bluuebunny/crossref_metadata_2025_split huggingface dataset:
Biogenesis of blowfly photoreceptor membranes is regulated by 11‐cis‐retinal
Authors: PAULSEN Reinhard , SCHWEMER Joachim 
Published: March 2005

Biogenesis of photoreceptor membranes has been investigated by analyzing the rhodopsin and opsin content of microvillar photoreceptor membranes after injecting retinal isomers and radioactive amino acids into the compound eyes of carotenoid‐deficient blowflies. The amount of rhodopsin in the membranes was measured photometrically in extracts of isolated rhabdoms. The opsin content of the membranes and the level of radioactive labelling of opsin were measured after separating the membrane proteins by dodecyl sulphate/polyacrylamide gel electrophoresis.1. In the photoreceptor membrane of carotenoid‐deficient flies the rhodopsin and opsin content is less than 4% of that in normal flies where opsin may constitute about 65% of the total membrane protein.2. In carotenoid‐deficient flies the incorporation of rhodopsin into photoreceptor membranes is triggered by 11‐cis‐retinal but not all‐trans retinal. After injection of 11‐cis‐retinal the opsin content of the photoreceptor membranes increases in parallel with the rhodopsin content.3. Radioactive labelling of opsin reveals that 11‐cis‐retinal triggers an incorporation of newly synthesized opsin into photoreceptor membranes whereas all‐trans retinal does not induce the assembly of opsin into photoreceptor membranes. Light‐dependent incorporation of radioactive labelled opsin into membranes with a high rhodopsin content shows that not only the visual pigment chromophore but also the opsin undergoes a light‐dependent turnover. The findings raise the possibility that opsin synthesis in blowfly photoreceptors and consequently the assembly of photoreceptor membranes is regulated by 11‐cis‐retinal.
Tertiary structure of animal tRNATrp in solution and interaction of tRNATrp with tryptophanyl‐tRNA synthetase
Authors: GARRET Maurice , LABOUESSE Bernard , LITVAK Simon , ROMBY Pascale , EBEL Jean Pierre , GIEGÉ Richard 
Published: March 2005

Alkylation in beef tRNATrp of phosphodiester bonds by ethylnitrosourea and of N‐7 in guanosines and N‐3 in cytidines by dimethyl sulfate and carbethoxylation of N‐7 in adenosines by diethyl pyrocarbonate were investigated under various conditions. This enabled us to probe the accessibility of tRNA functional groups and to investigate the structure of tRNATrp in solution as well as its interactions with tryptophanyl‐tRNA synthetase.The phosphate reactivity towards ethylnitrosourea of unfolded tRNA was compared to that of native tRNA. The pattern of phosphate alkylation of tRNATrp is very similar to that found with other tRNAs studied before using the same approach with protected phosphates mainly located in the D and TΨ arms. Base modification experiments showed a striking similarity in the reactivity of conserved bases known to be involved in secondary and tertiary interactions. Differences are found with yeast tRNAPhe since beef tRNATrp showed a more stable D stem and a less stable TΨ stem.When alkylation by ethylnitrosourea was studied with the tRNATrp· tryptophanyl‐tRNA synthetase complex we found that phosphates located at the 5′ side of the anticodon stem and in the anticodon loop were strongly protected against the reagent. The alkylation at the N‐3 position of the two cytidines in the CCA anticodon was clearly diminished in the synthetase · tRNA complex as compared with the modification in free tRNATrp; in contrast the two cytidines of the terminal CCA in the acceptor stem are not protected by the synthetase. The involvement of the anticodon region of tRNATrp in the recognition process with tryptophanyl‐tRNA synthetase was confirmed in nuclease S1 mapping experiments.
A rapidly light‐induced chloroplast protein with a high turnover coded for by pea nuclear DNA
Authors: MEYER Gabriele , KLOPPSTECH Klaus 
Published: March 2005

Among the translation products obtained in vitro with mRNAs isolated from etiolated grown pea, or after different times of illumination following the etiolation, a 24000‐Mr protein has been observed in the very early phase of greening; its occurrence culminates at 2–4 h after the start of illumination.From these data it is concluded that the corresponding mRNA appears in and disappears from the poly(A)‐containing RNA population within hours. The protein product has been characterized as the precursor for a 17000‐Mr, chloroplast protein; by means of post‐translational transport in vitro, the processed product becomes bound to chloroplast membranes.A product of the same size can also be labeled in vivo with a maximum of incorporation of label at 6–8 h after illumination. This product decays with a half‐life of about 5 h.These findings imply a regulatory function of the 17000‐Mr protein during the process of greening, possibly by synchronization of nuclear and chloroplast genomes. Other possibilities are considered.
The role of lipoteichoic acid biosynthesis in membrane lipid metabolism of growing Staphylococcus aureus
Authors: KOCH H. Uwe , HAAS Renate , FISCHER Werner 
Published: March 2005

Pulse‐chase experiments with [2‐3H]glycerol and [14C]acetate revealed that in Staphylococcus aureus lipoteichoic acid biosynthesis plays a dominant role in membrane lipid metabolism. In the chase, 90% of the glycerophosphate moiety of phosphatidylglycerol was incorporated into the polymer: 25 phosphatidylglycerol + diglucosyldiacylglycerol → (glycerophospho)25‐diglucosyldiacylglycerol + 25 diacylglycerol.Glycerophosphodiglucosyldiacylglycerol was shown to be an intermediate, confirming that the hydrophilic chain is polymerized on the final lipid anchor.Total phosphatidylglycerol served as the precursor pool and was estimated to turn over more than twice for lipoteichoic acid synthesis in one bacterial doubling. Of the resulting diacylglycerol approximately 10% was used for the synthesis of glycolipids and the lipid anchor of lipoteichoic acid. The majority of diacylglycerol recycled via phosphatidic acid to phosphatidylglycerol. Synthesis of bisphosphatidylglycerol was negligible and only a minor fraction of phosphatidylglycerol passed through the metabolically labile lysyl derivative.In contrast to normal growth, energy deprivation caused an immediate switch‐over from the synthesis of lipoteichoic acid to the synthesis of bisphosphatidylglycerol.
The T‐region of Ti plasmids codes for an enzyme synthesizing indole‐3‐acetic acid
Authors: SCHRÖDER Gudrun , WAFFENSCHMIDT Sabine , WEILER Elmar W. , SCHRÖDER Joachim 
Published: March 2005

Gene 2 from the T region of Ti plasmids appears to be expressed both in eucaryotic and in procaryotic systems. In transformed plant cells it participates in auxin‐controlled growth and differentiation, and in bacteria it is expressed into a defined protein of Mr 49000. We investigated the possibility that it codes for an enzyme involved in auxin biosynthesis.Only extracts from Escherichia coli cells expressing gene 2 hydrolyzed indole‐3‐acetamide into a substance which was unambiguously identified as indole‐3‐acetic acid. The same reaction was found in Agrobacteria containing gene 2, but not in strains lacking the gene. Extracts from tobacco crown gall cells, but not from non‐transformed cells, showed the same enzyme activity, and the reaction product was also identified as indole‐3‐acetic acid. The results indicate that gene 2 of the T region, which participates in tumorous growth of plant cells, codes both in bacteria and in plants for an amidohydrolase involved in the biosynthesis of the plant hormone indole‐3‐acetic acid.
Purification of the lactose:H+ carrier of Escherichia coli and characterization of galactoside binding and transport
Authors: WRIGHT J. Keith , OVERATH Peter 
Published: March 2005

The lactose carrier, a galactoside:H+ symporter in Escherichia coli, has been purified from cytoplasmic membranes by pre‐extraction of the membranes with 5‐sulfosalicylate, solubilization in dodecyl‐O‐β‐d‐maltoside, Ecteola‐column chromatography, and removal of residual impurities by anti‐impurity antibodies. Subsequently, the purified carrier was reincorporated into E. coli phospholipid vesicles.Purification was monitored by tracer N‐[3H]ethylmaleimide‐labeled carrier and by binding of the substrate p‐nitrophenyl‐α‐d‐galactopyranoside. All purified carrier molecules were active in substrate binding and the purified protein was at least 95% pure by several criteria.Substrate binding to the purified carrier in detergent micelles and in reconstituted proteoliposomes yielded a stoichiometry close to one molecule substrate bound per polypeptide chain.Large unilamellar proteoliposomes (1–5‐μm diameter) were prepared from initially small reconstituted vesicles by freeze‐thaw cycles and low‐speed centrifugation. These proteoliposomes catalyzed facilitated diffusion and active transport in response to artificially imposed electrochemical proton gradients (ΔpH+) or one of its components (ΔΨ or ΔpH).Comparison of the steady‐state level of galactoside accumulation and the nominal value of the driving gradients yielded cotransport stoichiometries up to 0.7 proton/galactoside, suggesting that the carrier protein is the only component required for active galactoside transport.The half‐saturation constants for active uptake of lactose (KT= 200μM) or β‐d‐galactosyl‐1‐thio‐β‐d‐galactoside (KT= 50–80 μM) by the purified carrier were found to be similar to those measured in cells or cytoplasmic membrane vesicles.The maximum rate for active transport expressed as a turnover number was similar in proteoliposomes and cytoplasmic membrane vesicles (kcat= 3–4s−1 for lactose) but considerably smaller than in cells (kcat= 40–60s−1). Possible reasons for this discrepancy are discussed.
Purification and properties of the basic lectins from winged bean seed [Psophocarpus tetragonolobus(L.) DC]
Authors: KORTT Alexander A. 
Published: March 2005

The seeds of winged bean, Psophocarpus tetragonolobus (L.) DC, contain a group of acidic lectins (pI ≊ 5.5) and a group of basic lectins (pI < 9.5) characterised by different erythrocyte hemagglutinating specificities. Three basic lectins were separated and purified to apparent homogeneity by chromatography on Ultrogel AcA44 and SP‐Sephadex C‐25. These lectins are glycoproteins with relative molecular mass of 58000. The total carbohydrate content of the major lectins, B2 and B3, was 5% and was comprised of mannose, N‐acetylglucosamine, fucose and xylose in amounts corresponding to 9, 8, 8 and 3 mol/58000g, respectively, for both lectins. Electrophoresis in dodecyl sulfate gave a single subunit of relative molecular mass 29000 for each lectin. Isoelectric focusing in 8 M urea revealed two differently charged subunits in each of the isolated lectins; a total of three different subunits in the three lectins was observed.The basic lectins have essentially the same amino acid composition and contain no half‐cystine. Two of the lectins (B2 and B3) showed identical amino‐terminal sequences and the sequence of lectin B3 to residue 40 revealed extensive homology with other legume lectins such as soybean lectin.The basic lectins agglutinated trypsinized rabbit and trypsinized human (type A and B) erythrocytes but not trypsinized human (type O) erythrocytes. The lectins were inhibited by N‐acetylgalactosamine, d‐galactose and d‐galactose containing disaccharides and trisaccharides. The specificity appears to be directed to glycosides having non‐reducing (terminal) α‐d‐galactopyranosyl groups. β‐d‐Galactosides, such as lactose, are poor inhibitors.
Guinea‐pig casein A cDNA
Authors: HALL Len , LAIRD Jane E. , PASCALL John C. , CRAIG Roger K. 
Published: March 2005

The nucleotide sequence (1036 bases) of guinea‐pig casein A mRNA has been determined. Two cDNA recombinant plasmids contained a total of 993 base pairs, including part of the 5′ noncoding region, and the complete coding and 3′ noncoding region. The remaining 5′ noncoding sequence was obtained by primer extension.The deduced 223‐amino‐acid‐coding sequence of guinea‐pig pre‐casein A exhibited 30% homology with bovine αs2 casein, the most striking similarities being in the locations of potential phosphorylation sites.
The effect of EDTA and related chelating agents on the oxidation of methanol by the methylotrophic bacterium, Methylophilus methylotrophus
Authors: CARVER Mark A. , HUMPHREY Kevin M. , PATCHETT Roy A. , JONES Colin W. 
Published: March 2005

The effects of EDTA and related metal‐chelating agents on the respiratory system of the methylotrophic bacterium Methylophilus methylotrophus have been investigated. EDTA completely inhibited whole cell methanol oxidase activity and concomitantly decreased the aerobic steady‐state reduction level of cytochrome c, but only partially inhibited methanol dehydrogenase activity.Analysis of the inhibition kinetics of EDTA and other chelating agents on methanol oxidase activity indicated that they were effective in the order CDTA < EDTA < HEDTA < EGTA < EDDA, and that they inhibited in an uncompetitive manner. Inhibition by EDTA varied as a function of the ambient cell mass in the assay, and could be partly reversed by the addition of divalent cations. EDTA had no effect on the activity of solubilised methanol dehydrogenase. These and other results indicate that EDTA binds a divalent metal ion, probably Mg2+, which is involved in the functional association of methanol dehydrogenase with the respiratory membrane.This concept is discussed in terms of the electron transfer properties and spatial organisation of the terminal respiratory chain of M. methylotrophus.
The arrangement of ribosomal RNA genes in Schistosoma mansoni
Authors: SIMPSON Andrew J. G. , DAME John B. , LEWIS Fred A. , McCUTCHAN Thomas F. 
Published: March 2005

The two large ribsomal RNA subunits of Schistosoma mansoni are encoded within a 10000‐base sequence, which is tandemly repeated in the schistosome genome. Restriction endonuclease digestion with Bam HI cuts the rRNA gene into three fragments, which have been cloned separately in pBR322 and used to constract a physical map of the gene.The sequence encoding the smaller rRNA subunit is about 2000 bases in length and is situated on the 5′ sie of the sequence encoding the larger subunit, which is about 4000 bases. Approximately 4000 bases of the rRNA gene are spacer and do not code for mature rRNA. There are approximately 100 copies of the rRNA gene per haploid genome of which about 10% exhibit length heterogeneity, as judged by the hybridization of the rDNA plasmids to restriction endonuclease digests of genomic DNA. These structural variants appear to contain additional DNA suquences at more than one site within the gene and are polymorphic within the species S. mansoni.
Response of Tetrahymena pyriformis to stress induced by starvation
Authors: GALEGO Lisete , BARAHONA Isabel , RODRIGUES‐POUSADA Claudina 
Published: March 2005

mRNA synthesis was studied in exponentially growing and starved Tetrahymena pyriformis. Poly(A)‐containing RNAs separated from total RNA by affinity chromatography on oligo(dT)‐cellulose were characterized by polyacrylamide gel electrophoresis; their template activity was assayed in a rabbit reticulocyte lysate system and their translation products were analysed using two‐dimensional electrophoresis according to O'Farrell.1. Polysome profiles show that the bulk of ribosomes are in 80S monosomes in starved cells, whereas less than 8% are present in the form of monosomes in exponentially growing cells, the rest being engaged in polysomes. Polysomes are almost completely reformed 30 min after addition of enriched medium to suspensions of starved cells. This polysome reformation is dependent on mRNA synthesis since we have shown that it is inhibited by actinomycin D.2. Electrophoretic profiles of poly(A)‐rich RNA isolated from cytoplasmic fractions of exponential and starved cells are indistinguishable except that in the latter state significant amounts of low‐molecular‐mass species are observed.3. Poly(A)‐rich RNAs isolated from polysomal and non‐polysomal (subpolysomal) fractions of exponential cells are equally able to promote protein synthesis. The corresponding poly(A)‐rich RNAs isolated from starved cells also possess equal template activities which are, however, 15% lower than those of the poly(A)‐rich RNAs of exponentially growing cells. We also present evidence that in the system used in vitro, polyadenylated RNA isolated from heavy polysomes of starved cells directs the synthesis of four sets of proteins with molecular masses around 100 kDa, 70 kDa, 50 kDa and 30 kDa. The former two groups of proteins are more abundant in the translation products of poly(A)‐rich RNA of starved than of normal cells, whereas the latter two groups are present only in the translation products of poly(A)‐rich RNA of starved cells. The fluorograms of the translation products obtained in vitro from subpolysomal poly(A)‐rich RNA are identical to those obtained from polysomal poly(A)‐rich RNA.4. Studies on starved cells in vivo show that polypeptides of 100 kDa, 70 kDa and 38 kDa are more strongly labelled and also revealed the specific presence of 85 kDa, 55 kDa, 50 kDa and 25 kDa proteins. These results lead us to the conclusion that this microorganism responds to depleted environmental conditions by regulating gene expression at the transcriptional level, but also at the translational level.
Sorbitol dehydrogenase
Authors: JEFFERY Jonathan , CEDERLUND Ella , JÖRNVALL Hans 
Published: March 2005

The first primary structure for a sorbitol dehydrogenase has been determined by analysis of the tetrameric enzyme from sheep liver. The [14C]carboxymethylated protein was cleaved with CNBr and proteolytic enzymes. Peptides were purified by several methods, often utilizing exclusion chromatography for pre‐fractionation and reverse‐phase high‐performance liquid chromatography for final purification. Different methods of sequence analysis complemented each other, mainly the manual dimethylaminoazobenzene isothiocyanate method and the use of liquid‐phase sequencer degradations.All eight major CNBr fragments were purified and form the basis of the work. Three minor CNBr fragments derived from an acid cleavage and from a partly resistant Met‐Thr bond were also obtained, as well as evidence for a contaminating homologous polypeptide. Most of the tryptic peptides were purified, including all with methionine residues, thus overlapping the CNBr fragments. Combined, all data permit the deduction of a 354‐residue amino acid sequence for the polypeptide chain of sorbitol dehydrogenase. The N terminus is acyl‐blocked, the C terminus is formed by a proline residue, tryptophan is the least common residue (two, at positions 50 and 301) and there are 10 cysteine residues, including the residue previously shown to be especially reactive (at postion 43). Similarities to ‘long’ alcohol dehydrogenases have functional implications.
Extensive variations and basic features in the alcohol dehydrogenase – sorbitol dehydrogenase family
Authors: JÖRNVALL Hans , von BAHR‐LINDSTRÖM Hedvig , JEFFERY Jonathan 
Published: March 2005

Structural comparisons of sorbitol dehydrogenase with zinc‐containing ‘long’ alcohol dehydrogenases reveal distant but clear relationships. An alignment suggests 93 positional identities with horse liver alcohol dehydrogenase (25% of 374 positions) and 73 identities with yeast alcohol dehydrogenase (20%). Sorbitol dehydrogenase forms a link between these distantly related alcohol dehydrogenases and is in some regions more similar to one of them that they are to each other. 43 residues (11%) are common to all three enzymes and include a heavy over‐representation of glycine (half of all glycine residues in sorbitol dehydrogenase), showing the importance of space restrictions in protein structures.Four regions are well conserved, two in each domain of horse liver alcohol dehydrogenase. They are two segments close to the active‐site zinc atom of the catalytic domain, and two in the central β‐pleated sheet strands of the coenzyme‐binding domain. These similarities demonstrate the general importance of internal and central building units in proteins.Large variations affect a region adjacent to the third protein ligand to the active‐site zinc atom in horse liver alcohol dehydrogenase. Such changes at active sites of related enzymes are unusual. Other large differences concern the segment around the non‐catalytic zinc atom of horse liver alcohol dehydrogenase; three of its four cysteine ligands are absent from sorbitol dehydrogenase. Three segments with several exchanges correspond to a continuous region with superficial areas, inter‐domain contacts and inter‐subunit interactions in the catalytic domain of alcohol dehydrogenase. They may correlate with the altered quaternary structure of sorbitol dehydrogenase. Regions corresponding to top and bottom β‐strands in the coenzyme‐binding domain of the alcohol dehydrogenase are also little conserved. Within sorbitol dehydrogenase, a large segment shows an internal similarity.The two distantly related alcohol dehydrogenases and sorbitol dehydrogenase form a triplet of enzymes illustrating basic protein relationships. They are ancestrally close enough to establish similarities, yet sufficiently divergent to illustrate changes in all but fundamental properties.
The role of Ca2+ uptake in the response of human platelets to adrenaline and to 1‐O‐alkyl‐2‐acetyl‐sn‐glycero‐3‐phosphocholine (platelet‐activating factor)
Authors: CLARE Katherine A. , SCRUTTON Michael C. 
Published: March 2005

Thrombin induces Ca2+ uptake into both stirred and unstirred human platelets in the presence or absence of acetylsalicylate. This Ca2+ uptake is closely correlated with adenine nucleotide secretion in accord with previous observations [Massini, P. and Lüscher, E. F. (1974) Biochim. Biophys. Acta 372, 109–121] but a low level of secretion is observed in the absence of significant Ca2+ uptake.
1‐O‐Alkyl‐2‐acetyl‐sn‐glycero‐3‐phosphocholine (1‐0‐alkylAcGEPC) also induces Ca2+ uptake into both stirred and unstirred human platelets in the presence and absence of acetylsalicylate. Aggregation and adenine nucleotide secretion induced by 1‐O‐alkylAcGEPC can be observed in the absence of added fibrinogen, but addition of fibrinogen causes a very marked shift to the left in the dose/response curves for aggregation and Ca2+ uptake induced by 1‐O‐alkylAcGEPC. In the absence of added fibrinogen a close correlation is observed between Ca2+ uptake and adenine nucleotide secretion induced by 1‐O‐alkylAcGEPC. In the presence of added fibrinogen significant aggregation can be observed in the absence of detectable Ca2+ uptake.
Adrenaline induces Ca2+ uptake only into stirred human platelets in the presence of added fibrinogen. Blockade of secretion, e.g. by addition of acetylsalicylate, also prevents Ca2+ uptake.
Addition of adrenaline fails to cause breakdown of phosphatidylinositol or phosphatidylcholine in acetylsalicylate‐treated platelets under conditions where such a response is observed on addition of thrombin.
We conclude that Ca2+ uptake into human platelets induced by thrombin, 1‐O‐alkylAcGEPC and adrenaline is closely associated with the secretory response and in some circumstances, e.g. stimulation by thrombin, is clearly a consequence of this latter response. Previous reports of Ca2+ uptake as an initiating event in the response of human platelets to adrenaline [Owen, N. E., Feinberg, H. and Le Breton, G. C. (1981) Am J. Physiol. 239, H483–488] have not been confirmed in this study.