bluuebunny/crossref_metadata_2025_split huggingface dataset:
Cross‐reconstitution of the FOF1‐ATP synthases of chloroplasts and Escherichia coli with special emphasis on subunit δ
Authors: ENGELBRECHT Siegfried , DECKERS‐HEBESTREIT Gabriele , ALTENDORF Karlheinz , JUNGE Wolfgang 
Published: March 2005

FOF1‐ATP synthases catalyse ATP formation from ADP and Pi by using the free energy supplied by the transmembrane electrochemical potential of the proton. The δ subunit of F1 plays an important role at the interface between the channel portion Fo and the catalytic portion F1. In chloroplasts it can plug the protonic conductance of CFo and in Escherichia coli it is required for binding of EF1 to EFo.We wanted to know whether or not δ of one species was effective between Fo and F1 of the other species and vice versa. To this end the respective coupling membrane (thylakoids, everted vesicles from E. coli) was (partially) depleted of F1 and purified F1, F1(‐δ), and δ were added in various combinations to the F1‐depleted membranes. The efficiency or reconstitution was measured in thylakoids via the rate of phenazinemethosulfate‐mediated cyclic photophosphorylation and in E. coli everted vesicles via the degree of 9‐amino‐6‐chloro‐2‐methoxyacridine fluorescence quenching.Addition of CF1 to partially CF1‐depleted thylakoid vesicles restored photophosphorylation to the highest extent. CF1(‐δ)+chloroplast δ, EF1, EF1(‐δ)+E. coliδ were also effective but to lesser extent. CF1(‐δ)+E. coliδ and EF1(‐δ)+chloroplast δ restored photophosphorylation to a small but still significant extent. With F1‐depleted everted vesicles prepared by repeated EDTA treatment of E. coli membranes, addition of CF1, CF1(‐δ)+chloroplast δ and CF1(‐δ)+E. coliδ gave approximately half the extent of 9‐amino‐6‐chloro‐2‐methoxyacridine fluorescence quenching as compared to EF1 or EF1(‐δ)+E. coliδ by energization of the vesicles with NADH, while EF1(‐δ)+chloroplast δ was ineffective. All ‘mixed’ combinations were probably reconstitutively active only by plugging the protonic leak through the exposed Fo (structural reconstitution) rather than by catalytic activity. Nevertheless, the cross‐reconstitution is stunning in view of the weak sequence similarity between chloroplast δ and E. coliδ. It favors a role of δ as a conformational transducer rather than as a proton conductor between F0 and F1.
Structural requirements for protein N‐glycosylation
Authors: ROITSCH Thomas , LEHLE Ludwig 
Published: March 2005

To understand better the structural requirements of the protein moiety important for N‐glycosylation, we have examined the influence of proline residues with respect to their position around the consensus sequence (or sequon) Asn‐Xaa‐Ser/Thr. In the first part of the paper, experiments are described using a cell‐free translation/glycosylation system from reticulocytes supplemented with dog pancreas microsomes to test the ability of potential acceptor peptides to interfere with glycosylation of nascent yeast invertase chains. It was found that peptides, being acceptors for oligosaccharide transferase in vitro, inhibit cotranslational glycosylation, whereas nonacceptors have no effect. Acceptor peptides do not abolish translocation of nascent chains into the endoplasmic reticulum. Results obtained with proline‐containing peptides are compatible with the notion that a proline residue in an N‐terminal position of a potential glycosylation site does not interfere with glycosylation, whereas in the position Xaa or at the C‐terminal of the sequon, proline prevents and does not favour oligosaccharide transfer, respectively.This statement was further substantiated by in vivo studies using site‐directed mutagenesis to introduce a proline residue at the C‐terminal of a selected glycosylation site of invertase. Expression of this mutation in three different systems, in yeast cells, frog oocytes and by cell‐free translation/glycosylation in reticulocytes supplemented with dog pancreas microsomes. leads to an inhibition of glycosylation with both qualitative and quantitative differences. This may indicate that host specific factors also contribute to glycosylation.
Evidence that the upstream stimulatory factor and the Sp1 transcription factor bind in vitro to the promoter of the human‐growth‐hormone gene
Authors: LEMAIGRE Frédéric P. , COURTOIS Stéphane J. , LAFONTAINE Dominique A. , ROUSSEAU Guy G. 
Published: March 2005

Expression of the human‐growth‐hormone gene is restricted to pituitary somatotrophs. Two protein‐DNA complexes that are specific to the pituitary, and two that are not, had been demonstrated in vitro on the promoter of this gene. The two pituitary‐specific footprints had been ascribed to a single protein called growth hormone factor 1. We have now characterized the factors responsible for the two other footprints by means of deoxyribonuclease‐I protection and gel‐retardation experiments.The first footprint, located between –257 and –290 relative to the transcription initiation site, involves at least two factors present in pituitary cells. One of these factors binds between nucleotides –257 and –267, and is indistinguishable from the upstream stimulatory factor, also called major late transcription factor or upstream element factor, initially described in HeLa cells. Earlier work by others had shown that the activator protein 2 purified from HeLa cells can bind to nucleotides –263 and –290. Our experiments suggest that a factor different from activator protein 2 is involved in the protection of this region against deoxyribonuclease I.The second footprint, located between nucleotides –116 and –140, involves only one factor. This factor, present in pituitary cells, recognizes a GC box and is indistinguishable from transcription factor Sp1, previously described in HeLa cells. The human‐growth‐hormone gene is therefore a candidate for regulation by these factors in vivo.
Ecdysteroid receptors of the blowfly Calliphora vicina
Authors: LEHMANN Michael , KOOLMAN Jan 
Published: March 2005

Ecdysteroids, the molting hormones of arthropods, act like vertebrate steroid hormones by binding to an intracellular receptor protein. We have recently isolated a protein from nuclei of blowfly larvae which has satisfied the requirements of an ecdysteroid receptor. The receptor was partially purified and its ecdysteroid‐binding properties were characterized. The availability of receptor preparations which have been stabilized by partial purification now enables us to study the general DNA‐binding properties of ecdysteroid receptors.DNA‐binding characterstics of ecdysteroid receptors were studied with calf thymus DNA. Affinity for DNA was observed both in the presence and in the absence of steroid ligand but the ligand clearly enhanced binding of receptors to DNA. Receptor preparations contained a heterogeneous mixture of receptors; up to 25% of DNA‐binding receptors, and nonbinding forms of ecdysteroid receptors. The ability to bind to DNA was subject to inactivation which was not affected by partial purification, but which could be decelerated by dilution of the receptor preparation. Thus, dilution resulted in a spurious activation of DNA binding. A genuine activation, which would have led to an increase in the percentage of the DNA‐binding form of the ecdysteroid receptor complex, was not observed.
Spectroscopic investigations of Panulirus interruptus hemocyanin in the crystalline state
Authors: VOLBEDA Anne , FEITERS Martin C. , VINCENT Michael G. , BOUWMAN Elisabeth , DOBSON Barry , KALK Kor H. , REEDIJK Jan , HÓL Wim G. J. 
Published: March 2005

Single‐crystal ultraviolet spectroscopy, X‐ray absorption spectroscopy and EPR measurements have been used to examine the oxidation and oxygenation state of the dinuclear copper site of several types of hemocyanin crystals. The crystals contain Panulirus interruptus hemocyanin which forms hexameric molecules with a molecular mass of approximately 470 kDa.Three types of crystals have been investigated. Type‐I monoclinic crystals, which have been used for the X‐ray structure determination, contain virtually only deoxyhemocyanin. Type‐II monoclinic crystals, which are less well ordered than the type‐I crystals, contain a mixture of deoxy, oxy and met forms. Older crystals contain relatively more methemocyanin. A third, hexagonal, crystal form is also partially oxygenated, and, like the type‐II monoclinic form, subject to gradual conversion to methemocyanin.
A common liver‐specific factor binds to the rat albumin and α‐foetoprotein promoters in vitro and acts as a positive trans‐acting factor in vivo
Authors: JOSE‐ESTANYOL Matilde , POLIARD Anne , FOIRET Danièle , DANAN Jean‐Louis 
Published: March 2005

Rat liver nuclear extracts were tested for the presence of factors which might be common to the transcriptional regulation of both the albumin and α‐foetoprotein genes. Gel shift assay showed the formation of three complexes (I, II and III) with the albumin probe. Two of them (I and III) could be displaced by the α‐foetoprotein promoter. Analysis of nuclear extracts from liver, kidney, spleen and brain and competition experiments using several oligonucleotides covering regions from the albumin and α‐foetoprotein promoters showed that complex III results from the binding of the ubiquitous nuclear factor 1, while complex II involves a CCAAT‐box‐binding protein also detected in brain and spleen extracts. Complex I is formed upon binding of a liver‐specific factor to a proximal element of the rat albumin promoter. This factor also binds to a similar sequence in the α‐foetoprotein promoter and is closely related to the hepatocyte nuclear factor 1, as shown by competition experiments using an oligonucleotide covering its target sequence on the β‐fibrinogen promoter.Transfection competition experiments indicated that, in vivo, this factor acts as a positive trans‐acting element in the expression of both the rat albumin and α‐foetoprotein genes.
The evolution of lysozyme and α‐lactalbumin
Authors: NITTA Katsutoshi , SUGAI Shintaro 
Published: March 2005

From the analysis of phylogenetic trees constructed from the amino acid sequences and metal‐binding properties of various lysozymes c and α‐lactalbumins, it was found that before the divergence of the lineages of birds and mammals, calcium‐binding lysozyme diverged from non‐calcium‐binding lysozyme. α‐Lactalbumin evolved from the calcium‐binding lysozyme along the mammalian lineage after the divergence of birds and mammals. Rapid evolution took place, not in the process of acquisition of the activity of α‐lactalbumin, but after the loss of lysozyme activity, due to the change in the distribution of selective pressure on each amino acid site.A general process for the change in function of a protein during evolution is suggested to be as follows: after duplication of the gene, one of their protein products acquires a new function, besides that already present; the old function is eventually lost.
Structure‐function relationships of elongation factor Tu
Authors: JENSEN Michael , COOL Robbert H. , MORTENSEN Kim K. , CLARK Brian F. C. , PARMEGGIANI Andrea 
Published: March 2005

The guanine‐nucleotide‐binding domain (G domain) of elongation factor Tu(EF‐Tu) consisting of 203 amino acid residues, corresponding to the N‐terminal half of the molecule, has been recently engineered by deleting part of the tufA gene and partially characterized [Parmeggiani, A., Swart, G. W. M., Mortensen, K. K., Jensen, M., Clark, B. F. C., Dente, L. and Cortese, R. (1987) Proc. Natl Acad. Sci. USA 84, 3141–3145]. In an extension of this project we describe here the purification steps leading to the isolation of highly purified G domain in preparative amounts and a number of functional properties. The G domain is a relatively stable protein, though less stable than EF‐Tu towards thermal denaturation (t50%= 41.3°C vs. 46°C, respectively). Unlike EF‐Tu, its affinity for GDP and GTP, as well as the association and dissociation rates of the relative complexes are similar, as determined under a number of different experimental conditions. Like EF‐Tu, the GTPase of the G domain is strongly enhanced by increasing concentrations of Li+, K+, Na+ or NH4+, up to the molar range. The effects of the specific cations shows similarities and diversities when compared to the effects on EF‐Tu. K+ and Na+ are the most active followed by NH4+ and Li+ whilst Cs+ is inactive. In the presence of divalent cations, optimum stimulation occurs in the range 3–5 mM, Mg2+ being more effective than Mn2+ and Ca2+. Monovalent and divalent cations are both necessary components for expressing the intrinsic GTPase activity of the G domain. The pH curve of the G domain GTPase displays an optimum at pH 7–8, similar to that of EF‐Tu.The 70‐S ribosome is the only EF‐Tu ligand affecting the G domain in the same manner as that observed with the intact molecule, although the extent of the stimulatory effect is lower. The rate of dissociation of the G domain complexes with GTP and GDP as well as the GTPase activity are also influenced by EF‐Ts and kirromycin, but the effects evoked are small and in most cases different from those exerted on EF‐Tu. The inability of the G domain to sustain poly(Phe) synthesis is in agreement with the apparent lack of formation of a ternary complex between the G domain GTP complex and aa‐tRNA. Our results indicate that the isolated G domain of EF‐Tu is a suitable model for studying the basic properties of the guanine nucleotide interaction and the catalytic activity of a guanine‐nucleotide‐binding protein.
Primary structure of spinach‐chloroplast thioredoxin f
Authors: KAMO Masaharu , TSUGITA Akira , WIESSNER Christoph , WEDEL Norbert , BARTLING Dieter , HERRMANN Reinhold G. , AGUILAR Fernando , GARDET‐SALVI Laura , SCHÜRMANN Peter 
Published: March 2005

The primary structure of thioredoxin f from spinach chloroplasts was determined by standard amino acid sequencing and furthermore by sequencing the corresponding nuclear genome region. The protein, with a calculated molecular mass of 12564 Da and a molar absorption coefficient at 280 nm of 17700 M−1 cm−1, consists of 113 residues and exhibits 24% residue identities with spinach chloroplast thioredoxin mb or Escherichia coli thioredoxin.A monospecific antibody elicited against thioredoxin f has been used to select recombinant phage from spinach cDNA libraries in λgt11. The inserts of positive clones were sequenced. They code for a polypeptide of 190 amino acids, composed of the thioredoxin f sequence (113 residues) and an upstream element (77 residues) which most probably consitutes the N‐terminal transit peptide that directs the polypeptide into chloroplasts. In vitro transcription and translation of this construct generates a polypeptide of approximately 21 kDa, which is imported by isolated spinach chloroplasts and processed to the mature 12.5‐kDa protein.
The coupling between protonmotive force and the NAD(P)+transhydrogenase in chromatophores from photosynthetic bacteria
Authors: COTTON Nicholas P. J. , LEVER Timothy M. , NORE Beston F. , JONES Michael R. , JACKSON J. Baz 
Published: March 2005

The activity of NAD(P)+transhydrogenase in chromatophores ofRhodobacter capsulatusrelaxed from a high rate during illumination to a lower rate after darkening with a half‐time of approximately 100 ms.The dissipative ionic current flowing across the chromatophore membrane was increased in the presence of transhydrogenase substrates. This is attributed to proton current through the transhydrogenase enzyme. Subject to the assumption that transhydrogenase does not conduct in the absence of nucleotide substrates, the ratio of protons translocated across the membrane per hydride ion transferred was 0.4±0.5. Within the error and uncertainities in the calibration procedure, this ratio may be consistent with a stoichiometry of one but higher values seem unlikely. The ratio of hydride ion transferred in the transhydrogenase to electrons transferred through the cyclic electron transport system was approximately 0.2.TheKmappvalues for the transhydrogenase substrates were determined for chromatophores in illuminated and darkened suspensions over a range of pH. These values are discussed in relation to the equivalent parameters reported for mitochondria transhydrogenase [Rydstrom. J. (1977)Biochim. Biophys. Acta 255, 9641–9646] and were used to calculate the concentrations of substrates which effectively saturate the enzyme.At substrate concentrations which were in excess of 8 xKmappthe dependence of transhydrogenase rate on the value of the membrane potential (zero pH gradient) was determined at pH 6.3, 6.9, 7.6 and 9.0. The relation was similar at pH 6.9 and 7.6. At alkaline pH the apparent threshold in the relation became more prominent as it was shifted to slightly higher values of membrane potential. At acid pH a shift in the opposite direction diminished the apparent threshold and saturation at high membrane potential became more dominant. We use these data in an attempt to discriminate between two models of energy transduction: (a) the driving force exerted by the membrane potential is mediated by a pH gradient formed through the operation of a proton well in the transhydrogenase; (b) the membrane potential increases a rate constant for charge translocation through transhydrogenase by decreasing the effective height of the Eyring barrier for charge transfer across the membrane through the enzyme. The second model leads to a more simple description than the first of the pH dependence of transhydrogenase rate on membrane potential.Divalent cations at very low concentrations (∼ 0.1 mM) or monovalent cations at higher concentrations (∼ 50 mM) stimulate considerably the transhydrogenase activity in chromatophores. The effect of monovalent and divalent cations on the relationship between the rate of transhydrogenase and membrane potential suggests that they do not influence the reaction by altering the membrane‐potential‐dependent rate constant.
Characterization of trehalose‐6‐phosphate synthase and trehalose‐6‐phosphate phosphatase of Saccharomyces cerevisiae
Authors: VANDERCAMMEN Annick , FRANÇOIS Jean , HERS Henri‐Géry 
Published: March 2005

The properties of yeast trehalose‐6‐phosphate synthase were reinvestigated in relation with the recent claim made by Panek et al. [Panek, A. C., de Araujo, P. S., Moura‐Neto, V. and Panek, A. D. (1987) Curr. Genet. 11, 459–465] that the enzyme would be stimulated by ATP and partially inactivated by cAMP‐dependent protein kinase. Trehalose‐6‐phosphate synthase activity was measured by the sum of [14C]trehalose 6‐phosphate and [14C]trehalose formed from UDP‐[14C]glucose and glucose 6‐phosphate. The activity measured in an extract of Saccharomyces cerevisiae was not affected by any treatment of the cells, such as incubation in the presence of glucose or of dinitrophenol, which are known to greatly increase the intracellular concentration of cAMP, nor by preincubation of the extract in the presence of ATP‐Mg, cAMP and bovine heart cAMP‐dependent protein kinase. The activity was also not significantly different in several mutants affected in the cAMP system. The kinetic properties of the partially purified enzyme were investigated; no effect of ATP could be detected but Pi acted as a potent noncompetitive inhibitor (Ki= 2 mM). The activity of trehalose‐6‐phosphate phosphatae was measured by the amount of [14C]trehalose formed from [14C]trehalose 6‐phosphate. The enzyme could be separated from other phosphatases and appeared to be highly specific for trehalose 6‐phosphate. It was Mg dependent and its kinetics for trehalose 6‐phosphate was hyperbolic. Studies performed with intact cells, crude extracts or the purified enzyme did not reveal any cAMP‐dependent change in its activity.Remarkably, trehalose‐6‐phosphate synthase and trehalose‐6‐phosphate phosphatase copurified in the course of different chromatographic procedures, suggesting that they are part of a single bifunctional protein. A 50‐fold purification of the two enzymes could be achieved with a yeild of only 2% by chromatography on Mono S followed by gel filtration on Superose 6B.
Chains and fragments of tetanus toxin
Authors: WELLER Ulrich , DAUZENROTH Marie‐Elisabeth , HERINGDORF Dagmar MEYER ZU , HABERMANN Ernst 
Published: March 2005

Tetanus toxin, as obtained from bacterial culture filtrates, consists of two chains. Since their roles in poisoning are unknown, we have made a detailed study of their preparation, reassociation and pharmacological activity.

Two‐chain tetanus toxin (pI 6.0) was subjected to isoelectric focussing under reducing conditions in 2 M urea. Both light (pI 4.8) and heavy (pI 7.2) chains separated as nearly homogeneous proteins of low toxicities. Upon removal of urea and reoxidation, partial homodimerization by formation of disulfide bonds took place in the purified fractions. The toxin was reconstituted nearly quantitatively by covalent heterodimerization of the complementary chains, as shown by SDS/gel electrophoresis, toxicity studies, inhibition of evoked [3H]‐noradrenaline release and binding to rat brain membranes.
Accordingly, fragment B (pI 5.6) resulting from papain hydrolysis, was separated into a light chain and the N‐terminal moiety of the heavy chain, called fragment β2 (pI 7.1 and 6.8, two maxima). Removal of urea and reoxidation led to reconstitution of fragment B. Covalent linkage did not occur between the two parts of the heavy chain, or between the light chain and the C‐terminal part of the heavy chain.
The heavy chain alone inhibited K+‐evoked [3H]noradrenaline release from a rat brain homogenate. However, the concentration‐response ratio was flat and 10–100‐fold higher concentrations were required than with native or reconstituted two‐chain toxin. The light chain was inactive. Purified heavy chain but not light chain decreased the [3H]noradrenaline content, whereas the two‐chain toxin increased it.

Binding to rat brain membranes was assessed by competition with 125I‐labelled two‐chain toxin. In hypotonic buffer, the heavy chain, the papain fragment C and native and reconstituted two‐chain toxin had comparable affinities to membranes. In isotonic buffer the heavy chain displayed an about 1000‐fold lower affinity than native or reconstituted two‐chain toxin. The light chain did not bind to membranes in either test.Our data indicate that (a) the light chain and the N‐terminal part of the heavy chain are held together not only by one disulfide bond but also by hydrogen bonds and ionic forces to yield a two‐chain toxin or fragment B and (b) both chains contribute to the actions of the toxin in vivo and in vitro, and to its binding.
Differences in hydrophobic properties for human α2‐macroglobulin and pregnancy zone protein as studied by affinity phase partitioning
Authors: BIRKENMEIER Gerd , CARLSSON‐BOSTEDT Lena , SHANBHAG Vasant , KRIEGEL Thomas , KOPPERSCHLÄGER Gerhard , SOTTRUP‐JENSEN Lars , STIGBRAND Torgny 
Published: March 2005

Human α2‐macroglobulin and pregnancy zone protein are related with regard to primary structure, physicochemical properties, and quarternary structure. Both proteins undergo conformational changes when they form complexes with proteinases or react with primary amines.The surface properties of the native, chymotrypsin‐treated and methylamine‐treated forms of α2‐macroglobulin and pregnancy zone protein were studied by partitioning in aqueous two‐phase systems composed of 7.5% dextran T70 and 5% poly(ethylene glycol) 8000. All proteins and their derivatives had a high potential for hydrophobic interaction as analyzed in terms of affinity for poly(ethylene glycol) esters of fatty acids included in the phase systems. Treatment of α2‐macroglobulin with methylamine or chymotrypsin increased the surface hydrophobicity significantly compared to that of the native protein. No difference in hydrophobic interaction was found for native and methylamine‐treated pregnancy zone protein, but the chymotrypsin‐treated protein showed a marked increase in binding to the hydrophobic ligand. The changes in surface hydrophobicity parallel changes in receptor binding properties of the derivatized forms of α2‐macroglobulin and could be a signal for binding to cell‐surface receptors, followed by internalization.