bluuebunny/crossref_metadata_2025 huggingface dataset:
Effect of the tyrosine 96 hydrogen bond on the inactivation of cytochromeP‐450caminduced by hydrostatic pressure
Authors: DI PRIMO Carmelo , HUI BON HOA Gaston , DOUZOU Pierre , SLIGAR Stephen
Published: March 2005

The effects of removal of the tyrosine 96 hydrogen bond on the stability and conformational events of cytochromeP‐450camare presented in this communication. Hydrostatic pressure has been used as a tool to perturbe the structure leading to the formation of cytochromeP‐420, an inactivated but soluble and undenatured form of the enzyme. We show that the spin transition of cytochromeP‐450cam, which is known to be influenced by hydrostatic pressure, is affected by this single mutation. The free energy of stabilisation of native substrate‐free cytochromeP‐450camis not affected by the removal of the tyrosine 96 hydrogen bond via mutagenesis to phenylalanine, whereas the substrate‐bound protein shows a difference of 21 kJ/mol. These results, as well as an observed 110 ml/mol difference for the volume of the inactivation reaction between substrate‐bound native and mutant proteins, have been interpreted in terms of a more hydrated heme pocket for the site‐directed mutant at position 96 compared to the wild‐type protein where camphor is tightly bound via the tyrosine 96 hydrogen bond and water excluded from the active site.
The role of myeloperoxidase in the oxidation of biologically active polyhydroxyphenols (substituted catechols)
Authors: METODIEWA Diana , DUNFORD H. Brian
Published: March 2005

The reaction of myeloperoxidase with biologically active polyhydroxyphenols (substituted catechols): catecholamine, norepinephrine and 2,4,5‐trihydroxyphenylalanine [Phe(OH)3] were investigated by using the ESR spin‐stabilization technique and rapid‐scan spectrophotometry in the millisecond time scale. The results presented here indicate that dihydroxyphenols and trihydroxyphenols are substrates in the myeloperoxidase reaction. The data of ESR and rapid‐scan optical investigation of the myleoperoxidase reaction with the dihydroxyphenols catecholamine and norepinephrine clearly indicate a normal peroxidase‐type pathway of catecholamine degradation. The first evidence of o‐semiquinone radical formation as a product of the enzymatic oxidation of catecholamine by myeloperoxidase is reported. The results obtained by rapid‐scan spectrophotometric investigation of enzyme intermediate formation and decay showed qualitative agreement with the spin‐stabilization studies. The first results on the reaction of myeloperoxidase with the trihydroxyphenol Phe(OH)3 presented here, indicate that it plays a role as an electron donor for myeloperoxidase I, but we were unable to obtain evidence that a normal peroxidase cycle is occurring. The inhibitory effect of superoxide dismutase on product formation was evident and indicate the involvement of superoxide radicals in the process. Attention is drawn to the biochemical and toxicological implications of these and other related studies of substituted catechol peroxidation by mammalian peroxidases.
Phosphorylation‐mediated changes in the electrophoretic mobility of CD5 molecules
Authors: LOZANO Francisco , ALBEROLA‐ILA José , PLACES Lourdes , GALLART Teresa , VIVES Jordi
Published: March 2005

This work shows that tumor promoter agents (TPA) induce the post‐translational modification of the human lymphocyte surface CD5 antigen (Tp67) in several cellular types. Treatment of [32P]orthophosphate‐ and [35S]cysteine‐labeled normal and lymphoblastoid T and B cells with active tumor promoters induced the rapid, transitory and dose‐dependent appearance of hyperphosphorylated CD5 forms with higher apparent molecular masses. These changes in the electrophoretic mobility of CD5 molecules were independent of RNA and protein synthesis, as well as of differences in neuraminic acid content. The inhibition of the TPA‐mediated changes by protein kinase C inhibitors (staurosporine and 1‐(5‐isoquinolylsulfonyl)‐2‐methylpiperazine) indicated its protein‐kinase‐C‐mediated nature. Phosphatase digestion of CD5 immunoprecipitates reverted the TPA‐medicated mobility changes showing its dependence on phosphorylation. Neuraminidase digestion of intact cells revealed that the target of the TPA effects are surface‐expressed CD5 molecules. In conclusion, we suggest that the heterogeneity in the electrophoretic mobility induced by TPA could reflect some structural and/or functional differences within CD5 molecules.
Activation of a serine/threonine kinase that phosphorylates microtubule‐associated protein 1B in vitro by growth factors and phorbol esters in quiescent rat fibroblastic cells
Authors: HOSHI Minako , NISHIDA Eisuke , INAGAKI Masaki , GOTOH Yukiko , SAKAI Hikoichi
Published: March 2005

We have previously found and characterized a mitogen‐activated, serine/threonine‐specific protein kinase that specifically phosphorylates microtubule‐associated protein 2 (MAP2) in vitro, which we call here MAP2 kinase [Hoshi, M., Nishida, E. & Sakai, H. (1988) J. Biol. Chem. 263, 5396–5401; Hoshi, M., Nishida, E. & Sakai, H. (1989) Eur. J. Biochem. 184, 477–486]. In this study, we have found another serine/threonine‐specific protein kinase that is activated by various mitogens. The activated kinase utilized microtubule‐associated protein 1B (MAP1B) as the major substrate in vitro, so we tentatively call it MAP1B kinase (M1BK). M1BK was maximally activated 20–30 min after treatment of quiescent rat fibroblastic 3Y1 cells with epidermal growth factor (EGF), while MAP2 kinase was maximally activated within 5–10 min of EGF treatment. The EGF‐activated M1BK was eluted at about 0.15 M NaCl on a DEAE‐cellulose column, while the activated MAP2 kinase was eluted at about 0.1 M NaCl under the conditions used. The EGF‐activated M1BK was eluted as a single peak just after the activated MAP2 kinase on an HPLC gel‐filtration column. Histone, casein and ribosomal protein S6 were very poor substrates for the M1BK, while MAP2 and myelin basic protein were moderate substrates. The M1BK activity in cell extracts was inhibited by Ca2+, glycerol 2‐phosphate and Zn2+, and slightly enhanced by heparin. These data suggested that M1BK is distinct from previously described mitogen‐activated kinases such as MAP2 kinase, casein kinase II and S6 kinase. Pretreatment with cycloheximide or puromycin did not block the M1BK activation by EGF. Furthermore, incubation of the EGF‐activated M1BK with acid phosphatase inactivated the kinase activity. Therefore, M1BK may be activated by phosphorylation in EGF‐treated cells. In addition to EGF, 12‐O‐tetradecanoylphorbol 13‐acetate, platelet‐derived growth factor and insulin‐like growth factor‐I also induced the activation of M1BK in quiescent cells.
Nuclear‐magnetic‐resonance investigations of the biliverdin – apomyoglobin complex
Authors: MARKO Helmut , MÜLLER Norbert , FALK Heinz
Published: March 2005

The recently described biliverdin – apomyoglobin complex has been investigated by two‐dimensional NMR methods and molecular modeling with respect to the geometry of the chromophore and its position within the myoglobin pocket. Nuclear Overhauser effect correlations between the ligand and apoprotein amino acid residues prove that the bile pigment assumes a cyclic helical conformation and a position similar to heme in native myoglobin.
Structural and functional aspects of calcium homeostasis in eukaryotic cells
Authors: PIETROBON Daniela , Di VIRGILIO Francesco , POZZAN Tullio
Published: March 2005

The maintenance of a low cytosolic free‐Ca2+ concentration, ([Ca2+]i) is a common feature of all eukaryotic cells. For this purpose a variety of mechanisms have developed during evolution to ensure the buffering of Ca2+ in the cytoplasm, its extrusion from the cell and/or its accumulation within organelles. Opening of plasma membrane channels or release of Ca2+ from intracellular pools leads to elevation of [Ca2+]i; as a result, Ca2+ binds to cytosolic proteins which translate the changes in [Ca2+]i into activation of a number of key cellular functions.

The purpose of this review is to provide a comprehensive description of the structural and functional characteristics of the various components of [Ca2+]i homeostasis in eukaryotes.
Okadaic acid activates microtubule‐associated protein kinase in quiescent fibroblastic cells
Authors: GOTOH Yukiko , NISHIDA Eisuke , SAKAI Hikoichi
Published: March 2005

Okadaic acid is a potent and specific inhibitor of protein phosphatases 1 and 2A, and is a strong tumor promoter that is not an activator of protein kinase C. Treatment of quiescent cultures of rat fibroblastic 3Y1 cells with okadaic acid induced marked activation of a kinase activity that phosphorylated microtubule‐associated protein (MAP) 2 and myelin basic protein, but not histone or casein, in vitro. This activated kinase eluted at ∼ 0.15 M NaCl on a DEAE‐cellulose column and its apparent molecular mass was determined to be ∼ 40 kDa by gel filtration. Detection of the kinase activity in polyacrylamide gels containing substrate proteins after sodium dodecyl sulfate gel electrophoresis revealed that the okadaic‐acid‐activated kinase activity resided mainly in two closely related polypeptides with apparent molecular mass ∼ 40 kDa. The characteristics of this kinase were indistinguishable from those of the mitogen‐activated MAP kinase in the same cells. The okadaic‐acid‐activated MAP kinase was deactivated by protein phosphatase 2A treatment in vitro. These results suggest that MAP kinase is negatively regulated by protein phosphatases 1 and/or 2A in quiescent cells and therefore can be activated by inhibiting these protein phosphatases. Interestingly, the okadaic‐acid‐induced activation of MAP kinase was transient and epidermal‐growth‐factor‐induced activation was also transient, even in the presence of okadaic acid. These data may imply that protein phosphatases 1 and 2A are not involved in the deactivation of MAP kinase in cells.
Structural study of gizzard caldesmon and its interaction with actin
Authors: LEVINE Barry A. , MOIR Arthur J. G. , AUDEMARD Etienne , MORNET Dominique , PATCHELL Valerie B. , PERRY S. Victor
Published: March 2005

The interaction between actin and caldesmon that is associated with the inhibition of actomyosin ATPase activity in smooth muscle has been studied using 1H‐NMR spectroscopy. Binding studies using the intact molecules were complemented by the use of thrombic cleavage fragments of both turkey and chicken gizzard caldesmon as well as defined peptides of actin, in order to investigate the conformational properties of caldesmon and to localise regions of the primary structures that participate in protein‐protein contacts. The binding of caldesmon is shown to involve distinct segments on the N‐terminal region (residues 1–44) of actin, as previously observed for the inhibitory component of the thin filament of striated muscle, troponin I [Levine et al. (1988) Eur. J. Biochem. 153, 389–397]. The comparable structural properties of these tissue‐specific inhibitors of actomyosin ATPase and the similarities in their mode of interaction at the N‐terminal region of actin suggest common aspects to the structural mechanism for thin‐filament regulation in smooth and striated muscle. Unlike the inhibitory interaction of troponin I, however, the binding of caldesmon to the N‐terminal region of actin directly involves groups within residues 20–41 of actin that are also recognised by myosin subfragment 1. The complementary segment of caldesmon has been localised to a 15‐kDa thrombic fragment (residues 483–578) derived from the N‐terminal portion of a 35‐kDa proteolytic cleavage product from the C‐terminal of caldesmon whose interaction with actin is modulated by calmodulin. The results are discussed in relation to the calcium‐mediated mechanism for thin‐filament regulation in smooth and striated muscle.
Patterns of histone acetylation
Authors: THORNE Alan W. , KMICIEK Daniel , MITCHELSON Keith , SAUTIERE Pierre , CRANE‐ROBINSON Colyn
Published: March 2005

The N‐terminal domains of all four core histones are subject to reversible acetylation at certain lysine residues. This modification has been functionally linked to transcription, histone deposition at replication and to histone removal during spermatogenesis. To increase understanding of the significance of this modification we have studied the specificity of site utilisation in the monoacetyl, diacetyl and triacetyl forms of histones H3, H4 and H2B (histone H2A has only a single modification site), using pig thymus and HeLa cells as the source of histones. The HeLa histones were extracted from cells grown both with and without butyrate treatment. It is found that for histone H3 there is a fairly strict order of site occupancy: Lys14, followed by Lys23, followed by Lys18 in both pig and HeLa histones. Since the order and specificity is the same when butyrate is added to the HeLa cell cultures, we conclude that addition of the fatty acid does not scramble the specificity of site utilisation, but merely allows more of the natural forms of modified histone to accumulate. For histone H4, the monoacetyl form is exclusively modified at Lys16, but further addition of acetyl groups is less specific and progresses through sites 12, 8 and 5 in an N‐terminal direction. Similar results were obtained for H4 from both pig thymus and butyrate‐treated HeLa cells. Histone H2B could be studied in detail only from butyrate‐treated HeLa cells and a much lower level of site specificity was found: sites 12 and 15 were preferred to the more N‐ and C‐terminal sites at Lys5 and Lys20. The data reinforces the view that lysine acetylation in core histones is a very specific phenomenon that plays several functionally distinct roles. The high degree of site specificity makes it unlikely that the structural effects of acetylation are mediated merely by a generalised reduction of charge in the histone N‐terminal domains.
The primary structure of artemin from Artemia cysts
Authors: DE GRAAF Jack , AMONS Reinout , MÖLLER Wim
Published: March 2005

The primary structure of artemin, a major protein isolated from Artemia cysts, has been determined by direct Edman degradation of the purified protein. The amino‐terminal acetylated protein has 229 amino acid residues and a high content of histidine and cysteine/cystine. A search in the GenBank Data Base at Los Alamos, using the FASTA program [Pearson, W. R. & Lipman, D. J. (1988) Proc. Natl Acad. Sci. USA 85, 2444–2448] revealed a limited but unmistakable similarity to ferritin from vertebrates.
Binding and activation of plasminogen at the surface of Staphylococcus aureus
Authors: KUUSELA Pentti , SAKSELA Olli
Published: March 2005

Untreated Staphylococcus aureus cells, strain Cowan I, specifically bound 125I‐Glu‐plasminogen. The binding was inhibited by both unlabeled Glu‐plasminogen and Glu‐plasmin. The Lys form of plasminogen, which lacks the 8‐kDa amino‐terminal activation peptide, was approximately 100‐fold more effective than the Glu form in competing with the binding of 125I‐labeled Glu‐plasminogen. This suggests an increase in binding affinity upon removal of the activation peptide. Fibronectin, fibrinogen and IgG, plasma components known to bind to the staphylococcal surface, did not significantly interfere with the binding. The competing activity in plasma was abolished by specifically absorbing plasminogen from the plasma sample. l‐Lysine and a fragment of plasminogen containing three of the first five protein attachment domains present in the molecule (kringle structures) also competed with plasminogen for binding suggesting that the lysine‐binding sites of plasminogen were involved in its interaction with staphylococci. Scatchard analysis revealed high‐ and low‐affinity binding sites. Kd and the number of high‐affinity binding sites were 1.7 nM and 780 binding sites/bacterial cell, respectively.

125I‐Glu‐plasminogen bound to staphylococcal surface was converted to plasmin by tissue‐type plasminogen activator. The conversion took place also in the presence of plasma. If the conversion was carried out in the absence of low‐molecular‐mass plasmin inhibitors such as aprotinin, the bound Glu‐plasmin was further converted to Lys‐plasmin. The surface‐bound plasmin was enzymically active, as judged by digestion of the synthetic substrate, S‐2251.

The plasminogen conversion shown by the present experiments not only leads to the surface‐bound plasmin but seems to considerably increase the affinity of plasmin for its binding site. This may represent a physiologically relevant method for a bacterial cell to retain surface‐bound active plasmin which is also protected from its soluble plasma inhibitors. This novel mechanism for staphylococci to adopt surface‐bound proteolytic activity, without the interference of plasma components, may have some role in the tissue penetration and invasion of microbes during infection.
Isolation and nucleotide sequence of cDNA clone for bovine pancreatic anionic trypsinogen
Authors: LE HUEROU Isabelle , WICKER Catherine , GUILLOTEAU Paul , TOULLEC René , PUIGSERVER Antoine
Published: March 2005

A cDNA clone encoding an anionic form of bovine trypsinogen was isolated from a pancreatic cDNA library. The corresponding 855‐nucleotide mRNA contains a short 5′ noncoding region of 8 nucleotides and a long 3′ noncoding region of 56 nucleotides in addition to a poly(A) tail of at least 50 nucleotides. The deduced amino acid sequence for the anionic pretrypsinogen (247 residues) includes the N‐terminal 15‐amino‐acid signal peptide followed by an 8‐amino‐acid activation peptide. The zymogen (232 residues) contains an additional C‐terminal serine, compared with the amino acid sequence of bovine cationic trypsinogen. The identity between the anionic and cationic forms of bovine trypsinogen (65%) is lower than that existing between the anionic protein and other mammalian anionic trypsinogens (73–85%), suggesting that trypsin gene duplication in mammals occurred prior to the evolutionary events responsible for the species divergence. Bovine pancreatic anionic trypsin possesses all the key amino acids characteristic of the serine protease family.
Evidence for two types of complexes formed by yeast tyrosyl‐tRNA synthetase with cognate and non‐cognate tRNA
Authors: RUBELJ Ivica , WEYGAND‐DURAŠEVIČ Ivana , KUŠAN Željko
Published: March 2005

Polyacrylamide gel electrophoresis at pH 8.3 was used to detect and quantitate the formation of the yeast tyrosyl‐tRNA synthetase (an α2‐type enzyme) complex with its cognate tRNA. Electrophoretic mobility of the complex is intermediate between the free enzyme and free tRNA; picomolar quantities can be readily detected by silver staining and quantitated by densitometry of autoradiograms when [32P]tRNA is used. Two kinds of complexes of Tyr‐tRNA synthetase with yeast tRNATyr were detected. A slower‐moving complex is formed at ratios of tRNATyr/enzyme ≤ 0.5; it is assigned the composition tRNA · (α2)2. At higher ratios, a faster‐moving complex is formed, approaching saturation at tRNATyr/enzyme = 1; any excess of tRNATyr remains unbound. This complex is assigned the composition tRNA ·α2. The slower, i.e. tRNA · (α2)2 complex, but not the faster complex, can be formed even with non‐cognate tRNAs. Competition experiments show that the affinity of the enzyme towards tRNATyr is at least 10‐fold higher than that for the non‐cognate tRNAs. ATP and GTP affect the electrophoretic mobility of the enzyme and prevent the formation of tRNA · (α2)2 complexes both with cognate and non‐cognate tRNAs, while neither tyrosine, as the third substrate of Tyr tRNA synthetase, nor AMP, AMP/PPi, or spermidine, have such effects. Hence, the ATP‐mediated formation of the α2 structure parallels the increase in specificity of the enzyme towards its cognate tRNA.
Two‐dimensional NMR studies and secondary structure of cobrotoxin in aqueous solution
Authors: YU Chin , LEE Chang‐Shin , CHUANG Li‐Chin , SHEI You‐Rhon , WANG Chi Ying
Published: March 2005

The 1H‐NMR spectra of cobrotoxin, a neurotoxic protein isolated from Formosan cobra Naja naja atra, have been studied by two‐dimensional NMR techniques. Of 62 amino acid residues in cobrotoxin, the complete assignments of 58 residues have been made. The resonances from several of the remaining residues have been identified but not yet specifically assigned. The secondary structure of an antiparallel triple‐ and double‐stranded β‐sheet has also been determined by observing the NOE.
Nuclear‐magnetic‐resonance studies of human epidermal growth factor
Authors: COOKE Robert M. , TAPPIN Michael J. , CAMPBELL Iain D. , KOHDA Daisuke , MIYAKE Tetsuo , FUWA Toru , MIYAZAWA Tatsuo , INAGAKI Fuyuhiko
Published: March 2005

The 1H‐NMR spectra of native human epidermal growth factor (EGF) and a derivative lacking the final five residues have been assigned by two‐dimensional methods, enabling their structures to be compared. The same structural features are observed for each protein, although the final five residues of native human EGF interact with residues earlier in the sequence. Comparison of the resonance shifts of human, rat and mouse EGF and human transforming growth factor α (TGFα) enables shifts characteristic of the EGF conformation to be identified, providing standards by which the structures of related proteins may be assessed.
Structural characterization of sialylated tetrasaccharides and pentasaccharides with blood group H and Lex activity isolated from bovine submaxillary mucin
Authors: SAVAGE Angela V. , DONOHUE John J. , KOELEMAN Carolien A. M. , van den EIJNDEN Dirk H.
Published: March 2005

In this study we have investigated the structures of a sialylated tetrasaccharide and two sialylated pentasaccharides released from bovine submaxillary mucin by alkaline borohydride treatment and isolated by high‐performance liquid chromatography. The tetrasaccharide contained NeuGc, while one of the pentasaccharides contained NeuAc and the other contained NeuGc. All three oligosaccharides contained the core type‐3 structure (GlcNAcβ1→3GalNAcol). The structures, determined by a combination of one‐ and two‐dimensional 1H‐NMR spectroscopy at 270 MHz and methylation analysis involving gas‐liquid chromatography/mass spectrometry, were as follows:

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The oligosaccharides occurred in the approximate molar ratios, 1.0:0.6:0.3. This is the first report of these oligosaccharides in bovine submaxillary mucin. 1H‐NMR data for structures A1/2c and A1/2e, which are novel structures, are presented for the first time. Oligosaccharide A1/2e contains the blood‐group‐H type‐2 antigenic determinant while oligosaccharide A1/2d contains the Lewisx determinant.
Ascorbyl free radical as a reliable indicator of free‐radical‐mediated myocardial ischemic and post‐ischemic injury
Authors: PIETRI Sylvia , CULCASI Marcel , STELLA Lucien , COZZONE Patrick J.
Published: March 2005

The real‐time kinetics of the release of ascorbyl free radicals in the coronary perfusate from isolated rat hearts submitted to an ischemia/reperfusion sequence has been achieved by continuous‐flow ESR using high‐speed acquisition techniques. Enhanced ESR detection of ascorbyl free radicals was obtained by addition of dimethyl sulfoxide (Me2SO), a strong cation chelator and oxidizing agent. A continuous‐flow device allowed a direct monitoring of the ascorbyl free radical and/or ascorbate leakage in coronary perfusate by observation of the ascorbyl radical doublet (aH= 0.188 mT and g= 2.0054).

The results showed that ascorbyl free radical release occurred mainly during sequences of low‐flow ischemia (90 min) coupled or not with 30 min of zero‐flow ischemia followed by reperfusion (60 min). The kinetic profiles of ascorbyl‐free‐radical detection confirm in quantitative terms the expected correlation between the duration of the ischemic insult and the magnitude of ascorbate extracellular release upon reperfusion. There is indication that ascorbyl free radical depletion could be secondary to oxygen‐derived‐free‐radical‐induced cellular damage.

The amount of residual ascorbic acid was quantitated on myocardial tissue at the end of reperfusion using Me2SO as extracting solvent. Intense oxidation of ascorbate and chemical stabilization of the resulting free radical species provided by Me2SO allowed ESR measurement of a marked tissue ascorbate depletion related to the duration of ischemia.

Perfusion of superoxide dismutase during low‐flow ischemia and the first 10 min of reperfusion greatly inhibited both extracellular release and endogenous ascorbate depletion. These results suggest that the ascorbate redox system constitutes a major protective mechanism against free‐radical‐induced myocardial injury.

The proposed direct ESR detection of ascorbyl free radicals in the coronary perfusates or in tissue extracts does not require extensive chemical preparation and conditioning of effluent or tissue samples. It provides an interesting straightforward alternative to the evaluation of detrimental free radical processes affecting the myocardium during ischemia and reperfusion.
A ΔφH clamp method for analysis of steady‐state kinetics of photophosphylation
Authors: STROTMANN Heinrich , THELEN Renate , MÜLLER Wolfgang , BAUM Wolfgang
Published: March 2005

An instrumental device is described which allows steady‐state kinetic measurements of photophosphorylation at adesired proton gradient which can be maintained throughout the course of the experiment (‘Δφ clamp’). This is achieved by electronic regulation of light intensity using the calibrated 9‐aminoacridine fluorescence signal as sensor of the gradient. The instrument is suitable for determination of kinetic parameters of the proton‐translocating ATPase in isolated envelope‐free chloroplasts under defined conditions.

At clamped Δφ, phosphorylation as a function of substrate concentration shows Michaelis‐Menten kinetics. The true Michaelis constantsand the dissociation constants for phosphate and ADP are reported. The Michaelis constants are not affected by the magnitude of the proton gradient in the investigated range. The significance of these results is discussed.
Reconstitution of CF0F1 into liposomes using a new reconstitution procedure
Authors: RICHARD Peter , RIGAUD Jean‐Louis , GRÄBER Peter
Published: March 2005

The H+‐ATPase (ATP synthase) from chloroplasts was isolated, purified and reconstituted into phosphatidylcholine/phosphatidic‐acid liposomes. Liposomes prepared by reverse‐phase evaporation were treated with various amounts of Triton X‐100 and protein incorporation was studied at each step of the solubilization process. After detergent removal by SM2‐Biobeads, the activities of the resulting proteoliposomes were measured indicating that the most efficient reconstitution was obtained by insertion of the protein into preformed, detergent‐saturated liposomes. The conditions for the reconstitution were optimized with regard to ATP synthesis driven by an artificially generated ΔpH/ΔΨ. An important benefit of the new reconstituted CF0F1 liposomes is the finding that the rate of ATP synthesis remains constant up to 10 s, indicating a low basal membrane permeability.
Amino acid sequence of a 12‐kDa inhibitor of protein kinase C
Authors: MOZIER Ned M. , ZÜRCHER‐NEELY Heidi A. , GUIDO David M. , MATHEWS W. Rodney , HEINRIKSON Robert L. , FRASER Elaine D. , WALSH Michael P. , PEARSON James D.
Published: March 2005

The complete primary structure of a bovine‐brain‐derived inhibitor of protein kinase C has been established. Fragments of the purified protein were obtained by cleavage with cyanogen bromide, Staphylococcus aureus V8 protease, trypsin and chymotrypsin. Subsequent analysis of the resulting fragments by fast‐atom‐bombardment mass spectrometry and Edman degradation revealed a calculated molecular mass of 11779 Da with the following 107‐amino‐acid sequence:

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This inhibitor does not share significant primary structural identity with any other known protein.
Ultimate limits for the reaction flux and metabolite levels that may be evolutionarily reached in a linear metabolic pathway
Authors: PETTERSSON Gösta , PETTERSSON Peter
Published: March 2005

A relationship is derived for the maximum steady‐state reaction rate that may be supported by an enzyme catalysing substrate/product interconversion by a generalized Michaelian mechanism for a single‐substrate reaction. This relationship is used to characterize the ultimate kinetic and thermodynamic limits for the evolutionary improvement of a linear metabolic sequence of reactions catalysed by Michaelian enzymes in response to a selective pressure in the direction of increased reaction flux. A mathematical analysis is presented which provides explicit expressions for the maximum reaction flux and metabolite concentrations that can be evolutionarily reached in such a pathway. These expressions may be used to obtain information on the reaction steps that represent ultimate bottlenecks for the attainment of high reaction flux in a certain pathway and to identify the enzymes that ultimately are likely to exert main flux control.
What metabolite levels may be evolutionarily reached in the glycolytic pathway?
Authors: PETTERSSON Gösta
Published: March 2005

The thermodynamic and kinetic properties of the glycolytic pathway have been analyzed in order to characterize the ultimate limits for the metabolite concentrations that may be evolutionarily reached in response to a selective pressure in the direction of increased glycolytic reaction flux. The results indicate that the chemical potential for conversion of glucose into pyruvate at presently observed levels of these two metabolites (and of NADH, NAD+, ATP, ADP and inorganic phosphate) is high enough to allow ultimately for an accumulation of fructose 1,6‐bisphosphate at concentrations which may be considered as indefinitely high from a practical point of view. This means that non‐kinetic factors such as metabolite solubility put a definite limit to the glycolytic reaction flux that may be evolutionarily reached.

With the reasonable assumption that evolution eventually may raise the concentration of fructose 1,6‐bisphosphate minimally to the same level as that presently attained by glucose, analytical evidence is presented to show that catalytic improvement of enzymes in the glycolytic pathway ultimately may result in glycerone phosphate levels that are at least two orders of magnitude higher than those presently observed. This argues strongly against the idea that triose‐phosphate isomerase represents a perfectly evolved catalyst in the sense that it should have reached the end of its evolutionary development.
Photoaffinity labelling of the purine – cytosine permease of Saccharomyces cerevisiae
Authors: CHIRIO Maria‐Chantal , BRÈTHES Daniel , NAPIAS Christian , GRANDIER‐VAZEILLE Xavier , RAKOTOMANANA Félicien , CHEVALLIER Jean
Published: March 2005

8‐Azidoadenine was used as a photoaffinity reagent to characterize the purine–cytosine permease of Saccharomyces cerevisiae. It is a potent competitive inhibitor of cytosine uptake and irradiation of the cells incubated with the label induced the irreversible inactivation of cytosine uptake. Addition of excess cytosine prevented this labelling which was restricted to the outer face of the plasma membrane since it was not accumulated by the cells.

In the strain with the amplified purine–cytosine permease gene the maximum cytosine uptake rate was increased 4–5‐fold relative to wild type without a modification of the Michaelis constant of uptake (Kt); no uptake could be measured in the deleted strain. The relative amounts of specific labelling determined for the cells and for membrane preparations were 0, 1 and 4 for the null, the wild‐type and the amplified strains, respectively. One major band specifically labelled by [3H]azidoadenine, corresponding to a polypeptide with an apparent molecular mass of 45 kDa, was observed in the wild type, amplified in the strain carrying the multicopy plasmid and not detected in the deleted strain. Therefore this polypeptide corresponds to the purine–cytosine permease.
Metabolism of cysteinyl leukotrienes in monkey and man
Authors: HUBER Michael , MÜLLER Juliane , LEIER Inka , JEDLITSCHKY Gabriele , BALL Howard A. , MOORE Kevin P. , TAYLOR Graham W. , WILLIAMS Roger , KEPPLER Dietrich
Published: March 2005

The proinflammatory cysteinyl leukotrienes are inactivated in primates by (a) intravascular degradation, (b) hepatic and renal uptake from the blood circulation, (c) intracellular metabolism of leukotriene E4 (LTE4), and (d) biliary and renal excretion of LTC4 degradation products. We have analyzed cysteinyl leukotriene metabolites excreted into bile and urine of the monkey Macaca fascicularis and of man. In both species, hepatobiliary leukotriene elimination predominated over renal excretion. In a representative healthy human subject at least 25% of the administered radioactivity were recovered from bile and 20% from urine within 24 h. In monkey and man intravenous administration of 14,15‐3H2‐labeled LTC4 resulted in the biliary and urinary excretion of labeled LTE4, ω‐hydroxy‐LTE4, ω‐carboxy‐LTE4, ω‐carboxy‐tetranor‐ dihydro‐LTE4, and ω‐carboxy‐tetranor‐dihydro‐LTE4. Small amounts of N‐acetyl‐LTE4 were detected in human urine only. Oxidative metabolism of LTE4 proceeded more rapidly in the monkey resulting in the formation of higher relative amounts of ω‐oxidized leukotrienes in this species as compared to man. [3H]H2O amounted to less than 2% of the administered dose in monkey and human bile and urine samples. Incubation of isolated human hepatocytes with [3H2]LTC4, [3H2]LTE4, and [3H2]LTD4 showed that only [3H2]LTE4 underwent intracellular oxidative metabolism resulting in the formation of ω‐ and β‐oxidation products. N‐Acetylated LTE4 derivatives were not detected as products formed by human hepatocytes.

By a combination of reversed‐phase high‐performance liquid chromatography and radioimmunoassay, endogenous LTE4 and N‐acetyl‐LTE4 were detected in human urine in concentrations of 220 ± 40 and 24 ± 3 pM, corresponding to 12 ± 1 and 1.5 ± 0.2 nmol/mol creatinine, respectively (mean ± SEM; n= 10). Endogenous LTD4 and LTE4 were detected in human bile (n= 3) in concentrations between 0.2–0.9 nM. Our results demonstrate that LTD4 and LTE4 are major LTC4 metabolites in human bile and/or urine and may serve as index metabolites for the measurement of endogenously generated cysteinyl leukotrienes. Moreover, ω‐oxidation and subsequent β‐oxidation from the ω‐end contribute to the metabolic degradation of LTE4 not only in monkey but also in man.
Self recognition by the immune system
Authors: VON BOEHMER Harald
Published: March 2005

In each organism, the immune system must acquire the ability to distinguish self from nonself. Experiments in T cell receptor transgenic mice indicate that this process involves the selection of lymphocytes in the thymus.
Eukaryotic DNA replication
Authors: THÖMMES Pia , HÜBSCHER Ulrich
Published: March 2005

A complex network of interacting proteins and enzymes is required for DNA replication. Much of our present understanding is derived from studies of the bacterium Escherichia coli and its bacteriophages T4 and T7. These results served as a guideline for the search and the purification of analogous proteins in eukaryotes. Model systems for replication, such as the simian virus 40 DNA, lead the way.

Generally, DNA replication follows a multistep enzymatic pathway. Separation of the double‐helical DNA is performed by DNA helicases. Synthesis of the two daughter strands is conducted by two different DNA polymerases: the leading strand is replicated continuously by DNA polymerase δ and the lagging strand discontinuously in small pieces by DNA polymerase α. The latter is complexed to DNA primase, an enzyme in charge of frequent RNA primer syntheses on the lagging strand. Both DNA polymerases require several auxiliary proteins. They appear to make the DNA polymerases processive and to coordinate their functional tasks at the replication fork. 3′→ 5′‐exonuclease, mostly part of the DNA polymerase δ polypeptide, can perform proof‐reading by excising incorrectly base‐paired nucleotides. The short DNA pieces of the lagging strand, called Okazaki fragments, are processed to a long DNA chain by the combined action of RNase H and 5′→ 3′‐exonuclease, removing the RNA primers, DNA polymerase α or β, filling the gap, and DNA ligase, sealing DNA pieces by phosphodiester bond formation. Torsional stress during DNA replication is released by DNA topoisomerases. In contrast to prokaryotes, DNA replication in eukaryotes not only has to create two identical daughter strands but also must conserve higher‐order structures like chromatin.
Construction, expression and unexpected regulatory properties of a tropomyosin mutant with a 31‐residue deletion at the C‐terminus (exon 9)
Authors: BARTEGI Aghleb , FERRAZ Conception , FATTOUM Abdellatif , SRI WIDADA Joannes , HEITZ Frédéric , KASSAB Ridha , LIAUTARD Jean‐Pierre
Published: March 2005

The cDNA coding for human skeletal muscle β‐tropomyosin was expressed in Escherichia coli to produce an unacetylated β‐tropomyosin. This cDNA was deleted from the sequence corresponding to the exon 9 and expressed in E. coli to produce an unacetylated β‐tropomyosin mutant lacking the C‐terminal residues 254–284. The main structural and functional properties of the two isolated proteins, designated tropomyosin‐1 and des‐(254–284)‐tropomyosin, respectively, were characterized in comparison with those of the genuine rabbit skeletal muscle αβ‐tropomyosin. The folding and thermal stability of the three tropomyosins were indistinguishable. Tropomyosin‐1, but not des‐(254–284)‐tropomyosin, was polymerized in the presence of troponin and did bind to actin in the presence of the troponin complex. Despite its weak binding to actin, des‐(254–284)‐tropomyosin displayed a regulatory function in the presence of troponin with a marked activation of the actomyosin subfragment‐1 ATPase in the presence of Ca2+ and low concentrations of subfragment‐1. The data were interpreted in the light of the allosteric models of regulation and suggest the involvement of the sequence coded by exon 9 in the stabilization by tropomyosin of the off state of the thin filament.
Over‐production, purification and properties of the uridine‐diphosphate‐N‐acetylmuramoyl‐l‐alanyl‐d‐glutamate: meso‐2,6‐diaminopimelate ligase from Escherichia coli
Authors: MICHAUD Catherine , MENGIN‐LECREULX Dominique , van HEIJENOORT Jean , BLANOT Didier
Published: March 2005

The UDP‐N‐acetylmuramoyl‐l‐alanyl‐d‐glutamate: meso‐2,6‐diaminopimelate ligase was over‐produced and purified from two plasmid‐harbouring strains of Escherichia coli. The first strain, E. coli JM83(pHE5), gave a 15‐fold over‐production relative to parental strain. The enzyme could be partially purified (8.8‐fold) by ion‐exchange chromatography. With the second strain, E. coli JM83(pMLD25), a very strong over‐production was obtained, since the enzyme represented about 20% of the cytoplasmic proteins. Purification yielded 77% protein homogeneity. However, the enzymatic activity, which was very unstable, was lost during the purification procedure. Several properties of the enzyme were studied. The enzyme gave maximal activity around pH 8. The isoelectric point was 5.2. The activity was increased by potassium phosphate. Reverse and exchange reactions could be catalysed. The N‐terminal sequence of the protein was determined and correlated with the nucleotide sequence of the mur E gene. The actual initiation codon was assigned.
Expression of human liver cytochrome P450 IIIA4 in yeast
Authors: RENAUD Jean‐Paul , CULLIN Christophe , POMPON Denis , BEAUNE Philippe , MANSUY Daniel
Published: March 2005

Cytochrome P‐450 (P450) NF, a member of the P450 IIIA subfamily, is the major contributor to the oxidation of the calcium‐channel blocker nifedipine in human liver microsomes. A cDNA clone designated NF25 encoding for human P450 NF was isolated from a bacteriophage λgt11 expression library [Beaune, P. H., Umbenhauer, D. R., Bork, R. W., Lloyd, R. S. & Guengerich, F. P. (1986) Proc. Natl Acad. Sci. USA 83, 8064–8068]. We have expressed NF25 cDNA in Saccharomyces cerevisiae using an expression vector constructed from pYeDP1/8–2 [Cullin, C. & Pompon, D. (1988) Gene 65, 203–217]. Yeast transformed with the plasmid containing the NF25 sequence (pVNF25) showed a ferrous – CO spectrum typical of cytochrome P‐450. Microsomal preparations contained a protein with an apparent molecular mass identical to that of P450‐5 (a form isolated from human liver indistinguishable from P450 NF) that was not present in microsomes from control yeast (transformed with pYeDP1/8–2 alone), as revealed by immunoblotting with anti‐P450–5 antibodies. On the other hand, antibodies raised in rabbits against human liver P450 IIC8–10 and rat liver P450 IA1 and P450 IIE1 did not recognize yeast‐expressed P450 NF25. The P450 NF25 content in microsomes was about 90 pmol/mg protein. Microsomal, yeast‐expressed P450 NF25 exhibited a high affinity for different substrates including macrolide antibiotics, dihydroergotamine and miconazole as shown by difference visible spectroscopy. Microsomal suspensions containing P450 NF25 were also able to catalyze several oxidation reactions that were expected from the activities of the protein isolated from human liver, including nifedipine 1,4‐oxidation, quinidine 3‐hydroxylation and N‐oxygenation, and N‐demethylation of the macrolide antibiotics erythromycin and troleandomycin. The yeast endogenous NADPH—cytochrome P‐450 reductase thus couples efficiently with the heterologous P450 NF25 though its level is far lower than that of its ortholog in human liver. Indeed addition of rabbit liver NADPH —cytochrome P‐450 reductase increased the oxidation rates. Rabbit liver cytochrome b5 also caused a marked enhancement of catalytic activities, as had been noted previously for this particular P450 enzyme in a reconstituted system involving the protein purified from human liver. Furthermore, the level of the yeast endogenous cytochrome P‐450 (lanosterol 14‐demethylase) has been found to be negligible compared to the heterologously expressed cytochrome P‐450 (30 times less). Thus, yeast microsomes containing P450 NF25 constitute by themselves a good functional model for studying the binding capacities and catalytic activities of this individual form of human hepatic cytochrome P‐450.
A highly active and oxidation‐resistant subtilisin‐like enzyme produced by a combination of site‐directed mutagenesis and chemical modification
Authors: GRØN Hanne , BECH Lene M. , BRANNER Sven , BREDDAM Klaus
Published: March 2005

The subtilisins are known to be susceptible to chemical oxidation due to the conversion of Met222 into the corresponding sulfoxide. A number of derivatives with resistance towards oxidation have previously been prepared by replacement of this group with the other 19 amino acid residues. Unfortunately, the activities of these enzymes were of the order of 1–10% of that obtained with the wild‐type enzyme. In contrast, the oxidation‐labile cysteine mutant exhibited much higher activity, suggesting that this is associated with the presence of a sulphur atom in the amino acid at position 222.

It is shown here that it is possible to maintain a sulphur atom in the amino acid at position 222 without the enzyme becoming labile towards oxidation. A subtilisin from Bacillus lentus, subtilisin 309, in which Met222 was replaced with a cysteinyl residue by site‐directed mutagenesis was modified with thioalkylating reagents. Treatment of such enzyme derivatives with H2O2 revealed that their stabilities towards oxidation had increased significantly compared to both wild‐type and unmodified [Cys222]subtilisin. One of the chemically modified enzyme derivatives, [Me‐S‐Cys222]subtilisin, exhibited a kcat/Km value of 56% of that obtained with the wild‐type enzyme when assayed against the substrate Suc‐Ala‐Ala‐Pro‐Phe‐NH‐Ph‐NO2 (Suc, succinyl) and it exhibited 89% activity when tested in an assay with dimethyl casein as a substrate. The corresponding values obtained for unmodified [Cys222]subtilisin were lower, i.e. 39% for the dimethyl casein activity and 46% for the kcat/Km for the hydrolysis of Suc‐Ala‐Ala‐Pro‐Phe‐NH‐Ph‐NO2. This demonstrates the feasibility of replacing the oxidation‐labile methionyl residue group in a subtilisin enzyme with a group stable towards oxidation without substantially reducing the activity.
Purification of a cysteine endopeptidase which is secreted with bioactive peptides from the epidermal glands of Xenopus laevis
Authors: DARBY Nigel J. , LACKEY David B. , SMYTH Derek G.
Published: March 2005

The purification is reported of an endopeptidase, XSCEP1 (Xenopus skin cysteine endopeptidase), present in skin secretions of Xenopus. The procedure involved an initial concentration of the enzyme by batchwise anion‐exchange chromatography and ammonium sulphate precipitation. The proteolytic activity, determined with Z‐Phe‐Arg‐Amc (Z, benzyloxycarbonyl; Amc, 7‐amidomethylcoumarin) as substrate, was fractionated by gradient ion‐exchange chromatography, yielding a major component which was purified to homogeneity by chromatography on an organomercury‐agarose column. SDS/PAGE demonstrated the presence of a single protein with a molecular mass of 27 kDa. The purified enzyme, which possesed a pH optimum of 5.5, exhibited the properties of a cysteine endopeptidase: it was activated by dithiothreitol and EDTA and inhibited by the mechanism‐based inhibitor trans‐epoxysuccinyl‐l‐leucylamido(4‐guanidino)butane. XSCEP1 exhibited a marked preference for substrates with a hydrophobic residue in the P1 position and arginine in the P2 position as opposed to a substrate with arginine residues in both positions. The enzyme was also able to cleave a Val‐Arg‐Gly sequence in a model substrate, reflecting cleavages undergone by a number of peptides present in Xenopus skin. The results point to a functional role for XSCEP1 as a putative processing enzyme.
Fe3+2‐transferrin and Fe3+2‐asialotransferrin deliver iron to hepatocytes by an identical mechanism
Authors: SHARMA Ravi J. , GRANT David A. W.
Published: March 2005

We have been unable to demonstrate the unequivocal presence of transferrin receptors on rat hepatocytes. The binding and presumed internalisation of 125I‐Fe3+2‐transferrin by freshly isolated hepatocytes was only partially inhibited by up to a 104‐fold molar excess of unlabelled ligand and was virtually insensitive to chloroquine. There would appear to be only a weak association between this ligand and some component of the hepatocyte cell surface. These results were not compatible with the commitment of Fe3+2‐transferrin to either a receptor‐mediated endocytotic pathway nor to a rapid recycling pathway through sorting endosomes. Desialylation of the biantennary oligosaccharide side chains of Fe3+2‐transferrin engendered a low affinity (Kd≥ 0.25 μM) for the asialoglycoprotein receptor. 125I‐Fe3+2‐asialotransferrin was only superficially internalised by isolated hepatocytes but this was characteristic of ligands for the asialoglycoprotein receptor which have only biantennary (or single triantennary) side chains. This was also incompatible with the delivery of the ligand to sorting endosomes where the release of iron has been presumed to occur. Despite the different properties of the two ligands, the rates of iron uptake from 59Fe3+2‐transferrin and 59Fe3+2‐asialotransferrin were identical, suggesting a common mechanism for the translocation of iron across the plasma (or possibly endosomal) membrane such as a transmembrane oxidoreductase. Competition studies with unlabelled ligand or impermeable ferric ammonium citrate gave an IC50 of 1 – 15 μg Fe3+/ml for this process. The absence of transferrin receptor from the surface of the terminally differentiated, quiescent hepatocyte would be compatible with the dual roles suggested for transferrin as an iron transport protein and as a growth factor. The release of iron at the hepatocyte cell surface would effectively uncouple the two functions and render the hepatocyte unresponsive to growth stimulation by transferrin.