bluuebunny/crossref_metadata_2025_split huggingface dataset:
High Variability of the Electron Carrier Plastocyanin in Microalgae
Authors: BOHNER Herbert , MERKLE Hellmut , KRONECK Peter , BÖGER Peter 
Published: March 2005

The plastocyanin content of Scenedesmus can be dramatically varied with the copper content of the culture medium. Figures as high as 7 mmol plastocyanin/mol chlorophyll are possible. Electron paramagnetic resonance (EPR) spectroscopy has been used to determine this physiological response quantitatively in intact cells having different amounts of plastocyanin. The results obtained by the EPR technique were compared with data on isolated plastocyanin determined either by spectro‐photometry or immunoelectrophoresis. Agreement was found for the amount of plastocyanin detected by the first two methods, whereas the last assay yielded data at least 25% higher on the average. Under all culture conditions a copper‐free plastocyanin precursor is present.
The EPR properties of purified plastocyanin and those of cellular plastocyanin located within the thylakoids are practically identical in terms of g‐values, hyperfine splittings, signal linewidths and saturation behavior at temperatures of 12‐15 K. Our data indicate that plastocyanin is not present in a membrane‐bound form but exists as a single soluble pool.
The studies have been extended to the algae Dunaliella parva and Bumilleriopsis filiformis. The first species exhibits a limited variation of plastocyanin with the copper content of the medium. Furthermore, no cytochrome c‐553 could be detected in Dunaliella even under conditions of copper deficiency. In contrast, Bumilleriopsis does not contain plastocyanin regardiess of the amount of copper offered.
Fragmentation of Crystalline β‐Haemocyanin of Helix pomatia with Plasmin and Trypsin. Location of the Fragments in the Polypeptide Chain
Authors: GIELENS Constant , VERSCHUEREN Laurent J. , PRÉAUX Gisèle , LONTIE René 
Published: March 2005

The action of plasmin on tenth molecules of βC‐haemocyanin of Helix pomatia at pH 8.2 yielde first a three‐domain fragment P3 and a five‐domain fragment P1 (present as a dimer at pH 8.2 Fragment P1 was further split by plasmin into a four‐domain fragment P2 and a one‐domai fragment P4 (also present as a dimer at pH 8.2). Trypsinolysis of P2 yielded T2 and fragment X, which was further split into T1C and T3.Fragments P3 and P4 corresponded respectively to the tryptic fragments T1A and T1B, als by their circular dichroic spectra.The determination of the N‐terminal groups and the order of splitting allowed the location of the fragments in the polypeptide chain: P3 (a–c), P2 (d–g), P4 (h); T1A (a–c), T3 (d), TIC (e–f) T2 (g), T1B (h).
Characterisation of Cucurbitin from Various Species of the Cucurbitaceae
Authors: BLAGROVE Robert J. , LILLEY Glenn G. 
Published: March 2005

There have been many studies of cucurbitin, the major seed storage globulin which can be isolated in crystalline form from several genera of cucurbits. However, recent literature reports have suggested a wide range of molecular weights for oligomeric cucurbitin at neutral pH in buffered aqueous salt solutions. We have measured the molecular weight of oligomeric cucurbitin isolated from mature pumpkin seeds by several methods in the analytical ultracentrifuge. The molecular weights of the major and minor components present are 325000 and 630000 respectively. In combination with dodecylsulphate gel results, these values show that cucurbitin is a hexamer composed of subunits of molecular weight 54000, while most preparations contain several percent of a dodecamer composed of the same subunits. The molecular weight of the major component was 320000 from analytical gel filtration on Sepharose CL‐6B.Aqueous salt solutions of cucurbitin isolated from mature seeds of marrow, cucumber, rock melon, water melon, and ornamental gourds are also composed of major (hexamer) and minor (dodecamer) components. The major fractions have similar elution volumes following chromatography over Sepharose CL‐6B, sedimentation coefficients in the range 12.7–12.9 S and molecular weights in the range 324000–327000 from meniscus depletion sedimentation equilibrium measurements. Although there is limited size heterogeneity between the cucurbitin subunits, there is considerable charge heterogeneity. This has been demonstrated by electrophoresis on cellulose acetate membranes in 8 M urea at pH 8.5 with and without reduction of disulphide bonds. The existence of two classes of subunit polypeptide (acidic Mr∼ 33000 and basic Mr∼ 22000), comparable to those for glycinin (soybean) or legumin (pea, broad bean), has been shown by two‐dimensional electrophoresis.
Glycolysis in Trypanosoma brucei
Authors: VISSER Nico , OPPERDOES Fred R. 
Published: March 2005

The possibility that the glycosomes present in the bloodstream form of Trypanosoma brucei [Opperdoes, F. R. and Borst, P. (1977) FEBS Lett. 80, 360–364] constitute a separate pool of glycolytic intermediates within the cell was investigated.In titrations of intact cells with digitonin, a differential activation of glycolytic enzymes was observed. Enolase, pyruvate kinase and the cell‐sap marker alanine aminotransferase were activated at 0.05 mg digitonin per mg protein. The nine glycosomal enzymes involved in the conversion of glucose and glycerol into 3‐phosphoglycerate were activated only at digitonin concentrations between 0.7 and 9.8 mg/mg protein. In subcellular fractions the activities of the latter enzymes were all latent between 70 and 92%. Latency was abolished by addition of 0.1% Triton X‐100 or partly by five cycles of freezing and thawing. We conclude that the glycosomal enzymes are surrounded by a membrane, which forms a permeability barrier to intermediates and co‐factors of glycolysis.The concentrations of glycolytic intermediates and of adenine nucleotides were measured under aerobic conditions as well as in the presence of 1 mM salicylhydroxamic acid, a respiratory inhibitor. Addition of salicylhydroxamic acid caused the following changes: (a) The levels of almost all glycolytic intermediates measured decreased. Glycerol‐3‐phosphate, however, increased fourfold, (b) The phosphate potential was drastically lowered from 2900 to 450 M −1. (c) The trypanosomes became more reduced, as monitored by a change in the apparent redox state of the NADH/NAD+ couple from E′h= ‐189 to E′h=–219 mV.From the high levels of metabolite concentrations found and from comparison of the apparent mass‐action ratios calculated for the separate glycolytic reactions with those for other organisms, we conclude that in bloodstream form T. brucei the glycolytic intermediates are present in the glycosomes as well as in the cytosol and that the two pools of intermediates equilibrate with each other, despite the presence of the glycosomal membrane.
Induction of Choline Transport and Its Role in the Stimulation of the Incorporation of Choline into Phosphatidylcholine by Polyamines in a Polyamine Auxotroph of Saccharomyces cerevisiae
Authors: HOSAKA Kohei , YAMASHITA Satoshi 
Published: March 2005

A mutant of Saccharomyces cerevisiae, defective in ornithine decarboxylase, was isolated. A prolonged culture of the mutant in a polyamine‐free medium resulted in a great decrease in the polyamine content and in cessation of growth. The addition of polyamines to the culture induced the growth after a lag period of 5–6.5 h. The growth rate in the presence of polyamine was comparable to that of the wild‐type strain. The effectiveness of polyamines was as follows: spermidine > putrescine ∼ spermine.
Phosphatidylcholine‐synthesizing activity during the lag phase of growth was determined by measuring the rate of incorporation of [14C]choline into phosphatidylcholine. The incorporation rate was markedly increased with time by polyamine prior to the initiation of cell division. Polyamines were effective in the following order: spermidine > putrescine ∼ spermine. Experiments whit methylgiyoxal bis(guanylhydrazone), an inhibitor of S‐adenosylmethionine decarboxylase, showed that putrescine stimulates cell growth and choline incorporation into phosphatidylcholine after it has been converted into spermidine in the cell.
The induction of the choline transport system was shown to be responsible for the increase in the rate of incorporation of [14C]choline into phosphatidylcholine effected by polyamines. A low concentration of cycloheximide completely prevented the induction of choline transport by polyamines. The levels of the CDP‐choline pathway enzymes such as choline kinase, cholinephosphate cytidyltransferase and cholinephosphotransferase were not significantly changed.
Isomers in Thioredoxins of Spinach Chloroplasts
Authors: SCHÜRMANN Peter , MAEDA Kayo , TSUGITA Akira 
Published: March 2005

We have developed a method for the concomitant purification of several components of the ferredoxin/thioredoxin system of spinach chloroplasts. By applying this method to spinach‐leaf extract or spinach‐chloroplast extract we separated and purified three thioredoxins indigenous to chloroplasts. The three thioredoxins, when reduced, will activate certain chloroplast enzymes such as fructose‐1,6‐bisphosphatase and NADP‐dependent malate dehydrogenase. Fructose‐ 1,6‐bisphosphatase is activated by thioredoxin f exclusively. Malate dehydrogenase is activated by thioredoxin mb and thioredoxin mb in a similar way, and it is also activated by thioredoxin f but with different kinetics.All three thioredoxins have very similar relative molecular masses of about 12000 but distinct isoelectric points of 6.1 (thioredoxin f), 5.2 (thioredoxin mb) and 5.0 (thioredoxin mc). The amino acid composition as well as the C‐terminal and N‐terminal sequences have been determined for each thioredoxin. Thioredoxin f exhibits clear differences in amino acid composition and terminal sequences when compared with the m‐type thioredoxins. Thioredoxin mb and thioredoxin mc, however, are very similar, the only difference being an additional lysine residue at the N‐terminus of thioredoxin mb.Amino acid analyses, terminal sequences, immunological tests and the activation properties of the thioredoxins support our conclusion that thioredoxins mb and me are N‐terminal redundant isomers coming from one gene whereas thioredoxin f is a different protein coded by a different gene.
Molecular Characteristics of a Non‐deletion α‐Thalassaemia of the Po River Delta
Authors: del SENNO Laura , BERNARDI Francesco , BUZZONI Daniele , MARCHETTI Giovanna , PERROTTA Carla , CONCONI Francesco 
Published: March 2005

The form of α‐thalassaemia of the Po river delta presents haematological and globin biosynthetic characteristics similar to α‐thalassaemia‐1 but never gives rise to Hb H disease nor to hydrops foetalis. In α‐thalassaemic subjects originally from this region globin mRNA translation and α‐globin gene arrangement have been investigated.The data obtained indicate that α‐globin synthesis and reticulocyte α‐globin mRNA are reduced by one fourth; in addition, since the defect in α‐globin synthesis does not change with cell ageing, a possible instability of α‐globin mRNA is excluded. Restriction enzyme analysis of the DNA shows a normal α‐globin gene organization. This form of α‐thalassaemia is therefore of the non‐deletion type; its molecular lesion is either at the level of α‐globin mRNA transcription or processing. The fact that this, as well as other forms of non‐4eletion α‐thalassaemia, have a phenotipic expression similar to α‐thalassaemia 1 is discussed.
Modification of the Thermodynamic Properties of the Electron‐Transferring Groups in Mitochondrial Succinate Dehydrogenase upon Binding of Succinate
Authors: BONOMI Franco , PAGANI Silvia , CERLETTI Paolo , GIORI Camillo 
Published: March 2005

The redox properties of the covalently‐bound flavin and of the tetrahedral iron‐sulfur center S1 of succinate dehydrogenase were studied as a function of the binding of different ligands to the enzyme. The midpoint potential of both flavin and S1 increases by some 200 mV when protein binds succinate to a site having KSuccd= 0.8–1.0 mM, thus different from the substrate binding site. Succinate binding increases the potential of the oxidized flavin/semiquinone half‐cell more than that of the semiquinone/reduced flavin one: this results in higher semiquinone formation with increasing succinate. Malonate and fumarate appear to mimic, in this regard, the effect of succinate.The increase in midpoint potential of S1 upon binding of dicarboxylic acid is related to an increase in hydrophobicity of the cluster environment.The possible molecular basis for the modulation of the flavin potential is discussed together with the significance of this shift on the catalytic behaviour of the protein.
Effect of Autonomic Denervation on DNA Synthesis during Liver Regeneration after Partial Hepatectomy
Authors: KATO Hideo , SHIMAZU Takashi 
Published: March 2005

The regulatory role of autonomic nerves in liver regeneration after partial hepatectomy was studied in rats by bilateral subdiaphragmatic vagotomy or splanchnicectomy.

In control rats the wet weight of the regenerating liver was restored to approximately 80% of the preoperative weight 72h after partial hepatectomy. Restoration of the liver weight was significantly impaired in vagotomy rats, but not in splanchnicectomy. Increases in the DNA and protein contents of the regenerating liver were also suppressed by vagotomy.
Hepatic DNA synthesis, measured as the incorporation of [methyl‐3H]thymidine into DNA at various times after partial hepatectomy, was significantly less in vagotomized rats, and slightly more in splanchnicectomized rats than in control rats. The onset of DNA synthesis triggered by partial hepatectomy was also delayed by vagotomy.
The increases in activities of hepatic aspartate transcarbamoylase and thymidine kinase, the key enzymes in synthesis of pyrimidine nucleotides via the de novo and salvage pathways respectively, during liver regeneration, were significantly suppressed and retarded in vagotomized rats. Conversely, splanchnicectomy tended to stimulate these enzyme inductions after partial hepatectomy.
During starvation the plasma insulin level decreased after partial hapatectomy in control and vagotomized rats, as in sham‐operated rats, but showed a transient increase 6 h after partial hepatectomy in splanchnicectomized rats.It is concluded that vagotomy inhibits and delays DNA synthesis and proliferation of liver cells after partial hepatectomy, whereas splanchnicectomy tends to stimulate these processes. The data also suggest that para‐sympathetic innervation of the liver may play an important regulatory role in liver regeneration.
Subunit Structure and Interactions of the Phloem Proteins of Cucurbita maxima (Pumpkin)
Authors: READ Steve M. , NORTHCOTE Don H. 
Published: March 2005

The two major proteins from the phloem exudate of Cucurbita maxima (pumpkin), PP1 and PP2, were stable in the absence of reducing agents after modification of their accessible cysteine residues with iodoacetamide. This permitted their purification without precautions to prevent oxidation.
PP2, a lectin specific for oligomers of N‐acetyl‐D‐glucosamine, was shown by sedimentation‐equilibrium ultracentrifugation to be a dimer of Mr, of 48000. Neither dithiothreitol nor tri‐(N‐acetyl‐D‐glucosamine) altered this value. The constituent polypeptides were linked by two buried disulphide bridges. PP2 behaved aberrantly on gel‐filtration on both Sephadex and Bio‐Gel unless tri‐(N‐acetyl‐D‐glucosamine) was added to the elution buffer; the Mr, was then measured as 46000. Other proteins which bind oligomers of N‐acetyl‐D‐glucosamine are also retarded on gel‐filtration.
Soluble phloem filaments were prepared by collection of exudate into deaerated buffer containing iodoacetamide but no reducing agent. Oxidative gellation of the filaments was prevented by rapid modification of their many accessible cysteine residues, and is assumed to have maintained the degree of polymerisation found in vivo. Those disulphide bridges which were present allowed the incorporation of approximatically 60% of the PP1 and 80% of the PP2 into polymeric material. It is concluded that PP1 and PP2 are both structural proteins present in the filaments observable in vivo.
PP2 had an elongated binding‐site for oligomers of N‐acetyl‐D‐glucosamine. It is suggested that this lectin immobilises bacteria and fungi to the cross‐linked filaments which seal wounded phloem sieve‐tubes, and thus maintains sterility.
Relationship between Oligosaccharide‐Lipid Synthesis and Protein Synthesis in Mouse LM Cells
Authors: GRANT Stephen R. , LENNARZ William J. 
Published: March 2005

Previous studies from several laboratories have reported that inhibition of protein synthesis results in a concomitant reduction in synthesis of oligosaccharide‐diphosphoryldolichol. We have investigated this phenomenon in LM cells grown in defined culture medium. The results of this study indicate that incubation of LM cells with cycloheximide at a concentration sufficient to totally arrest polypeptide synthesis, results in a rapid reduction in the synthesis of [3H]mannose‐labeled or [3H]glucosamine‐labeled oligosaccharide‐lipid within 2 min. Cycloheximide treatment had only a slight inhibitory effect on synthesis of GDP‐Man. Furthermore, during the first 5 min after the addition of cycloheximide to LM cells, when [3H]mannose incorporation into oligosaccharide‐lipid was maximally reduced, the specific activity of the GDP‐Man pool was identical to that observed in control cells. Labeling in vivo of the lipid‐linked saccharide precursors of oligosaccharide‐lipid revealed that cycloheximide had virtually no effect on the synthesis of Man‐P‐Dol, GlcNAc‐PP‐Dol, GlcNAc2‐PP‐Dol, and β‐Man‐(GlcNAc)2‐PP‐Dol (Dol = dolichol). In agreement with this finding, results of experiments in vitro using microsomes prepared from LM cells indicate that cycloheximide did not directly inhibit the enzymes responsible for the synthesis of these lipid‐linked saccharide precursors.Supplementation of mouse LM cells by preincubation for 1 h with dolichyl phosphate (5 μg/ml) resulted in a 300% stimulation of oligosaccharide‐lipid synthesis when compared to non‐supplemented cells. However, dolichyl phosphate supplementation of cycloheximide‐treated cells failed to restore oligosaccharide‐lipid synthesis to the level observed in control cells. In control cells pre‐labeled oligosaccharide‐lipid turned over rapidly and, as expected, cycloheximide addition to the chase medium significantly retarded this turnover process. However, preincubation of cells with exogenous dolichyl phosphate had little or no effect on the turnover of oligosaccharide‐lipid in control or cycloheximide‐treated cells. These findings argue against dolichyl phosphate deficiency as the primary cause of reduced oligosaccharide‐lipid synthesis when protein synthesis is blocked. The implications of these results, and an alternative hypothesis to explain the effect of inhibition of protein synthesis on oligosaccharide‐lipid synthesis based on elevation of intracellular GTP levels, are discussed.
Growth and adipose differentiation of sheep preadipocyte fibroblasts in serum‐free medium
Authors: BROAD Thomas E. , HAM Richard G. 
Published: March 2005

Fibroblasts from ovine skin, and from the perirenal and subcutaneous adipose tissues of sheep were grown at clonal densities in medium MCDB 202 supplemented with 1 μg/ml bovine insulin, 1 μM dexamethasone, 100 ng/ml fibroblast growth factor and 20 μg/ml of the lipid preparation described by Bettger, W. J., Boyce, S. T., Walthall, B. J. and Ham, R. G. [(1981) Proc. Natl Acad. Sci, USA, 78, 5588–5592].When maintained as a confluent monolayer in this medium, the fibroblasts from the adipose tissues spontaneously underwent an adipose differentiation. This was accelerated by substituting medium F12 for medium MCDB 202, and by raising the CO2 tension from 2% to 7.5% in air over the cultures. The differentiation was inhibited by deleting FGF from the growth medium, or by coating the culture surface with fibronectin or poly‐D‐lysine. Differentiation also failed to occur when the defined supplements were replaced with fetal bovine serum.The synthesis of triacylglycerol by the cells, as seen by the increased specific activity of [14C]acetate incorporated into this lipid class, was accompanied by an increase in the specific activity of glycerol‐3‐phosphate dehydrogenase.
The location of redox‐sensitive groups in the carrier protein of proline at the outer and inner surface of the membrane in Escherichia coli
Authors: POOLMAN Bert , KONINGS Wil N. , ROBILLARD George T. 
Published: March 2005

Evidence is presented in this report for the presence of two sets of dithiols associated with proline transport activity in Escherichia coli. One set is located at the outer surface, the other at the inner surface of the cytoplasmic membrane.Treatment of right‐side‐out membrane vesicles from E. coli ML 308–225 with the membrane‐impermeable oxidant ferricyanide resulted in inhibition of L‐proline uptake without having significant effect on the magnitude of the ΔμH+. Subsequent addition of reducing agents restored proline transport activity. The membrane‐impermeable SH‐reagent glutathione hexane maleimide inhibited proline transport in right‐side‐out membrane vesicles irreversibly. Pretreatment of the vesicles with ferricyanide protected the carrier against inactivation by glutathione hexane maleimide.Electron transfer in the respiratory chain of right‐side‐out vesicles led to the generation of a ΔμH+, interior negative and alkaline, and the conversion of a disulphide to a dithiol in the proline carrier as is shown by the increased inhibition of proline transport by the membrane impermeable dithiol reagent 4‐(2‐arsonophenyl)azo‐3‐hydroxy‐2, 7‐naphthalene disulphonic acid (thorin). The inhibition exerted by thorin was completely reversed by dithiothreitol. Pretreatment of the vesicles with thorin protected against glutathione hexane maleimide inhibition, indicating that both reagents react with the same group.Treatment of insideout membrane vesicles with ferricyanide inactivated the proline transport system reversibly. The oxidizing effect of ferricyanide in inside‐out vesicles resulted in protection against inhibition by glutathione hexane maleimide. Imposition in these vesicles of a ΔμH+, interior positive and acid, also protected the proline carrier against glutathione hexane maleimide inactivation, indicating that a dithiol is converted to a disulphide upon energization.