bluuebunny/crossref_metadata_2025 huggingface dataset:
Kinetic theory of enzymatic reactions in reversed micellar systems
Authors: KHMELNITSKY Yuri L. , NEVEROVA Irina N. , POLYAKOV Vyacheslav I. , GRINBERG Valeri Ya. , LEVASHOV Andrei V. , MARTINEK Karel
Published: March 2005

A kinetic theory is proposed for enzymatic reactions proceeding in reversed micellar systems in organic solvents, and involving substrates capable of partitioning among all pseudophases of the micellar system, i.e. aqueous cores of reversed micelles, micellar, membranes and organic solvent. The theory permits determination of true (i.e. with reference to the aqueous phase, where solubilized enzyme is localized) catalytic parameters of the enzyme, provided partition coefficients of the substrate between different phases are known. The validity of the kinetic theory was verified by the example of oxidation of aliphatic alcohols catalyzed by horse liver alcohol dehydrogenase in the system of reversed sodium bis(2‐ethylhexyl)sulfosuccinate (AOT, aerosol OT) micelles in octane. In order to determine partition coefficients of alcohols between phases of the micellar system, flow microcalorimetry technique was used. It was shown that in the first approximation, the partition coefficient of the substrate in a simple biphasic system consisting of water and corresponding organic solvent can be used as an estimate for the partition coefficient of the substrate between aqueous and organic solvent phases of the micellar system. True values of the Michaelis constant of alcohols in the micellar system, determined using suggested approach, are equal to those obtained in aqueous solution and differ from apparent values referred to the total volume of the system. The results clearly show that the previously reported shift in the substrate specificity of HLADH, observed on changing from aqueous solution to the system of reversed aerosol OT micelles in octane, is apparent and can be explained on the basis of partitioning effects of alcoholic substrates between phases of the micellar system.
Electron transfer reactions of metalloproteins at peptide‐modified gold electrodes
Authors: BARKER Paul D. , Di GLERIA Kati , HILL H. Allen O. , LOWE Valerie J.
Published: March 2005

The electron transfer reactions of four small redox proteins, cytochrome c, ferredoxin, plastocyanin and azurin, have been investigated at novel peptide‐modified gold electrodes. These proved to be effective and selective in facilitating electron transfer. Good, quasi‐reversible electron transfer was achieved selectively at different peptide‐protein configurations by changing the pH or the ionic strength of the solution. The use of peptides as promoters for protein electrochemistry opens up the possibility of designing very specific electrode surfaces for larger molecules like enzymes.
Activation of platelets induced by mAb P256 specific for glycoprotein IIb‐IIIa
Authors: BACHELOT Christilla , RENDU Francine , BOUCHEIX Claude , HOGG Nancy , LEVY‐TOLEDANO Sylviane
Published: March 2005

Monoclonal antibody P256, which is specific for glycoprotein IIb‐IIIa complex, was found to induce aggregation of normal platelets in plasma. The mechanism of platelet activation induced by this monoclonal antibody was thoroughly studied. The divalent binding to the IIb‐IIIa molecule was necessary for triggering aggregation since Fab′ fragments did not induce aggregation as did IgG and F(ab′)2 fragments; however, F(ab′)2 did not induce the release as did the whole IgG. P256‐induced aggregation was accompanied by release of all three granule constituents, namely dense granules, α‐granules and lysosomes, with parallel kinetics showing half‐maximum release 50 s after addition of P256. Thromboxane synthesis was initiated at the same time. Using 32P‐prelabeled platelets, no variation in level of [32P]phosphatidylinositol 4,5‐bisphosphate could be detected in the first minute after P256 addition, indicating no activation of the calcium‐independent phospholipase C specific for polyphosphoinositol phospholipid. P256 induced a calcium mobilization as measured by Indo‐1 fluorescence of about the third of that measured in the presence of a thrombin concentration giving the same intensity of aggregation. P256 induced phosphorylation of the myosin light chain p20 and of the main substrate of protein kinase C, p43. Addition of aspirin inhibited almost totally calcium mobilization and partially aggregation, release and protein phosphorylations. By contrast, in the absence of external calcium, although no aggregation could occur, the release reaction was only partially reduced. In this activation, the glycoprotein IIb‐IIIa complex thus appears to play a role in modulating platelet response, not only via calcium fluxes but also in activating protein kinase C responsible for p43 phosphorylation.
Nucleotide sequence and exon‐intron organization of the human proacrosin gene
Authors: KEIME Sabine , ADHAM Ibrahim M. , ENGEL Wolfgang
Published: March 2005

Acrosin is a serine proteinase and located in a zymogen form, proacrosin, in the acrosome of the sperm. As deduced from the cDNA sequences for human and boar proacrosin, the enzyme is synthesized as a preproenzyme, preproacrosin, which contains a hydrophobic leader sequence. Using cDNA clones as probes, we have isolated the gene coding for human proacrosin from a human leucocyte genomic library and a human cosmid library, respectively. The gene contains four introns between 0.2 kb–4.5 kb in length. Similar to other serine proteinases, the coding sequence of the preproacrosin gene is spread over all the five exons of the gene and the three active‐site residues His, Asp and Ser are encoded by three different exons. According to the exon‐intron structure, preproacrosin is suggested to be closely related to the serine proteinase subfamily containing trypsins and kallikrein. However, the light chain of proacrosin seems to be similar to that of chymotrypsin. The coding of the serine active‐site residue together with the proacrosin‐specific proline‐rich domain in one exon, namely exon E5, let us assume that the nucleotide sequence for the proline‐rich domain was generated during evolution by intron‐exon transfer from a foreign gene with subsequent intron excision. By primer extension analysis, the transcription initiation site of the preproacrosin mRNA could be assigned to the residue C at – 74 nucleotides upstream from the translation initiation codon ATG. In contrast to most other eucaryotic genes, including the known testis‐specific genes, typical TATA and CAAT box sequences in convential distances from the 5′ end of the transcription start site could not be evaluated in the proacrosin gene.
A critical evaluation of the hydropathy profile of membrane proteins
Authors: DEGLI ESPOSTI Mauro , CRIMI Massimo , VENTUROLI Giovanni
Published: March 2005

New membrane‐preference scales are introduced for categories of membrane proteins with different functions. A statistical analysis is carried out with several scales to verify the relative accuracy in the prediction of the transmembrane segments of polytopic membrane proteins. The correlation between some of the scales most used and those calculated here provides criteria for selecting the most appropriate methods for a given type of protein. The parameters used in the evaluation of the hydropathy profiles have been carefully ascertained in order to develop a reliable methodology for hydropathy analysis. Finally, an integrated hydropathy analysis using different methods has been applied to several sequences of related proteins.

The above analysis indicates that (a) microsomal cytochrome P450 contains only one hydrophobic region at the N‐terminus that is consistently predicted to transverse the membrane; (b) only four of the six or seven putative transmembrane helices of cytochrome oxidase subunit III are predicted and correspond to helices I, III, V and VI of the previous nomenclature; (c) the product of the mitochondrial ATPase‐6 gene (or the chloroplast ATPase‐IV gene) of F0‐F1‐ATPase shows that helix IV is not consistently predicted to traverse the membrane, suggesting a four‐helix model for this family of proteins.
Subcellular distribution of acetylcholinesterase forms in chromaffin cells
Authors: BON Suzanne , BADER Marie‐France , AUNIS Dominique , MASSOULIÉ Jean , HENRY Jean‐Pierre
Published: March 2005

The presence of acetylcholinesterase (AChE) in chromaffin granules has been controversial for a long time. We therefore undertook a study of AChE molecular forms in chromaffin cells and of their distribution during subcellular fractionation. We characterized four main AChE forms, three amphiphilic forms (G1a· G2a and G4a), and one non‐amphiphilic form (G4na). Each form shows the same molecular characteristics (sedimentation, electrophoretic migration, lectin interactions) in the different subcellular fractions. All forms are glycosylated and seem to possess both N‐linked and O‐linked carbohydrate chains. There are differences in the structure of the glycans carried by the different forms, as indicated by their interaction with some lectins. Glycophosphatidylinositol‐specific phospholipases C converted the G2a form, but not the other amphiphilic forms, into non‐amphiphilic derivatives. The distinct patterns of AChE molecular forms observed in various subcellular compartments indicate the existence of an active sorting process. G4na was concentrated in fractions of high density, containing chromaffin granules. We obtained evidence for the existence of a lighter fraction also containing chromogranin A, tetrabenazine‐binding sites and G4na AChE, which may correspond to immature, incompletely loaded granules or to partially emptied granules. The distribution of G4na during subcellular fractionation suggested that this form is largely, but not exclusively, contained in chromaffin granules, the membranes of which may contain low levels of the three amphiphilic forms.
Three human interferon‐α2 subvariants disclose structural and functional differences
Authors: von GABAIN Alex , LUNDGREN Erik , OHLSSON Monica , HOLMGREN Erik , JOSEPHSSON Staffan , ALKAN Sefik S.
Published: March 2005

The human interferon‐α2 subvariants 2a, 2b and 2c differ by only one or two amino acids at positions 23 and/or 34 of the mature protein. In this study, the coding regions of the three interferon‐α2 subvariants were derived from the cDNA of interferon‐α2c by site‐directed in vitro mutagenesis. The interferon‐α subvariants were synthesized using the same Escherichia coli strain for production and were subsequently purified. Comparative studies revealed that they differ significantly in their biological and antigenic properties. Therefore, amino acid positions 23 and 34 seem to be crucial for structure/function of human interferon‐α. Furthermore, the study points to the importance of defining, whether such minor structural variants of naturally occurring polypeptides represent functional variants.
Binding of nuclear proteins to the enhancer elements of the rat apolipoprotein A‐I gene
Authors: DAI Pei‐hua , LAN Shirley S.‐F. , DING Xiao‐hua , CHAO Yu‐sheng
Published: March 2005

To determine the cis‐ and trans‐regulatory elements which control the expression of the apolipoprotein (apo) A‐I gene, several DNA‐protein binding assays, namely, gel mobility shift, exonuclease III protection, and exonuclease III footprinting assays, were employed to identify these elements. It is demonstrated that nuclear proteins of Hep G2 cells bind to five regions of DNA sequences between 252 and 149 base pairs upstream from the transcription initiation site of the rat apo A‐I gene. Using South‐Western blot analysis, it is determined that DNA‐binding protein has a molecular mass of approximately 90 kDa. It is also shown that the DNA‐binding protein was present in Hep G2 cells and rat livers but absent in rabbit livers. The results suggest that the lack of expression of the apo A‐I gene in rabbit livers is due to the absence of this DNA‐binding protein.
Characterization of purified pp60c‐src protein tyrosine kinase from human platelets
Authors: REUTER Christoph , FINDIK Duygu , PRESEK Peter
Published: March 2005

Intact pp60c‐src, the cellular homologue of the transforming protein of Rous sarcoma virus, was purified from human platelets. The purified fractions also contained small amounts of a 54‐kDa proteolytic degradation product of pp60c‐src. We investigated some of the biochemical and kinetic properties of pp60c‐src protein tyrosine kinase. Maximum kinase activity occurred at pH 6.5 and required a mixture of 2 mM Mn2+/Mg2+ as divalent cations. The enzyme most strongly phosphorylated casein, followed by enolase and alcohol dehydrogenase. The Km value for ATP was 4 μM for substrate phosphorylation and for autophosphorylation. Using casein, we determined a Vmax for substrate phosphorylation by pp60c‐src in the range of 1.9–3.4 nmol · min−1· mg−1. Since the Vmax value for the purified 54‐kDa fragment of pp60c‐src was also included in this value, we conclude that proteolytic degradation of a 6‐kDa fragment from the N‐terminus of pp60c‐src did not affect its kinase activity. Tryptic phosphopeptide analysis identified Tyr‐416 as the major autophosphorylation site. Preincubation of purified pp60c‐src with ATP increased the amount of autophosphorylation accompanied by an increase in Vmax, whereas the Km values were not altered. Our data directly demonstrate that autophosphorylation at Tyr‐416 exerts, in contrast to phosphorylation at Tyr‐527, a positive regulatory effect on the pp60c‐src kinase activity.
Effects of nutrients and hormones on transcriptional and post‐transcriptional regulation of acetyl‐CoA carboxylase in rat liver
Authors: KATSURADA Akihiko , IRITANI Nobuko , FUKUDA Hitomi , MATSUMURA Yohko , NISHIMOTO Naomi , NOGUCHI Tamio , TANAKA Takehiko
Published: March 2005

The effects of nutrients and hormones on transcriptional and post‐transcriptional regulation of acetyl‐CoA carboxylase in rat liver were investigated following a cDNA cloning. After refeeding a carbohydrate/protein diet to fasted rats, the transcriptional rate was increased 2.5‐fold in only 1 h. The mRNA concentration reached a maximal level of 9–12‐fold increase in 8–16 h, and the enzyme induction increased 10‐fold in 48 h. By a carbohydrate diet without protein, the transcriptional rate, mRNA concentration and enzyme induction were similarly increased to the levels in the carbohydrate/protein diet. It appears that protein feeding is not necessary to induce acetyl‐CoA carboxylase. Corn oil feeding decreased the transcriptional rate. In diabetic rats, the transcriptional rate, mRNA concentration and enzyme induction were very low in comparison with the normal. After insulin treatment, the transcriptional rate was increased 2‐fold (the normal level) in 2 h in diabetic rats. By fructose feeding to diabetic rats, the transcriptional rate and mRNA concentration were increased similarly to the levels reached by insulin treatment, while the enzyme induction was increased by only 60%. Thus, it is suggested that insulin is importantly involved in the transcription and also translation of acetyl‐CoA carboxylase. On the other hand, triiodothyronine treatment increased the mRNA and enzyme levels in diabetic and normal rats, and somewhat increased the transcriptional rate only in diabetic rats. Triiodothyronine appears to stabilize the mRNA besides having an insulin‐like action in acetyl‐CoA carboxylase transcription.
Implications for a reduced DNA‐elongation rate in polyamine‐depleted cells
Authors: OREDSSON Stina M. , NICANDER Björn , HEBY Olle
Published: March 2005

Treatment of Ehrlich ascites tumor cells with 2‐difluoromethylornithine (F2MeOrn), an enzyme‐activated irreversible inhibitor of ornithine decarboxylase, resulted in depleted putrescine and spermidine content, and reduced growth rate. We have previously shown that adenine ribonucleotide levels are substantially increased in these polyamine‐depleted cells. The present paper addresses the question whether the elevated ATP pool is accompanied by a concomitant increase in the dATP pool. If this is the case, the observed growth inhibition could be explained by the well‐known dATP‐mediated feedback inhibition of ribonucleotide reductase. We found that dNTP pools were not unbalanced and that dNTP synthesis was not arrested in polyamine‐depleted cells. Moreover, the dNTP content and the activity of ribonucleotide reductase (CDP reduction) and thymidylate synthase, remained elevated despite the fact that the cells were inhibited in their growth by F2MeOrn treatment. Incorporation of a radiolabeled precursor into DNA was initially lower in F2MeOrn‐treated cells than in control cells. However, while incorporation of a radiolabeled precursor into DNA decreased markedly in plateau‐phase control cells, it remained at a higher level in cells inhibited in growth by polyamine depletion. This discrepancy may be explained by the fact that polyamine‐depleted cells accumulated in the S phase, and that they had an increased content of acid‐soluble radiolabeled DNA precursor. Our data indicate that polyamine depletion adversely affects the DNA synthetic machinery by reducing the rate of elongation.
Coupling of insulin‐responsive glucose transport to receptors for insulin‐like growth factor 1 in primary human fibroblasts
Authors: MAASSEN J. Antonie , van der ZON Gerard C. M.
Published: March 2005

We have recently described an insulin‐resistant patient with leprechaunism (leprechaun G.) having a homozygous leucine → proline mutation at amino acid position 233 in the α‐chain of the insulin receptor. The mutation results in a loss of insulin binding to cultured fibroblasts. Fibroblasts from the patient and control individuals were used to quantify the stimulation of 2‐deoxyglucose uptake by insulin and insulin‐like growth factor 1 (IGF‐1). Insulin hardly stimulates basal 2‐deoxyglucose uptake in the patient's fibroblasts whereas in control fibroblasts the uptake of 2‐deoxyglucose is stimulated by insulin approximately 1.7 times. In contrast, IGF‐1 stimulates hexose uptake in the patient's fibroblasts 1.8 times, a similar value to that obtained by stimulation of control fibroblasts with insulin or IGF‐1. With both types of fibroblasts, maximal IGF‐1 response is reached at about 10 nM IGF‐1, the ED50 being approximately 4 nM.

The results indicate that the insulin responsive glucose transport in primary fibroblasts is functionally linked to the receptor for IGF‐1. Insulin binds with an approximately 200‐fold lower affinity to IGF‐1 receptors, compared to homologous IGF‐1 binding. As an insulin concentration of 10 μM is unable to give maximal stimulation of glucose uptake in the patient's fibroblasts, which is already seen with 10 nM IGF‐1, it seems that occupation of IGF‐1 receptors by insulin on the patient's cells is less efficient at stimulating hexose uptake compared to homologous activation.
Bile acids mobilise internal Ca2+ independently of external Ca2+ in rat hepatocytes
Authors: COMBETTES Laurent , BERTHON Brigitte , DOUCET Edith , ERLINGER Serge , CLARET Michel
Published: March 2005

In the present study, we investigated the possible role of external Ca2+ in the rise of the cytosolic Ca+ concentration induced by the monohydroxy bile acid taurolithocholate in isolated rat liver cells. The results showed that: (a) the bile acid promotes the same dose‐dependent increase in the cytosolic Ca+ concentration (half‐maximal effect at 23 μM) in hepatocytes incubated in the presence of 1.2 mM Ca2+ or 6 μM Ca2+; (b) taurolithocholate is able to activate the Ca2+‐dependent glycogen phosphorylase a by 6.3‐fold and 6.0‐fold in high and low Ca2+ media, respectively; (c) [14C]taurolithocholate influx is not affected by external Ca2+, and 45Ca2+ influx is not altered by taurolithocholate. These results establish that the effects of taurolithocholate on cell Ca2+ do not require extracellular Ca2+ and are consistent with the view that monohydroxy bile acids primarily release Ca2+ from the endoplasmic reticulum in the liver.
Different classes of tryptophan residues involved in the conformational changes characteristic of the sarcoplasmic reticulum Ca2+‐ATPase cycle
Authors: de FORESTA Béatrice , CHAMPEIL Philippe , le MAIRE Marc
Published: March 2005

Various classes of tryptophan residues in the Ca2+‐ATPase of sarcoplasmic reticulum membranes have been distinguished on the basis of their sensitivities to certain fluorescence quenchers: the brominated phospholipid 1,2‐bis(9,10‐dibromostearoyl)‐sn‐glycero(3)phosphocholine, the calcium ionophore calcimycin (A23187) and its brominated analog (4‐bromo‐A23187), and the nucleotide analog 2′(3′)‐O‐(2,4,6‐trinitrophenyl)‐adenosine 5′‐triphosphate. We show that tryptophans located at the protein‐lipid interface are the main contributors to the well‐known fluorescence intensity change occurring in parallel with the conformational rearrangement induced by addition of calcium to the ATPase or its removal; Trp‐794 on the ATPase chain may be one of these tryptophans. We also show that tryptophans more deeply embedded in the transmembrane protein structure contribute to the fluorescence change observed upon phosphorylation from inorganic phosphate of the calcium‐free ATPase. This phosphorylation step involves opposite changes in the fluorescence quantum yield of tryptophans located in the membrane and in the cytoplasmic regions of the ATPase. This result is in agreement with models in which phosphorylation from inorganic phosphate not only changes the ATPase conformation locally around the catalytic center, but also reorganizes the membrane portion of the ATPase by long‐range action, allowing, for instance, the calcium sites to become accessible from the luminal medium.
The ATPase of Bacillus alcalophilus
Authors: HOFFMANN Artur , DIMROTH Peter
Published: March 2005

The ATPase of Bacillus alcalophilus was extracted from the bacterial membranes with Triton X‐100 and purified by hydroxyapatite column chromatography. SDS gel electrophoresis of the purified protein indicated the typical subunit pattern of an F1F0 structure with five F1 subunits (α,β,γ,δ,ɛ) and three F0 subunits (a,b,c). The α and β subunits were antigens for an antiserum against the corresponding subunits of the ATPase of Escherichia coli. Subunit c was extracted from the bacterial membranes with chloroform/methanol. Its amino acid composition was in the range of subunits c from other ATPases. Maximal ATPase activity was observed in the presence of 2–5 mM MgCl2, an ATP/Mg2+ ratio of 2:1 and 25% methanol. In the absence of methanol, only about 1% of the maximal activity was observed. The enzyme was also activated by Ca2+ (in the absence of methanol), reaching about 30% of the maximal activity. The dependence of initial velocity versus ATP of the Ca2+‐activated but not of the Mg2+/methanol‐activated enzyme indicated cooperativity with three strongly cooperative binding sites.
Thermodynamics of information transfer between subunits in oligomeric enzymes and kinetic cooperativity
Authors: GIUDICI‐ORTICONI Marie‐Thérèse , BUC Jean , BIDAUD Michel , RICARD Jacques
Published: March 2005

The theory and the methods that have been described in the two preceding papers in this journal have been used to analyze the kinetic properties of chloroplast fructose bisphosphatase. The enzyme is a tetramer made up of apparently identical subunits and displays a sigmoidal kinetics with respect to its substrate, fructose bis‐phosphate. The free ionic species, magnesium and fructose bisphosphate bind to the enzyme and the chelate fructosé‐bisphosphate‐magnesium does not affect the sigmoidicity of the rate curves.

The Hill coefficient with respect to free fructose bisphosphate is equal to 2.3, which is indeed incompatible with the view that the enzyme behaves as a dimer of dimers. This conclusion is confirmed by direct analysis of the rate curve. On the basis of the sum of the residuals, their sum of squares, the standard error of the kinetic parameters of the equation, the kinetic scheme associated with a dimer of dimers may be ruled out. On the basis of the same criteria, the fit of an Adair equation to the rate data cannot be retained as satisfactory. This is a direct proof that neither the Monod nor the Koshland model can correctly fit these kinetic data. In fact the model that fits these data best is a structural kinetic scheme where information transfer occurs between each subunit and its three neighbors (‘tetrahedral’ mode of information transfer).

The fit of these models to a large number of kinetic data allows one to compute the free energy profile during the successive binding processes of the four substrate molecules to the enzyme. Whereas the first two steps are associated with an increase of free energy, all the other subsequent steps are associated with a decrease of free energy.
A plant serpin gene
Authors: BRANDT Anders , SVENDSEN Ib , HEJGAARD Jørn
Published: March 2005

A 3133‐bp nucleotide sequence of the gene Paz1 on chromosome 4 of barley, encoding endosperm protein Z4, has been determined. The sequence includes 1079 bp 5′ upstream and 523 bp 3′ downstream of the coding region. The 1079‐bp 5′ upstream region of the gene shows little similarity to 5′ regions of other sequenced genes expressed in the developing cereal endosperm. The coding sequence is interrupted by one 334‐bp‐long intron (bases 1497–1830). The deduced amino acid sequence, which was corroborated by peptide sequences, consists of 399 amino acids and has a molecular mass of 43128 Da. This sequence confirms protein Z4 to be a member of the serpin superfamily of proteins. The similarity with other members of the family expressed as amino acids in identical positions is in the order of 25–30% and pronounced in the carboxy‐terminal half of the molecule. Sequence residues assumed to form clusters stabilizing the tertiary structure are highly conserved. Protein Z4 is synthesized in the developing endosperm without a signal peptide and protein Z4 mRNA was evenly distributed among the free and membrane‐bound polyribosomes of the endosperm cell. An internal hydrophobic region of 21 amino acids (residues 36–56) may serve as a signal for targeting the polypeptide into the lumen of the endoplasmic reticulum. The gene for protein Z4 could not be detected in the barley variety Maskin and some of its descendants. The ‘high‐lysine’ allees, lysl (Hiproly barley) and lys3a (Bomi mutant 1508) on chromosome 7, enhance and repress, respectively, the expression of the protein Z4 gene. Also, 1554 bp of another 8‐kbp fragment of the barley genome PazΨ, similar to the protein‐Z4‐coding region, have been determined. Small insertions and deletions and the presence of an internal stop codon identify this fragment as part of a pseudogene related to the protein Z4 gene.
Isolation, characterization and chromosomal localization of cDNA clones for the E1β subunit of the pyruvate dehydrogenase complex
Authors: CHUN Kathy , MACKAY Nevi , WILLARD Huntington F. , ROBINSON Brian H.
Published: March 2005

A full‐length cDNA clone for the E1β subunit of the human pyruvate dehydrogenase (PyrDH) complex was isolated from a human skin fibroblast cDNA library. When sequenced, it showed differences from the nucleotide sequence already published [Koike, K., Ohta, S., Urata, Y., Kagawa, Y. & Koike, M. (1988) Proc. Natl Acad. Sci. USA 85, 41–45], such that 19 amino acids were different in the translated open reading frame. Northern blotting of human fibroblast cell lines revealed a major mRNA species of 1.6 kb and a weaker band of 5.5 kb. In a series of nine PyrDH‐complex‐deficient cell lines from patients with this deficiency, no patients had severely reduced amounts of mRNA, but there was one patient cell line with an increased amount of abnormal‐size mRNA. Chromosome localization carried out with DNA blots from man‐mouse hybrid cell lines indicated that the E1β subunit of pyruvate dehydrogenase is located on chromosome 3. A motif AXGXXXXGL(R/K) X15(D/E)Q was found in common with a variety of other oxo‐acid oxidoreductases, but its function is not known.
Purification and fractionation of lipopolysaccharide from Gram‐negative bacteria by hydrophobic interaction chromatography
Authors: FISCHER Werner
Published: March 2005

By hydrophobic interaction chromatography on octyl‐Sepharose, lipopolysaccharide (LPS) of Escherichia coli Re mutant and of wild‐type smooth‐form (S‐form) Salmonella typhimurium and Salmonella abortus equi is fractionated according to increasing amount of fatty acids. Thereby a fractionation of S‐form LPS according to the length of the O‐polysaccharide chain also occurs, because with increasing of fatty acids there is a decrease in the mean length of the O‐polysaccharide chain from approximately 30 to 4 repeating units. Molecular species of Re‐mutant LPS contain four 3‐hydroxytetradecanoyl residues in addition to which dodecanoic, tetradecanoic and possibly hexadecanoic acid, appear in this sequence. Among the molecular species of S‐form LPS, dodecanoic, tetradecanoic and hexadecanoic acids appear in the same order, but in contrast to Re‐mutant LPS a significant fraction of S‐form LPS contains less than four 3‐hydroxytetradecanoyl residues.

Hydrophobic interaction chromatography also proved an effective one‐step purification procedure of LPS as was shown with a crude preparation from S‐form S. typhimurium.
Temperature‐induced expression of proteins in Leishmania mexicana amazonensis
Authors: PINELLI Elena , SHAPIRA Michal
Published: March 2005

Temperature increase is an integral part of Leishmania life cycle, and plays a major role in stage transformation. Analysis of the temperature‐dependent pattern of protein synthesis on two‐dimensional gel electrophoresis shows that, in addition to the conserved heat‐shock type of response in which expression of the major 70‐kDa and 83‐kDa heat‐shock proteins is observed, a group of low‐molecular‐mass (17–40 kDa) proteins is induced in promastigotes of Leishmania mexicana amazonensis at elevated temperatures. Immuno‐gold labelling with antibodies raised against the heat‐induced 22‐kDa proteins was localized mainly in the mitochondrion of Leishmania parasites, though labelling was observed also in the nucleus. The correlation of this finding with various reports on induction of mitochondrial enzymes in response to temperature stress in other organisms is discussed.
Isolation and characterization of a tumor‐derived human protein related to chromogranin A and its in vitro conversion to human pancreastatin‐48
Authors: TAMAMURA Hirokazu , OHTA Mitsuhiro , YOSHIZAWA Kumi , ONO Yoshiyuki , FUNAKOSHI Akihiro , MIYASAKA Kyoko , TATEISHI Kayoko , JIMI Atsuo , YAJIMA Haruaki , FUJII Nobutaka , FUNAKOSHI Susumu
Published: March 2005

A protein with pancreastatin‐like immunoreactivity has been isolated and purified from liver metastasis of a patient with insulinoma. NH2‐terminal residue analysis, in conjunction with the use of antibodies that are specific for the C‐terminal amide peptide of porcine pancreastatin, identified this protein as a 186‐amino‐acid protein corresponding to human chromogranin A‐116‐301 (the fragment corresponding to the positions from 116 to 301 of human chromogranin A). Digestion of this protein with trypsin yielded a 48‐amino‐acid peptide with the retention of full pancreastatin activity. Serum from patient with insulinoma contains a peptide specie(s) that comigrates with the 48‐amino‐acid pancreastatin, suggesting that this peptide might be a physiologically important circulation form of pancreastatin in humans.

A sensitive radioimmunoassay was established using antibody developed against a synthetic 29‐amino‐acid peptide amide of pancreastatin. Immunocytochemical staining revealed that a major population of human pancreatic islet cells were immunoreactive to the antiserum but with varying intensity of staining. Pancreastatin‐like immunoreactivity was not observed in exocrine acinar cells.
Staphyloferrin A: a structurally new siderophore from staphylococci
Authors: KONETSCHNY‐RAPP Silvia , JUNG Günther , MEIWES Johannes , ZÄHNER Hans
Published: March 2005

Two ferric ion‐binding compounds, designated staphyloferrin A and B, were detected in the culture filtrates of staphylococci‐grown under iron‐deficient conditions. Staphyloferrin A was isolated from cultures ofStaphylococcus hyicusDSM 20459. The structural elucidation of this highly hydrophilic, acid‐labile compound revealed a novel siderophore,N2,N5‐di‐(l‐oxo‐3‐hydroxy‐3,4‐dicarboxybutyl)‐D‐ornithine, which consists of one ornithine and two citric acid residues linked by two amide bonds. The two citric acid components of staphyloferrin A provide two tridentate pendant ligands, comprising of a β‐hydroxy, β‐carboxy‐substituted carboxylic acid derivative, for octahedral metal chelation. The CD spectrum of the staphyloferrin A ferric complex indicates a predominant A configuration about the ferric ion center. The uptake of ferric staphyloferrin A byS. hyicusobeys Michaelis‐Menten kinetics (Km= 0.246 μM;vmax= 82 pmol · mg−1· min−1), indicating active transport of this siderophore. The staphyloferrin A transport system is different from that of the ferrioxamines as shown by an antagonism test. Production of staphyloferrin A is strongly iron‐dependent and is stimulated by supplementation of the medium with either D‐ or L‐ornithine. DL‐[5‐14C]ornithine was incorporated into staphyloferrin A, demonstrating that ornithine is an intermediate in staphyloferrin A biosynthesis.
Valyl‐tRNA synthetase from yeast
Authors: FREIST Wolfgang , CRAMER Friedrich
Published: March 2005

For discrimination between valine and the 19 naturally occurring noncognate amino acids, as well as between valine and 2‐amino‐isobutyric acid by valyl‐tRNA synthetase from baker's yeast, discrimination factors (D) have been determined from kcat and Km values in aminoacylation of tRNAVal‐C‐C‐A. The lowest values were found for Trp, Ser, Cys, Lys, Mct and Thr (D = 90–870), showing that valine is 90–870 times more frequently attached to tRNAVal‐C‐C‐A than the noncognate amino acids at the same amino acid concentrations. The other amino acids exhibit D values between 1100 and 6200. Generally, valyl‐tRNA synthetase is considerably less specific than isoleucyl‐tRNA synthetase, but this may be partly compensated in the cell by valine concentrations higher than those of noncognate acids.

In aminoacylation of tRNAVal‐C‐C‐A(3′NH2 discrimination factors D1 are in the range of 40–1260. From D values and AMP formation stoichiometry, pretransfer proof‐reading factors II1 were determined; post‐transfer proof‐reading factors II2 were determined from D values and AMP formation stoichiometry in acylation of tRNAVal‐C‐C‐A.II1 values (7–168) show that pretransfer proof‐reading is the main correction step, posttransfer proof‐reading (II2=1–7) is less effective and in some cases negligible.

Initial discrimination factors were calculated from discrimination and proof‐reading factors according to a two‐step binding process. These factors, due to different Gibbs free energies of binding can be related to hydrophobic interaction forces, and a hypothetical ‘stopper’ model of the amino‐acid‐binding site is discussed.
Two‐dimensional NMR studies on des‐pentapeptide‐insulin
Authors: BOELENS Rolf , GANADU Maria Luisa , VERHEYDEN Patricia , KAPTEIN Robert
Published: March 2005

The shortened analogue of insulin, des‐(B26 B30)‐pentapeptide insulin, has been characterized by two‐dimensional 1H NMR. The 1H resonance assignments and the secondary structure in water solution are discussed. The results indicate that the secondary structure in solution is very similar to that reported for the crystalline state. A high flexibility of both A and B chains is observed. Of the two conformations seen in the 2‐Zn insulin crystals and indicated as molecules 1 and 2 (Chinese nomenclature), the structure of the analogue is more similar to that of molecule 1.
Molecular characterization of nuclear basic protein HPI1, a putative precursor of human sperm protamines HP2 and HP3
Authors: MARTINAGE Arlette , ARKHIS Ahmed , ALIMI Elisabeth , SAUTIÈRE Pierre , CHEVAILLIER Philippe
Published: March 2005

The largest intermediate basic protein HPI1 (101 residues) from human sperm chromatin was isolated and characterized. The amino acid composition and sequence analysis of the protein and of tryptic peptides together with peptide mapping of endoproteinases Lys‐C and Glu‐C hydrolysates showed that the C‐terminal region (residues 45 – 101) of HPI1 is identical to protamine HP2. These structural data strongly suggest that protein HPI1 is a precursor of human sperm protamines HP2 and HP3 (57 and 54 residues, respectively) as well as of two other intermediate basic proteins HPS1 and HPS2 (69 and 66 residues, respectively) sequenced previously.
Purification and characterization of a ribonuclease from human spleen
Authors: YASUDA Toshihiro , MIZUTA Keiko , SATO Wataru , KISHI Koichiro
Published: March 2005

A ribonuclease has been isolated from human spleen (RNase HS) by means of acid extraction, ammonium sulphate fractionation, successive column chromatographies on CM‐cellulose, heparin‐actigel, and poly(G)‐agarose, and double gel‐filtration on Sephadex G‐75. The purified preparation was homogeneous as judged by SDS/PAGE. RNase HS was found to be a glycoprotein, containing three fucose, one mannose and five glucosamine residues/molecule, with a molecular mass of 17 kDa as determined by both SDS/PAGE and gel filtration. The catalytic properties and structural features, including its amino acid composition and the amino acid sequence of the N‐terminal 35 residues, indicated that the enzyme was strictly related to nonsecretory RNase isolated from human urine and liver. In particular, the amino acid sequence of the N‐terminal was identical with that of urine nonsecretory RNase and eosinophil‐derived neurotoxin. Furthermore, analyses using three different antibodies specific to RNase HS, urine nonsecretory RNase and urine secretory RNase, indicated that RNase HS was not immunologically distinguishable from urine nonsecretory RNase, but clearly so from urine secretory RNase. However, the carbohydrate compositions of RNase HS and urine nonsecretory RNase were found to differ. It therefore remains to be resolved whether or not the tissue of origin of nonsecretory RNase in urine is the spleen.
Molecular cloning and nucleotide sequence of full‐length cDNA for ascorbate oxidase from cultured pumpkin cells
Authors: ESAKA Muneharu , HATTORI Tsukaho , FUJISAWA Kouichi , SAKAJO Shigeru , ASAHI Tadashi
Published: March 2005

A λgt11 cDNA expression library was constructed from size‐fractionated poly(A)‐rich RNA of cultured pumpkin cells. A full‐length cDNA clone for ascorbate oxidase mRNA was selected from the library by screening with synthetic oligonucleotides designed from the amino‐terminal sequence of ascorbate oxidase protein. The identity of the clone was confirmed by comparing the amino acid sequence deduced by nucleotide sequence analysis with that determined for the amino‐terminal sequence of pumpkin ascorbate oxidase. The nucleotide sequence of the cDNA insert was found to contain an open reading frame of 579 codons corresponding to a signal peptide of 30 amino acids and the mature 549‐residue ascorbate oxidase. Furthermore, it was found that the amino acid sequence deduced from the nucleotide sequence of the cDNA insert contained four potential N‐glycosylation sites and copper‐binding amino acid residues located in four regions where the sequence was identical or nearly identical to those of the other known blue multicopper oxidases Neurospora crassa laccase and human ceruloplasmin.
Ubiquitin in Chlamydomonas reinhardii
Authors: WETTERN Michael , PARAG Hadas A. , POLLMANN Lutz , OHAD Itzhak , KULKA Richard G.
Published: March 2005

Ubiquitin, a highly conserved 76‐amino‐acid protein, is involved in the response of many types of eukaryotic cells to stress but little is known about its role in lower plants. In the present study we have investigated the distribution of ubiquitin in the unicellular alga Chlamydomonas reinhardii as well as the effect of heat and light stress on its conjugation to cellular proteins. Immunoelectron microscopy shows that ubiquitin is located in the chloroplast, nucleus, cytoplasm, pyrenoid and on the plasma membrane. The location of ubiquitin within chloroplasts has not been observed previously. In immunoblots of whole cell extracts with an antibody to ubiquitin a prominent conjugate band with an apparent molecular mass of 29 kDa and a broad region of high‐molecular‐mass conjugates (apparent molecular mass > 45 kDa) were observed. Exposure of cells to a 41.5°C heat shock in both the dark and light caused the disappearance of the 29‐kDa conjugate and an increase in the high‐molecular‐mass conjugates. After step down to 25°C the 29‐kDa conjugate reappeared while the levels of high‐molecular‐mass conjugates decreased. In light, the recovery of the 29‐kDa band was more rapid than in the dark. Photoinhibition alters the ubiquitin conjugation pattern similarly to heat shock, but to a lesser degree. These observations imply that, in Chlamydomonas, ubiquitin has a role in the chloroplast and in the response to heat and light stress.
Virus‐vs endotoxin‐induced activation of liver macrophages
Authors: BUSAM Klaus Johannes , HOMFELD Angela , ZAWATZKY Rainer , KÄSTNER Stefanie , BAUER Joachim , GEROK Wolfgang , DECKER Karl
Published: March 2005

The response of liver macrophages (Kupffer cells) to distinct pathogenic material was investigated by comparing virus‐ and endotoxin‐induced macrophage activation. Endotoxin‐induced stimulation and induction with Newcastle disease virus (NDV) or Sendai virus led to the release of the same pattern of prostanoids characterized by a predominant production of prostaglandin E2(PGE2). With respect to peptide mediators, hepatic macrophages secreted tumor necrosis factor‐α (TNF‐α) and interleukin‐6 after viral induction and endotoxin treatment, respectively. In response to viruses, however, much more interleukin‐6 and TNF‐α was detected than after endotoxin stimulation. Interferon type I (interferon‐α/β), on the other hand, was only detected in the supernatants of macrophages infected with viruses, but not of those exposed to endotoxin. This study also revealed that rat TNF‐α exists in several soluble species, some of which are glycosylated.
Interdependence of tumor necrosis factor, prostaglandin E2, and protein synthesis in lipopolysaccharide‐exposed rat Kupffer cells
Authors: PETERS Thomas , KARCK Ulrich , DECKER Karl
Published: March 2005

Kupffer cells are the main producers of tumor necrosis factor‐α (TNF; cachectin) and eicosanoids in the liver exposed to lipopolysaccharide (endotoxin; LPS). A very rapid but transient release of TNF is followed by a slow, steady synthesis of prostaglandin E2 (PGE2). TNF itself is able to provoke eicosanoid synthesis in Kupffer cells; the rate and pattern of prostaglandin production are similar to those observed after treatment with LPS. Anti‐TNF antibodies completely neutralize TNF action on Kupffer cells, thus ruling out any participation of contaminating LPS. LPS stimulation of PGE2 production in Kupffer cells is reduced by the antiserum to 50%, indicating an involvement of TNF in the stimulatory action of LPS. On the other hand, PGE2, a potent inhibitor of LPS‐elicited TNF release, is able to suppress LPS‐ but not TNF‐stimulated eicosanoid synthesis in rat Kupffer cells.

In addition to this autocrine circuit, extrahepatic factors participate in the regulation of Kupffer cell activation: glucocorticoids not only inhibit TNF or prostaglandin production, they also reverse the LPS‐specific changes in the prostaglandin pattern of Kupffer cells.

LPS, TNF or cycloheximide when given alone in the concentration range applied in this study do not affect the viability of rat Kupffer cells. However, the combinations of cycloheximide and either LPS or TNF cause rapid death of the cultured cells. The cytolytic potential of either combination cannot be alleviated by treatment with glucocorticoids.
Effect of mRNA secondary structure on the efficiency of translational initiation by eukaryotic ribosomes
Authors: ROY Patrice , RONDEAU Stéphane B. , VÉZINA Claire , BOILEAU Guy
Published: March 2005

We have used site‐directed mutagenesis to determine whether the structural context surrounding the AUG triplet influences its ability to be selected as an initiation codon by the eukaryotic preinitiation complex. AUG triplets were introduced in a loop and stem structure naturally occurring at the midpoint of the 129‐nucleotides‐long 5′‐untranslated region of the porcine proopiomelanocortin mRNA; one AUG triplet was inserted in the loop while another was inserted in the stem of the hairpin structure. The proopiomelanocortin cDNA and the mutant cDNAs were inserted downstream from the early promoter of an expression vector derived from simian virus 40 (SV40) and transfected into monkey kidney COS‐1 cells. Analysis of the proopiomeianocortin‐related peptides present in the culture medium 72 h after transfection revealed that both mutant cDNAs direct the synthesis of more proopiomelanocortin than the non‐mutant cDNA. The increased translational efficiency observed with both mutants is probably due to the decreased secondary structures of the shortened 5′‐untranslated region. In addition, comparison of the two mutants indicates that the mutant mRNA with the AUG triplet inserted in the loop region of the hairpin structure directs the synthesis of approximately 75% more proopiomelanocortin than the mutant mRNA with the AUG triplet inserted in the stem region of the same hairpin structure, supporting a role for the structural context in the efficiency of translational initiation.
The 1H‐NMR assignments of the aromatic resonances in complexes of Lactobacillus casei dihydrofolate reductase and the origins of their chemical shifts
Authors: BIRDSALL Berry , ARNOLD John R. P. , JIMENEZ‐BARBERO Jesus , FRENKIEL Thomas A. , BAUER Christopher J. , TENDLER Saul J. B. , CARR Mark D. , THOMAS Janette A. , ROBERTS Gordon C. K. , FEENEY James
Published: March 2005

All the aromatic proton resonances in the 500‐MHzNMR spectra of Lactobacillus casei dihydrofolate reductase have been assigned for several of its complexes with inhibitors. For the complexes with methotrexate and trimethoprim this was achieved by using a combination of NMR techniques in conjunction with a selectively deuterated protein designed to simplify the spectra such that nuclear Overhauser effect (NOE) connections could be detected with greater ease and certainty. By correlating these NOE data with crystal structure data on related complexes it was possible to assign all the aromatic resonances and to extend these assignments to spectra of other complexes of dihydrofolate reductase.

The conformation‐dependent chemical shifts observed for many of the resonances could be explained qualita‐tively, but not quantitatively, in terms of ring‐current shifts. The discrepancies between calculated ring‐current shifts and the observed conformation‐dependent shifts could not in general be accounted for satisfactorily in terms of carbonyl‐group anisotropic shielding contributions calculated using presently available models. In the case of the Hδ1,δ2 protons of Phe30 some of the discrepancy probably results from a difference in the conformation of the Phe ring between the solution and crystal states.
Fructose‐1,6‐bisphosphate‐activated pyruvate kinase from Escherichia coli
Authors: SPERANZA Maria L. , VALENTINI Giovanna , MALCOVATI Massimo
Published: March 2005

The allosteric properties of the fructose‐1,6‐bis‐phosphate‐activated pyruvate kinase from Escherichia coli were examined in the presence of a number of fructose bisphosphate analogues, as well as of increased ionic strength (NaCl) and of the hydrogen‐bond‐breaking agent, formamide.

Fructose 2,6‐bisphosphate, ribulose 1,5‐bisphosphate and 5‐phosphorylribose 1‐pyrophosphate gave allosteric activation (additive to that of fructose 1,6‐bisphosphate).

Formamide always decreased Vmax, but left unchanged the Km for phosphoenopyruvate, while it decreased the concentration of fructose bisphosphate required to give half‐maximal activity (K0.5).

NaCl increased the K0.5 for both phosphoenolpyruvate and fructose bisphosphate, leaving Vmax unchanged.

These results are consistent with ionic binding of fructose bisphosphate through phosphates and with a critical role of hydrogen bonds in stabilizing both the inactive and the active enzyme conformers.