bluuebunny/crossref_metadata_2025_split huggingface dataset:
The function of cytoplasmic membrane of Paracoccus denitrificans in controlling the rate of reduction of terminal acceptors
Authors: KUČERA Igor , LAUČÍK Jaroslav , DADÁK Vladimír 
Published: March 2005

The rate of reduction of terminal acceptors (nitrate, nitrite, and oxygen) in anaerobically grown cells of Paracoccus denitrificans increased on permeabilization of cytoplasmic membrane. It was proved that under aerobic conditions the increase of the rate of nitrate reduction was caused by: (i) the abolishment of the permeability barrier for nitrate, (ii) the enhancement of the influx of redox equivalents to the respiratory chain due to the stimulation of succinate dehydrogenase reaction, and (iii) the inhibition of electron flow to oxygen by endogenously formed nitrite.Nitrite inhibits oxygen reduction by its interaction with the terminal part of the respiratory chain (I50= 15 μM) localized at the inner aspect of the cytoplasmic membrane. The distribution of nitrite between intact cells and the suspension medium follows the Nernst equation for monovalent anion. The possible physiological consequences of the low intracellular nitrite concentration are discussed.
Influence of species and sex on cholesterol 7α‐hydroxylase activity in experimental animals
Authors: BOSISIO Enrica , GALLI Giovanni , GALLI KIENLE Marzia 
Published: March 2005

Cholesterol 7α‐hydroxylase activity was evaluated measuring by selected ion monitoring the amount of 7α‐hydroxycholesterol formed from endogenous microsomal cholesterol and from exogenously added [4‐14C]cholesterol in incubations of liver microsomes.The enzyme activity was determined in female rats, hamsters and guinea pigs and results were compared with those obtained in male rats.The results suggest that there is no difference between male and females as far as the activity of the enzyme from endogenous cholesterol is concerned at least in rat liver. Moreover, saturation of the enzyme by the endogenous substrate was observed in all female animals in contrast to the male rat for which the enzyme was found to be not saturated, as already reported by others.From molar radioactivity of 7α‐hydroxycholesterol obtained during incubations with [4‐14C]cholesterol, differences were also evidenced in the equilibration of the substrate with the endogenous pool of cholesterol. In the male rat, equilibration depended upon the concentration of radioactive cholesterol added to the medium and reached completion at 60 μM concentration. In female animals, molar radioactivity of 7α‐hydroxycholesterol was always linearly correlated with that of cholesterol in the medium but it was always lower than that one. This indicates that in the tested female animals only part of exogenously added cholesterol is accessible to the substrate pool of the enzyme.
Primary structure of protamine from the Northern pike Esox lucius
Authors: SPECKERT Wally , KENNEDY Brian , DAISLEY St Laurent , DAVIES Peter 
Published: March 2005

The basic nuclear protein in the sperm of the Northern pike is a protamine, 32‐residues long, which behaved as a single component during ion‐exchange chromatography and gel electrophoresis. Amino acid analysis gave close to molar ratios for the eight different residues with no evidence of microheterogeneity. However, the presence of sequence variants was revealed following a combination of automated protein sequencing and cleavage of the protamine by CNBr, endoproteinase Lys‐C and thermolysin.At position 28 there is an equal probability of having serine or glycine. At position 9 glycine is found more frequently than serine. The reciprocal nature of the substitutions results in glycine and serine contents which are close to a 4:2 ratio. Pike protamines are homologous to those of the trout but show less sequence variation between components.
Isolation and structural characterization of five major sialyloligosaccharides and a sialylglycopeptide from normal human urine
Authors: PARKKINEN Jaakko , FINNE Jukka 
Published: March 2005

Five major sialyloligosaccharides and a sialylglycopeptide have been isolated from normal human urine by charcoal adsorption, gel filtration, ion‐exchange chromatography, and paper chromatography. Structural studies including gas‐liquid chromatography of monosaccharide and disaccharide derivatives, methylation analysis, glycosidase treatments, and CrO3 oxidation indicated the following structures for the compounds: 1, NeuAc(α2‐6)Gal(β1‐4)Glc; 2, NeuAc(α2‐6)Gal(β1‐4)GlcNAc; 3, NeuAc(α2‐3)Gal(β1‐4)Glc; 4, NeuAc(α2‐3)Gal(β1‐4)GlcNAc; 5, NeuAc(α2‐3)Gal(β1‐3) [NeuAc(α2‐6)]GalNAc; and 6, NeuAc(α2‐3)Gal(β1‐3) [NeuAc(α2‐6)]GalNAc(α1‐O)Ser. Compounds 4, 5, and 6 have not been described in a free form before.The presence of compound 5 in urine may suggest that it derives from glycoproteins through a catabolic pathway involving cleavage of the carbohydrate‐peptide linkage by an endo‐N‐acetylgalactosaminidase. The predominating sialyloligosaccharides in urine were compounds 3 and 4. The predominance of the compounds with the sialyl(α2‐3) linkage is of interest in view of the recent discovery of uropathogenic Escherichia coli strains with binding specificity for sialyl(α2‐3)galactosides.
DNA topoisomerase I from calf thymus is inhibited in vitro by poly(ADP‐ribosylation)
Authors: JONGSTRA‐BILEN Jenny , ITTEL Marie‐Elisabeth , NIEDERGANG Claude , VOSBERG Hans‐Peter , MANDEL Paul 
Published: March 2005

A slight DNA topoisomerase I activity was detected in highly purified poly(ADP‐Rib)polymerase prepared from calf thymus. This copurified activity was found to be suppressed under conditions where the poly(ADP‐ribosylation) reaction occurs in the presence of NAD.Purified topoisomerase I from calf thymus was shown to be ADP‐ribosylated by poly(ADP‐Rib) polymerase purified from the same tissue. Poly(ADP‐ribosylation) of topoisomerase I produces an inhibition of the enzymatic activity in parallel to the extent of ADP‐ribosylation.The fact that a slight poly(ADP‐Rib) polymerase activity was also found to copurify with a topoisomerase I preparation and that toposiomerase I activity can be modified by ADP‐ribosylation, may suggest a spacial and functional correlation of these two enzymes in chromatin.
Hepatocyte heterogeneity in glutamate uptake by isolated perfused rat liver
Authors: HÄUSSINGER Dieter , GEROK Wolfgang 
Published: March 2005

Glutamate is simultaneously taken up and released by perfused rat liver, as shown by 14CO2 production from [1‐14C]glutamate in the presence of a net glutamate release by the liver, turning to a net glutamate uptake at portal glutamate concentrations above 0.3 mM.
14CO2 production from portal [1‐14C]glutamate is decreased by about 60% in the presence of ammonium ions. This effect is not observed during inhibition of glutamine synthetase by methionine sulfoximine. 14CO2 production from [1‐14C]glutamate is not influenced by glutamine. Also, when glutamate accumulates intracellularly during the metabolism of glutamine (added at high concentrations, 5 mM), 14CO2 production from [1‐14C]glutamate is not affected.
If labeled glutamate is generated intracellularly from added [U‐14C]proline, stimulation of glutamine synthesis by ammonium ions did not affect 14CO2 production from [U‐14C]proline.
After induction of a perivenous liver cell necrosis by CCl4, i.e. conditions associated with an almost complete loss of perivenous glutamine synthesis but no effect on periportal urea synthesis, 14CO2 production from [1‐14C]glutamate is decreased by about 70%.
The results are explained by hepatocyte heterogeneity in glutamate metabolism and indicate a predominant uptake of glutamate (that reaches the liver by the vena portae) by the small perivenous population of glutamine‐synthesizing hepatocytes, whereas glutamate production from glutamine or proline is predominantly periportal. In view of the size of the glutamine synthetase‐containing hepatocyte pool [Gebhardt, R. and Mecke, D. (1983) EMBO J. 2, 567–570], glutamate transport capacity of these hepatocytes would be about 20‐fold higher as compared to other hepatocytes.
Purification, characterisation and reconstitution of glutaconyl‐CoA decarboxylase, a biotin‐dependent sodium pump from anaerobic bacteria
Authors: BUCKEL Wolfgang , SEMMLER Roswitha 
Published: March 2005

Glutaconyl‐CoA decarboxylase from Acidaminococcus fermentans is a biotin enzyme, which is integrated into membranes. It is activated by Triton X‐100 and inhibited by avidin. The results obtained by a combination of both agents indicate that biotin and the substrate‐binding site are located on the same side of the membrane.
The decarboxylase was solubilized with Triton X‐100 and purified by affinity chromatography on monomeric avidin‐Sepharose. The enzyme is composed of three types of polypeptides: the group of α chains (Mr 120000–140000) containing the biotin, the β chain (60000) and an apparently hydrophobic γ chain (35000). Sodium ions specifically protected the latter chain from tryptic digestion. It was supposed, therefore, that this chain might function as the Na+ channel. The β and γ chains but not the α chain could be labelled by N‐ethyl‐[14C]maleimide. Similar decarboxylases but with much smaller biotin peptides (Mr 15000–20000) were isolated from Peptococcus aerogenes and Clostridium symbiosum.
The decarboxylases from all three organisms could be reconstituted to active sodium pumps by incubation with phospholipid vesicles and octylglucoside followed by dilution. The Na+ uptake catalysed by the enzyme from A. fermentans was completely inhibited by monensin and activated twofold by valinomycin/K+ indicating an electrogenic Na+ pump. The coupling between Na+ transport and decarboxylation was not tight. During the reaction the ratio decreased from initially 1 to 0.2.
The three organisms mentioned above and Clostridium tetanomorphum without glutaconyl‐CoA decarboxylase are able to ferment glutamate and require 10 mM Na+ for rapid growth. There is no correlation between the concentration of monensin necessary to inhibit growth and the presence of decarboxylase in these organisms.
Purification and characterization of proteases from the polychaete annelid Sabellaria alveolata (L.)
Authors: PEAUCELLIER Gérard 
Published: March 2005

Eleven proteases have been purified to electrophoretic homogeneity from crude digestive fluid of polychaete annelids, Sabellaria alveolata. Purification steps were Sephadex G‐100 gel filtration, benzamidine‐cellulose and SBTI‐Sepharose (SBTI = soybean trypsin inhibitor) affinity chromatography, CM‐Sepharose and DEAE‐Sepharose ion‐exchange chromatography.Nine proteases have been purified in sufficient quantities for characterization. All are active at basic pH and are probably serine proteases, since they are inhibited by phenylmethylsulfonyl fluoride, specific chloromethyl ketone amino acids derivatives, but not by EDTA and p‐chloromercuribenzoate. They do not hydrolyse exopeptidase substrates.From their properties, they can be divided into five classes.

A trypsin‐like protease, which hydrolyses only trypsin substrates and is inhibited by N‐tosyl‐l‐lysine chloromethyl ketone (TosLysCH2Cl), leupeptin and antipain. It differs from bovine trypsin by its very acidic isoelectric point (below 3.3) and its higher Mr (35000).
A chymotrypsin‐like protease which hydrolyses only chymotrypsin substrates and is inhibited by TosPheCH2Cl, Z‐PheCH2Cl, chymostatin but only slightly by leupeptin and antipain. Its isoelectric point is below 3.3 and its Mr 31000.
Two minor chymotrypsin‐like proteases with slightly broader specificity, since they hydrolyse trypsin substrates significantly and are much more inhibited by leupeptin. They have acidic isoelectric points (3.3 and 3.5) and slightly lower Mr (27000).
Four proteases hydrolyse trypsin and chymotrypsin substrates equally well. Their chymotryptic character is, however, predominant since they are inhibited by TosPheCH2Cl and Z‐PheCH2Cl but not TosLysCH2Cl. They have similar Mr (27000) but isoelectric points ranging from 4.0 to above 9.1.
The last one is very similar but has lower esterolytic activities.

These proteases of broad specificity do not resemble any known serine protease since they differ from subtilisins by their sensitivity to TosPheCH2Cl.
Repair of O6‐ethylguanine DNA lesions in isolated cell nuclei
Authors: RENARD André , VERLY Walter G. 
Published: March 2005

Cell nuclei prepared from rat liver were alkylated in vitro with ethylnitrosourea; the nuclear DNA was found to lose O6‐ethylguanine and 7‐ethylguanine during a subsequent incubation at 37°C. The rate of O6‐ethylguanine loss is comparable to that observed in vivo, indicating that no cytoplasmic component is needed for the repair; no free O6‐ethylguanine was found in the incubation medium of the ethylated nuclei. The rate of 7‐ethylguanine loss is higher than the spontaneous depurination in vitro and an amount of free 7‐ethylguanine equivalent to that lost by the nuclear DNA was found in the incubation medium; these results suggest that this DNA lesion is excised by a DNA glycosylase.The proteins of the chromatin prepared from the isolated nuclei induced the disappearance of O6‐ethylguanine from an added ethylated DNA. No free O6‐ethylguanine was released indicating that the repair is not catalyzed by a DNA glycosylase; no oligonucleotides enriched in O6‐ethylguanine were released either, indicating that the disappearance of O6‐ethylguanine from DNA is not the result of the cooperative action of a specific endonuclease and an exonuclease.Activities capable of removing O6‐ethylguanine from DNA were found in other cell compartments; most of it, however, is in the nucleus where the main location is chromatin. A pretreatment of the rats with daily low doses of diethylnitrosamine during 3 or 4 weeks increased 2–3‐times the repair activity of the chromatin proteins.
Kinetic and thermodynamic evidence of a molybdate interaction with glucocorticoid receptor in calf thymus
Authors: CARDO Paolo P. , GAMBETTI Maurizio , VIGNALE Bruno , DIVANO Maria Carla 
Published: March 2005

The effect of molybdate on the kinetic and thermodynamic properties of the dexamethasone‐receptor interaction was studied in calf thymus cytosol.In the presence of molybdate both the equilibrium binding studies and the association and dissociation experiments reveal a significantly lower affinity of the receptor for [3H]dexamethasone. At 0°C the equilibrium dissociation constant increases from 0.8 nM to 1.8 nM, the association rate constant shifts from 1.5 × 108 M−1 h−1 to 0.2 × 108 M−1 h−1, whereas the rate of dissociation of the untransformed receptor increases from 0.04 h−1 to 1.1 h−1 in the molybdate‐containing buffer. All these effects appear dependent on the concentration of molybdate but the dissociation of the transformed receptor (0.01 h−1) is unaffected. The enthalpy for the association, ΔH+, increases at least twofold whereas the entropy, both for the association (ΔS*=−25 to +104 J K−1 mol−1) and for the equilibrium (ΔS°=−100 to +38 J K−1 mol−1), is markedly influenced by the presence of molybdate.Taken all together these data suggest that molybdate interacts with the receptor molecule turning it into a form that displays low affinity for steroid, in addition to the well‐documented incapacity to transform itself. This fact leads us to think that both the binding and the transformation are the expression of conformational modifications involving molybdate‐sensitive groups.
The presence of the iron‐sulfur protein (subunit V) of complex III in mitochondria of heme‐deficient yeast cells lacking iron‐sulfur clusters detectable by electron paramagnetic resonance
Authors: LIN Ching‐I. P. , OHNISHI Tomoko , CLEJAN Liviu , BEATTIE Diana S. 
Published: March 2005

The presence of subunit V, the iron‐sulfur protein, of complex III has been demonstrated in mitochondria from a mutant of Saccharomyces cerevisiae which lacks 5‐aminolevulinic acid synthase and, hence, is devoid of heme. The mature form (24 kDa) of the iron‐sulfur protein was observed in equal amounts in the heme‐deficient and heme‐sufficient cells with antiserum against subunit V and either the sensitive immuno‐transfer technique or immunoprecipitation from dodecylsulfate‐solubilized mitochondria. In addition, a slight shoulder with a molecular mass 1.5 kDa larger than the mature form was present in mitochondria from the heme‐deficient cells.Electron paramagnetic resonance spectroscopy revealed the absence of iron‐sulfur signals due to clusters S‐1, S‐2 and S‐3 of succinate dehydrogenase or to Rieske's iron‐sulfur cluster of complex III in mitochondria from the hemedeficient cells. The lack of iron‐sulfur centers in these cells may be a consequence of the absence of sulfite reductase in the cells without heme.
Casein kinase activity in rat mammary gland Golgi vesicles
Authors: WEST David W. , CLEGG Roger A. 
Published: March 2005

A Golgi vesicle preparation isolated from the mammary tissue of rats in mid‐lactation has been shown to contain the caseins of rat milk. These proteins were phosphorylated when the Golgi vesicles were incubated in the presence of [γ‐32P] ATP. Although this phosphorylation occurred when the physical integrity of the vesicles was maintained, it was markedly increased when the membrane structure was disrupted by hypoosmotic conditions or by use of detergents. The kinase responsible has been shown to be responsive to the intravesicular concentration of Ca2+ and to the extravesicular concentration of Mg2+. These results have been interpreted in terms of a model suggesting a transmembrane location for the enzyme with binding sites on the cytosolic membrane face for Mg2+ and possibly also for ATP and on the luminal surface for Ca2+ and the caseins.Others have postulated that the assembly of caseins into micelles occurs in Golgi vesicles and requires both prior phosphorylation of the proteins and the presence of Ca2+. In this investigation we demonstrate that treatments which increase the intravesicular casein phosphorylation also alter the Ca2+ balance within the vesicle lumen. These results are discussed in relation to the ATP‐dependent accumulation of Ca2+ by the mammary gland Golgi vesicles.
Effects of the skin mitogens tumor‐promotor 12‐O‐tetradecanoylphorbol 13‐acetate and divalent‐cation‐ionophore A23187 on ion fluxes and membrane potential in a murine epidermal cell line (HEL30) and in 3T3 fibroblasts
Authors: SEEMANN Dietmar , FÜRSTENBERGER Gerhard , MARKS Friedrich 
Published: March 2005

The transmembrane potential of HEL30 keratinocytes and 3T3 fibroblasts has been determined by measuring the distribution of labelled triphenylmethylphosphonium bromide. The tumor‐promotor 12‐O‐tetradecanoylphorbol 13‐acetate (1–5μM) induces hyperpolarization in 3T3 cells but does not exert any effect on the membrane potential of keratinocytes, whereas the divalent cation ionophore A23187 (0.5–1 μM) hyperpolarizes keratinocytes and probably also 3T3 cells.Studies on Na+ and Rb+ fluxes, as well as with different inhibitors, indicate that the hyperpolarizing effect is the consequence of an increased Na+ influx which in turn stimulates the Na+/K+‐dependent ATPase. No causal relationship seems to exist between the change of the membrane potential and arachidonic acid release (and subsequent prostaglandin synthesis) which is induced by both drugs in both cell lines.Since the induction of the arachidonic cascade (by both agents) as well as the stimulation of Na+ influx (by A23187) are found to be critically dependent on extracellular Ca2+ and are inhibited by ‘Ca2+‐blockers’, it is concluded that both reactions are triggered by the same event (Ca2+ translocation) but proceed independently of each other. The release of arachidonic acid is already stimulated under conditions where a measurable influx of Ca2+ is not yet observed. This indicates a local mobilization of Ca2+, perhaps across the plasma membrane.It is concluded that monovalent cation fluxes and changes of the membrane potential are not critically involved in the stimulation of the arachidonic acid cascade and cellular proliferation by agents which induce epidermal hyperplasia in vivo.
Conformational properties of phospholipases A2
Authors: DUFTON Mark J. , EAKER David , HIDER Robert C. 
Published: March 2005

The sequences of 32 phospholipases A2 (EC 3.1.1.4) were analysed by secondary‐structure prediction and the results were compared with the available crystallographic data. Good agreement is evident between prediction and experiment, especially for helical structure. Circular dichroic spectra were also determined for six enzymes from Elapid snake venom and these, in association with previously published spectra, confirm the main implication of the predictions, namely that all the homologues have qualitatively similar tertiary structures.Consideration was then given to possible structure/activity relationships in the light of the above findings. The relative hydrophobicity/hydrophilicity of the area of the enzyme thought to interact with lipid/water interfaces was predicted and certain correlations were noted with relative penetrating power, species of origin and the presence of β‐neurotoxic properties.
The expression of different monooxygenases supported by cytochrome P‐450 in neonatal rats and in primary fetal hepatocytes in culture
Authors: KREMERS Pierre , LETAWE‐GOUJON Francine , DE GRAEVE Jean , DUVIVIER Joseph , GIELEN Jacques E. , BASTIN Michèle , FRANKINET‐COLLIGNON Colette , WOLFF Dany 
Published: March 2005

In rat liver, the perinatal development of various monooxygenase activities follows different patterns, depending upon the reaction studied. The ontogeny of the 6β‐, 7α‐ and 16α‐testosterone hydroxylase activities differs very significantly.Aldrin epoxidase and steroid‐metabolizing monooxygenases are expressed in primary fetal rat liver cells in culture after treatment in vitro with dexamethasone. Testosterone is not metabolized by the control cells and is hydroxylated on the 6β and 16α positions following the addition of corticoids to the culture medium. The dose and time curves vary according to the hydroxylated position of the steroid. Aldrin epoxidase activity is nearly undetectable in the control cells, but is present and is inducible by phenobarbital following treatment with the corticoid. Phenobarbital induces aldrin epoxidase in the absence of dexamethasone in the culture medium, providing that the cells are pretreated with the corticoid for 48 h. The use of antibodies against the main cytochrome P‐450 species purified from adult and phenobarbital‐treated rats confirms that a similar cytochrome P‐450 can be induced in fetal cells in culture.The perinatal regulation of biological events, such as the expression of the monooxygenases, can be reproduced in fetal rat liver cells in culture; such a model constitutes a unique tool for studying the biochemical mechanisms which control these phenomena.