bluuebunny/crossref_metadata_2025_split huggingface dataset:
Optical Study of the Light‐Induced Protonation Changes Associated with the Metarhodopsin II Intermediate in Rod‐Outer‐Segment Membranes
Authors: BENNETT Nelly 
Published: July 2010

The kinetics of the protonation changes and of the formation of the metarhodopsin II intermediate are measured in the presence of the pH indicator dye bromocresol purple after two consecutive flashes on the same suspension of right‐side‐out vesicles from cattle rod‐outer‐segment membranes. A rapid proton binding followed by partial proton release is observed. The formation of metarhodopsin II is found to be biphasic. The rapid phase of metarhodopsin II formation is slightly faster than the proton binding, and the kinetics of the slow phase are similar to that of the proton release. The protein groups which are protonated and deprotonated are shown to be situated on the outside of the vesicles.These results provide evidence that a structural change of the protein involving pK changes occurs without spectral modification before the formation of metarhodopsin III (half‐time of the order of seconds).
Light‐Induced Appearance of Polysomal Poly(A)‐Rich Messenger RNA during Greening of Barley Plants
Authors: HEINZE Hartmut , HERZFELD Frank , KIPER Manuel 
Published: July 2010

Changes in polysotnal poly(A)‐rich tnRNA during greening of etiolated barley plants were studied by the technique of cDNA‐mRNA hybridization. Hybridization data of the homologous reactions reveal that in etiolated as well as in greened shoots a complexity of 5 × 107 nucleotides or about 33000 different average‐sized mRNAs are present. These are organized in different abundancy classes with 94% of the total complexicity present in each of the slowest reacting class representing rare messengers.Heterologous hybridizations indicate that 92% of all polysotnal poly(A)‐rich mRNAs in etiolated shoots are complementary to those of greened and 82%, of ‘green’ poly(A)‐rich mRNAs are complementary to white ones.It is shown that the abundant mRNA classes are essentially responsible for these diferences. The prevalent classes making up 15% (‘white’) and 31% (‘green’) of the poly(A)‐rich mRNA mass but comprising only a complexity of 1.8 × 104 and 2.1 × 104 nucleotides are identical to 50% with each other. Hybridization of isolated prevalent ‘green’ cDNA with whole ‘white’ poly(A)‐rich mRNA indicates that the additionally appearing 50% prevalent green messengers must be regarded as greenspecific, only present in polysomal poly(A)‐rich mRNA after illumination.This conclusion is underlined by the hybridization of the ‘green’ cDNA with total polysomal RNA of etiolated shoots. Evidently appearance of these prevalent messengers in functional polysomes is not caused by a shift from poly(A)‐free mRNA to poly(A)‐rich mRNA.The results clearly demonstrate that light induces greening by turning on genes or influencing post‐transcriptional processing to produce mature green‐specific poly(A)‐rich tnRNA.
Cytochrome b‐565 in Saccharomyces cerevisiae: Use of Mutants in the Cob‐Box Region of the Mitochondrial DNA to Study the Functional Role of this Spectral Species of Cytochrome b
Authors: CHEVILLOTTE‐BRIVET Paule , MEUNIER‐LEMESLE Danielle 
Published: July 2010

A wild‐type strain of Saccharomyces cerevisiae and temperature‐dependent revertants of isonuclear box mutants partially or completely devoid of cytochrome b‐565, were used to study the role of this cytochrome in oxidative phosphorylation.At the mitochondrial level, the phosphorylating data and the succinate oxidase activity at three different temperatures (16° C, 28° C and 36° C) were measured in these strains showing various cytochrome b‐565 contents. It is concluded that this cytochrome b‐565 is not in the main pathway of the electron transfer chain.At the optimum growth temperature (28° C), the measurements of the P/O ratio for the wild phenotype strains, with ethanol as substrate, led to the conclusions that two phosphorylation sites of the respiratory chain are functional in these strains.The growth yields for wild phenotype strains and revertants grown in vivo in complex media, with ethanol or galactose as the energy source (at 16° C, 28° C and 36° C), were compared with the cytochrome b‐565 content.The growth yields showed small variations if compared to the reference strain, when the cytochrome b‐565 content was greatly diminished or absent.Thus the ATP production at site II is independent of the cytochrome b‐565 content. Cytochrome b‐565 does not play an essential role in oxidative phosphorylation.
Cross‐linking of Histone H1 in Chromatin
Authors: THOMAS Jean O. , KHABAZA Andrew J. A. 
Published: July 2010

Arrays of neighbouring histone H1 molecules are present in rat liver chromatin, and can be cross‐linked to each other by bisimidoesters. The product, poly(H1), can be extracted from crosslinked chromatin in high yield with 5% perchloric acid and thus distinguished from cross‐linked oligomers of the core histones. On analysis in dodecyl sulphate/polyacrylamide gels poly(H1) gives a striking pattern of alternating strong and weak bands which have been shown, by the use of cleavable cross‐linked reagents and two‐dimensional gel electrophoresis, to arise respectively from H1 homopolymers and H1 polymers linked to the nucleosome core histones.H1‐H1 proximities as measured by cross‐linking exist both at low ionic strength where the nucleosome filament is extended, and at higher ionic strengths at which it is folded into a 30‐nm‐diameter fibre, probably in the form of a solenoid, although some additional H1‐H1 contacts seem to occur in the folded form. A similar pattern of H1‐H1 cross‐linking is observed for nucleosome oligomers too short to form a complete turn of a solenoid. The lack of any strong dependence of the H1 cross‐linking pattern on ionic strength, and the results for short oligomers, suggest that in a soleniud the major H1‐H1 interactions are lateral (i.e. they occur between neighbouring nucleosomes along a turn) although additional vertical contacts (between successive turns) are not excluded. For a short nucleosome oligomer containing n nucleosomes, the number of H1 molecules that may be cross‐linked into an array at low ionic strength (∼ 15 mM) is n, consistent with the presence of one H1 per nucleosome.H1‐H1 cross‐linking occurs only within the chromatin framework; when cross‐linking is carried out in 0.5 M NaCl so that H1 is dissociated from chromatin, no poly(H1) is formed. When H1 which has been dissociated in 0.5 M NaCl is allowed to reassociate by gradual lowering of the ionic strength, the poiy(H1) is identical with that in native chromatin, suggesting faithful rebinding of H1 molecules. H1‐H1 proximities do not seem to be mediated by the presence of bound high‐mobility‐group non‐histone proteins because the poly(H1) pattern persists when cross‐linking is carried out at 365 mM ionic strength, when these high‐mobility‐group proteins are dissociated from chromatin.The H1 cross‐linking pattern described here could prove to be a useful assay for the native arrangement of H1 molecules in reconstituted chromatin and for changes in H1‐H1 contacts that might result from cell‐cycle modifications (e.g. phosphorylation) of H1. It may not prove a good assay for higher‐of der structure because of the relative insensitivity to ionic strength of the poly(H1) pattern as studied here. A detailed analysis will be necessary to determine whether there are any subtle differences in the interactions between H1 molecules in the folded and extended nucleosome filament.
Non‐specific Lanosterol and Hopanoid Biosynthesis by a Cell‐Free System from the Bacterium Methylococcus capsulatus
Authors: ROHMER Michel , BOUVIER Pierrette , OURISSON Guy 
Published: July 2010

A cell‐free system from the bacterium Methylocoecus capsulatus was incubated with [12‐3H]squalene; diploptene and diplopterol, normally present in the bacterium, were labelled.
The same cell‐free system was incubated with (RS)‐2,3‐epoxy‐2,3‐dihydro‐[3‐3H]squalene. Several radioactive 3‐hydroxytriterpenes were purified. Lanosterol, which is normally present in this bacterium, was found labelled as well as 3‐epilanosterol. In addition, radioactive 3α‐hydroxy and 3β‐hydroxydiploptene were formed.
These data may be explained by the coexistence of two cyclases in M. capsulatus: a squalene/hopane cyclase and a squalene epoxide/lanosterol cyclase. The squalene cyclase exhibits the same lack of substrate specificity as those of Acetobacter pasteurianum and Tetrahymena pyriformis, i. e. in addition to its normal substrate squalene, it can cyclize the two enantiomers of squalene epoxide into 3‐hydroxyhopanoids.
The presence of a squalene epoxide/lanosterol cyclase activity, which was suspected in view of the unique 3β‐hydroxy 4α‐methyl steroids of M. capsulatus, was demonstrated by the labelling of lanosterol. More surprisingly 3‐epilanosterol was also present and labelled. We showed that this does not derive from lanosterol by isomerization via a 3‐oxo compound. Therefore the squalene epoxide cyclase of M. capsulatus, like the one of eukaryotes cyclizes the (3S) enantiomer of squalene epoxide into lanosterol. But it is definitely less substrate‐specific as it can also cyclize the (3R) enantiomer into 3‐epilanosterol.
Bovine Kidney Pyruvate Dehydrogenase Complex
Authors: KRESZE Georg‐B. , RONFT Hedwig 
Published: July 2010

Bovine kidney pyruvate dehydrogenase multienzyme complex is inactivated by elastase in a similar manner as described earlier for papain. The core component, lipoate acetyltransferase, is cleaved by elastase into an active fragment (Mr 26000) and a fragment with apparent Mr of 45000 as analyzed by dodecylsulfate gel electrophoresis. Due to the fragmentation of the core, the enzyme complex is disassembled into its component enzymes which retain their complete enzymatic activities as assayed separately.
A different mechanism was found for the inactivation of pyruvate dehydrogenase complex with trypsin and some other proteases (chymotrypsin, clostripain). In these cases, the pyruvate dehydrogenase component is inactivated rapidly by limited proteolysis. More slowly, the enzyme complex is disassembled simultaneously with fragmentation of the lipoate acetyltransferase which again results in an active fragment of Mr 26000 and another fragment of apparent Mr 45000. Upon prolonged proteolysis, the latter fragment is cleaved further to give products of Mr 36000 or lower.
The enzyme‐bound lipoyl residues of the pyruvate dehydrogenase complex have been labelled covalently by incubation with [2‐14C]pyruvate. After treatment of this [14C]acetyl‐enzyme with papain, elastase, or trypsin, radioactivity was associated exclusively with the 45000‐Mr and 36000‐Mr fragments but not with the active 26000‐Mr fragment.
It is concluded that the bovine kidney lipoate acetyltransferase core is composed of 60 subunits each consisting of two dissimilar folding domains. One of these contains the intersubunit binding sites as well as the active center foi‐transacylation whereas the other possesses the enzyme‐bound lipoyl residues.
Microtubule‐Associated‐Protein MAP1 is not Implicated in the Polymerization of Microtubules
Authors: VILLASANTE Alfredo , de la TORRE Javier , MANSO‐MARTINEZ Rafael , AVILA Jesús 
Published: July 2010

Addition of DNA to microtubule protein solutions results in a decrease of the amount of the high‐molecular‐weight microtubule‐associated proteins (MAP1 and MAP2) available for polymerization. Complete removal of MAP1 from microtubules requires lower concentrations of DNA compared to MAP2. Removal of MAP1 from microtubules has little or no effect on microtubule polymerization (nucleation and propagation). In contrast, removal of MAP2 produces a marked decrease in microtubule polymerization.Incubation of microtubular protein with DNA results in complete disappearance of the 30‐S ring‐shaped oligomers at concentrations of DNA which are not markedly inhibitory of polymerization.
Identification and Characterization of the Translationally Repressed Cytoplasmic Globin Messenger‐Ribonucleoprotein Particles from Duck Erythroblasts
Authors: VINCENT Alain , CIVELLI Olivier , MAUNDRELL Kinsey , SCHERRER Klaus 
Published: July 2010

Globin messenger ribonucleoprotein (mRNP) particles which have been isolated from duck erythroblast post‐polyribosomal supernatant are translationally inactive in vivo and in vitro but contain translatable mRNA active after deproteinisation. They were characterized following pruification by successive sucrose gradient sedimentation in a buffer containing 0.05 M KCI. The complex, which sediments homogeneously at about 20 S, has a density of 1.39 g/cm3 and thus consists of four parts protein to one part RNA; 40% of this RNA is globin mRNA and no other mRNA could be detected.Sedimentation of the purified globin mRNP on sucrose gradients in 0.5 M KCl produced four components while polyacrylamide gel electrophoresis in non‐denaturing conditions and in the presence of EDTA resulted in the separation of three components. Hybridization to globin cDNA and translation in vitro of the RNA extracted from these subparticles revealed the existence of two core parcticles containing globin mRNA with nominal sedimentation coefficients of 13 S and 16 S.Analysis of the protein components of the isolated sub‐complexes by dodecyl sulfate and bidimensional gel electrophoresis indicated a very characteristic protein composition for each of these complexes. The 16‐S and 13‐S globin mRNPs differed essentially by the presence in the 13‐S mRNP only of a group of major polypeptides. Of the other two sub‐complexes, one consisted of 90% small RNA in the 4‐S range; the second sedimented ahead of the globin mRNP core particles at about 19 S and consisted of a very characteristic set of about 14 polypeptides. The polyrisohomal 73000‐Mr poly(A)‐binding protein was not detected in the purified free globin mRNP although the PIRNA in the untranslatable particle is polyadenylated.The presence in the cytoplasm of duck erythroblasts of two forms of untranslated globin messenger ribonucleoprotein particles, distinct in their protein composition from polyribosomal globin mRNP, suggests that they may have a specific role in the regulation of translation of globin mRNA.
Translation of Zea mays Endosperm Sucrose‐Synthase mRNA in vitro
Authors: WÖSTEMEYER Johannes , BEHRENS Ute , MERCKELBACH Armin , MÜLLER Markus , STARLINGER Peter 
Published: July 2010

mRNA of 23‐day‐old maize endosperm was translated both in wheat germ extracts and rabbit reticulocyte lysates. A protein with an apparent molecular weight of 88000 comigrates in dodecylsulfate/polyacrylamide electrophoresis with sucrose synthase. This protein is precipitated with an antiserum against sucrose synthase and shows the same protease digestion pattern as the enzyme. It is not synthesized with mRNA extracted from sh/sh mutant kernels lacking sucrose synthase. By these criteria, the protein is the translation product in vitro of sucrose synthase mRNA.The separation of mRNA in methylmercury‐hydroxide‐agarose gels and subsequent translation indicates a length of sucrose synthase mRNA of 2800 nucleotides which is compatible with the coding length necessary for a protein with a molecular weight of 88000 plus untranslated sequences.
Spectroscopic Properties of Light‐Chain Derivatives of Murine MOPC‐315 Immunoglobulin A
Authors: ZIDOVETZKI Raphael , FARVER Ole , PECHT Israel 
Published: July 2010

Three light‐chain derivatives of the homogeneous IgA, secreted by the mouse myeloma MOPC‐315, were studied employing circular dichroism and thermal‐perturbation spectroscopy: (a) the light‐chain dimer with intact native inter‐chain disulfide bond, L2.cov (b) the light‐chain dimer with this bond reduced and alkylated, LL2.ncov and (c) the dimer of only the variable regions of the light chains, (VL)2. Comparison of the well resolved circular dichroism spectra of these derivatives allowed the assignments of the bands above 290 nm to the following chromophores: Trp‐35L and Trp‐91L in the variable domains, and Trp‐148L Trp‐185L and the disulfide of Cys‐214L in the constant domains. The differences in the spectral characteristics of L2.cov as compared to those of L2.ncov and (VL)2 illustrate the significant influence of the disulfide bridge on the conformation of the L2.cov. Pronounced differences are found between these light‐chain derivatives and light chain‐heavy chain associates, namely the intact protein M‐315 and its Fv fragment.The comparison between the CD spectra of the free and the hapten‐bound L2.cov, L2.ncov and (VL)2 directly demonstrates the existence of the conformational transitions in these proteins induced by hapten binding.
A Native‐Like Intermediate on the Ribonuclease A Folding Pathway
Authors: SCHMID Franz X. 
Published: July 2010

Unfolded ribonuclease A consists of a mixture of fast‐folding (UF) and slow‐folding species (US); US folds slowly because it contains non‐native proline isomers. The folding kinetics of US have been determined by measuring the changes in tyrosine fluorescence upon refolding. Under conditions where the energy of stabilization of the native protein is small (in marginally native conditions) the fluorescence‐detected kinetics of folding coincide with the ones measured by other probes. However, when refolding is carried out under conditions where the native protein is highly stabilized (in strongly native conditions), the fluorescence changes become progressively slower than folding monitored by tyrosine absorbance or by 2′CMP binding. Under these conditions the fluorescence‐detected kinetics of folding show properties of proline isomerization reactions. In addition they match proline isomerization measured during refolding of Us by a two‐step assay for non‐native proline isomers.The following conclusions are drawn. (1) There is a kinetic intermediate between US and the native enzyme in the folding pathway of RNase A. This has been shown by the divergent progress curves monitored by different probes. Although the intermediate contains non‐native proline isomers, it is folded and hence sensitive to agents that affect the stability of native proteins. (2) Fluorescence‐detected folding closely follows proline isomerization during folding. Refolding of US is not blocked by incorrect prolinc isomers, but US is able to fold to a structural intermediate before proline isomerization occurs. (3) The quantum yield of the fluorescence of native RNase A depends on the precise spatial location of the tyrosine side chains with respect to their hydrogen bond acceptors and to the disulfide bonds. Therefore the native fluorescence is not regained until all essential proline peptide bonds are in the right configuration.
A Native‐Like Intermediate on the Ribonuclease A Folding Pathway
Authors: SCHMID Franz X. , BLASCHEK Heidi 
Published: July 2010

A folded intermediate IN has been detected in the slow refolding reaction of ribonuclease A, US→N, where US is an unfolded species which contains non‐native isomers of X‐Pro peptide bonds and N is the native enzyme. IN has been characterized previously as a structured folding intermediate: it shows a tyrosine absorbance like native ribonuclease, but it still contains at least one non‐native proline peptide bond and its tyrosine fluorescence differs from the native enzyme.Here we have used three probes to characterize further the structure of IN in comparison to native ribonuclease A. (1) The accessibility of the tyrosine hydroxyl groups to ionization has been investigated in IN. (2) The binding of the specific inhibitor cytidine 2′‐phosphate (2′CMP) to IN was measured. (3) The regain of enzymatic activity during folding was monitored to decide whether IN is an enzymatically active intermediate.The results indicate that IN possesses a compact folded structure which shields part of the tyrosine residues from the aqueous environment like native ribonuclease A. The active‐site region of IN has folded to the native conformation. IN binds 2′CMP as well as native ribonuclease A does and it shows enzymatic activity similar to the native enzyme.We conclude that unfolded molecules, which have only proline‐93 in the non‐native trans configuration, can refold to the native‐like intermediate IN before proline isomerization takes place. Proline‐93 is located far away from the active‐site region in an external bend and therefore does not block folding. It isomerizes in the folded molecule to the native cis configuration.
360‐MHz Nuclear Magnetic Resonance and Laser Photochemically Induced Dynamic Nuclear Polarization Studies of Bile Salt Interaction with Porcine Colipase A
Authors: COZZONE Patrick J. , CANIONI Paul , SARDA Louis , KAPTEIN Robert 
Published: July 2010

Porcine pancreatic colipase possesses a particular three‐dimensional surface domain containing six out of the seven aromatic residues of the molecule in a highly hydrophobic environment [P. Canioni and P. Cozzone (1979) Biochimie (Paris) 61, 343–354; J. Wieloch, B. Borgström, K. E. Falk and S. Forsen (1979) Biochemistry, 18, 1622–1628]. The domain corresponds to the 49–57 and 77–86 β‐sheet fragments brought into spatial proximity by protein folding [P. Canioni, P. Cozzone and L. Sarda (1980) Biochim. Biophys. Acta, 621, 29–42]. The identification of this specific domain as being the lipid binding site on colipase is proposed on the basis of the NMR and photochemically induced dynamic nuclear polarization (photo‐CIDNP) studies of the complexes of colipase with organized bile salt micelles.The binding of taurodeoxycholate micelles specifically perturbs the proton NMR ring resonances of Tyr‐I and Tyr‐II (Tyr‐56 and Tyr‐57) and His‐II (His‐86) together with several aliphatic resonances, reflecting the involvement of the hydrophobic aromatic domain in micelle fixation. In the presence of colipase, specific shifts and broadening of the methyl groups at position 18 and 21 of the sterane ring of taurodeoxycholate are observed and suggest that the hydrophobic side of the bile salt is primarily involved. The strong photo‐CIDNP effects of Tyr‐I and Tyr‐II in the presence of lumiflavin dye, which have been described on free colipase [P. Canioni, P. Cozzone and R. Kaptein (1980) FFBS Lett. 111, 219–222] are totally suppressed in the presence of taurodeoxycholate or chenocholamine micelles, indicating that due to the protection by the bile salt, the aromatic surface residues are no longer accessible. The pH dependence of the NMR perturbations and photo‐CIDNP effects observed in the colipase‐micelle complexes confirms that (1) the hydrophobic aromatic domain is directly involved and (2) the driving force of the primary micelle binding is essentially hydrophobic.A general model for micelle binding to colipase is proposed and involves a two‐step mechanism. Initially, the lipid hydrophobic binding site participates in the building of the bound taurodeoxycholate aggregate to form a mixed micelle through hydrophobic surface interactions (stoichiometric complex). At higher bile salt concentrations, polar forces might account for further growth of the micellar structure bound on colipase.