bluuebunny/crossref_metadata_2025 huggingface dataset:
Comparative evaluation of gene expression in archaebacteria
Authors: ZILLIG Wolfram , PALM Peter , REITER Wolf‐Dieter , GROPP Felix , PÜHLER Gabriela , KLENK Hans‐Peter
Published: March 2005

Gene organization, gene structure, especially regarding transcription and translation signals, and the structure of essential components of the gene expression machinery of archaebacteria are compared with those of eubacteria and eukaryotes. Many features of the genetic machinery of archaebacteria are shared either with eubacteria or with eukaryotes. For example, the translation signals including ribosome‐binding sites are the same as in eubacteria, but the consensus sequence of archaebacterial promoters closely resembles that of the eukaryotic polymerase II promoters.

Archaebacterial genes can be organized in transcription units resembling those of eubacteria. But the sequences of several protein components of the genetic machinery have strikingly more homology with those of their eukaryotic than with those of their eubacterial correspondents.

The sequences of the large components of DNA‐dependent RNA polymerases of archaebacteria closely resemble those of the eukaryotic RNA polymerases II and, somewhat less, III. In a dendrogram calculated from percentage homology data, the eukaryotic RNA polymerase I component A shares a branching point with the eubacterial component. The implications of these findings for the origin and the evolution of the eukryotic ancestry are discussed.
Nucleotide sequence of the gene for aqualysin I (a thermophilic alkaline serine protease) of Thermus aquaticus YT‐1 and characteristics of the deduced primary structure of the enzyme
Authors: KWON Suk‐Tae , TERADA Ichiro , MATSUZAWA Hiroshi , OHTA Takahisa
Published: March 2005

Aqualysin I is an alkaline serine protease which is secreted into the culture medium by Thermus aquaticus YT‐1, an extreme thermophile [Matsuzawa, H., Hamaoki, M. & Ohta, T. (1983) Agric. Biol. Chem. 47, 25–28]. The gene encoding aqualysin I was cloned into Escherichia coli using synthetic oligodeoxyribonucleotides as hybridization probes. The nucleotide sequence of the cloned DNA was determined. The primary structure of aqualysin I, deduced from the nucleotide sequence, agreed with the NH2‐terminal sequence previously reported and the determined amino acid sequences, including the COOH‐terminal sequence, of the tryptic peptides derived from aqualysin I. Aqualysin I comprised 281 amino acid residues and its molecular mass was determined to be 28350. On alignment of the whole amino acid sequence, aqualysin I showed high sequence homology with the subtilisin‐type serine proteases, and 43% identity with proteinase K, 37–39% with subtilisins and 34% with thermitase. Extremely high sequence identity was observed in the regions containing the active‐site residues, corresponding to Asp32, His64 and Ser221 of subtilisin BPN'.

The nucleotide sequence of the cloned DNA (1105 nucleotides) revealed that it contains the entire gene encoding aqualysin I and one open reading frame without a translational stop codon. Therefore, aqualysin I was considered to be produced as a large precursor, which contains a NH2‐terminal portion, the protease and a COOH‐terminal portion. The G + C content of the coding region for aqualysin I was 64.6%, which is lower than those of other Thermus genes (68–74%). The codon usage in the aqualysin I gene was rather random in comparison with that in other Thermus genes.
The ‘CC.Ar.GG’ box
Authors: PHAN‐DINH‐TUY Françoise , TUIL David , SCHWEIGHOFFER Fabien , PINSET Christian , KAHN Axel , MINTY Adrian
Published: March 2005

We have previously suggested that a repeated sequence motif in the upstream region of the human cardiac actin gene ‘CC.Ar.GG’, where Ar is an (A + T)‐rich six‐base‐pair‐sequence, may be important in the muscle‐specific expression of this gene [Minty, A. & Kedes, L. (1986) Mol. Cell Biol. 6, 2125–2136]. Here we show that this sequence binds a nuclear protein, and that binding is abolished by mutating either the CC and GG dinucleotides or the (A + T)‐rich centre. Mutation of the CC and GG nucleotides also abolishes the transcription‐stimulating activity of this sequence on the cardiac actin promoter. A similar sequence has been implicated in the serum‐response of the c‐fos gene [Treisman, R. (1986) Cell 46, 567–574]. We show that this c‐fos‘CC.Ar.GG’ sequence competes with the cardiac actin sequence for factor binding. Our results suggest that the minimum sequence requirements for binding of the serum response factor may correspond to the ‘CC.Ar.GG’ box sequence. Using this criterion, we predict and confirm the existence of such a binding site in a regulatory region of the interleukin‐2 receptor gene. It appears therefore that interactions between ‘CC.Ar.GG’ boxes and similar proteic factors could be involved in the control of different genes responding to different stimuli, e.g. muscle differentiation (cardiac actin gene) or growth stimulation (c‐fos, cytoskeletal actin or interleukin‐2 receptor genes).
Proximity between fluorescent probes attached to four essential lysyl residues in phosphoenolpyruvate carboxylase
Authors: WAGNER Richard , PODESTÁ Florencio E. , GONZÁLEZ Daniel H. , ANDREO Carlos S.
Published: March 2005

Phosphoenolpyruvate carboxylase, purified from maize leaves, is rapidly inactivated by the fluorescence probe dansyl chloride. The loss of activity can be ascribed to the covalent modification of an R‐NH2 group, presumably the ɛ‐NH2 group of lysine. Analysis of the data by the statistical method of Tsou [Sci. Sin. 11, 1535–1558 (1962)] provides clear evidence that at pH 8 eight R‐NH2 groups can be modified in the tetrameric form of the enzyme, four of which are essential for catalytic activity. Essential groups are modified about five times more rapidly than the non‐essential ones. The enzyme was completely protected against inactivation by Mg2+ plus phosphoenolpyruvate and consequently binding of the modifier to the essential groups is completely abolished. Hence the four essential groups seemed to be located at or near the active site(s).

One of the four essential groups was modified with dansyl chloride and the other three progressively with eosin isothiocyanate.

In the doubly labeled protein non‐radiative singlet‐singlet energy transfer between dansyl chloride (donor) and eosin isothiocyanate (acceptor) was observed. The low variance (± 5%) in the efficiency of energy transfer obtained at a particular acceptor stoichiometry (0.8–1.1, 1.9–2.1, 2.9–3.1) in triplicate samples provided confidence that the measured transfer efficiency may be interpreted as transfer between specific sites. The distances calculated from the efficiency of resonance energy transfer revealed two acceptor sites, equally separated, 4.8–5.1 nm from the donor site and the third site being 6.4 nm apart from the donor.

Under conditions where the tetrameric enzyme dissociates into the monomers, no transfer of resonance energy between the protein‐bound dansyl chloride and eosin isothiocyanate was observed.

Most likely the four essential lysyl residues in the tetrameric enzyme are located in different subunits of the enzyme, hence each of the subunits would contain a substrate‐binding site with one lysyl residue crucial for activity.
Uridine(5′)diphospho(1)‐α‐d‐glucose
Authors: OIKONOMAKOS Nikos G. , ACHARYA K. Ravindra , STUART David I. , MELPIDOU Angeliki E. , McLAUGHLIN Paul J. , JOHNSON Louise N.
Published: March 2005

UDP‐glucose is an R‐state inhibitor of glycogen phosphorylase b, competitive with the substrate, glucose 1‐phosphate and noncompetitive with the allosteric activator, AMP. Diffusion of 100 mM UDP‐glucose into crystals of phosphorylase b resulted in a difference Fourier synthesis at 0.3‐nm resolution that showed two peaks: (a) binding at the allosteric site and (b) binding at the catalytic site.

At the allosteric site the whole of the UDP‐glucose molecule can be located. It is in a well defined folded conformation with its uracil portion in a similar position to that observed for the adenine of AMP. The uracil and the glucose moieties stack against the aromatic side chains of Tyr‐75 and Phe‐196, respectively. The phosphates of the pyrophosphate component interact with Arg‐242, Arg‐309 and Arg‐310.

At the catalytic site, the glucose‐1‐P component of UDP‐glucose is firmly bound in a position similar to that observed for glucose 1‐phosphate. The pyrophosphate is also well located with the glucose phosphate interacting with the main‐chain NH groups at the start of the glycine‐loop α helix and the uridine phosphate interacting through a water molecule with the 5′‐phosphate of the cofactor pyridoxal phosphate and with the side chains of residues Tyr‐573, Lys‐574 and probably Arg‐569. However the position of the uridine cannot be located although analysis by thin‐layer chromatography showed that no degradation had taken place. Binding of UDP‐glucose to the catalytic site promotes extensive conformational changes. The loop 279–288 which links the catalytic site to the nucleoside inhibitor site is displaced and becomes mobile. Concomitant movements of residues His‐571, Arg‐569, and the loop 378–383, together with the major loop displacement, result in an open channel to the catalytic site. Comparison with other structural results shows that these changes form an essential feature of the T to R transition. They allow formation of the phosphate recognition site at the catalytic site and destroy the nucleoside inhibitor site. Kinetic experiments demonstrate that UDP‐glucose activates the enzyme in the presence of high concentrations of the weak activator IMP, because of its ability to decrease the affinity of IMP for the inhibitor site.
The stoichiometry of charge translocation by cytochrome oxidase and the cytochrome bc1 complex of mitochondria at high membrane potential
Authors: MURPHY Michael P. , BRAND Martin D.
Published: March 2005

The q+/2e stoichiometries (number of charges translocated per electron pair transferred) of cytochrome oxidase and the cytochrome bc1 complex in rat liver mitochondria were determined at a range of membrane potentials up to 180 mV. The method used was similar to the one used in the preceding paper by us in this journal to determine the q+/O stoichiometry of the mitochondrial electron transport chain from succinate to oxygen. The measured q+/2e stoichiometry of cytochrome oxidase was 3.5 positive charges per O atom reduced at low membrane potential (120 mV) and it decreased to about 1.5 at high membrane potential (180 mV). The measured q+/2e stoichiometry of the cytochrome bc1 complex was between 1 and 1.25 positive charges ejected per electron pair and did not change significantly as ΔΨ was varied from 85 mV to 157 mV. The sum of the q+/2e stoichiometries of cytochrome oxidase and the cytochrome bc1 complex determined separately was similar to their value determined together for electron transport from succinate to oxygen over the range of membrane potentials studied. The most probable interpretation of these results is that the stoichiometry of the cytochrome bc1 complex is invariant over a range of membrane potentials and that the q+/2e stoichiometry of cytochrome oxidase decreases from 4 at low membrane potential to 2 at high membrane potential.
Polymerization of actin modified with fluorescein isothiocyanate
Authors: MILLER Larry , PHILLIPS Martin , REISLER Emil
Published: March 2005

Solution properties of skeletal muscle actin, modified at lysine‐61 with fluorescein isothiocyanate (FITC) [Burtnick, L.D. (1984) Biochim. Biophys. Acta 791, 57–62], were re‐examined in this work by light scattering, analytical ultracentrifugation, fluorescence, electron microscopy and myosin ATPase activity measurements. Fluorescence measurements using trace amounts of actin labeled with N‐(1‐pyrenyl)iodoacetamide showed that the FITC modification inhibited but did not block completely the polymerization of actin by KCl and MgCl2. Sedimentation velocity runs of FITC‐actin, incubated with 100 mM KCl and 2 mM MgCl2, revealed the presence in these solutions of polymeric, oligomeric and monomeric species. The critical concentration for FITC‐actin polymerization under these conditions was 12 μM. As judged by electron microscopy, FITC‐actin polymers were similar to but generally shorter than standard F‐actin filaments. Light scattering measurements indicated that FITC modification inhibited also the polymerization of actin by myosin subfragment 1 (S1) but the resulting complexes were indistinguishable from standard, decorated actin filaments. MgATPase measurements showed that FITC‐actin, polymerized by preincubation with S1, activated the MgATPase activity of S1 while the monomeric labeled protein did not. Thus, in analogy to native actin, the activating function of FITC‐actin depended on the formation of actin filaments. Results presented in this study suggest that the region around lysine‐61 of actin plays an important role in actin‐actin contact and is less crucial to actomyosin interaction.
Potentiation and suppression of mouse liver cytochrome P‐450 isozymes during the acute‐phase response induced by bacterial endotoxin
Authors: STANLEY Lesley A. , ADAMS David J. , LINDSAY Robert , MEEHAN Richard R. , LIAO Warren , WOLF C. Roland
Published: March 2005

Infection and inflammation are known to affect the metabolism and disposition of drugs and carcinogens. We report a detailed study of the effects of bacterial endotoxin on the constitutive and inducible expression and activities of cytochrome P‐450 isozymes from families P‐4501, P‐450IIB, P‐450IIC and P‐450III. In general high doses of high endotoxin caused very marked suppression of P‐450 isozymes and associated activities. However, this effect was differential, the expression of certain isozymes being only slightly reduced whereas others were suppressed to almost undetectable levels. Low doses of endotoxin also gave differential effects on cytochrome P‐450 expression. Of particular interest was the very marked potentiation of the inductive effect of both 3‐methylcholanthrene and phenobarbital. In the case of 3‐methylcholanthrene the 10‐fold induction of activity was increased to 24‐fold by concomitant endotoxin administration. In this regard it was interesting that 3‐methylcholanthrene was an effective inducer of a wide variety of acute‐phase proteins including metallothionein, serum amyloid A, fibrinogen and hemopexin.

These data show that endotoxin, and therefore bacterial infection and inflammation, can have profound and differential effects on components of the cytochrome‐P‐450 monooxygenase system which could result in significant changes in susceptibility to the effects of drugs, chemical toxins and carcinogens.
A novel fungal enzyme, NADPH‐dependent carbonyl reductase, showing high specificity to conjugated polyketones
Authors: SHIMIZU Sakayu , HATTORI Shizuo , HATA Hiroyuki , YAMADA Hideaki
Published: March 2005

A novel enzyme which specifically catalyzes the reduction of conjugated polyketones was purified to homogeneity from cells of Mucor ambiguus AKU 3006. The enzyme has a strict requirement for NADPH and irreversibly reduces a number of quinones such as p‐benzoquinone, α‐naphthoquinone and acenaphthenequinone. The enzyme also reduces polyketones such as isatin and ketopantoyl lactone, and their derivatives. The apparent Km values for isatin and ketopantoyl lactone are 49.9 μM and 714 μM, respectively. The reduction of ketopantoyl lactone proceeds stereospecifically to yield l‐(+)‐pantoyl lactone. The pro‐S(A) hydrogen at C‐4 of NADPH is transferred to the substrate. The enzyme is not a flavoprotein and consists of two polypeptide chains with an identical relative molecular mass of 27 500. Quercetin, dicoumarol and some SH reagents inhibit the enzyme activity. 3‐Methyl‐1,2‐cyclopentanedione and 1,3‐cyclohexanedione are uncompetitive inhibitors with Ki values of 80.9 μM and 64.5 μM, respectively, to ketopantoyl lactone.
Species differences in the molecular characteristics of vasoactive‐intestinal‐peptide receptors in the pancreas from rat and guinea‐pig
Authors: SVOBODA Michal , POLOCZEK Piotr , WINAND Jacques , ROBBERECHT Patrick , CHRISTOPHE Jean
Published: March 2005

The receptors for vasoactive intestinal peptide (VIP) present in dispersed acini and membranes from rat and guinea‐pig pancreas differed in selectivity pattern, i.e. in the displacement of [125I]iodo‐VIP by parent peptides, as revealed by a VIP:secretin IC50 ratio at least ten times higher in rat than in guinea‐pig preparations. The molecular properties of these VIP receptors were therefore investigated.

When comparing six succinimidyl ester cross‐linkers, bis[2‐(succinimidooxycarbonyloxy)ethyl]sulfone proved to be the most universal [125I]iodo‐VIP cross‐linker for all pancreatic preparations.

In intact rat acini the main labeled peptide had an Mr of 80000, whereas the main labeled peptide in intact guinea‐pig acini was a smear of Mr 160000. In both rat and guinea‐pig pancreatic membranes, the main labeled peptide ([125I]iodo‐VIP–binding‐protein complex) had an Mr of 66000. In addition, variable proportions of an Mr‐80000 peptide and an Mr‐83000 peptide were visualized in, respectively, rat and guinea‐pig membranes. The labeling of all peptides was suppressed by VIP and by GTP. Reducing conditions allowed only a better resolution, making the presence of intermolecular disulfide bridges unlikely.

Taking into account the Mr of VIP it is thus plausible that the main native Mr‐77000 VIP‐binding site present in rat acini could be easily converted to an Mr‐63000 peptide during membrane preparation, while in guinea‐pig acini Mr‐80000 and/or Mr‐63000 VIP‐binding sites were often closely associated with another membrane component in the native state. These molecular differences between VIP receptors in intact rat and guinea‐pig acini are in keeping with functional differences.
13C‐NMR study of glycerol metabolism in rabbit renal cells of proximal convoluted tubules
Authors: JANS Arnold W. H. , WILLEM Rudolph
Published: March 2005

Perchloric acid extracts of rabbit renal proximal convoluted tubular cells (PCT) incubated with [2‐13C]glycerol and [1,3‐13C]glycerol were investigated by 13C‐NMR spectroscopy. These 13C‐NMR spectra enabled us to determine cell metabolic pathways of glycerol in PCT cells. The main percentage of 13C‐label, arising from 13C‐enriched glycerol, was found in glucose, lactate, glutamine and glutamate. So far it can be concluded that glycerol is a suitable substrate for PCT cells and is involved in gluconeogenesis and glycolysis as well in the Krebs cycle intermediates.

Label exchange and label enrichment in 13C‐labelled glucose, arising from [2‐13C]glycerol and [1,3‐13C]glycerol, is explained by label scrambling through the pentose shunt and a label exchange in the triose phosphate pool. From relative enrichments it is estimated that the ratio of the pyruvate kinase flux to the gluconeogenetic flux is 0.97:1 and that the ratio of pyruvate carboxylase activity relative to pyruvate dehydrogenase activity is 2.0:1. Our results show that 13C‐NMR spectroscopy, using 13C‐labelled substrates, is a powerful tool for the examination of renal metabolism.
Regeneration after cardiotoxin injury of innervated and denervated slow and fast muscles of mammals
Authors: d'ALBIS Anne , COUTEAUX René , JANMOT Chantal , ROULET Agnès , MIRA Jean‐Claude
Published: March 2005

The regeneration of adult rat and mouse slow (soleus) and fast (sternomastoid) muscles was examined after the degeneration of myofibers had been achieved by a snake venom cardiotoxin, under experimental conditions devised to spare as far as possible the satellite cells, the nerves, and the blood vessels of the muscles.

Three days after the injury, no myosin was detectable in selected portions of the muscles. New myosins of embryonic, neonatal, and adult types started to be synthesized during the following two days. Adult myosins thus appeared more precociously than in development, which implies that the synthesis of myosin isoforms during regeneration does not entirely ‘recapitulate’ the sequence of myosin transitions observed during normal development.

Two weeks after the injury, the isomyosin electrophoretic pattern displayed by regenerated muscles was already the same as that of control muscles; the normal adult pattern was therefore expressed more rapidly in regenerating than in developing muscles.

Except for the synthesis of the slow isoform which was generally inhibited in denervated muscles, the same types of myosins were expressed during the early stages of regeneration in denervated as in innervated muscles; long‐term denervation prevented however the qualitative and quantitative recovery of the normal myosin pattern.
Monoclonal antibodies reveal evolutionary conservation of alternative splicing of the αA‐crystallin primary transcript
Authors: HENDRIKS Wiljan , SANDERS Jo , de LEIJ Loe , RAMAEKERS Frans , BLOEMENDAL Hans , de JONG Wilfried W.
Published: March 2005

Because of their specificity and sensitivity, monoclonal antibodies are powerful tools in studies of protein structure and function. Therefore, we raised monoclonal antibodies against αA‐crystallin and identified the antigenic determinant for two of these antibodies. Applying limited‐digestion methods, we show that the region spanning residues 158–168 of αA‐crystallin contains the epitope for the two monoclonal antibodies. These monoclonals were then used to study the occurrence in the lenses of different vertebrates of the elongated αAins‐crystallin chain, a product of alternative splicing. It appears that the mutational event resulting in the alternative splicing pattern of the αA‐crystallin gene took place at least 70 million years ago. This alternative splicing phenomenon has been maintained in rodents and some other, unrelated mammals, but disappeared again in most mammalian lineages.
Properties of N‐terminal truncated yeast aspartyl‐tRNA synthetase and structural characteristics of the cleaved domain
Authors: LORBER Bernard , MEJDOUB Hafedh , REINBOLT Joseph , BOULANGER Yves , GIEGÉ Richard
Published: March 2005

Cytoplasmic aspartyl‐tRNA synthetase from Saccharomyces cerevisiae is a dimer made up of identical subunits of Mr, 64000 as shown by biochemical and crystallographic analyses. Previous studies have emphasized the high sensitivity of the amino‐terminal region (residues 1–32) to proteolytic enzymes. This work reports the results of limited tryptic or chymotryptic digestion of the purified enzyme which gives rise to a truncated species that has lost the first 50–64 residues with full retention of both the activity and the dimeric structure. In contrast the larger tryptic fragment is distinguished from the whole enzyme by its weaker retention on heparin‐substituted agarose gels. The cleaved N‐terminal part presents peculiar structural features, such as a high content in lysine residues arranged in a palindromic fashion. The properties of the trypsin‐modified enzyme and of the cleaved amino‐terminal region are discussed in relation to the known structural characteristics of aspartyl‐tRNA synthetase and of other eukaryotic aminoacyl‐tRNA synthetases.
Isolation and characterization of porcine diazepam‐binding inhibitor, a polypeptide not only of cerebral occurrence but also common in intestinal tissues and with effects on regulation of insulin release
Authors: CHEN Zheng‐wang , AGERBERTH Birgitta , GELL Katarina , ANDERSSON Mats , MUTT Viktor , ÖSTENSON Claes‐Göran , EFENDIĆ Suad , BARROS‐SÖDERLING Jane , PERSSON Bengt , JÖRNVALL Hans
Published: March 2005

The polypeptide DBI (diazepam‐binding inhibitor) has been purified from the porcine upper intestine, where it is abundant. Porcine mature DBI is composed of 86 amino acid residues and has a blocked N‐terminus. The primary structure, the first DBI structure determined at the protein level, differs from those indirectly deduced for human and rat DBI at 11 and 17 positions, respectively. In total, the three mammalian DBIs differ at 22 positions but have exactly identical C‐terminal 11‐residue segments, highly charged and ending with C‐terminal isoleucine. The porcine DBI inhibits both the early and the late phase of glucose‐induced insulin release from the isolated perfused rat pancreas. Thus, the results identify by direct analysis the presence of DBI at a non‐cerebral localization (gut), establish a novel structural form (porcine) and demonstrate a novel bioactivity (on insulin release). These aspects are of special interest in relation to the conserved segments, including the one at the C‐terminal end, which may constitute functionally important parts of the polypeptide. It is possible that DBI belongs to a new family of gut polypeptides which inhibit glucose‐mediated insulin release by hormonal and/or neurocrine mechanisms.
Postnatal development of Ca2+‐sequestration by the sarcoplasmic reticulum of fast and slow muscles in normal and dystrophic mice
Authors: LEBERER Ekkehard , HÄRTNER Karl‐Thomas , PETTE Dirk
Published: March 2005

Ca2+‐uptake activities of the sarcoplasmic reticulum (SR) were determined with a Ca2+ ‐sensitive electrode in homogenates from fast‐ and slow‐twitch muscles from both normal and dystrophic mice (C57BL/6J strain) of different ages. Immunochemical quantification of tissue Ca2+‐ATPase content allowed determination of the specific Ca2+ ‐transport activity of the enzyme. In 3‐week‐old mice of the dystrophic strain specific Ca2+ transport was already significantly lower than in the normal strain. It progressively decreased with maturation and reached only 40–50% and 30–50% of the normal values in fast‐ and slow‐twitch muscles of adult dystrophic animals, respectively. Tissue contents of calsequestrin were reduced in both types of muscle leading to an increased Ca2+‐ATPase to calsequestrin protein ratio. Equal amounts of the Ca2+ ‐ATPase protein (detected by Coomassie blue staining of polyacrylamide gels) were present in SR vesicles isolated by Ca2+ ‐oxalate loading from adult normal and dystrophic fast‐twitch muscles. However, the specific ATP‐hydrolysing activity of the enzyme was approximately 50% lower in dystrophic than in normal SR. The reduced ATP‐hydrolysing activity was correlated with decreased Ca2+ ‐transport activity, phosphoprotein formation and fluorescein isothiocyanate labeling as determined in total microsomal and heavy SR fractions. Although the Ca2+ and ATP affinities of the enzyme were unaltered, its ATPase activity was reduced at all levels of ATP in the dystrophic SR. Taken together, these findings point to a markedly impaired function of the SR and an increase in the population of inactive SR Ca2+ ‐ATPase molecules in murine muscular dystrophy.
Purification and properties of a specific primase‐stimulating factor of bovine thymus
Authors: ITAYA Asako , HIRONAKA Takashi , TANAKA Yasuharu , YOSHIHARA Koichiro , KAMIYA Tomoya
Published: March 2005

The DNA replicase activity of the complex between bovine thymus DNA polymerase α and RNA primase was markedly decreased after the purification by ssDNA‐cellulose column chromatography. In an attempt to restore the activity by supplementing some fractions eliminated from the purified enzyme, we found that a fraction eluted from the column by increasing salt concentration and 30% ammonium sulfate precipitates of the phosphocellulose‐step enzyme possessed a high ability to restore the replicase activity. Thus, the factors were purified to near homogeneity from the two sources and the properties were examined. Both factors were heat‐labile and trypsin‐sensitive, possessed a native molecular mass of approximately 150–200 kDa as judged by Sephacryl S‐200 column chromatography, and were composed of two polypeptides of 146 kDa and 47 kDa on SDS/polyacrylamide gel electrophoresis, indicating that they were an identical protein. The factor, which did not show any DNA polymerase or primase activities by itself, stimulated approximately 20‐fold the replicase activity of purified DNA‐polymerase‐α–primase at a very low concentration (10 ng/50 μl). The factor did not affect the deoxyribonucleotide polymerizing activity of the enzyme complex at all, but specifically stimulated the primase activity only. Thus, we designated the factor as primase‐stimulating factor. Although varying the template concentration did not significantly affect the mode of stimulation, increasing the concentration of substrate for primer synthesis (ATP) markedly decreased the extent of stimulation. Thus, the stimulating factor seems to decrease the substrate concentration required for the primase reaction as well as increasing threefold the maximum activity attained by varying the substrate concentration. So far, no ATPase activity has been detected in the factor.
NADH oxidase from the extreme thermophile Thermus aquaticus YT‐1
Authors: COCCO Dina , RINALDI Augusto , SAVINI Isabella , COOPER Jonathan M. , BANNISTER Joe V.
Published: March 2005

A protein with NADH oxidase activity from the extreme thermophile Thermus aquaticus YT‐1 was purified and characterised. The enzyme was found to have a relative molecular mass of 110000 and be composed of two subunits of identical size. FAD was found to be present at a concentration of 0.7 mol/mol dimer and was required for activity. During the oxidation of NADH, oxygen uptake takes place with the production of hydrogen peroxide. The enzyme had, with the exception of a higher glutamic acid and tryptophan content, a similar amino acid composition as the NADH oxidase isolated from the mesophile Bacillus megaterium. Purified NADH oxidase was found to have a Km, of 39 μM for β‐NADH and a VmaX of 4.68 μmol NADH mg‐−1 min−1 and was still active at 95°C. Enzymatic activity was found to be independent of pH between 5.0 and 10.5.
Electron transfer between the hydrogenase from Desulfovibrio vulgaris (Hildenborough) and viologens
Authors: HOOGVLIET Johan C. , LIEVENSE Lou C. , van DIJK Cees , VEEGER Cees
Published: March 2005

The electron transfer kinetics between the hydrogenase from Desulfovibrio vulgaris (strain Hildenborough) and the mediators methyl viologen, di‐(n‐aminopropyl) viologen and propyl viologen sulfonate have been investigated by chronoamperometry. Second‐order rate constants were calculated on basis of the theory for a simple catalytic mechanism and are compared with the results obtained before by cyclic voltammetry (preceding paper in this journal). From the ionic‐strength dependence and the observed differences in the rate constants for the differently charged viologens, the existence of an electrostatic interaction between mediator and a negatively charged part of the protein is confirmed. Chronoamperometry (computer‐controlled) was found to possess advantages over cyclic voltammetry in the determination of homogeneous rate constants (faster, more accurate, and better reproducibility).
The protection by ascorbate and glutathione against microsomal lipid peroxidation is dependent on vitamin E
Authors: WEFERS Heribert , SIES Helmut
Published: March 2005

Lipid peroxidation of rat liver microsomal fractions was monitored by its low‐level chemilunminescence in preparations from controls and vitamin‐E‐deficient animals. Measurements were made (a) of the duration of the lag phase τ0 after initiation with NADPH/iron‐ADP and (b) of the slope of the chemiluminescence increase.

In microsomes with normal vitamin E (α‐tocopherol) level the lag phase τ0 was substantially increased by ascorbate; in contrast, even an enhanced peroxidation was observed with ascorbate in vitamin‐E‐deficient microsomes. Therefore, the ascorbate‐mediated protection of microsomal membranes against lipid peroxidation is dependent on vitamin E in the membrane. In vitamin E deficiency the pro‐oxidant effect of ascorbate was abolished when glutathione (GSH) was present.

Likewise, GSH does not prolong the lag phase τ0 in vitamin E deficiency. However, GSH (but not cysteine) exerts an antioxidant effect both in controls and in vitamin E deficiency by decreasing the slope of the chemiluminescence increase during lipid peroxidation. The involvement of GSH in an enzyme‐dependent mechanism is suggested.
The bovine cardiac receptor for calcium channel blockers is a 195‐kDa protein
Authors: SCHNEIDER Toni , HOFMANN Franz
Published: March 2005

The cardiac receptor for calcium channel blockers was purified from bovine microsomal membranes which contained 235 ± 33 fmol nimodipine‐binding sites/mg protein (mean ± SEM of nine preparations). To identify the receptor during the purification 20% of its binding sites were prelabeled with (+)[3H]PN200‐110. The receptor was solubilized with 0.6% digitonin and was purified to a specific density of 157 pmol/mg using a combination of ion‐exchange, wheat‐germ‐agglutinin – Sepharose chromatography and sucrose density gradient centrifugation. In the last sucrose gradient bound (+)[3H]PN200‐110 comigrated with a 195‐kDa protein. (±)[3H]Azidopine and [3H]ludopamil, the photoaffinity ligands for the dihydropyridine and phenylalkylamine‐binding site of the calcium channel, were incorporated specifically into the 195‐kDa protein. These data indicate that the bovine cardiac receptor for calcium channel blockers is a 195‐kDa protein. Its molecular mass suggests that the bovine cardiac receptor differs considerably from the rabbit skeletal muscle receptor protein for calcium channel blockers.
Structural organization of satellite I chromatin of calf liver
Authors: PAGÈS Michel , ROIZÈS Gérard
Published: March 2005

The organization of liver calf satellite DNA I chromatin has been studied. Precisely seven nucleosomes per DNA satellite repeat (1.4 kb) are shown to be present. Several strict positionings of the nucleosomes along the satellite DNA repeat have been described. One of these positionings, the major phase, was shown to be preferentially organized in clustered repeats. Most of the non‐methylated CpG restriction sites were shown to be located in the linker DNAs of the satellite I nucleosomes, whilst, accordingly, methylated HpaII sites were found in the core satellite I DNA. The significance of these results is discussed.
Induction and repression of cellular gene transcription during Herpes simplex virus infection are mediated by different viral immediate‐early gene products
Authors: KEMP Lynn M. , LATCHMAN David S.
Published: March 2005

Nuclear run‐off and pulse‐labelling techniques have been used to study the changes in transcription rates of a number of cellular genes during infection with Herpes simplex virus. The majority of these genes show a decrease in transcription rate to about 60% of that observed prior to infection. In contrast, a small number of genes are transcriptionally activated during infection. These effects, which occur at a point in infection after the synthesis of viral proteins but prior to the onset of viral DNA synthesis, are mediated by different immediate‐early proteins of the virus. Thus we show that, whilst transcriptional activation requires a functional ICP4 protein, repression is dependent upon the presence of another immediate early protein‐ICP22.
Expression of a DNA‐ligase‐stimulatory factor in Bloom's syndrome cell line GM1492
Authors: KENNE Kerstin , LJUNGQUIST Siv
Published: March 2005

An increased DNA ligase activity is observed in extracts of Bloom's syndrome (BS) fibroblast cell line GM1492. The activity is 2–3‐fold higher in this cell line compared to normal human fibroblasts, and 5–20‐fold higher than in three other BS cell lines investigated. The DNA ligase activity in GM1492 cells is promoted by a heat‐resistant, protease‐sensitive factor comigrating with DNA ligases on single‐stranded‐DNA–cellulose. The factor stimulates DNA ligase I as well as DNA ligase II, and is not identical to the activity‐promoting homologous DNA pairing, which is also enhanced in GM1492 cell extracts.
Selective loss of binding sites for the iodinated α‐neurotoxin I from Naja mossambica mossambica venom upon enzymatic deglycosylation of Torpedo electric organ membranes
Authors: ZEGHLOUL Saâdia , MARCHOT Pascale , BOUGIS Pierre E. , RONIN Catherine
Published: March 2005

Removal of asparagine‐linked carbohydrate chains from Torpedo marmorata electric organ membranes was found to inhibit the binding of the iodinated α‐neurotoxin I from Naja mossambica mossambica snake venom to its receptor. Optimal deglycosylation of membranes by endoglycosidase F resulted in a 55% inhibition of α‐neurotoxin‐I‐saturable binding. Under these conditions, up to 70% of concanavalin A binding was also lost, indicating an efficient removal of mannose‐rich carbohydrate chains. Saturation binding experiments at equilibrium on membranes incubated in the absence of endoglycosidase F indicated, when analyzed by Scatchard plots, the presence of two classes of high‐affinity binding sites for α‐neurotoxin I (Kd= 9 pM and 68 pM respectively) with capacities of 24 and 14 pmol/mg membrane proteins, respectively. After endoglycosidase F treatment, only the former class of binding sites (Kd= 11 pM) was recovered together with a 45% reduction in the number of total binding sites. Dissociation experiments further confirmed the presence of two types of toxin‐receptor complexes in control membranes and the selective loss of the rapidly dissociating component upon deglycosylation. The binding of α‐neurotoxin I to its receptor, deglycosylated or not, was totally inhibited by carbamoylcholine, d‐tubocurarine or α‐bungarotoxin. These findings show that the neurotoxin binding sites present on the acetylcholine receptor can be discriminated on the basis of their differential susceptibility to the removal of asparagine‐linked carbohydrate chains.
Amino acid sequence of a calcium‐binding protein (TCBP‐10) from Tetrahymena
Authors: KOBAYASHI Tomoyoshi , TAKAGI Takashi , KONISHI Kazuhiko , OHNISHI Kazuo , WATANABE Yoshio
Published: March 2005

The amino acid sequence of a calcium‐binding protein obtained from the cilium and cell body of Tetrahymena, designated as TCBP‐10 (Tetrahymena calcium‐binding protein; molecular mass = 10 kDa [Ohnishi, K. and Watanabe, Y. (1983) J. Biol. Chem. 258, 13978–13985] was determined. It is composed of 102 amino acid residues. The exact molecular mass is calculated to be 11563 Da. From the amino acid sequence analysis, it has two EF‐hand‐type calcium‐binding sites.
The dihydrolipoyltransacetylase component of the pyruvate dehydrogenase complex from Azotobacter vinelandii
Authors: HANEMAAIJER Roeland , JANSSEN Anja , de KOK Arie , VEEGER Cees
Published: March 2005

The gene encoding the dihydrolipoyltransacetylase component (E2) of the pyruvate dehydrogenase complex from Azotobacter vinelandii has been cloned in Escherichia coli. A plasmid containing a 2.8‐kbp insert of A. vinelandii chromosomal DNA was obtained and its nucleotide sequence determined.

The gene comprises 1911 base pairs, 637 codons excluding the initiation codon GUG and stop codon UGA. It is preceded by the gene encoding the pyruvate dehydrogenase component (E1) of pyruvate dehydrogenase complex and by an intercistronic region of 11 base pairs containing a good ribosome binding site. The gene is followed downstream by a strong terminating sequence. The relative molecular mass (64913), amino acid composition and N‐terminal sequence are in good agreement with information obtained from studies on the purified enzyme. Approximately the first half of the gene codes for the lipoyl domain. Three very homologous sequences are present, which are translated in three almost identical units, alternated with non‐homologous regions which are very rich in alanyl and prolyl residues. The N‐terminus of the catalytic domain is sited at residue 381. Between the lipoyl domain and the catalytic domain, a region of about 50 residues is found containing many charged amino acid residues. This region is characterized as a hinge region and is involved in the binding of the pyruvate dehydrogenase and lipoamide dehydrogenase components. The homology with the dihydrolipoyltransacetylase from E. coli is high: 50% amino acid residues are identical.
cAMP‐dependent protein phosphorylation of membrane proteins in the parotid gland, platelets and liver
Authors: THIEL Gerald , SÖLING Hans‐Dieter
Published: March 2005

Stimulation of secretion in exocrine secretory glands leads to the phosphorylation of a 22‐kDa membrane protein (protein III) whose function is still unknown [Jahn et al. (1980) Eur. J. Biochem. 112, 345–352; Jahn & Söling (1980) Proc. Natl Acad. Sci. USA 78, 6903–6906]. This report describes the comparison of this protein with phosphorylated membrane proteins of similar molecular mass in platelets and liver. Incubation of platelets with agents which raise the intracellular cAMP concentration results in the phosphorylation of a 22‐kDa protein which is also phosphorylated in membrane preparations by endogenous kinases or by exogenous cAMP‐dependent protein kinase. It is shown that this protein is distinct from protein III although both proteins have the same molecular mass and are substrates of cAMP‐dependent protein kinase. In contrast to platelets, protein III could be demonstrated in liver microsomes. This indicates that the function of protein III is not exclusively linked to the stimulus‐secretion coupling in exocrine cells.
Conformational alterations detected by circular dichroism induced in the normal ras p21 protein by activating point mutations at position 12, 59, or 61
Authors: VALENCIA Alfonso , SERRANO Luis , CABALLERO Rafael , ANDERSON Paul S. , LACAL Juan Carlos
Published: March 2005

Activation of the oncogenic potential of ras oncogenes occurs by point mutations at codons 12, 13, 59, 61, and 63 of the sequences that codify for its product, a 21‐kDa protein designated as p21. This activation has been postulated by computer models as modifiers of the structure of the protein, which may alter its biochemical and biological activities. We have expressed in bacteria the normal ras p21 and five mutated p21 proteins with mutations at positions 12, 59, 61, 12 plus 59, and 12 plus 61. Purification was carried out by solubilization from bacterial pellets in 7 M urea and chromatography through a Sephadex G‐100 column to obtain >95% purified proteins. Circular dichroic (CD) spectra showed that the normal protein and that activated by substitution of Ala59 to Thr59 are very similar in their overall structure. By contrast, point mutations affecting either 12 or 61 residues substantially altered the structure of the proteins. When the parameters of Chen et al. [Biochemistry 11, 4120–4131 (1972)] were applied to the CD spectra, both normal and thr59‐mutated ras proteins showed a less organized structure than mutated proteins at position 12 or 61. Since the Thr59 mutant has a more similar transforming activity than other activated proteins, but a GTPase activity similar to that of the normal protein, our results support the hypothesis that there is more than one mechanism of activation of the ras p21 protein. One of these mechanisms involves important structural alterations by point mutations at position 12 or 61 which reduce the GTPase activity of the protein. Another mechanism will be that induced by a substitution of Ala59 to Thr59 which does not substantially alter the protein conformation. A putative alternative mechanism for the activation of this mutant is discussed.
Studies of a novel repetitive sequence family in the genome of mice
Authors: DECOVILLE Martine , MALFOY Bernard , LOCKER Daniel
Published: March 2005

A new middle repetitive sequence is described in the mouse genome. It has been revealed with a recombinant clone isolated from a Mus musculus BamHI gene library constructed in pBR322 and containing an insertion of 1.73 kb. When digests of genomic DNA were subjected to Southern blot hybridization, using the 1.73‐kb insert as probe, we obtained a light smear and discrete bands, indicating a dispersion in the mouse genome of this sequence. This 1.73‐kb sequence seems to be a part of a greater repetitive sequence at least 6 kb in length.

The sizes of the bands hybridizing with the 1.73‐kb insert are similar when compared between different laboratory strains but differ remarkably between the two species M. musculus and Mus caroli. We have shown also a great variation in the copy number of the sequence studied between these two species. When rat DNA is probed with the 1.73‐kb insert, no hybridization is observed.

Subcloning of the 1.73‐kb sequence in three fragments has pointed out that the reiteration was not homogeneous along the 1.73‐kb sequence.

The 1.73‐kb clone was sequenced and compared with other interspersed repetitive sequences, previously described in the rodent genome, and no homology was found.
Role of the N‐terminus of rat pheochromocytoma tyrosine hydroxylase in the regulation of the enzyme's activity
Authors: BONNEFOY Eliette , FERRARA Pascual , ROHRER Hermann , GROS François , THIBAULT Jean
Published: March 2005

Activation of rat pheochromocytoma tyrosine hydroxylase by limited tryptic proteolysis was investigated. The modifications produced upon the enzyme's structure were analyzed with the use of sodium dodecyl sulfate/polyacrylamide gel electrophoresis and tyrosine hydroxylase activity was measured all through the digestion. During the proteolysis the activity of tyrosine hydroxylase was elevated threefold at the same time as a 56‐kDa tryptic fragment was formed. When the enzyme was phosphorylated, at its N‐terminal region, by a kinase copurified with tyrosine hydroxylase, the major 56‐kDa species did not appear to be phosphorylated on the autoradiograph, suggesting that it was derived from the native subunit by cleavage of the N‐terminal of the protein. The reactivity of the 2/40/15 anti‐(tyrosine hydroxylase) monoclonal antibody with the N‐terminal of tyrosine hydroxylase was also investigated, using the Western‐blot technique. This antibody reacted with the 62‐kDa hydroxylase subunit but not with the 60‐kDa tryptic fragment; the amino acid sequences of these two species showed that the 60‐kDa fragment lacked the first 16 N‐terminal amino acids of the native molecule. These results suggest that the N‐terminal region of tyrosine hydroxylase is apparently responsible for an inhibition of the hydroxylase activity and that the first N‐terminal amino acids of the hydroxylase are necessary for the recognition of the enzyme by its antibody.