bluuebunny/crossref_metadata_2025_split huggingface dataset:
On the oxidation pathways of the mitochondrial bc1 complex from beef heart
Authors: ESPOSTI Mauro DEGLI , TSAI Ah‐Lim , PALMER Graham , LENAZ Giorgio 
Published: March 2005

We have investigated the oxidation of the reduced ubiquinol: cytochrome c (bc1 complex) isolated from beef heart mitochondria. The oxidation of cytochrome c1 by both potassium ferricyanide and cytochrome c in the ascorbate‐reduced bc1 complex is not a first‐order process. This is taken as evidence that cytochrome c1 is in rapid equilibrium with the Rieske iron‐sulphur center. Among the several inhibitors tested, only 5‐n‐undecyl‐6‐hydroxy‐4,7‐dioxobenzothiazole and stigmatellin are seen to affect this redox equilibrium between the high‐potential centers of the beef heart bc1 complex.The oxidation of cytochrome b by cytochrome c in both the succinate‐reduced and the fully reduced bc1 complex is blocked by all the inhibitors tested. This inhibition occurs simultaneoulsy with an acceleration in the oxidation of cytochrome c1, even after extraction of the endogenous ubiquinone which is present in the bc1 preparation. Almost complete extraction of ubiquinone from the bc1 complex has no effect upon the rapid phase of cytochrome b oxidation, nor does it alter the inhibition of cytochrome b oxidation by the various inhibitors.The oxidation of cytochrome b by exogenous ubiquinones is stimualted by myxothiazol and partially inhibited by antimycin. However, the addition of both these inhibitors together completely blocks the oxidation of cytochrome b by quinones. In contrast, the simultaneous addition of antimycin and myxothiazol has no such synergistic effect upon the oxidation of cytochrome b by cytochrome c.Our data show that intramolecular electron transfer from cytochrome(s) b to the Rieske iron‐sulphur center can take place in the bc1 complex without involvement of endogenous ubiquinone‐10. This electron pathway is sensitive to all the inhibitors of the enzyme.
The proton channel, CF0, in thylakoid membranes
Authors: LILL Holger , ENGELBRECHT Siegfried , SCHÖNKNECHT Gerald , JUNGE Wolfgang 
Published: March 2005

We investigated the conductance of pea thylakoid membranes and their capacity for photophosphorylation as function of the extraction of chloroplast coupling factor CF1. The degree of extraction was varied via the incubation time in EDTA‐containing hypo‐osmolar medium and was measured by rocket electroimmunodiffusion. The conductance of thylakoid membranes was measured by flash kinetic spectrophotometry.The time course of extraction followed the time course of thylakoid swelling. Contrary to expectation increasing loss of CF1 did not primarily increase the velocity of proton efflux from each vesicle. Instead proton‐tight vesicles were converted to leaky ones, which lost phosphorylating activity. Two subpopulations occurred, although both types of vesicles, leaky and proton‐tight ones, were CF1‐depleted to a similar degree. This implied that only a small fraction of CF1‐lacking CF0 was functional as a proton channel. Tight vesicles had no functional channels while leaky ones had at least one. We determined the proportion of tight vesicles in three independent ways: via the residual phosphorylation activity, via measurements of proton efflux and via measurements of the electric relaxation across the membrane. The results obtained were identical. A statistical evaluation of the data led us to the following conclusions. (a) EDTA treatment produced vesicles containing approximately 105 chlorophyll molecules, equivalent to a total of approximately 100 CF0CF1 per vesicle. (b) Even at the highest degree of extraction (75% of total CF1 extracted) only 2.5 out of 75 exposed CF0 per vesicle were proton‐conducting. (c) The unit conductance of one open CF0 channel was 169 ± 18 fS at pH 7.5 and room temperature. At an electrical driving force of 100 mV this was equivalent to the passage of approximately 105 protons/s.The most important consequence of this relatively high unit conductance was that a single open CF0 channel was capable of dissipating the protonmotive force of one vesicle, thereby deactivating the whole remaining catalytic capacity of this vesicle.
Reconstitution of CF1‐depleted thylakoid membranes with complete and fragmented chloroplast ATPase
Authors: ENGELBRECHT Siegfried , LILL Holger , JUNGE Wolfgang 
Published: March 2005

Chloroplast ATPase (CF1) was isolated from spinach, pea and maize thylakoids by EDTA extraction followed by anion‐exchange chromatography. CF1 was purified and resolved by HPLC into integral CF1, and CF1 lacking the δ & ɛ subunits: CF1(–δ) and CF1(–ɛ). Washing Mono‐Q‐bound CF1 with alcohol‐containing buffers followed by elution without alcohol produced the β subunit and in separate peaks CF1(–δ) and CF1(–ɛ). Elution from Mono Q in the presence of tenside yielded a βδ fragment, CF1(–δ) and CF1(–δɛ).Chloroplasts were CF1‐depleted by EDTA extraction. Reconstitution of photophosphorylation in these ‘EDTA vesicles’ was obtained by addition of CF1 and its fragments. CF1, CF1(–δ) and CF1(–δɛ) were active with cross‐reactivity between spinach, pea and maize. δ‐containing CF1 always reconstituted higher activities than δ‐deficient CF1. The βδ fragment and dicyclohexylcarbodiimide (DCCD)‐inhibited CF1 also were reconstitutively active while β and DCCD‐inhibited CF1(–δ) were not.These results support the notion that subunit δ can function as a stopcock to the CF0 proton channel as proposed by Junge, W., Hong, Y. Q., Qian, L. P. and Viale, A. [(1984) Proc. Natl Acad. Sci. USA 81, 3078–3082].
Reduced stability of RNA coding for yolk polypeptide 3 in Drosophila melanogaster ovary
Authors: WILLIAMS John Lewis , BOWNES Mary 
Published: March 2005

In Drosophila three yolk polypeptides (YP1, YP2 and YP3) are synthesised at two sites in the adult female: in the fat body tissue, from which they are transported via the haemolymph to the ovary, and in the ovarian follicle cells which surround the developing oocytes. All three yolk polypeptides are synthesised at equal levels in the fat body. In this paper we show that the steady‐state level of YP3 RNA is significantly reduced in the ovary in comparison with the fat body, and that none of the yolk protein genes is amplified either in the fat body or the follicle cells.In order to determine the basis of the reduced level of YP3 RNA in the ovary, which could result from a lower rate of transcription or through a decreased stability of the RNA, we have devised an in vivo method of determining relative rates of gene transcription. In both the fat body and the ovary all three yolk proteins are transcribed at similar rates. Thus we infer that YP3 RNA is destablished in the ovary, accounting for the reduction in its steadystate level.
Core region of Citrobacter lipopolysaccharide from strain PCM 1487
Authors: ROMANOWSKA Elzbieta , GAMIAN Andrzej , DABROWSKI Janusz 
Published: March 2005

The core structure of Citrobacter PCM 1487 lipopolysaccharide has been established using methylation analysis/mass spectrometry, chemical degradations and one‐ and two‐dimensional 1H‐NMR spectroscopy at 500 MHz. 1H‐NMR assignments are given for all sugar components of the core oligosaccharide. In the formula shown below, the alternative locations of branch terminal heptose (LD Hep) and diphosphorylethanolamine (PPEtN) residues are marked by dashed lines; dOclA stands for 3‐deoxy‐D‐manno‐octulosonic acid.
image

The sample of the core oligosaccharide showed some microheterogeneity due to a slightly incomplete substitution by terminal N‐acetylgalactosamine and a partial splitting of diphosphorylethanolamine residues.
Hexokinase PII from Saccharomyces cerevisiae is regulated by changes in the cytosolic Mg2+‐free ATP concentration
Authors: MORENO Fernando , FERNANDEZ Teresa , FERNANDEZ Rosa , HERRERO Pilar 
Published: March 2005

Hexokinase PII is not inhibited by high Mg‐ATP concentrations if the Mg2+‐free ATP is kept at low levels (0.01 mM) in the assay mixture. Hexokinase PI activity is not affected either by Mg2+‐free ATP nor by free Mg2+ in the assay mixture. Thus, hexokinase PI and PII activities appear not to be regulated by substrate inhibition as proposed previously [Kopetzki, E. & Entian, K. D. (1985) Eur. J. Biochem. 146, 657–662]. However, the level of Mg2+‐free ATP in the hexokinase PII assay mixture strongly affects the enzyme activity by decreasing the Vmax and increasing the Km value for Mg‐ATP from 0.15 mM to 5.0 mM.The physiological role of this inhibition, which has not been described previously, was investigated by determining the cytosolic ATP and Mg2+ concentrations in yeast cells grown under derepressing and repressing conditions. Derepression is accompanied by an important loss of Mg2+ from the cells, maintaining the ATP concentration constant. This produces an increase of Mg2+‐free ATP in the cytosol from 0.01 mM to 0.1 mM. This free ATP concentration would lead to a maximal inhibition of hexokinase PII.
Chemical characterization of enterobacterial common antigen isolated from Plesiomonas shigelloides ATCC 14029
Authors: BASU Sumanta , KUHN Hella‐M. , NESZMELYI András , HIMMELSPACH Karl , MAYER Hubert 
Published: March 2005

Serologically characterized samples of enterobacterial common antigen (ECA) from Plesiomonas shigelloides, Salmonella montevideo and Shigella sonnei were investigated by chemical methods including methylation and NMR techniques. All showed the same sugar composition and contained a lipid moiety with palmitic acid as main fatty acid and with a phosphodiester group. Additional enzymatic studies, reported in the preceding paper, provided evidence that the lipid moiety is an L‐glycerophosphatidyl residue attached via a phosphodiester linkage to C‐1 of GlcNAc as the reducing end of the ECA sugar chain.ECA of P. shigelloides showed the best‐resolved 13C‐NMR spectra, especially after the removal of non‐stoichiometric O‐acetyl groups at C‐6 of GlcNAc of the ECA repeating unit and of the lipid moiety by mild acid hydrolysis (0.01 M HCl, 100°C, 10 min). Subsequent 13C‐NMR studies were therefore carried out with the mild‐acid‐treated ECA of P. shigelloides which allowed a tentative assignment of all resonances of the ECA repeating unit. 13C‐NMR spectra of Salmonella and Shigella ECA were essentially the same as those obtained with Plesiomonas ECA.The same trisaccharide repeating unit was encountered as demonstrated previously in the cyclic form of ECA isolated from S. sonnei by Dell et al. [Carbohydr. Res. 133, 95–104 (1984)]. Methylation analysis, however, afforded small amounts of terminal GlcNAc thus proving, in combination with the demonstration of the attached lipid moiety, an acyclic nature of ECA from P. shigelloides and from the two enterobacterial species. The question of whether the cyclic form co‐exists in S. sonnei phase I and possibly in other enterobacterial species or, whether it had been formed during extraction as an artifact, has not yet been answered. The way in which ECA was isolated in our studies would preclude the presence of a non‐amphiphilic (cyclic) polysaccharide.The finding that the sugar chain of ECA is attached to an L‐glycerophosphatidyl residue is in full corroboration with serological, enzymatical and gel electrophoretic studies shown in the preceding paper and with the character of ECA as a surface antigen being anchored by hydrophobic interactions in the outer membrane of Entero‐bacteriaceae and P. shigelloides.
The Ca2+‐dependent actions of the α‐adrenergic agonist phenylephrine on hepatic glycogenolysis differ from those of vasopressin and angiotensin
Authors: KLEINEKE Jochen , SÖLING Hans‐Dieter 
Published: March 2005

The stimulation of hepatic glycogenolysis by the Ca2+‐dependent hormones phenylephrine, vasopressin and angiotensin II was studied as a function of intracellular and extracellular Ca2+.

In the isolated perfused rat liver the decline in glucose formation was monophasic (‘half‐life’∼ 3 min) with vasopressin (1 nM) or angiotensin II (0.05 μM), but biphasic (half‐life of 4.8 min and 17.6 min) in the presence of the α‐agonist phenylephrine (0.01 mM), indicating either a different mode of mobilization or the mobilization of additional intracellular calcium stores.
Under comparable conditions an elevated [Ca2+] level was maintained in the cytosol of hepatocytes for at least 10 min in the presence of phenylephrine, but not vasopressin.
Titration experiments performed in the isolated perfused liver to restore cellular calcium revealed differences in the hormone‐mediated uptake of Ca2+. The onset in glucose formation above that seen in the absence of exogenous calcium occurred at approximately 30 μM or 70–80 μM Ca2+ in the presence of phenylephrine or vasopressin respectively. The shape of the response curve was sigmoidal for vasopressin and angiotensin II, but showed a distinct plateau between 0.09 mM and 0.18 mM in the presence ofphenylephrine. The plateau was also observed at phenylephrine concentrations as low as 0.5 μM. The formation of plateaus observed after treatment of the liver with A 23187, but not after EGTA, is taken as an indication that intracellular calcium stores are replenished.
A participation of the mitochondrial compartment could be excluded by pretreatment of the liver with the uncoupler 2,4‐dinitrophenol.
Differences in the Ca2+ dependence of the glycogenolytic effects of these hormones were also revealed by kinetic analysis.
It is concluded that phenylephrine differs from vasopressin and angiotensin II in that, in addition to a more common, non‐mitochondrial pool, which is also responsive to the vasoactive peptides, the agonist mobilizes Ca2+ from a second, non‐mitochondrial pool.

The results are consistent with the proposal that Ca2+ transport across subcellular membranes may be subject to different hormonal control.
The phosphoglycerate kinases from Trypanosoma brucei
Authors: MISSET Onno , OPPERDOES Fred R. 
Published: March 2005

Trypanosoma brucei has two phosphoglycerate kinase (PGK) isoenzymes, one is particle‐bound and localized in glycosomes while the other is present in the cytosol. The cytosolic isoenzyme (cPGK) was 900‐fold purified from cultured procyclic trypanosomes by hydrophobic interaction chromatography on phenyl‐Sepharose followed by affinity chromatography on 2′,3′‐ATP‐Sepharose and had a specific activity of 275 units/mg protein. cPGK was compared with the purified glycosomal isoenzyme (gPGK) from bloodstream‐form trypanosomes as well as with the commercially available PGKs from yeast, rabbit muscle and Spirulina platensis, a blue‐green alga.Like all other PGKs, cPGK was a monomeric protein with a molecular mass of approximately 45 kDa similar to that of the PGKs from other organisms but 2 kDa smaller than that of gPGK. Despite this difference in length and a great difference in isoelectric point, the two trypanosome isoenzymes strongly resembled each other in several respects. (a) The kinetic parameters did not differ significantly from each other or from the PGKs of other organisms. (b) Both trypanosome enzymes resembled the enzyme from S. platensis in that they had an almost absolute requirement for ATP, contrary to the enzymes from yeast and rabbit muscle, which were capable of utilizing GTP and ITP also. This difference in substrate specificity may be related to the amino acid substitutions, Trp 308→His and Ala 306→Glu in the adenine‐binding site, which are only found in the two Trypanosoma isoenzymes. Kinetic analysis showed that these substitutions do not prevent binding of the ATP analogoues, but probably prevent phosphoryl‐group transfer. (c) Both isoenzymes displayed an activity optimum at pH 6.0—9.0 similar to that for the enzyme of yeast.Both gPGK and cPGK were inhibited by the trypanocidal drug Suramin. This inhibition could be described as competitive both with ATP and 3‐phosphoglycerate with two inhibitor molecules binding to one molecule of enzyme. The gPGK, however, was much more sensitive (Kappi= 8.0 μM) to Suramin than either the cPGK (Kapp.i= 20 μM) or the enzymes from rabbit muscle (Kapp.i= 55 μM), yeast (Kapp.i= 167 μM) or S. platensis (Kapp.i= 250 μM). It is suggested that positive charges on the enzyme's surface may play an important role in the potentiation of the binding of the negatively charged Suramin molecule.
The complete amino acid sequence of rubredoxin from the green phototrophic bacterium Chlorobium thiosulphatophilum strain PM
Authors: WOOLLEY Kevin J. , MEYER Terry E. 
Published: March 2005

A complete amino acid sequence for the rubredoxin from the photosynthetic bacterium Chlorobium thiosulphatophilum is proposed. The sequence, a single polypeptide chain of 53 amino acids, was deduced from the sequences of peptides obtained by chymotryptic, tryptic, thermolytic or mild acid digestion.The rubredoxin shows a high degree of sequence homology with rubredoxins from non‐photosynthetic bacteria, and the evolutionary implications of this are considered.
Regulation of synthesis and cell content of the low‐density‐lipoprotein receptor protein in cultured fibroblasts from normal and familial hypercholesterolaemic subjects
Authors: KNIGHT Brian L. , PATEL Dilip D. , SOUTAR Anne K. 
Published: March 2005

Incorporation of [35S]methionine into low‐density‐lipoprotein (LDL) receptors by normal fibroblasts and those from a homozygous familial hypercholesterolaemic (FH) subject who produced defective but immunoprecipitable receptor proteins of normal size, was compared with the ability of the cells to bind LDL and their content of LDL receptor protein determined using a double‐antibody radioimmunoassay. The FH cells produced precursor protein with a longer half‐life (3–4 h) than normal cells (40 min), most of which was eventually processed to a mature form of the receptor. Total receptor half‐life was similar to normal (approx. 12 h) and LDL binding about 20% of normal.Incubation of normal fibroblasts with lipoprotein‐deficient serum (LPDS) led to an increase in the amount of LDL receptor protein in the cells which was closely followed by the increase in their ability to bind LDL. Receptor synthesis increased rapidly at first, but then fell by more than 60% before remaining constant. The peak of synthesis coincided with the greatest rate of increase in receptor content. At equilibrium in LPDS receptor synthesis, LDL binding and receptor protein content were all approximately 3.3‐fold higher than in cells maintained in 10% foetal calf serum (FCS). The FH cells also responded to LPDS with a rise and fall in the rate of receptor synthesis. They did not compensate for their inefficiency in producing active receptors with an increase in total receptor content. In LPDS, peak synthesis and maximum receptor content of the FH cells were similar to normal. In FCS receptor synthesis and content were well below maximum so that they did not fully employ even the low capacity for LDL uptake of which they were capable. With both types of cell, inhibition of mevalonic acid and cholesterol synthesis with compactin delayed, but did not prevent the secondary fall in the rate of receptor synthesis, again suggesting a regulatory role for some factor not directly related to cholesterol metabolism.
Receptor‐mediated endocytosis of apamin by liver cells
Authors: STRONG Peter N. , EVANS W. Howard 
Published: March 2005

The binding and uptake of the bee venom toxin apamin, by guinea‐pig and rat liver were studied.Guinea‐pig liver plasma membranes contain inhibitable, high‐affinity binding sites for [125I]monoiodoapamin: Kd= 12.6±0.8 pM (SE); Bmax= 4.2±0.2 fmol/mg protein. No binding sites for [125I]monoiodoapamin on rat liver plasma membranes were detected in agreement with the absence of a physiological response to the toxin by rat hepatocytes.[125I]Monoiodoapamin, injected into the portal vein of guinea‐pigs, was recovered in an undegraded form in a liver endosome fraction. The uptake of [125I]monoiodoapamin by rat livers was less than 4% of that taken up by guinea‐pig livers and there was little evidence of radiolabelled toxin appearing in isolated rat endocytic vesicles.Inhibitable, high‐affinity binding sites for [125I]monoiodoapamin were also identified on isolated guinea‐pig liver endosomal membranes; Kd= 10.6±3.3 pM; Bmax= 2.5±0.6 fmol/mg protein. No inhibitable apamin binding sites were detected on rat endosomal membranes.Plasma membranes and endosomal membranes isolated from guinea‐pig liver showed a similar spectrum of polypeptides to that previously reported for plasma membranes and endosomal membranes isolated from rat liver. The enzymatic composition of guinea‐pig endosomes was also similar to that previously reported for rat endosomes.The results indicate that apamin was internalised by receptor‐mediated endocytosis by guinea‐pig liver cells in an analogous manner to that already shown for a variety of endogenous ligands.
The two fast‐reacting thiols of 3‐phosphoglycerate kinase are structurally juxtaposed
Authors: VAS Mária , CSANÁDY György 
Published: March 2005

The two fast‐reacting thiol groups of pig muscle 3‐phosphoglycerate kinase can be simultaneously blocked by one mole equivalent of bifunctional reagent: either mercuric chloride (HgCl2) or 1,4‐bis(bromomercuri)butane. The reactions are accompained by an enzyme activity loss of about 50–70% and 60–80% with mercuric chloride and 1,4‐bis(bromomercuri)butane respectively. Removal of either of the reagents with excess cysteine leads to the recovery of at least 70–90% of the original enzymic activity.Gel chromatographic analysis revealed no change in the molecular mass of the enzyme modified with mercuric chloride, while an increase of about 30% of the apparent molecular mass was observed after the reaction with 1,4‐bis(bromomercuri)butane. Since no dimer formation could be detected by independent crosslinking, the increase of the apparent molecular mass is probably due to modification causing protein conformational change. The results strongly suggest that the fast‐reacting thiols are intramolecúlarly connected by either of the above bifunctional reagents. In the light of the known structural data on the enzyme, it may follow that the two fast‐reacting thiols belong to the two sequentially neighbouring cysteinyl residues.
Etude structurale et métabolique des acides mycoliques de Mycobacterium fortuitum
Authors: LACAVE Charlotte , LANÉELLE Marie‐Antoinette , DAFFÉ Mamadou , MONTROZIER Henri , ROLS Marie‐Pierre , ASSELINEAU Cécile 
Published: March 2005

Structural and metabolic studies of the mycolic acids of Mycobacterium fortuitum. The biosynthesis of mycolic acids was studied in whole cells of Mycobacterium fortuitum. At first the structures of the main mycolates produced by the used strain were established as diunsaturated and epoxymycolates. By using [1‐14C]acetate as a radiotracer of the lipid synthesis, it was observed that the turnover of the mycolates during the exponential phase of growth of M. fortuitum is fast enough to make very difficult the identification of their precursors.If the growth of the bacterial cells is stopped or highly diminished, by the removal of a large part of their nutritional medium, mycolate synthesis, in contrast to the synthesis of other fatty acids, is stopped as shown by incubation of the concentrated bacterial culture with [1‐14C]acetate. After removal of aliquots of the sedimented bacteria at intervals, during several hours, mycolate synthesis resumes when the cell concentration becomes lighter. In these conditions the sequence of radiolabeling of mycolates and of their potential precursors (tetracosanoate and meromycolates) can be observed. In spite of their low accumulation, tetracosanoate and meromycolates were isolated and purified and their specific radioactivity, after different incubation times, could be measured.The results are in agreement with the hypothesis that meromycolates are condensed with tetracosanoate to produce mycolates. However, because of the large differences of isotopic dilution of these two precursors inside the mycolate molecule, this hypothesis, generally taken as evidence, has to be modified. A hypothetical pathway of the mycolate synthesis is proposed, taking into account all these observations.