bluuebunny/crossref_metadata_2025 huggingface dataset:
Specific synthesis of adenosine(5′)tetraphospho(5′)nucleoside and adenosine(5′)oligophospho(5′)adenosine (n > 4) catalyzed by firefly luciverase
Authors: ORTIZ Begoña , SILLERO Antonio , GÜNTHER SILLERO María A.
Published: March 2005

Luciferase catalyzes the preferential synthesis of adenosine(5′)tetraphospho(5′)nucleoside (Ap4N) in the presence of luciferin (LH2), adenosine 5′‐[γ‐thio]triphosphate (ATP[γS]) and NTP (other than ATP), with very low, or undetectable synthesis of Ap4A or Np4N, because ATP[γS] is a good adenylyl donor for the formation of the E‐LH2‐AMP complex, but a poor adenylyl acceptor from the complex, and NTP, other than ATP, are bad nucleotidyl donors, but good acceptors of the AMP moiety of the E‐LH2‐AMP complex. Synthesis of the corresponding Ap4N (or Ap5G in the case of p4G were obtained in the presence of ATP[γS] and GTP, UTP, CTP, XTP, dTTP, ITP, dGTP, dCTP, dITP, ɛATP (ɛA, N6‐ethenoadenosine) or p4G. The yield of synthesis of Ap4N was at least 50% of that theoretically expected. The process can be easily scaled‐up, which allows synthesis of at least 1–5 μmol Ap4N. Further evidence for the synthesis of Ap4G from ATP[γS] and GTP was obtained by 1H‐NMR and 31P‐NMR spectroscopy. Synthesis of Ap4N in yields lower than those above, can also be obtained in the presence of ADP and NTP; synthesis is due to the presence in commercial luciferase of enzymes (adenylate kinase and NDP kinase) that catalyze the synthesis of ATP from ADP and NTP. In the presence of ATP and polyphosphates, luciferase catalyzes the synthesis of a variety of compounds of adenosine 5′‐polyphosphates (pnA; n= 3–20 and ApnA; n= 4–16). In the presence of P3 or P4, preferential synthesis of p4A and Ap5A or p5A and Ap6A were obtained, respectively, showing that both polyphosphates accept the adenylyl moiety of the E‐LH2‐AMP complex. Polyphosphates of chain length 5, 15 and 35 elicited the synthesis of a variety of PnA and ApnA. Ap4A is also split by luciferase in the presence of P3 or P4 (but not in the presence of P5) yielding preferential synthesis of p4A and Ap5A, or p5A and Ap6A, respectively.
Carotenoid‐binding proteins of photosystem II
Authors: BASSI Roberto , PINEAU Bernard , DAINESE Paola , MARQUARDT Jürgen
Published: March 2005

The distribution of the photosynthetic pigments of the chlorophyll‐binding proteins or photosystem‐II membranes, isolated from dark‐adapted maize leaves was determined. Most (80%) of a xanthophyll, violaxanthin, was found in the three minor chlorophyll‐a/bproteins CP24, CP26 and CP29 whose function is unknown. Violaxanthin is the precursor of zeaxanthin, which is involved in dissipating excess excitation energy into heat [Demming‐Adams, B. (1991)Biochim. Biophys. Acta 1020, 1–24] under conditions of high transmembrane pH gradient [Gilmore, A. M. & Yamamoto, H. Y. (1992)Proc. Natl Acad. Sci. USA 89, 1899–1903]. We propose that a role for the minor photosystem‐II chlorophyll‐a/bproteins is the regulation of energy transfer to the reaction centre. It was also confirmed that the photosystem II reaction centre (D1‐D2‐cytochromeb559) contains β‐carotene as the only carotenoid. However, the two other chlorophyll‐a‐binding proteins of photosystem II, CP47 and CP43, bind not only β‐carotene, but also the xanthophyll lutein, previously thought to be restricted to chlorophyll‐a/bproteins.
Synthetic immunogens.
Authors: MORODER Luis , MUSIOL Hans‐Jürgen , KÖCHER Klaus , BALI Jean P. , SCHNEIDER Conrad H. , GUBA Wolfgang , MÜLLER Gerhard , MIERKE Dale F. , KESSLER Horst
Published: March 2005

Chimeras of the double chain bis‐cystinyl hinge fragment 225–232/225′–232′ of human IgG1 and of peptides related to human little‐gastrin were synthesized, whereby the fully bioactive gastrin sequences 2–17 and 5–17 were amide‐bond‐linked N‐ and N‐ or C‐terminally, respectively, to the hinge peptide. All the dimeric constructs proved to be efficient immunogens; however, both the configuration of the constructs and the length of the haptenic gastrin molecule were found to drastically affect the specificity of the antibody response and, thus, the type of dominant immune epitope expressed. The different degree of accessibility of the gastrin chains in the dimers is similarly reflected by their binding affinities to gastrin receptors and their bioactivities in vivo. Molecular dynamics simulations of the chimeric compounds clearly revealed that the conformational space of the gastrin peptide chains 2–17 and 5–17 is strongly restricted upon linkage to the hinge peptide. Only in the gastrin‐(2–17) construct does sufficient free conformational space seem to be retained, at least for one of the two gastrin chains, in order to allow folding into the bioactive structure. This also agrees with the observation that the dimeric gastrin‐(2–17) behaves like a gastrin monomer in terms of receptor binding affinity and biopotency in vivo; but it could additionally explain why an antibody response of gastrin receptor‐like specificity could only be induced with this construct. The experimental data may therefore suggest a high degree of parallelism between the mechanism of recognition of the gastrin peptides in the dimeric constructs as hormonal ligands by the gastrin receptors and as haptens by the immune competent cells.
Defective biosynthesis of the lipid A component of temperature‐sensitive firA (omsA) Mutant of Escherichia coli
Authors: HELANDER Ilkka M. , LINDNER Buko , SEYDEL Ulrich , VAARA Martti
Published: March 2005

The biosynthesis of lipid A component was shown to be defective in a temperature‐senitive firA mutant of Escherichia coli. Cells were biosynthetically labelled with [14C]acetate and incorporation of radioactivity into the glycerophospholipid compared to lipid A fractions was measured. The lipid A/glycerophospholipid biosynthesis ratio of the firA mutant at 37°C was approximately 50% and at the nonpermissive temperature of 42°C was less than 20% of that observed in the corresponding wild‐type strain. Analysis of radiolabelled lipid A 4′‐monophosphate derivatives and glycerophospholipids by thin‐layer chromatography revealed that the firA mutant at 42°C elaborated an altered lipid A, and its phosphatidylglycerol content was low. The chemical composition of the extracted lipopolysaccharides differed significantly between the firA and the wild‐type strain only in the proportion of hexadecanoic acid, which was minimal in the wild type grown at 37°C and 42°C and in firA lipopolysaccharide grown at 37°C. In the firA mutant lipopolysaccharide produced at 42°C, hexadecanoic acid was present in approximately every third molecule, attached to the hydroxyl group of the amide‐linked (R)‐3‐hydroxytetradecanoic acid at the reducing glucosamine of lipid A. Inspection of dephosphorylated free lipid A preparations by laser‐desorption mass spectrometry confirmed that significant amounts of heptaacyl lipid A was elaborated by the firA strain grown at 42°C.
Selective eicosanoid formation during HL‐60 macrophage differentiation
Authors: NÜSING R. , GOERIG M. , HABENICHT A. J. R. , ULLRICH V.
Published: March 2005

Earlier studies on HL‐60 cells induced to differentiate into macrophages by phorbol esters have shown a selective stimulation of thromboxane (Tx) formation from endoperoxide prostagiandin (PG) H2, indicating that Tx synthesis is regulated at the level of Tx synthase (TxS), one of the peripheral enzymes of the PGH‐synthase pathway. We now report on the regulation of TxS during HL‐60 macrophage differentiation using monoclonal anti‐TxS serum and comparing turnover rates of TxS and its biological activity with those of other enzymes of arachidonic acid metabolism. Western‐blot analysis, enzyme‐linked immunosorbent assay, immunohistochemical staining and [35S]methionine‐labeling experiments suggested a phorbol‐ester‐dependent early induction of synthesis of TxS. [35S]Methionine incorporation into TxS was stimulated within 4 h after initiation of differentiation and was associated with a major rise in the TxS catalytical activity. Pulse‐chase experiments showed a half life for the TxS protein of 16.4 h in both control and phorbol‐ester‐treated cells. The biologicalhalf life of TxS was 10.5 h, as determined by PGH2 incorporation into TxB2 after cycloheximide treatment. In contrast, the biological half lives of PGH synthase, prostacyclin synthase and 5‐lipoxy‐genase were significantly shorter and were 3, 2.5 and 2.5 h, respectively. These results reveal that Tx synthesis in macrophages is mediated by at least two distinct mechanisms; a protein‐kinase‐C‐dependent induction of de novo synthesis of TxS and the selective resistance of the enzyme against the activity of protein kinase C.
N‐(7‐Nitrobenz‐2‐oxa‐1,3‐diazol‐4‐yI)colcemid, a probe for different classes of colchincine‐binding site on tubulin
Authors: SENGUPTA Suparna , PURI Kamal Deep , SUROLIA Avadhesha , ROY Siddhartha , BHATTACHARYYA Bhabatarak
Published: March 2005

The nature of binding of 7‐nitrobenz‐2‐oxa‐1,3‐diazol‐4‐yl‐colcemid (NBD‐colcemid), an environment‐sensitive fluorescent analogue of colchicine, to tubulin was tested. This article reports the first fluorometric study where two types of binding site of colchincine analogue on tubulin were detected. Binding of NBD‐colcemid to one of these sites equilibrates slsowly. NBD‐colcemid competes with colchicine for this site. Binding of NBD‐colcemid to this site also causes inhibition of tubulin self‐assembly. In contrast, NBD‐colcemid binding to the other site is characterised by rapid equilibration and lack of competition with colchicine. Nevertheless, binding to this site is highly specific for the cholchicine nucleus, as alkyl‐NBD analogues have no significant binding activity. Fast‐reaction‐kinetic studies gave 1.76 × 105 M–1 s–1 for the association and 0.79 s–1 for the dissociation rate constants for the binding of NBD‐colcemid to the fast site of tubulin. The association rate constants for the two phases of the slow site are 0.016 × 10–4 M–1 s–1 and 3.5 × 10–4 M–1 respectively. These two sites may be related to the two sites of colchicine reported earlier, with binding characteristics altered by the increased hydrophobic nature of NBD‐colcemid.
Purification and some properties of 11‐hydroxythromboxane B2 dehydrogenase from porcine kidney
Authors: WESTLUND Pär
Published: March 2005

A protein with NAD‐dependent 11‐hydroxythromboxane B2 dehydrogenase activity was purified to apparent homogeneity from porcine kidney using a relatively simple purification procedure, involving precipitation, anion‐exchange chromatography (diethylaminoethyl‐cellulose), affinity chromatography (5′‐AMP–Sepharose) and gel‐filtration chromatography (Protein Pak 125). The dehydrogenase was found to have a molecular mass of 50–55 kDa as determined by comparison with standards on SDS/PAGE. The molecular mass on gel‐filtration chromatography was dependent on the ionic strength of the buffer. The apparent Km and Vmax values for thromboxane B2 were also dependent of the ionic strength with a Vmax of 214 nmol min−1 mg−1 using 250 mM Tris/HCl, pH 8.0, and a corresponding Km of 2.9 mM. The enzyme was NAD dependent and was clearly separated from the proteins with 15‐hydroxyprostaglandin dehydrogenase activity also present in the kidney. Furthermore, it was found that 11‐hydroxythromboxane B2 dehydrogenase did not utilize prostaglandin D2, prostaglandin E2, prostaglandin F2α or cholic acid as substrate, and that the enzyme did not catalyse the reverse reaction, conversion of 11‐dehydrothromboxane B2 to thromboxane B2.
Glycerophosphocholine release in human erythrocytes
Authors: SELLE Hartmut , CHAPMAN Bogdan E. , KUCHEL Philip W.
Published: March 2005

The direct techniques of 1H spin‐echo and 31P‐NMR spectroscopy made it possible to monitor the release of glyccrophosphocholine from lysophosphatidylcholine in lysates from human red blood cells. Thus, the existence of a lysophospholipase in human erythrocytes was confirmed using a new more direct method. No evidence for a phospholipase A2 activity in the haemolysates was found with the same approach; since this enzyme is present in leukocytes, the absence of activity helped verify the purity of the erythrocyte preparation. The lysophospholipase may constitute, with the earlier described glycerophosphocholine phosphodiesterase activity, a metabolic unit for the removal of haemolytic lysophosphatidylcholine which is formed in the erythrocyte membranes as well as taken up from the plasma.
In vitro pore‐forming activity of the lantibiotic nisin
Authors: GARCERÁ Maria J. García , ELFERINK Marieke G. L. , DRIESSEN Arnold J. M. , KONINGS Wil N.
Published: March 2005

Nisin is a lantibiotic produced by some strains of Lactococcus lactis subsp. lactis. The target for nisin action is the cytoplasmic membrane of Gram‐positive bacteria. Nisin dissipates the membrane potential (Ψ) and induces efflux of low‐molecular‐mass compounds. Evidence has been presented that a Ψ is needed for nisin action. The in vitro action of nisin was studied on liposomes loaded with the fluorophore carboxyfluorescein. Nisin‐induced efflux of carboxyfluorescein was observed in the absence of a Ψ from liposomes composed of Escherichia coli lipids or dioleoylgly‐cerophosphocholine (Ole2GroPCho) at low nisin/lipid ratios. The initial rate of carboxyfluorescein efflux is dependent on the nisin/lipid ratio and saturates at high ratios. Both Ψ (inside negative) and φ (inside alkaline) enhance the action of nisin, while nisin is more potent at acidic external pH values. Efficient carboxyfluorescein efflux is observed with the zwitterionic phospholipid Ole2GroPCho or mixtures of Ole2GroPCho with dioleoylglycerophosphoethanolamine and neutral glycolipids, while anionic phospholipids are strongly inhibitory. It is concluded that a Ψ is not essential, but that the total protonmotive force stimulates the action of nisin.
Complete primary structure of chicken collagen XIV
Authors: WÄLCHLI Chantal , TRUEB Judith , KESSLER Benedikt , WINTERHALTER Kaspar H. , TRUEB Beat
Published: March 2005

We have isolated and characterized several overlapping cDNA clones for chicken collagen XIV which span a total of 6.5 kbp. These clones contain an open reading frame of 5571 bp encoding the entire collagen XIV polypeptide. The predicted polypeptide has an estimated molecular mass of 205 kDa in its glycosylated form. It is composed of 1857 amino acids which are arranged in 16 individual subdomains, including a signal peptide of 28 residues. The large amino‐terminal globular domain of collagen XIV (NC3) comprises 11 of these 16 subdomains. Two of them are related to the A modules of von Willebrand factor, eight show some relationship to the type III‐repeats of fibronectin and one is similar to the NC4 domain of collagen IX. The carboxy‐terminal triple‐helical domain is composed of two collagenous segments (COL1 and COL2), which make up less than 14% of the entire molecular mass, and of two short non‐collagenous domains (NC1 and NC2). A detailed analysis of our cDNA clones indicates that collagen XIV exists in two alternatively spliced forms which differ by 31 amino acids in their NC1 domain. The variant form of the polypeptide contains 1888 amino acids with a total molecular mass of 208 kDa. Our results demonstrate that collagen XIV displays a complex multidomain structure resembling that proposed for collagen XII.
Tumor necrosis factor cytotoxicity is associated with phospholipase D activation
Authors: DE VALCK Dirk , BEYAERT Rudi , VAN ROY Frans , FIERS Walter
Published: March 2005

The activation of phospholipase D in different cell lines treated with recombinant human tumor necrosis factor (TNF) has been investigated. When the murine fibrosarcoma cell lines L929 and WEHI164cl13, as well as the human promonocytic cell line U937, were prelabeled with [14C]palmitic acid or [3H]arachidonic acid, and treated with TNF in the presence of ethanol, TNF induced the synthesis of [14C]phosphatidylethanol or [3H]phosphatidylethanol, respectively, as the result of a phospholipase‐D‐catalyzed transphosphatidylation. TNF‐induced phospholipase D activity was observed 1 h before the onset of cell killing and gradually incresed thereafter. Subclones selected for resistance to TNF cytotoxicity did not show phospholipase D activation in response to TNF. In contrast, when these subclones were treated with TNF in the presence of actinomycin D, TNF cytotoxicity as well as TNF‐induced phospholipase D activity could be restored. TNF cytotoxicity and TNF‐induced phospholipase D activity were equally modulated by various drugs known to interfere with different steps in the TNF‐signaling pathway. Phospholipase D activation was found not to be the result of cell killing per se, as a number of other cytotoxic reagents were unable to activate phospholipase D. Prelabeling of cells with [14C]lysophosphatidylcholine indicated phosphatidylcholine as one of the substrates for TNF‐activated phospholipase D. In conclusion, this study demonstrates that TNF‐induced cytotoxicity is associated with activation of phospholipase D.
Physico‐chemical characterisation of membrane‐bound and water‐soluble forms of Bacillus subtilis cytochrome c‐550
Authors: von WACHENFELDT Claes , HEDERSTEDT Lars
Published: March 2005

Cytochrome c‐550 of the Gram‐positive bacterium, Bacillus subtilis, is a membrane‐bound 13‐kDa protein encoded by the cccA gene. The cytochrome has been proposed to be comprised of an N‐terminal membrane anchor domain (about 30 residues) which spans the cytoplasmic membrane in an α‐helical conformation and a C‐terminal heme domain (about 70 residues) which is located on the outside of the cytoplasmic membrane. Cytochrome c‐550 was purified in the presence of Triton X‐100 and characterised. In the reduced state it shows absorption maxima at 415, 521, 550 nm and in the oxidised state a Soret band at 408 nm and a weak band at about 695 nm. The latter absorption band, together with data from amino acid sequence comparisons, strongly suggest His64 and Met99 as the fifth and sixth axial ligands to the heme iron in cytochrome c‐550. The midpoint redox potential of the cytochrome, +178 mV, was pH‐independent in the pH range 6.0–7.9. Oxidised cytochrome c‐550 showed an EPR signal at gmax= 3.41, which is unusual for low‐spin cytochromes c with His/Met axial ligation. The heme domain was isolated as a tryptic fragment of 74 residues and as a protein‐A–cytochrome‐c‐550 hybrid protein. Both these forms were water‐soluble and showed thermodynamic and spectroscopic properties indistinguishable from the membrane‐bound form of cytochrome c‐550 and are suitable for structural analysis of the heme domain by X‐ray crystallography or NMR techniques. Polypeptide analysis of the membrane‐bound and water‐soluble tryptic fragment confirmed that B. subtilis cytochrome c‐550 in the membrane consists of 120 amino acid residues and has a two‐domain structure.
Effects of anions on the binding of the cAMP receptor protein to the lactose promoter
Authors: HUDSON J. Michael , CROWE Michael , FRIED Michael G.
Published: March 2005

The DNA binding affinities of several gene‐regulatory proteins, restriction endonucleases and the Escherichia coli RNA polymerase have previously been found to be dependent on the nature of the dominant buffer anion. To discover whether the E. coli cAMP receptor protein (CAP) exhibits a similar dependency, we measured its affinity for its primary lactose promoter binding site (lac site 1) in buffers in which the principal anion was chloride, phosphate, sulfate, acetate, or glutamate. We found that the affinity of CAP for lac site 1 is affected only slightly by changes in the dominant buffer anion. The binding of cAMP is similarly insentive to buffer anion type, indicating that specific protein—anion interactions, if they occur, must be similar for the free and cAMP‐bound forms of the protein. The effect of anion substitution on the ability of acrylamide to quench the intrinsic fluorescence of tryptophanyl residuces of CAP is also small, suggesting that changes in buffer anion composition have minimal effect on the conformation of tryptophan‐proximal regions of CAP. This conclusion is extended by the finding that anion substitution has a relatively small effect on the urea‐concentration dependence of CAP denaturation. Taken together, these results support the notion that neither CAP nor CAP · cAMP nor the CAP · cAMP complex with lac promoter DNA interact selectively with anions present in the surrounding buffer. A possible role for this anion‐insensitivity in the in vivo function of CAP is suggested.
Nuclear matrix of the lower eukaryote Physarum polycephalum and the mammalian epithelial LLC‐PK1 cell line
Authors: EBERHARTER Anton , GRABHER Angelika , GSTRAUNTHALER Gerhard , LOIDL Peter
Published: March 2005

Agarose‐encapsulated nuclear matrix preparations of the lower eukaryote Physarum polycephalum and the mammalian renal epithelial LLC‐PK1 cell line were analyzed after various experimental protocols with respect to the protein composition. The effect of the mode of deproteinization (2 M NaCl, 0.25 M ammonium sulfate or 25 mM lithium diiodosalicylate), presence of 2‐mercaptoethanol, Ca2+, Cu2+, chelating agents, the sequence of protein extraction and nuclease digestion, the use of RNase, the temperature at which the experimental manipulations were performed and the use of hypotonic or isotonic conditions was investigated. No significant differences in the final nuclear matrix composition could be observed, regardless of the experimental procedure applied. In Physarum, the major nuclear matrix proteins range over 12–70 kDa with prominent bands at 24, 31, 37 and 45 kDa; the proteins of the matrix in LLC‐PK1 cells extend predominantly over 40–80 kDa. Furthermore, no essential differences in the protein composition could be observed when type I and type II nuclear matrices from the highly differentiated LLC‐PK1 cell line were compared. The same was found for analogous matrix preparations of Physarum. Therefore, in both systems a distinction between type I/II matrix is questionable. Immunoblotting of the matrix preparations with a variety of antibodies against intermediate filament proteins and with antinuclear autoantibodies revealed the presence of intermediate filament proteins as components of the nuclear matrix. We conclude that the nuclear matrix represents a much more stable and reproducible structure than has been proposed so far, largely independent of changes in the preparation protocol.
Regulation of HSP70 gene expression during the life cycle of the parasitic helminth Schistosoma mansoni
Authors: NEUMANN Sylvia , ZIV Etty , LANTNER Frida , SCHECHTER Israel
Published: March 2005

Analyses of RNA from different developmental stages of Schistosoma mansoni showed stage‐specific expression of heat‐shock protein 70 (hsp70), which is regulated by a developmental program and by stress. The developmental program, common to hsp70 and other genes (e. g. paramyosin), refers to constitutive expression in miracidia sporocyst and adult worm but not in cercariae, and to the termination of hsp70 gene transcription during sporocyst/cercaria transformation. Stress induction, specific to hsp70, refers to transient accumulation of high levels of hsp70 mRNA during cercariae/schistosomula transformation and in adult worms after heat shock (42 °C). Cercariae/schistosomula transformation can be visualized as a physiological stress involving shifts in temperature (23–37 °C) and in salt concentration (from water to isotonic medium), as well as removal of tails from cercariae to yield isolated bodies that transform into schistosomula. It was found that temperature is an important factor, but not sufficient for strong induction of the hsp70 genes of schistosomula. Tail removal is an obligatory step for full induction of the hsp70 genes of schistosomula, in response to a temperature shift from 23–37 °C. The hsp70 genes in cercariae and isolated tails do not respond to stimuli (salt and temperature increases) that strongly activate the genes in isolated bodies (i. e., schistosomula). We speculate that the hsp70 genes in intact cercariae are not inducible because the tails can produce inhibitory signals that diffuse to the bodies and suppress their hsp70 genes. This hypothesis is useful to explain the termination of hsp70 gene transcription during sporocyst/cercaria transformation by the inhibitory effect of the growing tail.
Ligand binding to the haem‐copper binuclear catalytic site of cytochrome bo, a respiratory quinol oxidase from Escherichia coli
Authors: INGLEDEW W. John , HORROCKS Janet , SALERNO John C.
Published: March 2005

The Escherichia coli quinol oxidase, cytochrome bo, is closely related to the cytochrome‐c oxidase, cytochrome aa3, and reacts with ligands to the high‐spin ferric haem or the high‐spin ferric‐cupric binuclear catalytic site in similar ways. Cyanide reacts with the isolated enzyme to give a low‐spin complex, manifested by a red shift in the Soret band, the loss of an absorption band at 630 nm and the appearance of a low‐spin ferric haem EPR resonance at g = 3.3. Sulphide also elicits a low‐spin complex, whereas azide gives a mixture of low‐spin and high‐spin species. Formate and fluoride (and azide) give a blue shift in the Soret band and the development of a modified absorption band in the 600–650 nm range. These latter species are attributed to an integral spin compound involving the binuclear centre.
N‐Carbamoyl‐d‐amino acid amidohydrolase from Comamonas sp. E222c Purification and characterization
Authors: OGAWA Jun , SHIMIZU Sakayu , YAMADA Hideaki
Published: March 2005

N‐Carbamoyl‐d‐amino acid amidohydrolase was purified 119‐fold, with 36% overall recovery from a cell‐free extract of Comamonas sp. E222c. The purified enzyme was homogeneous as judged by SDS/PAGE. The relative molecular mass of the native enzyme was 120 000 and that of the subunit was 40 000. The purified enzyme hydrolyzed various N‐carbamoyl‐d‐amino acids to d‐amino acids, ammonia and carbon dioxide. N‐Carbamoyl‐d‐amino acids having hydrophobic groups served as good substrates for the enzyme. The Km and Vmax values for N‐carbamoyl‐d‐phenylalanine were 19.7 mM and 13.1 units/mg, respectively, and those for N‐carbamoyl‐d‐p‐hydroxyphenylglycine were 13.1 mM and 0.56 units/mg, respectively. The enzyme strictly recognized the configuration of the substrate and only the d‐enantiomer of the N‐carbamoyl amino acid was hydrolyzed. The enzyme activity was not significantly affected by N‐carbamoyl‐l‐amino acids and ammonia. The enzyme was sensitive to thiol reagents and did not require metal ions for its activity. The enzyme did not hydrolyze N‐carbamoyl‐β‐alanine or N‐carbamoyl‐dl‐aspartate suggesting that the enzyme is different from the N‐carbamoylamide‐hydrolyzing enzymes involved in the pyrimidine degradation pathway. The enzyme did not hydrolyze allantoin and allantoic acid, which are intermediates in purine degradation, N‐carbamoylsarcosine and citrulline, suggesting that it is a novel N‐carbamoylamide amidohydrolase.
Epitope mapping and accessibility of immunodominant regions of yeast plasma membrane H+‐ATPase
Authors: SERRANO Ramón , MONK Brian C. , VILLALBA José M. , MONTESINOS Consuelo , WEILER Elmar W.
Published: March 2005

Immunodominant regions of yeast plasma membrane H+‐ATPase have been mapped by two different approaches. A rabbit polyclonal antibody was used to screen a library of random fragments of the ATPase gene in a bacterial expression plasmid. In addition, the epitopes recognized by a panel of mouse monoclonal antibodies against the ATPase were mapped by reactions with defined fragments of the enzyme expressed in Escherichia coli. Both methodologies indicated that two regions within the amino‐terminal part of the ATPase (at amino acid positions 5–105 and 168–255) contain most of the antigenic determinants. The accessibility of the monoclonal antibodies to their epitopes in native and solvent‐perturbed ATPase preparations was investigated by immunofluo‐rescence studies on yeast protoplasts. Cells fixed and permeabilized with formaldehyde were either treated with or without detergents and organic solvents. ELISA competition tests with plasma membrane vesicles and with detergent‐purified ATPase incubated in solution with the monoclonal antibodies gave similar results. All the epitopes were accessible in detergent‐treated ATPase preparations. In contrast, only the epitopes at amino acids 24–56 were accessible in ATPase preparations not treated with detergents or organic solvents. These epitopes were cytoplasmic because protoplast permeabilization was required for decoration by the reactive monoclonal antibodies.
Influence of the immunization against heterologous alcohol dehydrogenase on liver alcohol dehydrogenase isozymes and alcohol abuse of rats
Authors: PSHEZHETSKY Alexey V. , DANILOVA Raisa A. , FEDOROVA Irina M. , SAGIMBAEVA Shalpan K. , PERVUSHINA Svetlana V. , OBUCHOVA Maria A. , ŠVEDAS Vytas K. , ASHMARIN Igor P.
Published: March 2005

The possible biochemical and immunological mechanisms of the previously discovered phenomenon of significant and prolonged decrease of alcohol abuse of albino rats after the intravenous injection of the heterologous alcohol dehydrogenase (ADH) were studied. It was found that immunization of rats against horse liver S‐ADH result in the appearance of a high litre of antibodies against rat liver ADH‐3 isozyme. This isozyme is responsible for the metabolism of 90% of alcohol administrated in vivo. It is shown that immunization against horse liver S‐ADH leads also to a significant (2–3‐fold) lowering of the activity level of ADH‐3 in rat liver. The activity level of the other rat ADH isozymes, ADH‐1 and ADH‐2, was not found to be changed. The intravenous injection of the specific anti‐(horse liver S‐ADH) serum, purified by affinity chromatography on immobilized horse liver S‐ADH, resulted in a significant decrease of alcohol abuse in alcohol‐dependent animals. These results allow us to assume an important role of rat liver ADH‐3 in the mechanism of alcohol addiction.
Human pre‐α‐trypsin inhibitor‐precursor heavy chain cDNA and deduced amino‐acid sequence
Authors: BOURGUIGNON Jeannette , DIARRA‐MEHRPOUR Maryam , THIBERVILLE Luc , BOST Frédéric , SESBOÜÉ Richard , MARTIN Jean‐Pierre
Published: March 2005

Pre‐α‐trypsin inhibitor (PαI) is a serine‐proteinase inhibitor of Mr 130000 found in human serum. This protein belongs to the family of proteins called inter‐ã‐trypsin inhibitor (ITI). PαI is composed of a heavy chain (HC3) and of a light chain (bikunin), synthesized by two separate mRNA. Bikunin is identical to the ITI light chain, the structure of which has already been established. The HC3 is obtained from a precursor called H3. The bikunin is covalently linked to HC3 by a chondroitin‐4‐sulfate glycosaminoglycan. We report here the H3 full‐length cDNA sequence and the deduced amino‐acid sequence of the heavy‐chain H3 precursor. The high degree of similarity between the nucleotide and amino‐acid sequences of ITI heavy‐chain families H1, H2, H3 is examined with respect to their probable structure and assembly with bikunin in the final proteins, PαI and ITI.
Removal of N‐terminal formyl groups and deblocking of pyrrolidone carboxylic acid of proteins with anhydrous hydrazine vapor
Authors: MIYATAKE Norifumi , KAMO Masaharu , SATAKE Kazuo , UCHIYAMA Yohtaro , TSUGITA Akira
Published: March 2005

Many proteins have a blocked α‐amino group which renders them inaccessible to sequence analysis by the classical Edman degradation procedure. Blockage typically occurs when the α‐amino groups are acylated with acetyl or formyl groups or when the N‐terminal residue is pyrrolidone carboxylic acid formed by cyclization of glutamine. We have found that N‐formyl groups of proteins and peptides can be removed by exposure to hydrazine vapor at‐5°C for 8 h. Under these conditions, peptide‐bond cleavage or modification of the constituent amino‐acid residues does not occur. Deblocking of N‐terminal pyrrolidone carboxylate residues by conversion to γ‐hydrazidyl glutamic acid can be achieved by exposure to hydrazine vapor at 20°C for 4 h. These conditions cause partial modification of asparagine and glutamine residues to their corresponding hydrazides, and conversion of arginine residues to ornithine.
The sum of the control coefficients of all enzymes on the flux through a group‐transfer pathway can be as high as two
Authors: van DAM Karel , van der VLAG Johan , KHOLODENKO Boris N. , WESTERHOFF Hans V.
Published: March 2005

In simple metabolic pathways the control exerted by enzyme concentrations on the pathway flux adds up to one when the control is quantified in terms of control coefficients. In this paper we demonstrate that this classical summation theorem has to be modified in pathways where the enzymes participate by transferring a group between each other. We derive the corresponding new control theorem and show how it is consistent with standard metabolic control analysis. In group‐transfer pathways lacking enzyme complexes, the sum of the flux control by enzyme concentrations and by the donor and acceptor couples of the pathway, equals two. In group‐transfer pathways with enzyme‐enzyme interactions the flux control by the dissociation rate constants of the enzyme‐enzyme complexes must be added to obtain this sum of two. In all cases, the sum of the controls by all reaction activities remains one. Both by using the new theorem and by numerical simulations, we then demonstrate that, in group‐transfer pathways with or without enzyme interactions, the sum of the control of enzymes on the pathway flux is higher than one and can reach a value of two. The total control of all enzymes on the concentration of any intermediate either with or without the transferred group can be equal to one, rather than to the zero found in the classical case. Examples of group‐transfer pathways are the bacterial phosphoenolpyruvate:sugar phosphotransferase system, the main pathway for uptake of sugars in Enterobacteriaceae, and the electron‐transfer chain in free‐energy transducing membranes.
Nicotinoprotein [NAD(P)‐containing] alcohol/aldehyde oxidoreductases
Authors: VAN OPHEM Peter W. , VAN BEEUMEN Jozef , DUINE Johannis A.
Published: March 2005

Extracts of Gram‐positive bacteria like Rhodococcus rhodochrous, Rhodococcus erythropolis and Amycolatopsis methanolica, but not those of several Gram‐negative ones, showed dehydrogenase activity for ethanol as well as for methanol when 4‐nitroso‐N, N‐dimethylaniline (NDMA) was used as electron acceptor. Chromatography of extracts of the first two organisms revealed one activity for both substrates, that of A. methanolica two activities, one of which is able to oxidize methanol and has been purified (Bystrykh, L. V., Govorukhina, N. I., van Ophem, P. W., Hektor, H. J., Dijkhuizen, L. and Duine, J. A., unpublished results). The other, indicated as NDMA‐dependent alcohol dehydrogenase (NDMA‐ADH), was purified to homogeneity. It is a trimeric enzyme consisting of subunits of 39 kDa and one firmly bound NAD as cofactor. Although NDMA‐ADH shows structural similarity with the long‐chain, zinc‐containing, NAD(P)‐dependent alcohol dehydrogenases with respect to the N‐terminal sequence up to residue 41 (56% identity with horse liver alcohol dehydrogenase), the enzymes are catalytically different since NDMA‐ADH is unable to use NAD(P)(H) as a coenzyme and NAD(P)‐dependent alcohol dehydrogenases are inactive with NDMA (in the absence of NAD). Comparison of the NDMA‐ADH properties with those of the methanol‐oxidizing enzyme of A. methanolica, Mycobacterium gastri and Bacillus methanolica C1, and formaldehyde dismutase of Pseudomonas putida F61 revealed large differences in structural as well as catalytic properties, in spite of the fact that all are nicotinoproteins [enzymes which have bound NAD(P) as a cofactor]. It is concluded, therefore, that NDMA‐ADH is a novel type of nicotinoprotein alcohol dehydrogenase.
Heavy‐chain isoforms of non‐muscle myosin in human tissues
Authors: KIMURA Akihiko , NAKASHIMA Shoichi , UDA Tomoji , IKEDA Haruhiko , YASUDA Seiji , TSUJI Tsutomu , MATSUMURA Sueo
Published: March 2005

The heavy‐chain isoforms of myosin in human non‐muscle and smooth muscle tissues were analyzed by means of SDS/PAGE and using three distinct newly developed monoclonal anti‐(human cerebrum myosin) Ig (HBM‐1, HBM‐3 and HBM‐4). Purified cerebrum myosin contained three electrophoretic variants of non‐muscle myosin heavy chain (NM1, NM2 and NM3, with apparent molecular masses of about 200, 198 and 196 kDa, respectively). Both NM1 and NM2 were recognizable by the brain‐specific antibody HBM‐1, while NM3 was recognizable by HBM‐3. Each of the variants reacted with HBM‐4 to a similar extent. Purified cerebellum myosin gave three electropho retic variants of the heavy chain which were indistinguishable electrophoretically or immunologically from those of cerebrum myosin. Aortic myosin contained four electrophoretic variants, including the two smooth muscle myosin heavy chain isoforms and NM2‐like and NM3‐like heavy chains. Liver, platelet and kidney myosins contained a heavy chain very similar to NM3. Kidney myosin also contained a small fraction of an NM2‐like electrophoretic variant. In addition, cerebrum, kidney, liver and platelet myosins appeared to contain minor, 194‐kDa myosin heavy‐chain‐like polypeptide(s) (NM4). NM1, as well as NM2 and NM3, thus appear to be the brain‐type and non‐brain‐type non‐muscle myosin heavy‐chain isoforms, respectively, and additional minor heavy‐chain isoforms are also likely to be present in human tissues.
Evidence for a common structure for a class of membrane channels
Authors: HOLZENBURG Andreas , JONES Phil C. , FRANKLIN Tim , PALI Tibor , HEIMBURG Thomas , MARSH Derek , FINDLAY John B. C. , FINBOW Malcolm E.
Published: March 2005

Electron microscopic analysis of gap‐junction‐like structures isolated from an arthropod (Nephrops norvegicus) and composed of a 16‐kDa polypeptide, show the functional unit to be a star‐shaped hexamer of protein arranged around a central channel which runs perpendicular to the plane of the membrane. Estimations of the molecular volume carried out on an averaged projection are consistent with a subunit mass of 16–18 kDa. Fourier transform infrared spectroscopy indicates a high α‐helical content for the protein, supporting secondary‐structure predictions of four transmembrane α helices/monomer. The averaged projection shows a close resemblance to a hexamer of the 16‐kDa protein built on the basis of a four α‐helical bundle [Finbow, M. E., Eliopoulos, E. E., Jackson, P. J., Keen, J. N., Meagher, L., Thompson, P., Jones, P. C. & Findlay, J. B. C. (1992) Protein Eng. 5, 7–15]. The reconstructed image is also similar to that obtained for gap‐junction‐like channels isolated from a related arthropod [Homarus americanus; Sikerwar, S. S., Downing, K. H. & Glaeser, R. M. (1991) J. Struct. Biol. 106, 255–263] whose protein content was unknown but which we demonstrate may be composed of a related 16‐kDa protein.

Previous studies have shown a high sequence identity of the Nephrops 16‐kDa protein with the 16‐kDa proteolipid subunit c of the vacuolar H+‐ATPase, both of which in turn bear similarity to the 8‐kDa proteolipid subunit of the F1F0‐ATP synthase. Expression of cDNA coding for the Nephrops 16‐kDa protein in Saccharomyces cerevisiae, in which the endogenous gene coding for the V‐ATPase proteolipid has been inactivated, restores V‐ATPase activity and cell growth.
Cardiac α1‐adrenoceptors stimulate a high‐affinity GTPase activity in sarcolemmal membranes from rabbit atrial and ventricular myocytes
Authors: BRAUN Andrew P. , WALSH Michael P.
Published: March 2005

The interaction between cardiac α1‐adrenoceptors and GTP‐binding regulatory proteins was characterized in isolated rabbit cardiac myocytes (thereby avoiding interference by other cell types present in the myocardium) by examining the α1‐adrenergic stimulation of GTPase activity in sarcolemma‐enriched membrane fractions. Stimulation of membrane‐associated GTPase activity in both atrial and ventricular myocyte preparations by the α1‐adrenergic agonists 1‐noradrenaline and methoxamine (in the presence of propranolol) was observed to be both linear with time and saturable. α1‐adrenergic stimulation did not change the Km for GTP (0.14–0.21 μM), but increased the Vmax by 39% and 72% above basal levels in atrial and ventricular membranes, respectively. Stimulation of GTPase activity by α1‐agonists occurred in a concentration‐dependent fashion and was blocked in the presence of the α‐adrenoceptor antagonists phentolamine and prazosin, but not yohimbine. Prior treatment of myocytes with pertussis toxin had no effect on the α1‐adrenergic stimulation of GTPase activity, but inhibited stimulation by muscarinic‐receptor activation with carbachol. Finally, photoaffinity labelling of an approximately 75‐kDa membrane‐bound protein with [α‐32P]GTP was enhanced in the presence of the α1‐agonist methoxamine and abolished by addition of excess nonlabelled GTP, suggesting that this GTP‐binding protein may interact with cardiac α1‐adrenoceptors; a similar GTP‐binding protein which may be coupled to α1‐adrenoceptors has been reported in rat liver plasma membranes (Im, M. J. & Graham, R. M. (1990) J. Biol. Chem. 265, 18944–18951).
Channelling can affect concentrations of metabolic intermediates at constant net flux: artefact or reality?
Authors: CORNISH‐BOWDEN Athel , CÁRDENAS María Luz
Published: March 2005

We show that if a metabolic intermediate is directly transferred (‘channelled’) from an enzyme that catalyses its production to another that uses it as substrate, there is no change in its free concentration compared with a system with the same net flux in which there is no direct transfer. Thus the widespread idea that channelling provides a mechanism for decreasing metabolite concentrations at constant flux is false. Results from computer simulation that suggest otherwise [Mendes, P., Kell, D. B. & Westerhoff, H. V. (1992) Eur. J. Biochem. 204, 257–266] are artefacts either of variations in flux or of alterations in opposite directions of the activities of the relevant enzymes.
Characterization and photoaffinity labeling of the ATP binding site of the ryanodine receptor from skeletal muscle
Authors: ZARKA Aliza , SHOSHAN‐BARMATZ Varda
Published: March 2005

The photoaffinity analog of ATP, 3′‐O‐(4‐benzoyl)benzoyl‐adenosine 5′‐triphosphate (Bz2ATP) was used to covalently label and to identify the ATP binding site of the skeletal muscle ryanodine receptor. Like ATP, Bz2ATP stimulates up to fivefold the binding of ryanodine to its receptor. Photoactivation by ultraviolet light of the benzophenone group in the [α‐32P]Bz2ATP results in covalent binding of [α‐32P]Bz2ATP to the 450‐kDa polypeptide, the ryanodine receptor's subunit. An apparent molar stiochiometry of Bz2ATP to the tetrameric ryanodine receptor complex of 1.146±0.087 (n= 2) was estimated. The covalent binding of [α‐32P]Bz2ATP was inhibited by ATP and analogous compounds in the order: ATP = AdoPP[CH2]P= ADP = Ado = cAMP > AMP > ITP = GTP. Similar specificity was obtained for the stimulation of ryanodine binding by these nucleotides. ATP increased the ryanodine binding affinity by about sixfold.

The polycationic dye ruthenium red, known as an inhibitor of Ca2+ release and ryanodine binding, inhibited the labeling of the ryanodine receptor by [α‐32P]Bz2ATP. Tryptic digestion of the ryanodine receptor revealed a [α‐32P]Bz2ATP‐labeled 76‐kDa tryptic fragment. Digestion of either the [α‐32P]Bz2ATP‐labeled 450‐kDa or the 76‐kDa polypeptides with S. aureus resulted in the appearance of four labeled fragments of 39, 33, 27 and 13 kDa, where the 39‐kDa fragment is the precursor of the 27‐kDa and 13‐kDa fragments. The results suggest that the regulation of Ca2+ release by ATP involves an ATP binding site(s) located on the 27‐kDa and 13‐kDa fragments of the ryanodine receptor protein.
Involvement of the 20S proteasome in the degradation of ornithine decarboxylase
Authors: BERCOVICH Zippy , KAHANA Chaim
Published: March 2005

Eukaryotic cells have been shown to contain two high‐molecular‐mass proteases of 700 kDa and 1400 kDa (20S and 26S proteases, respectively). It has been suggested that the 20S protease, also known as proteasome, may constitute the catalytic core of the 26S protease. While the role of the free 20S protease in intracellular protein degradation is unclear, the 26S protease is implicated in the degradation of ubiquinated proteins. We have recently demonstrated, that ornithine decarboxylase (ODC), one of the most labile proteins in mammalian cells, is degraded via an ATP‐dependent but ubiquitin‐independent proteolytic pathway. Here we extend these observations by demonstrating that in reticulocyte lysate ODC degradation is inhibited by antibodies raised against the C9 subunit of rat proteasome. Partial fractionation of the lysate demonstrated preferential degradation of ODC in the fraction of the lysate proteins that are precipitated by 38% ammonium sulfate. Since it was demonstrated that the 26S protease precipitates at this concentration of ammonium sulfate while the 20S proteasome remains soluble, our results suggest that the 26S protease is the one degrading ODC.
Human‐liver cytochromes P‐450 expressed in yeast as tools for reactive‐metabolite formation studies
Authors: LOPEZ GARCIA M. Pilar , DANSETTE Patrick M. , VALADON Philippe , AMAR Claudine , BEAUNE Philippe H. , GUENGERICH F. Peter , MANSUY Daniel
Published: March 2005

Human liver cytochromes P‐450 (P450) 2C9 and 2C10 expressed in yeast reproduce all the metabolic features of the oxidation of tienilic acid (2‐aryloxo‐thiophene) and its isomer (3‐aroyl‐thiophene) by human liver microsomes. Microsomes of yeast expressing either P450 2C9 or P450 2C10 catalyze (a) the 5‐hydroxylation of tienilic acid by NADPH and O2 (Km= 6 μM, Vmax= 2.5 turnover/min), (b) the activation of tienilic acid and its isomer into electrophilic metabolites which covalently bind to proteins, and (c) the formation of a mercaptoethanol adduct which results from the trapping of the tienilic acid isomer sulfoxide by this thiol. Microsomes of yeast expressing human liver P450 3A4, 1A1 and 1A2 are unable to catalyze these reactions. There is a striking similarity between the quantitative characteristics of the oxidation of tienilic acid (and its isomer) by yeast‐expressed P450 2C9 (or 2C10) and by human liver microsomes: (a) analogous Km values (around 10 μM) for tienilic acid 5‐hydroxylation, (b) a strong inhibition of tienilic acid oxidation by human sera containing anti‐(liver kidney microsomes type 2) (anti‐LKM2) antibodies, and (c) almost identical relative ratios of tienilic acid metabolic activation/5‐hydroxylation and of tienilic acid activation/the activation of its isomer with both systems. Rates of oxidation of tienilic acid (and its isomer) by yeast microsomes are 6–8‐fold higher than those found in human liver microsomes, which would be in agreement with the previously reported amount of P450 2C9 in human liver. These results not only suggest the important role of P450 2C9 in the oxidative metabolism of tienilic acid in human liver, but also indicate that the 5‐hydroxylation reaction could be a useful marker for P450 2C9 activity and underline the interest of human liver P450s expressed in yeast as tools for studying the formation of reactive metabolites.