitochondrial protein) at 100 protection correlated well with published data from the literature.
Gel filtration of the labeled protein in the presence of non‐ionic detergents showed a molecular weight of at least twice of that obtained by gel electrophoresis in the presence of sodium dodecylsulfate.
Purification and Characterization of a Chymotrypsin‐Like Enzyme (Protease‐S) in Thermally Injured Rat Skin
Authors: MIYAKE Yoshimasha , ITO Toshiko 
Published: March 2005

Protease‐S was extracted from thermally injured rat skin, and partially purified by column chromatography using Sephadex G‐50, CM‐Sephadex (A‐50), Sephadex G‐75 gel filtration.The optimum pH of this enzyme was 8.6–8.8, and the molecular weight determined by Sephadex G‐75 gel filtration was approximately 30000. This enzyme is active on the N‐acetyl‐L‐tyrosine ethyl ester, N‐succinyl‐L‐phenylalanine‐p‐nitroanilide (of chymotrypsin substrate) but not N‐tosyl‐L‐arginine methyl ester, N‐benzoyl‐L‐arginine‐p‐nitroanilide. Also, protease‐S was completely inhibited by diisopropylfluorophosphate (1 mM) or phenylmethylsulfonylfluoride (10 μM), and N‐tosyl‐L‐phenylalanine chloromethylketone (1 mM). These results are very similar to those obtained with bovine chymotrypsin. But the enzyme is not identical with the chymotrypsin‐like proteases in mast cells and leukocyte granules. When protease‐S was measured during the inflammatory reaction in vivo, maximal activity was found after 8 h, at the end of inflammation.
Identification of the Amino Acid Residue Modified in Bacillus stearothermophilus Alcohol Dehydrogenase by the NAD+ Analogue 4‐(3‐Bromoacetylpyridinio)butyldiphosphoadenosine
Authors: JECK Reinhard , WOENCKHAUS Christoph , HARRIS J. Ieuan , RUNSWICK Michael J. 
Published: March 2005

4‐(3‐Bromoacetylpyridinio)butyldiphosphoadenosine was synthesized with a [carbonyl‐14C]acetyl label. The reactive coenzyme analogue inactivates alcohol dehydrogenase from Bacillus stearothermophilus by forming a covalent enzyme‐coenzyme compound. The inactivation kinetics as well as the spectral properties of the modified enzyme after treatment with sodium hyposulphite suggest that the analogue is bound at the coenzyme binding site. B. stearothermophilus alcohol dehydrogenase modified with 14C‐labelled coenzyme analogue and subsequently carboxymethylated with unlabelled iodoacetic acid was digested with trypsin. The radioactive peptide was isolated and sequenced in parallel with the corresponding peptide similarly isolated from unmodified enzyme that had instead been carboxymethylated with iodo[14C]acetic acid. Amino acid and sequence analysis show that Cys‐38 of the B. stearothermophilus alcohol dehydrogenase was modified by the reactive coenzyme analogue. This residue is homologous to Cys‐43 in yeast alcohol dehydrogenase and Cys‐46 in the horse liver enzyme but, unlike the latter two, Cys‐38 is not reactive towards iodoacetate in the native bacterial enzyme.
Affinity Labelling of Yeast and Liver Alcohol Dehydrogenases with the NAD Analogue 4‐(3‐Bromoacetylpyridinio)butyldiphosphoadenosine
Authors: WOENCKHAUS Christoph , JECK Reinhard , JÖRNVALL Hans 
Published: March 2005

The NAD analogue 4‐(3‐bromoacetylpyridinio)butyldiphosphoadenosine inactivates alcohol dehydrogenases from horse liver and yeast by modification of amino acid side chains at the active sites of the proteins. In the presence of excess inactivator the reaction is pseudo first order. The stoichiometry is one mole inactivator incorporated per mole enzyme subunit.The liver enzyme is inactivated by ketoalkylation of the essential cysteine residue at position 46. No intermediate reactions of other residues are detected, and added cysteine does not influence the modification. In contrast, the labelling results with the yeast enzyme depend on cysteine treatment. The only radioactive peptide isolated is labelled on the essential cysteine residue 43.
The Complete Amino‐Acid Sequence of Histone H2B from the Mollusc Patella granatina
Authors: van HELDEN Paul D. , STRICKLAND Walter N. , BRANDT Wolf F. , von HOLT Claus 
Published: March 2005

1. From the marine mollusc, Patella granatina, a histone has been isolated. Its primary structure has been established and it has been designated histone H2Bpatella. It consists of a polypeptide chain of 121 amino acids.2. In the carboxy‐terminal two thirds of the molecule there is a high degree of sequence homology to the corresponding region in calf histone H2B with identical residues in 95% of the positions.3. In the N‐terminal 22 amino acids histone H2Bpatella differs considerably from the mammalian histone H2B and it is shorter by four residues.
Phase Behaviour of Cord Factor and Related Bacterial Glycolipid Toxins
Authors: DURAND Elisabeth , WELBY Michele , LANEELLE Gilbert , TOCANNE Jean‐François 
Published: March 2005

C‐6 esters of methyl α‐D‐glucoside and C‐6, C‐6′ diesters of α,α′‐D‐trehalose with C18 and C32threo and erythro mycolic acids (from chemical source) and of C80‐erythro‐mycolic acid (from natural source) have been synthesized. Esters of a C32 deoxy analogue were prepared as well.Throughout a monolayer study at the air‐water interface, these glycolipids are shown to form well organized phases in which the two hydrocarbon chains of mycoloyl residues must be in interaction.Compression isotherms of C32 esters suggested a transition between liquid‐expanded and liquid‐condensed states. Latent heats Qc and entropy changes ΔS associated with these phase transitions as well as the critical temperature at which they occur have been measured. Within the monolayer, the molecular packing of these glycolipids depends on the presence of the hydroxyl group of mycoloyl residues and on its stereochemistry. In particular, intermolecular hydrogen bonds between these groups are postulated in the case of the bis(C32‐erythro‐mycoloyl)‐trehalose.On the other hand, short chain C18 esters form fluid phases (t > 10°C) whereas very long chain C80 mycoloyl esters of trehalose exist in a condensed state (t= 20°C).These glycolipids were found to interact strongly with dipalmitoylphosphatidylcholine and egg yolk lecithins (3‐sn‐phosphatidylcholine). Their phase behaviours are discussed in connection with hypotheses concerning the way they can interact with mitochondrial membranes.
Dissociation and Association of Fumarase Subunits with Special Reference to the Formation of a Functional Tetramer
Authors: YAMATO Susumu , MURACHI Takashi 
Published: March 2005

Swine heart fumarase is known to exist as a tetramer which can be dissociated with denaturing agents into inactive subunits. We have succeeded, for the first time, in reverting once‐dissociated subunits to form a functional tetramer, and studied the mechanism of the restoration of its activity.Incubation of fumarase in a medium containing 50 mM sodium phosphate buffer, pH 7.3, 10 mM dithiothreitol, 0.1 mM EDTA · 2 Na, 10% glycerol, 200 mM KCl and 6 M urea at 25°C for 30 min resulted in dissociation of the tetrameric enzyme into monomeric subunits which were totally inactive and lost most α‐helical structure, with all sulfhydryl groups freely accessible to the medium. The denatured subunits could then be reassociated by dialysis at 4°C for 24 h against the same medium as above with omission of 6 M urea. The product was found to be an inactive dimer which could be transformed into an enzymatically active tetramer only after incubation at 25°C for 3 h. The final product was indistinguishable from the native fumarase in physical, chemical and enzymatic properties.All the ingredients of the media were found to be necassary to achieve freely reversible and complete conversion of monomer to tetramer. An intermediate product, a dimer, showed fluorescence and circular dichroism spectra almost indistinguishable from those of a tetramer, but it was highly susceptible to tryptic digestion while the tetramer was strongly resistant. It is concluded that a small but very important conformational change associates with the conversion of dimers to tetramers, finalizing proper orientation of the active site residues.
NADPH‐Cytochrome c Reductase of Candida tropicalis Grown on Alkane
Authors: BERTRAND Jean Claude , BAZIN Hermine , ZACEK Marcelle , GILEWICZ Michèle , AZOULAY Edgard 
Published: March 2005

During growth on n‐tetradecane Candida tropicalis synthesizes a NADPH‐cytochrome c reductase whose level is 4‐times higher than that of cells grown on glycerol and 20‐times higher than that of cells grown on glucose. An original method of solubilisation of this enzyme based on an osmotic shock is described. Studies of the purified enzyme showed that it is a flavoprotein having an apparent molecular weight of 67000 (± 1000) containing 1 mol each of FMN and FAD per mol of enzyme. Dichloroindophenol and ferricyanide are reduced by this enzyme. NADPH is reduced in the presence of menadione. The Michaelis constant for cytochrome c and NADPH are respectively equal to 4.55 μM and 29 μM. The maximal activity is obtained at a pH comprised between 7.6 and 8. It is influenced by ionic strength and inhibited by thiol inhibitors.The NADPH‐cytochrome c reductase present in the cells grown on glycerol was isolated and purified. Its properties were found identical to those of the enzyme present in the cells grown on tetradecane, including its apparent molecular weight. However, its physiological function remains to be determined. Effectively we have shown that the cells grown on glycerol do not synthesize cytochrome P‐450 and the microsomal fraction prepared from these cells does not have any hydroxylase activity with dodecanoic acid as substrate.This flavoprotein involved in the hydroxylation of the alkanes is a cytochrome c reductase, probably a cytochrome P‐450 reductase.
Binding of Oligonucleotides to the Disk of Tobacco‐Mosaic‐Virus Protein
Authors: GRAHAM James , BUTLER P. Jonathan G. 
Published: March 2005

The trinucleoside diphosphate A‐A‐G and the hexanucleotide fraction from a ribonuclease I digest of yeast RNA have been soaked into crystals of the disk aggregate of tobacco mosaic virus protein. At high concentrations these cause disruption of the crystal, probably by mimicking the normal nucleation of assembly.At lower nucleotide concentrations the crystals remain intact and the differences caused by nucleotide binding have been studied by X‐ray diffraction. The most obvious change is an upwards movement of about 0.3 nm at the low‐radius end of the left radial helix in the protein with some stiffening of the helix so that it now extends visibly in from 4 nm to 3 nm radius. Similar shifts also occur in the right radial and left slewed helices.A positive peak, which is tentatively identified with the bound oligonucleotide, is seen around 4 nm radius and below the right radial helix. The amino acid residues in possible contact with this feature are discussed.
Cobalt(III) Labeling of Methionyl‐tRNA Synthetase from Escherichia coli
Authors: KALOGERAKOS Théodore , BLANQUET Sylvain , WALLER Jean‐Pierre 
Published: March 2005

Native and trypsin‐modified methionyl‐tRNA synthetases from Escherichia coli were found to be inactivated by incubation in the presence of Co(III) complexes of ATP, stabilized either by imidazole or phenanthroline, or by oxidation in situ to Co(III) of the substrate ATP‐Co(II).It has been shown that the inactivation proceeds by specific labeling of the catalytic ATP‐Mg(II) site of the synthetases. The enzymes are completely inactivated by the incorporation of one cobalt atom and one ATP molecule per active site.The inactivated enzymes may be stored for a long period without significant reactivation or removal of the cobalt label. In the presence of dithiothreitol or 2‐mercaptoethanol, the labeled enzymes recover full activity with concomittant release of the bound label molecules.
Defect in the Split Proteins of 30‐S Ribosomal Subunits and Under‐Methylation of 16‐S Ribosomal RNA in a Polymamine‐Requiring Mutant of Escherichia coli Grown in the Absence of Polymines
Authors: IGARASHI Kazuei , KASHIWAGI Keiko , KISHIDA Kazuo , WATANABE Yasuhiro , KOGO Atsuko , HIROSE Seiyu 
Published: March 2005

Polyphenylalanine synthesis was carried out with Escherichia coli Q13 50‐S ribosomal subunits and reconstituted 30‐S particles containing different combinations of 23‐S core particles and 30‐S subunit split proteins obtained from a polyamine‐requiring mutant of E. coli during its growth in the presence or absence of putrescine. It was concluded that the defect in the amount of some kinds of 30‐S subunit split proteins was responsible for the decrease of polypeptide synthesis in a polyamine‐requiring mutant of E. coli grown in the absence of polyamines.The methylation of 16‐S RNA during growth in the absence of putrescine was decreased, while the degree of methylation of 23‐S RNA did not change significantly. The decrease in methylation of 16‐S RNA in the absence of putrescine was due mainly to a decrease of methylation of adenine.The relationship between the decrease of polypeptide synthetic activity of 30‐S ribosomal subunits obtained from a polyamine‐requiring mutant of E. coli grown in the absence of polyamines and the decrease of methylation of 16‐S RNA is discussed.
Properties of Reconstituted Cholesterol 7α‐Hydroxylase System from Rat and Rabbit Liver Microsomes
Authors: HANSSON Ronnie , WIKVALL Kjell 
Published: March 2005

7α‐Hydroxylation of cholesterol was studied in reconstituted systems containing cytochrome p‐450 and NADPH‐cytochrome P‐450 reductase from rat and rabbit liver microsomes. NADPH‐cytochrome P‐450 reductase was prepared with a biospecific affinity chromatographic method. Cytochrome P‐450 was prepared by solubilization with non‐ionic detergents and various chromatographic procedures. After the last purification step two fractions of cytochrome P‐450 from rat liver were analyzed for cholesterol 7α‐hydroxylase activity. One fraction had low or no detectable 7α‐hydroxylase activity but was able to catalyze 7α‐hydroxylation of taurodeoxycholic acid, and 11‐hydroxylation and 12‐hydroxylation of lauric acid. The other fraction catalyzed an efficient 7α‐hydroxylation of cholesterol as well as 7α‐hydroxylation of cholestanol. This fraction also catalyzed 12α‐hydroxylation and 26‐hydroxylation of 5β‐cholestane‐3α,7α‐diol, 7α‐hydroxylation of taurodeoxycholic acid and, 11‐hydroxylation and 12‐hydroxylation of lauric acid. Three cytochrome P‐450 fractions from rabbit liver, obtained after the final purification step, were analyzed for cholesterol 7α‐hydroxylase activity. Two fractions had no detectable 7α‐hydroxylase activity but were able to catalyze 12α‐hydroxylation and 25‐hydroxylation of 5β‐cholestane‐3α,7α‐diol, 25‐hydroxylation of 5β‐cholestane‐3α,7α,12α‐triol and 11‐hydroxylation and 12‐hydroxylation of lauric acid. The third fraction catalyzed in addition 7α‐hydroxylation of cholesterol and cholestanol.The cholesterol 7α‐hydroxylase activity showed an absolute requirement for cytochome P‐450 and NADPH‐cytochrome P‐450 reductase. Phospholipid had no significant effect on cholesterol 7α‐hydroxylation.
An Apolipoprotein Homolog of Rat Apolipoprotein A‐IV in Human Plasma
Authors: BEISIEGEL Ulrike , UTERMANN Gerd 
Published: March 2005

A protein with properties similar to apoprotein A‐IV of Rat high‐density lipoproteins has been isolated from the lipoprotein fraction (δ < 1.006 g/ml) of individual non‐fasting hypertriglyceridaemic subjects. The Mr of human apolipoprotein A‐IV determined by polyacrylamide‐gel electrophoresis in sodium dodecylsulfate and by column chromatography in 6 M guanidine hydrochloride is 46000. The amino acid composition is significantly different from any previously characterised human apolipoprotein but resembles that of rat apolipoprotein A‐IV. The N‐terminal amino acid of human and rat apolipoprotein A‐IV is glutamate or glutamine. The pI of the major human A‐IV band in 6 M urea is ∼ 5.15. Immunochemically apolipoprotein A‐IV is not identical with any of the well known human apolipoproteins. Human apolipoprotein A‐IV is present in all fractions of δ < 1.006 g/ml isolated by a single ultracentrifugal spin from non‐fasting subjects, but is lost from lipoproteins of δ < 1.006 g/ml upon recentrifugation.Immunoelectrophoretic studies show that most of human apolipoprotein A‐IV is present in the fraction of δ > 1.21 g/ml and is unassociated with the major lipoprotein fractions in serum. Charge‐shift electrophoresis however clearly demonstrates the hydrophobic character of human apolipoprotein A‐IV. There is evidence that apolipoprotein A‐IV is a constituent of chylomicrons that is removed from the particle surface when chylomicrons enter the plasma compartment.
Immunological Cross‐Reactivity between Electric‐Eel Acetylcholinesterase and Rat‐Tail‐Tendon Collagen
Authors: ANGLISTER Lili , TARRAB‐HAZDAI Rebecca , FUCHS Sara , SILMAN Israel 
Published: March 2005

Immunological cross‐reactivity between acetylcholinesterase from the electric organ of the electric eel and rat tail tendon collagen was examined both on the cellular and humoral levels.

Guinea pigs immunized with rat tail tendon collagen displayed a strong delayed‐type skin reaction when tested with the elongated acetylcholinesterase preparation (i.e. 14‐S + 18‐S molecular forms). However, when the glubular 11‐S enzyme was tested, almost no cross‐reactivity was obtained. Similarly, guinea pigs immunized with 14‐S + 18‐S preparation exhibited skin sensitization to rat tail tendon collagen.
Using a radioimmunoassay, it was observed that 125I‐labeled 14‐S + 18‐S acetylcholinesterase binds efficiently to rabbit antiserum elicited against rat tail tendon collagen, whereas 125I‐labeled 11‐S enzyme does not bind at all to this antiserum. Similar results were obtained by passive hemagglutination assay.

The experiments suggest that 14‐S + 18‐S acetylcholinesterase, but not 11‐S enzyme, which is devoid of the tail structure, has antigenic determinants in common with collagen from rat tail tendon.
The ‘DNA‐Membrane Complex’ of Escherichia coli B/r
Authors: ROBERTSON William , WATKINS David 
Published: March 2005

The composition and properties of the ‘DNA‐membrane complex’ of Escherichia coli B/r have been investigated. The ‘complexes’ contain most of the DNA and membrane of the cells, and about 50% and 25% of the RNA and protein respectively. The properties of DNA synthesized by the ‘complexes’ are described and the process is concluded to be largely mediated through polymerase I.Nascent DNA synthesized by the ‘DNA‐membrane complexes’ was of two main classes, one of molecular weight around 600000–800000 and the other of higher molecular weight. Polynucleotide ligase activity was not detectable. The onset of synthesis coincided with the dissociation of at least 70% of the genome DNA and all of the nascent DNA from the ‘complexes’ and was concomitant with the action of a nuclease on parental DNA. This nuclease activity was not ATP‐dependent.
Receptor‐Mediated Gonadotropin Action in Ovary
Authors: AZHAR Salman , MENON K. M. Jairam 
Published: March 2005

The effect of gangliosides and cholera enterotoxin on 125I choriogonadotropin binding, cAMP formation, protein kinase activation and progesterone production have been studied in isolated rat ovarian interstitial cells. A close correlation was observed between the amount of adenosine 3′:5′‐monophosphate (cAMP) produced in the cells and the extent of the activation of protein kinase in response to cholera enterotoxin and choriogonadotropin. Similarly cholera enterotoxin, like choriogonadotropin, stimulated steroidogenesis in a dose‐dependent and time‐dependent manner.Addition of highly purified ganglioside mixtures abolished the stimulatory effect of cholera enterotoxin on cAMP and progesterone responses, while these agents had no effect on choriogonadotropin‐stimulated responses. Similar results were also obtained when different lutropin preparations were tested. Pretreatment of ovarian cells with gangliosides did not show any enhancement in the extent of steroidgenesis stimulated by toxin or trophic hormones compared to that seen with untreated cells.Both gangliosides and cholera enterotoxin failed to block 125I‐labelled choriogonadotropin binding to ovarian cells. Individual gangliosides GD1a, GD1b, GT1 and GM1 [designated according to Svennerholm (1963) J. Neurochem. 10, 613–623] did not modify gonadotropin binding to ovarian cells. Similarly gangliosides isolated from rat ovary, bovine corpus luteum and rat brain also lacked the ability to modify 125I‐labelled choriogonadotropin binding to cells. Analysis of ovarian cells for gangliosides indicated that major ganglioside was GM3 with trace amounts of other gangliosides including GM1 and more complex gangliosides.The results presented in this communication suggest that gonadotropin binding and subsequent cAMP and progesterone production are not influenced directly by gangliosides. In contrast, choleragen‐stimulated cellular events in the ovarian cells were completely inhibited by gangliosides. This suggests that these two agents exert their effects on the responsive system by interacting through separate components on the cell surface.