bluuebunny/crossref_metadata_2025_split huggingface dataset:
Regulation of the expression of the phosphoenolpyruvate carboxykinase gene in cultured rat hepatocytes by glucagon and insulin
Authors: CHRIST Bruno , NATH Annegret , BASTIAN Helge , JUNGERMANN Kurt 
Published: March 2005

The induction of phosphoenolpyruvate carboxykinase (PEPCK) by glucagon was studied in primary rat hepatocyte cultures by determining the time course of the sequential events, increases in the enzyme's mRNA abundance, synthesis rate, amount and activity, and by investigating the antagonistic action of insulin on the induction by glucagon.

The mRNA of PEPCK was induced maximally 2–3 h after addition of 10 nM glucagon, as detected by Northern‐blot analysis after hybridization with a biotinylated antisense RNA of PEPCK.
The synthesis rate of PEPCK increased maximally 2–3 h after application of glucagon as revealed by pansorbin‐linked immunoprecipitation of [35S]methionine‐labelled PEPCK.
The enzyme amount and activity was maximally induced 4 h after glucagon application.
The mRNA of PEPCK was half‐maximally induced by 0.1 nM and maximally by 1 nM and 10 nM glucagon. The half‐maximal induction by 0.1 nM glucagon was antagonized almost totally, and the maximal induction by 1 nM glucagon partially, while the maximal induction by 10 nM glucagon remained unaffected by 10 nM insulin.

The results show that in cultured rat hepatocytes physiological concentrations of glucagon stimulated the induction of PEPCK by an increase in mRNA, that the glucagon‐dependent increase in mRNA and enzyme‐synthesis rate occurred in parallel and preceded the increase of enzyme amount and activity by 1–1.5 h, and that physiological levels of insulin antagonized the induction by glucagon in the physiological concentration range with glucagon being the dominant hormone.
Fidelity of aminoacylation by rat‐liver tyrosyl‐tRNA synthetase
Authors: TAKAHASHI Ryoya , GOTO Sataro 
Published: March 2005

The possibility of change in the rate of misrecognition of amino acids by rat liver tyrosyl‐tRNA synthetase during aging was investigated. Frequency of misrecognition of phenylalanine vs tyrosine was determined at two temperatures by competitive assay using partially purified enzymes. At 25°C, the error frequencies were 5.17 × 10−8 and 8.24 × 10−8 in young and old animals, respectively. These values are much below the reported error frequencies for the prokaryotic enzymes: i.e. ∼ 5 × 10−6. Although the fidelity of tyrosyl‐tRNA synthetase from old animals appeared to be slightly lower, the difference was not statistically significant. At 37°C, the error frequencies were increased 1.3–1.5‐fold, but again the difference between young and old animals was not significant.To our knowledge, this is the first report in which fidelity of aminoacyl‐tRNA synthetase of animals of various ages has been compared using natural amino acids.
Panulirus interruptus hemocyanin
Authors: JEKEL Peter A. , BAK Henk J. , SOETER Nell M. , VEREIJKEN Johan M. , BEINTEMA Jaap J. 
Published: March 2005

The primary structure of subunit b of Panulirus interruptus hemocyanin has been derived from two digests (trypsin and CNBr) and, in some cases, with aid from the similarity with the sequence of subunit a. Differences between the amidation states of Asx and Glx residues in subunit b relative to a were investigated more thoroughly. When compared to the sequence of subunit a, 18 differences (2.7%) were found and certain heterogeneities, indicating the presence of a minor subunit b′, were observed.Several differences in properties between subunits a and b, including their anomalous behaviour on SDS/ polyacrylamide gel electrophoresis, could be explained by amino acid replacements.
Erythrocyte adducin
Authors: WASEEM Ahmad , PALFREY H. Clive 
Published: March 2005

Two major substrates for human erythrocyte protein kinase C (PK‐C) of Mr 120 000 and 110 000, previously named PKC‐1 and PKC‐2 [Palfrey, H. C. & Waseem, A. (1985) J. Biol. Chem. 260, 16021–16029] have been found to be identical to CaM‐BP103/97 or ‘adducin’, recently described by K. Gardner and V. Bennett [(1986) J. Biol. Chem. 261, 1339–1348; (1987) Nature (Lond.) 328, 359–362]. These proteins have been purified from the membrane skeleton by high‐salt extraction, ion‐exchange and gel filtration chromatography. The two proteins co‐fractionate in a ratio of ∼ 1:1 under a number of conditions suggesting that they exist as a complex. Physicochemical data indicate that the native adducin complex is probably an asymmetric heterodimer of α and β subunits. Adducin binds to a calmodulin (CaM) affinity matrix in a Ca2+‐dependent manner and is specifically eluted with EGTA. Fingerprinting of the iodinated peptides derived from the α and β subunits using three different proteases yields 16–37% overlapping peptides, indicating limited similarity between the two polypeptides. Affinity‐purified polyclonal antibodies against each protein show little or no cross‐reactivity with the other, indicating that the β subunit is not derived from the α subunit or vice versa. Proteins reactive with both anti‐(α‐adducin) and anti‐(β‐adducin) antibodies are found in erythrocytes from rat, rabbit, pig, ferret and duck. Immunoblots of adducin after non‐ionic detergent extraction of ghosts reveal that a significant fraction of the protein may associate with non‐skeleton membrane components.The phosphorylation of adducin is stimulated by both phorbol esters and cAMP analogues in intact erythrocytes. Fingerprinting suggests that protein kinase C preferentially phosphorylates four distinct sites on the two proteins. Phosphopeptide maps of α‐adducin are virtually identical to those of β‐adducin after phorbol ester stimulation of intact cells, or after PK‐C‐catalyzed phosphorylation of the purified protein, indicating strong local similarities in the two proteins. Such maps also suggest that cAMP‐dependent protein kinase (cAMP‐PK) modifies adducin at some similar and some distinct sites as those modified by PK‐C. In vitro phosphorylation of isolated adducin by purified PK‐C results in rapid incorporation of phosphate to a final level of ∼ 1.5 mol/mol in both α and β subunits. Phosphorylation by the catalytic subunit of cAMP‐PK is equally rapid, with α‐ adducin being preferentially phosphorylated to a level of ∼ 1.8 mol/mol. Increased phosphorylation enhances the extraction of adducin from the membrane by non‐ionic detergent.The multivalent properties of this protein complex suggest that it may play a role in mediating the interactions of components of the membrane skeleton with the inner surface of the membrane and that CaM and/or phosphorylation may modulate this function.
Structural studies on the neutral polysaccharide of Bacillus subtilis AHU 1219 cell walls
Authors: IWASAKI Hiroyoshi , ARAKI Yoshio , KAYA Shunji , ITO Eiji 
Published: March 2005

A neutral and an acidic polysaccharide with molecular masses of about 22 kDa and 45 kDa, respectively, were isolated from the N‐acetylated cell walls of Bacillus subtilis AHU 1219 by heating at pH 2.5, followed by separation of the water‐soluble product by ion‐exchange chromatography and gel chromatography. The neutral polysaccharide, accounting for 40% of the mass of the cell walls, contained glucose, N‐acetylglucosamine, N‐acetylgalactosamine and N‐acetylmannosamine in a molar ratio of 1:2:1:1. The minor, acidic polysaccharide contained glucuronic acid, glucose, galactose, L‐serine and L‐threonine in an approximate molar ratio of 1:1:1:0.5:0.5. Lysozyme digestion of the N‐acetylated cell walls gave a polymer containing the neutral polysaccharide and glycopeptide components and another polymer which contained the acidic polysaccharide components together with small proportions of the neutral polysaccharide and glycopeptide components. Thus, the neutral and acidic polysaccharide chains seem to be attached to peptidoglycan through acid‐labile linkages in the cell walls of this strain.Structural analysis of the neutral‐polysaccharide preparation, involving 1H‐NMR and 13C‐NMR measurement. methylation and Smith degradation, led to the most likely structure, →6)[Glc(β1 → 3)]GalNAc(α1 → 4)‐[GlcNAc(β1 → 3)]ManNAc(β1 → 4)GlcNAc(β1 →, for the repeating units of this polysaccharide.
Regulation of gene expression at the translational level
Authors: GANOZA M. Clelia , KNAUBER Donna C. , MURPHY Brian , FALLAVOLITA Dino , CHUNG Dae‐Gyun 
Published: March 2005

Extracts of the conditionally‐lethal mutant Escherichia coli N4316 are defective in a newly described translation factor, the rescue protein. We have analyzed the in vitro translation products of this mutant by gel electrophoresis during normal and arrested synthesis at the permissive and non‐permissive temperatures. Translation programmed with MS2 bacteriophage RNA at the non‐permissive temperature results in highly reduced synthesis of the coat protein with no detectable levels of the maturation and replicase products. Thus the relative number of copies of proteins synthesized by the ribosomes is altered in this mutant. In addition, there is mistranslation of the coat gene which results in the overproduction of the phage encoded no. 7 protein. Aberrant synthesis is also reflected in the increased read‐through of termination codons during synthesis directed by phage RNAs harbouring amber mutations in the coat cistron. The rescue protein, purified from the parental strain, is able to complement the thermosensitive defect and restore proper synthesis.Biochemical characterization of the defect in the absence of rescue shows no detectable deficiency in the extent of initiation complex formation in reactions inhibited with sparsomycin. Peptidyltransferase is fully active as judged by the kinetics of formylmethionine‐puromycin formation. However, rescue does exert an effect at the level of termination. In addition, the thermolability of the mutant can be reversed by dissociating 70S ribosomes into 30S and 50S subunits. Based on these and other observations, we propose tht rescue mediates a novel function in the association/dissociation of ribosomal subunits which is essential to the accuracy and efficiency of translation.
Sequence analysis and expression of the arginine‐deiminase and carbamate‐kinase genes of Pseudomonas aeruginosa
Authors: BAUR Heinz , LUETHI Ernst , STALON Victor , MERCENIER Annick , HAAS Dieter 
Published: March 2005

The arcABC operon of Pseudomonas aeruginosa encodes arginine deiminase, catabolic ornithine carbamolytransferase and carbamate kinase, respectively. We have determined the nucleotide sequences of the arcA and arcC genes. The arcA open reading frame specifies a polypeptide of 46.3 kDa. The same molecular mass was obtained for the subunit of purified arginine deiminase after electrophoresis under denaturing conditions. The N‐terminal amino acid sequence of arginine deiminase was in agreement with the corresponding nucleotide sequence. The native arginine deiminase had an estimated molecular mass of 175–180 kDa, suggesting a tetrametric structure. The enzyme was activated by Mg2+ or Mn2+ and strongly inhibited by Zn2+. The apparent Km for L‐arginine was 0.04 mM in the presence of Mg2+ and 0.47 mM without Mg2+. The arcC open reading frame codes for a 33‐kDa protein, confirming the molecular mass previously reported for the subunit of carbamate kinase. The translation‐initiation site of arcC was determined by deletion mapping. Two regions of dyad symmetry found between arcA and arcC might stabilize the putative arcABC transcript in the upstream (arcA) region; this might contribute to the high level of arcA expression as compared to the moderate level of arcC expression.Carbamate kinase had 37% sequence similarity (and 13.5% identity) with the C‐terminal part of carbamoylphosphate synthetase (large subunit) from Escherichia coli. Arginine deiminase had no apparent similarity with argininosuccinate lyase. Thus, the arcA and arcC genes do not appear to be closely related to arginine biosynthetic genes, whereas it had previously been shown that the arcB gene has a high degree of identity with the arginine biosynthetic argF genes of P. aeruginosa and E. coli.
Molecular cloning of rat human type IX collagen cDNA and localization of the α1(IX) gene on the human chromosome 6
Authors: KIMURA Tomoastsu , MATTEI Marie‐Geneviève , STEVENS Jeff W. , GOLDRING Mary B. , NINOMIYA Yoshifumi , OLSEN Bjorn R. 
Published: March 2005

Type IX collagen is found in hyaline cartilage, where it is asociated with type II collagen lin quarter‐stagered collagen firils. Chicken type IX collagen has been extensively characterized and shown to contain molecules with three tripole‐helical domains, interspersed with non‐triple‐helical sequences. The molecule contains three, genetically disstinct, subunits and one of these subunits carries a covalently bound glycosaminoglycan side chain.In the present report, we describe for the first time the primary structure of mammalian type IX collagen chains, based on cloning and sequencing of cDNA from rat and human cDNA libraries. The results suggest that mammalian α1(IX) chains have the same multi‐domain structure as the avian protein. We also demonstrate, by in situ hybridization of chromosome spreads, that the human α1(IX) collagen gene is located on the long arm of chromosome 6. The cloning of human type IX collagon cDNA provides a probe for molecular studies of human chondrodysplasias that may involve abnormalities in this extracellular collagen‐proteoglycan.
Chloroplast ATP synthase contains one single copy of subunit δ that is indispensable for photophosphorylation
Authors: ENGELBRECHT Siegfried , SCHÜRMANN Karin , JUNGE Wolfgang 
Published: March 2005

F0F1 ATP synthases synthesize ATP in their F1 portion at the expense of free energy supplied by proton flow which enters the enzyme through their channel portion F0. The smaller subunits of F1, especially subunit δ, may act as energy transducers between these rather distant functional units. We have previously shown that chloroplast δ, when added to thylakoids partially depleted of the coupling factor CF1, can reconstitute photophosphorylation by inhibiting proton leakage through exposed coupling factor CF0. In view of controversies in the lieterature, we reinvestigated two further aspects related to subunit δ, namely (a) its stoichiometry in CF0CF1 and (b) whether or not δ is required for photophosphorylation.By rocket immunoelectrophoresis of thylakoid membrances and calibration against purified δ, we confirmed a stoichiometry of one δ per CF0CF1. In CF1‐depleted thylakoids photophosphorylation could be reconstituted not only by adding CF1 and subunit δ but, surprisingly, also by CF1(−δ). We found that the latter was attributable to a contamination of CF1(−δ) preparations with integral CF1. To Lesser extent CF1(−δ) acted by complementary rebinding to CF0 channels that were closed because they contained δ [CF0(+δ)]. This added catalytic capacity to proton‐tight thylakoid vesicles.The ability of subunit δ to control proton flow through CF0 and the absolute requirement for δ in restoration of photophosphorylation suggest an essential role of this small subunit at the interface between the large portions of ATP synthase: δ may be part of the couping site between electrochemical, conformational and chemical events in this enzyme.
Specific S‐thiolation of a 30‐kDa cytosolic protein from rat liver under oxidative stress
Authors: ROKUTAN Kazuhito , THOMAS James A. , SIES Helmut 
Published: March 2005

Thin‐gel isoelectric focusing (IEF) is a simple and sensitive method of quantifying S‐thiolation of individual proteins (protein mixed‐disulfide formation). IEF of rat liver cytosol identified one major protein (pI 7.0) which underwent S‐thiolation with glutathione disulfide to produce two acidic bands with pIs 6.4 and 6.1. The S‐thiolated forms of the protein were purified by preparative isoelectric focusing. An apparent molecular mass of 30 kDa was determined by SDS/polyacrylamide gel electrophoresis. The 30‐kDa protein amounted to 7 ± 2% of the total cytosolic protein on IEF.The most abundant soluble protein of freshly isolated hepatocytes, with an identical isoelectric point to the liver 30‐kDa protein, was modified in a similar manner in response to oxidative stress induced by model compounds. Addition of 50 μM tert‐butyl hydroperoxide, 50 μM diamide [1,1‐azobis(N,N′‐dimethylformamide)] or 20 μM menadione (2‐methyl‐1,4‐naphthoquinone) initiated the S‐thiolation within less than 2 min in the hepatocytes. These compounds, at the concentrations employed, did not result in cell death. Menadione produced slowly progressive S‐thiolation of the protein, while tert‐butyl hydroperoxide or diamide produced rapid S‐thiolation that decreased quickly after 2 min.
The quaternary structure of the dihydrolipoyl transacetylase component of the pyruvate dehydrogenase complex from Azotobacter vinelandii
Authors: HANEMAAIJER Roeland , WESTPHAL Adrie H. , VAN DER HEIDEN Tini , DE KOK Arie , VEEGER Cees 
Published: March 2005

After limited proteolysis of the dihydrolipoyl transacetylase component (E2) of Azotobacter vinelandii pyruvate dehydrogenase complex (PDC), a C‐terminal domain was obtained which retained the transacetylase active site and the quaternary structure of E2 but had lost the lipoyl‐containing N‐terminal part of the chain and the binding sites for the peripheral components, pyruvate dehydrogenase and lipoamide dehydrogenase. The C‐terminus of this domain was determined by treatment with carboxypeptidase Y and shown to be identical with the C‐terminus of E2. Together with the previously determined N‐terminus and the known amino acid sequence of E2, a molecular mass of 27.5 kDa was calculated. From the molecular mass of the native catalytic domain, 530 kDa, and the symmetry of the cubic structures observed on electron micrographs, a 24‐meric structure is concluded instead of the 32‐meric structure proposed previously.From the effect of guanidine hydrochloride on the light‐scattering of intact E2 it was concluded that dissociation occurs in a two‐step reaction resulting in particles with an average mass 1/6 that of the original mass before the N → D transition takes place. Cross‐linking experiments with the catalytic domain indicated that the multimeric E2 is built from tetramers and that the tetramers are arranged as a dimer of dimers.A model for the quaternary structure of E2 is given, in which it is assumed that the tetrameric E2 core of PDC is formed from each of the six morphological subunits located at the lateral face of the cube. Binding of peripheral components to a site that interferes with the cubic assembly causes dissociation, resulting in the unique small PDC of A. vinelandii.
Preparation and kinetic properties of 5‐ethylphenazine – poly(ethylene glycol) – NAD+ conjugate, a unique catalyst having an intramolecular reaction step
Authors: YOMO Tetsuya , SAWAI Haruyo , URABE Itaru , OKADA Hirosuke 
Published: March 2005

5‐Ethylphenazine ‐ poly(ethylene glycol) ‐ NAD+ conjugate (EP+‐PEG‐NAD+) was prepared by linking 1‐(3‐carboxypropyloxy)‐5‐ethylphenazine (I) to poly(ethylene glycol)‐bound NAD+ (PEG‐NAD+) and its kinetic properties were studied. As a reference compound, poly(ethylene glycol)‐bound 5‐ethylphenazine derivative (III) was also prepared and the effects of poly(ethylene glycol) on the reaction rate of the 5‐ethylphenazine moiety with NADH was investigated. The second‐order rate constant, k1, of the reaction of III with NADH is 2.78 mM−1 s−1 and is about 1.7 times that of 1‐(3‐ethoxycarbonylpropyloxy)‐5‐ethylphenazine (II) with NADH. A similar effect of the attached poly(ethylene glycol) was observed for the reaction of PEG‐NADH with I or II. The second‐order rate constants, k2 and k3, of the reactions of the reduced form of III with oxygen and with 3‐(4′,5′‐dimethylthiazole‐2‐yl)‐2,5‐diphenyltetrazolium ion, respectively, were k2= 1.22 mM−1 s−1 and k3= 32 mM−1 s−1; the k2 value is not changed but the k3 value is decreased by the attachment of the polymer.EP+‐PEG‐NAD+ works as a unique catalyst having an intramolecular reaction step within its turnover cycle in a coupled multi‐step reaction system containing malate dehydrogenase, malate, EP+‐PEG‐NAD+, a tetrazolium salt and oxygen. The first‐order rate constant, k4, of the intramolecular reaction was 1.1 s‐1. The effects of the covalent linking of the 5‐ethylphenazine and the NAD+ moieties were estimated by comparing the value of k4 with that of k1 for the reaction of III with NADH; the effective concentration of the NADH moiety for the 5‐ethylphenazine moiety on the same EP+‐PEG‐NADH molecule (or vice versa) was calculated to be 0.40 mM from the ratio of k4/k1. The values of the rate constants in the coupled multi‐step reaction system enable us to understand the dynamic features of the system and the characteristics of EP+‐PEG‐NAD+ as a catalyst are discussed.
Modulation of the affinity of the single‐stranded DNA‐binding protein of Escherichia coli (E. coli SSB) to poly(dT) by site‐directed mutagenesis
Authors: BAYER Irene , FLIESS Anja , GREIPEL Joachim , URBANKE Claus , MAASS Günter 
Published: March 2005

A vector for site‐directed mutagenesis and overproduction of the Escherichia coli single‐stranded‐DNA‐binding protein (E. coli SSB) was constructed. An E. coli strain carrying this vector produces up to 400 mg pure protein from 25 g wet cells. The vector was used to mutate specifically the Phe60 residue of E. coli SSB. Phe60 had been proposed to be located near the single‐stranded‐DNA‐binding site.Substitution of the Phe60 residue by Val, Ser, Leu, His, Tyr and Trp gave proteins with no or only minor conformational changes, as detected by NMR spectroscopy.The affinity of the mutant E. coli SSB proteins for single‐stranded DNA decreased in the order Trp > Phe (wild‐type) > Tyr > Leu > His > Val > Ser, leading to the conclusion that position 60 is a site of hydrophobic interaction of the protein with DNA.
Nucleotide sequence of the glyceraldehyde‐3‐phosphate dehydrogenase gene from the mesophilic methanogenic archaebacteria Methanobacterium bryantii and Methanobacterium formicicum
Authors: FABRY Stefan , LANG Jutta , Niermann Thomas , Vingron Martin , HENSEL Reinhard 
Published: March 2005

The genes for glyceraldehyde‐3‐phosphate dehydrogenase (gap genes) from the mesophilic methanogenic archaebacteria Methanobacterium formicicum and Methanobacterium bryantii were cloned and sequenced. The deduced amino acid sequences show 95% identity to each other and about 70% identity to the glyceraldehyde‐3‐phosphate dehydrogenase from the thermophilic methanogenic archaebacterium Methanothermus fervidus. Although the sequence similarity between the archaebacterial glyceraldehyde‐3‐phosphate dehydrogenase and the homologous enzyme of eubacteria and eukaryotes is low, an equivalent secondary‐structural arrangement can be deduced from the profiles of the physical parameters hydropathy, chain flexibility and amphipathy.In order to find possible thermophile‐specific structural features of the enzyme from M. fervidus, a comparative primary‐sequence analysis was performed. Amino acid exchanges leading, to a stabilization of the main‐chain conformation, could be found throughout the sequence of the thermophile enzyme. Striking features of the thermophile sequence are the preference for isoleucine, especially in β‐sheets, and a low arginine/lysine ratio of 0.54.