bluuebunny/crossref_metadata_2025 huggingface dataset:
The complete amino acid sequence of ribosomal protein S12 from Bacillus stearothermophilus
Authors: Kimura Makoto , Kimura Junko
Published: July 2002

The amino acid sequence of ribosomal protein S12 from Bacillus stearothermophilus has been completely determined. The sequence data were mainly obtained by manual sequencing of peptides derived from digestion with trypsin, Staphylococcus aureas protease and pepsin. A few overlaps of tryptic peptides were established by DNA sequence analysis of a chromosomal fragment containing the rpsL gene coding for ribosomal protein S12. The protein contains 138 amino acid residues and has an M
r of 15208. Comparison of this sequence with the sequences of the ribosomal S12 proteins from E. coli as well as from Euglena, tobacco and liverwort chloroplasts shows that 75% of the amino acid residues are identical within the S12 proteins of all four species. Therefore, S12 is the most strongly conserved ribosomal protein known so far.
Production of a specific antibody against pyruvate kinase type M2 using a synthetic peptide
Authors: Oude Weernink Paschal A. , Rijksen Gert , Staal Gerard E.J.
Published: July 2002

The pyruvate kinase isozymes M1 and M2 are structurally and immunologically closely related. To obtain an antibody which discriminates between these two forms, a synthetic tetradecapeptide with a sequence specific for pyruvate kinase type M2 from rats was constructed. Antisera from rabbits, immunized with this peptide, reacted specifically with the M2‐type holoenzyme of both rat and human origin, and did not cross‐react with the M1‐type isozyme. This was established by immunoblot analysis, both under dissociating and non‐dissociating conditions.
Nucleotide sequence of a cDNA for the dihydrolipoamide acetyltransferase component of human pyruvate dehydrogenase complex
Authors: Thekkumkara Thomas J. , Ho Lap , Wexler Isaiah D. , Pons Gabriel , Te-Chung Liu , Patel Mulchand S.
Published: July 2002

Deoxynucleotide sequencing of a cDNA for the dihydrolipoamide acetyltransferase (PDC‐E2) component of human pyruvate dehydrogenase complex (PDC) revealed an open reading frame of 1848 base pairs corresponding to a leader sequence of 54 amino acids and a mature protein of 561 amino acids (59 551 Da). Both an amino‐terminal lipoyl‐bearing domain and a carboxy‐terminal catalytic domain are present in the deduced amino acid sequence. The lipoyl‐bearing domain contains two repeating units of 127 amino acids, each harboring one lipoic acid‐binding lysine. Thus, mammalian PDC‐E2 differs as to the number of lipoic acid‐binding sites from other dihydrolipoamide acyltransferases in both prokaryotic and eukaryotic organisms.
The redox potential of the primary quinone QA of bacterial photosynthesis is independent of the divalent metal ion
Authors: Buchanan Susan K. , Dismukes G.Charles , Prince Roger C.
Published: July 2002

The function of the ferrous ion which links the primary and secondary quinone electron acceptors, QA and QB, via histidine ligands from the reaction center protein of the photosynthetic bacterium Rbodobacter sphaeroids (Y) has been investigated by substitution with Mn(II), Cu(II) and Zn(II), using biosynthetic incorporation of the metal ion. The midpoint potential and pH dependence for reduction of QA in chromatophore membranes were found to be indistinguishable for all four divalent ions, and in agreement with earlier results on the R26 and Ga strains of R. sphaeroides containing only iron. We therefore conclude that the divalent ion contributes no functionally significant covalent coupling to the primary acceptor and that these metals have no differential electrostatic influence. Furthermore, inhibition of photoreduction of QB by the herbicide ametryne was found to be the same for all four samples. Thus, these four divalent ions exhibit no differential influence on the binding properties of this herbicide in the QB site.
Bacterial Na+ energetics
Authors: Skulachev Vladimir P.
Published: July 2002

Novel observations related to the Na+‐linked energy transduction in bacterial membranes are considered. It is concluded that, besides the well‐known systems based on the circulation of protons, there are those based on the circulation of Na+. In some cases, H+ and Na+ cycles co‐exist in one and the same membrane. Representatives of the ‘sodium world’, i.e. cells possessing primary Na+ pumps (ΔΔµ̃Na generators and consumers) are found in many genera of bacteria. Among the ΔΔµ̃Na generators, one should mention Na+‐NADH‐quinone reductase and Na+‐terminal oxidase of the respiratory chain, Na+‐decarboxylases and Na+‐ATPases. For Δµ̃Na consumers, there are Na+‐ATP‐synthases, Na+‐metabolite symporters and Na+ motors. Sometimes, one and the same enzyme can transport H+ or, alternatively, Na+. For instance, an Na+‐ATP‐synthase of the F0F1 type translocates H+ when Na+ is absent. Employment of the Na+ cycle, apart from or instead of the H+ cycle, increases the resistance of bacteria to alkaline or protonophore‐containing media and, apparently, to some other unfavourable conditions.
Effects of iron‐EDTA on uroporphyrinogen oxidation by liver microsomes
Authors: Jacobs Judith M. , Sinclair Peter R. , Lambrecht Richard W. , Sinclair Jacqueline F.
Published: July 2002

Uroporphyrinogen oxidation by hepatic microsomes from chick embryos or mice pretreated with methylcholanthrene was increased by addition of iron‐EDTA. This increase was partially prevented by catalase, mannitol, ketoconazole and piperonyl butoxide, whereas only ketoconazole and piperonyl butoxide inhibited the oxidation in the presence and absence of iron‐EDTA. These data suggest that the oxidations of uroporphyrinogen in the presence and absence of added iron occur by different mechanisms.
Radiation inactivation analysis of the A1 adenosine receptor of rat brain Decrease in radiation inactivation size in the presence of guanine nucleotide
Authors: Reddington Martin , Klotz Karl-Norbert , Lohse Martin J. , Hietel Bernhard
Published: July 2002

Radiation inactivation analysis of the binding of the A1 adenosine receptor antagonist, 8‐cyclopentyl‐1,3‐dipropylxanthine to rat brain membranes yielded a radiation inactivation size of 58 kDa. In the presence of GTPγS this was reduced to 33 kDa, in good agreement with the size of the ligand‐binding subunit detected after photoaffinity labelling. The data indicate that the structural association of A1 adenosine receptors with G‐protein components is altered in situ in the presence of guanine nucleotides.
The O2.− generating oxidase activation of bovine neutrophils
Authors: Pilloud Marie-Claire , Doussiere Jacques , Vignais Pierre V.
Published: July 2002

Respiratory burst; Oxidase activation; Neutrophil; Superoxide ion
Purification of GTP‐binding proteins from bovine brain membranes
Authors: Kobayashi Ichiro , Shibasaki Hisayuki , Takahashi Katsunobu , Kikkawa Satoshi , Ui Michio , Katada Toshiaki
Published: July 2002

ADP‐ribosylation; GTP‐binding protein; Islet‐activating protein
35S‐Atractyloside binding affinity to the inner mitochondrial membrane
Authors: Vignais P.V. , Vignais P.M. , Colomb M.G.
Published: July 2002

Isolated inner mitochondrial membrane contains a small number of binding sites for atractyloside (of the order of 0.1 nmole/mg of protein) with very high binding affinity (half saturation at 0.014 μM atractyloside). The high affinity binding ability of the inner mitochondrial membrane is markedly decreased upon aging, acidification of the medium or addition of ADP, but remains unchanged in the presence of uncouplers such as FCCP. Added ADP causes a two‐step transition from the high affinity binding to low affinity binding (K
d > 0.50 μM) and concomitantly a significant increase of the measured number of binding sites (about a doubling). The half maximum effect in the first step transition is given by 1 μM ADP. The use of 35S‐atractyloside as a probe of the inner mitochondrial membrane conformation specifically related to the adenine nucleotide translocation is discussed.
Does a 2 ← 5β‐turn structure exist in enkephalins?
Authors: Moret E. , Gerothanassis I.P. , Hunston R.N. , Lauterwein J.
Published: July 2002

The 17O chemical shifts of the Gly‐2 and Gly‐3 oxygens of a fully protected Leu‐enkephalin were measured to be identical in acetone solution. This allows the conclusion that neither of these peptide oxygens is hydrogen bonded and that no specific 2 ← 5 β‐turn structure exists to an appreciable extent.
A fourth subunit is present in cytochrome c oxidase from the thermophilic bacterium PS3
Authors: Sone Nobuhito , Shimada Shin-ichi , Ohmori Tamiya , Souma Yukiko , Gonda Masahiro , Ishizuka Mono
Published: July 2002

A new putative subunit was found in cytochrome c oxidase (aa3
‐type) of the thermophilic bacterium PS3. The N‐terminal amino acid sequence of this 12 kDa protein coincides with the deduced sequence of an open reading frame found downstream from the gene encoding subunit I of the PS3 cytochrome oxidase [(1988) J. Biochem. 103, 606‐610]. This small hydrophobic protein, composed of 109 amino acid residues after the initial methionine residue has been processed, shows homology with the subunit IV (cyoD product) of cytochrome bo‐type quinol oxidase of Escherichia coli.
Presence of a differentially expressed U3A RNA variant in mouse
Authors: Mazan S. , Qu L.H. , Sri-Widada J. , Nicoloso M. , Bachellerie J.P.
Published: July 2002

A U3 RNA variant has been identified in mouse, the abundance of which relative to the previously characterized major form (U3B) appears to vary to a large extent depending upon the cell origin. Its partial sequence analysis shows that it is clearly related to the U3A form previously described in rat. Sequence comparisons suggest that the separation of the two forms of U3 genes now found in rat and mouse represent a relatively ancient event in rodent evolution. While mouse U3B RNA is encoded by four clustered genes, the U3A variant is encoded by a unique gene. Both mouse U3 RNAs differ substantially in primary structure (more than 10% divergence). Although rodent U3 RNAs exhibit a largely similar secondary structure, a specific difference between the A and B form can nevertheless be observed.
Evidence for a unique Rieske iron‐sulphur centre in Heliobacterium chlorum
Authors: Liebl Ursula , Rutherford A.William , Nitschke Wolfgang
Published: July 2002

An iron sulphur centre (g
z = 2.035, g
y =1.89, g
x =1.81), which can be observed in both whole cells and isolated plasma membranes of Heliobacterium chlorum, was identified as a Rieske centre on the basis of its sensitivity to the inhibitors 2,5‐dibromo‐3‐methyl‐6‐isopropyl‐benzoquinone (DBMIB), 5‐(n‐undecyl)‐6‐hydroxy‐4,7‐dioxobenzothiazole (UHDBT) and stigmatellin as well as on the basis of its orientation (gx, is oriented perpendicular and g
y is oriented parallel to the membrane plane). Its midpoint potential is unusually low (E
m,7= +120 ± 10 mV) and does not depend on pH in the range between pH 6.5 and pH 8.0. The effects of the inhibitors on the EPR spectrum are altered compared to Rieske centres in other systems. The significance of the low E
m is discussed with regard to the overall midpoint potentials of the electron transfer chain in Heliobacterium chlorum.
Myosin structure in the eel (Anguilla anguilla L)
Authors: Chanoine C. , Saadi A. , Guyot-Lenfant M. , Hebbrecht C. , Gallien Cl.L.
Published: July 2002

Myosin extracts from central white fibers and peripheral red fibers of the lateral muscle of eel (Anguilla anguilla) were analysed by electrophoresis under non‐dissociating conditions, which demonstrated a polymorphism of myosin isoforms. The light and heavy subunit content of the isomyosins was established using SDS‐PAGE and two‐dimensional eleetrophoresis. In the central white muscle, 3 myosin isoforms FM3, FM2, FMI, were characterized by 3 types of fast light chain and one fast heavy chain HCf; the existence of a fourth isomyosin is discussed. In the peripheral red muscle, two myosin isoforms were found, SM1 and SM2, each characterized by a specific heavy chain, HCs1 or HCs2, and containing the same slow light chain content. This work demonstrates for the first time the existence of 3 heavy chains in the skeletal muscle of a fish.
The apparent molecular size of native α‐crystallin B in non‐lenticular tissues
Authors: Chiesa Raúl , McDermott Martin J. , Mann Eric , Spector Abraham
Published: July 2002

The apparent molecular size of the native α‐crystallin B in cytosol preparations from rat heart, brain and retina was determined by gel permeation chromatography, detecting the protein by immunochemical assay (ELISA), using an α‐crystallin specific antiserum. Native α‐crystallin from cytosol preparations of rat lens cortex was used as a reference. α‐Crystallin B present in all three cytosol preparations from non‐lenticular tissues eluted in a single symmetrical peak, with the same elution volume as α‐crystallin from lens cortex cytosol preparations, corresponding to an apparent average molecular size of 0.8 × 106 Da. No other species could be detected. The results indicate that the α‐crystallin aggregates characterized by an apparent average molecular mass of 0.8 × 106 Da, and considered to be the native, physiological form of the protein in the lens, are indeed not specific to lens tissue. Furthermore, the size of these α‐crystallin aggregates is independent of their polypeptide composition. Aggregates found in the lens, composed of αA and αB polypeptides and their respective phosphorylated forms αAp and αBp, are similar in size to those found in heart, brain and retina, containing the αB but not the αA polypeptide.
β‐Turns as structural motifs for the proteolytic processing of seed proteins
Authors: Monsalve Rafael I. , Menéndez-Arias Luis , López-Otin Carlos , Rodríguez Rosalía
Published: July 2002

These facts are interpreted in terms of the existence of a β‐turn specific endoprotease activity involved in the maturation process of 2 S albumins.
Detection of heterogeneity of Cu, Zn‐superoxide dismutase with monoclonal antibodies and the establishment of a highly sensitive fluorescence sandwich enzyme‐linked immunosorbent assay
Authors: Ono Kumeo , Kimura Satsuki , Nakano Minoru , Naruse Takuji
Published: July 2002

Murine monoclonal antibodies directed against a native form of Cu, Zn‐superoxide dismutase (SOD) were produced by immunizing SOD purified from human erythrocytes. The monoclonal antibodies able to bind SOD were further screened for their ability to absorb SOD activity using antimouse 1gG conjugated iron beads as solid supports in magnetic separation. This new screening method revealed the heterogeneity of native SOD in the reactivity with the antibodies. One monoclonal antibody succesfully absorbed the entire activity of SOD detected by an inhibition assay of cypridina luciferin analog (MCLA)‐dependent chemilluminescence induced by superoxide anion production, while others absorbed only a part of the SOD activity. The evidence that all of the latter antibodies failed to react with recombinant artificial SOD free of charge isomers suggested correlation of the heterogeneity with the presence of charge isomeric forms. The former antibody was further used to establish a fluorescence sandwich enzyme immunoassay, and this provided a very sensitive detection limit as low as 100 pg/ml.
Immunologic mapping of the amino‐ and carboxy‐termini of the turkey erythrocyte β‐adrenergic receptor; Selective proteolysis of both domains
Authors: Luxembourg Alain , Hekman Mirko , Ross Elliott M.
Published: July 2002

Peptide‐directed antibodies were used to map the N‐ and C‐termini of the turkey erythrocyte β‐adrenergic receptor, the full length recombinant receptor expressed in Sf9 cells, and a mutant that terminates after residue 424 (T424). Both forms of the natural receptor (P40 and P50): were proteolytically clipped between residues 419 and 424, P40, but not P50, is also proteolyzed between residues 14 and 28. Truncation mutants, but not full length receptors, also display both large and small forms. The short form or T424 is formed by proteolysis after residue 14, but neither form is proteolyzed in the C‐terminal region. The wild type recombinant receptor is not proteolyzed.
Selective effect of inhibitors on inner mitochondrial membrane channels
Authors: Antonenko Yuri N. , Kinnally Kathleen W. , Perini Sean , Tedeschi Henry
Published: July 2002

The effect of amphiphilic cationic drugs on the channel activity of the mitochondrial inner membrane was examined with patch‐clamp techniques. The therapeutic drugs amiodarone, propranolol and quinine reduced the probability of being open for the multiconductance channel (MCC) activity (levels from 30 pS to over I nS). While amiodarone decreased the probability of being open for the voltage dependent ∼ 100 pS channel, it increased the conductance 42 ± 20% (mean ± SD, n = 6) with no significant change in mean open time. Similar results were obtained with propranolol. These data indicate that the ∼ 100 pS channel is distinct from MCC activity.
In vitro activation of the Saccharomyces cerevisiae Ras/adenylate cyclase system by glucose and some of its analogues
Authors: Pardo Luis Angel , Sánchez Luis Maria , Lazo Pedro S. , Ramos Sofia
Published: July 2002

Using crude membrane preparations of Saccharomyces cerevisiae, we have demonstrated that glucose and glucose analogues which are not efficiently phosphorylated activate the guanine nucleotide‐dependent adenylate cyclase in vitro. The activation appears to be mediated by the Ras proteins. Moreover, data are presented indicating that glucose and its analogues activate adenylate cyclase by stimulating the exchange of guanine nucleotides at its regulatory component. Thus, it has been possible to show the action of a physiological effector on the nucleotide exchange reaction in a member of the ras superfamily.
Formation and crystallization of Thermus thermophilus 70S ribosome/tRNA complexes
Authors: Yusupova Gulnara , Yusupov Marta , Spirin Alexander , Ebel Jean-Pierre , Moras Dino , Ehresmann Chantal , Ehresmann Bernard
Published: July 2002

70S ribosomes from Thermus thermophilus are able to form ternary complexes with N‐AcPhe‐tRNAPhe from either Thermus thermophilus or Escherichia coli, in the presence of a short oligo(U) of six or nine uridines. A complex of N‐AcPhe‐tRNAPhe/(U)9/70S ribosome from Th. thermophilus was crystallized under the same conditions used for the growth of crystals from isolated ribosomes (S.D. Trakhanov, et al., (1987) FEBS Lett. 220, 319–322).
Asparaginyl‐tRNA synthetase from the Escherichia coli temperature‐sensitive strain HO202 A proline replacement in motif 2 is responsible for a large increase in Km for asparagine and ATP
Authors: Madern Dominique , Anselme Jocelyne , Härtlein Michael
Published: July 2002

The Escherichia coli K12 mutant gene, asnS40, coding for asparaginyl‐tRNA synthetase (AsnRS) in the temperature‐sensitive strain HO202, was isolated from genomic DNA using the Polymerase Chain Reaction. DNA sequencing revealed that the mutant enzyme differs from the wild‐type AsnRS by two amino acids, but only the P231L replacement leads to a change in aminoacylation activity. In the ATP‐PPi exchange reaction at 37°C the purified P231L enzyme has a more than 50‐fold increased K
m value for asparagine compared to the wild‐type enzyme, while the K
m value for ATP is increased 8‐fold. In the aminoacylation reaction the mutant enzyme shows also significantly increased K
m values for asparagine and ATP. Interestingly Pro‐231 is part of the conserved motif 2 in class II aminoacyl‐tRNA synthetases (Eriani, G., Delarue, M., Poch, O., Gangloff, J. and Moras, D. (1990) Nature 347, 203–206), indicating that this motif might be involved in all class II enzymes in amino acid activation.
Cytochrome oxidase‐catalyzed superoxide generation from hydrogen peroxide
Authors: Ksenzenko M.Yu. , Vygodina T.V. , Berka V. , Ruuge E.K. , Konstantinov A.A.
Published: July 2002

Superoxide dismutase is shown to affect spectral changes observed upon cytochroma c oxidase reaction with H2O2, which indicates a possibility of O−
2 radicals being formed in the reaction. Using DMPO as a spin trap, generation of superoxide radicals from H2O2 in the presence of cytochrome oxidase is directly demonstrated. The process is inhibited by cyanide and is not observed with a licat‐denatured enzyme pointing to a speciric reaction in the oxygen‐reducing centre of cytochrome c oxidase. The data support a hypothesis on a catalase cycle catalysed by cylochrome c oxidase in the presence of excess H2O2 (Vygodina and Konstantinov (1988) Ann. NY Acad. Sci., 550, 124‐138);
Multisite phosphorylation of the 80 kDa (MARCKS) protein kinase C substrate in C3H/10T1/2 fibroblasts Quantitative analysis of individual sites by solid‐phase microsequencing
Authors: Arness Bob , Manjarrez-Hernandez H.Angel , Howell Steve A. , Learmonth Michele , Aitken Alastair
Published: July 2002

A synthetic peptide, KKKKRFSFKKSFKLSGFSFKK, containing the phosphorylation sites of the acidic 80–87 kDa protein kinase C substrate was used to identify phosphopeptides in enzyme digests of this protein from mouse fibroblast C3H/10T1/2 cells. Stimulation of phosphorylation occurred, in vivo, with TPA at Ser7, Ser11 and Ser18, and, will two less potent phorbol esters, at Ser7 and Ser18. Okadaic acid effected a net phosphorylation of Ser7 and/or Ser11. Solid‐phase sequencing showed that, in vitro, the order of initial rate of phosphorylation was Ser11 > Ser7 > Ser18, while Ser18 was preferentially phosphorylated when either Ser7 or Ser11 was occupied. No significant phosphorylation of Ser15 was detected.
The jasmonate precursor, 12‐oxo‐phytodienoic acid. Induces phytoalexin synthesis in Petroselinum crispum cell cultures
Authors: Dittrich Heinz , Kutchan Toni M. , Zenk Meinhart H.
Published: July 2002

The pentacyclic biosynthetic precursor of jasmonic acid, 12‐oxo‐phytodienoic acid, was found to induce synthesis of the major flavonoid, apiin. in cell suspension cultures of Petroselinum crispum. The accumulation of apiin was preceded by an increase in the relative levels of poly (A)* RNAs that code for the flavonoid biosynthetic enzymes phenylalanine ammonia lyase, 4‐coumarate:CoA ligase and chalcone synthase, Poly (A)* RNAs reached maximal levels at approximately 4–6 h after the addition of elicitor while flavonoids continued to accumulate in the cultures for at least 6 days. 12‐Oxo‐phytodienoic acid is the first pentacyclic precursor in the jasmonic acid biosynthetic chain which functions as a signal transducer for phytoalexin induction.
Fourier transform infrared (FTIR) spectroscopic investigation of the nicotinic acetylcholine receptor (nAChR) Investigation of agonist binding and receptor conformational changes by flash‐induced release of ‘caged’ carbamoylcholine
Authors: Görne-Tschelnokow Ute , Hucho Ferdinand , Naumann Dieter , Barth Andreas , Mäntele Werner
Published: July 2002

The binding and interaction of carbamoylcholine with the nicotinic acetylcholine receptor was investigated using photolytically release carbamoylcholine (‘caged’ carbamoylcholine). Upon UV flash activation of this photolabile substrate analog, characteristic changes in the IR absorbance spectrum were detected. Apart from difference bands arising from the changes of molecular structure upon photolytical release, spectral features can be attributed to the agonist upon binding to the receptor as well as to conformational changes of the receptor itself. The two photo‐labile agonist analogs N‐[1‐(2‐nitrophenyl)ethyl] carbamoylcholine iodide (cage I) and N‐(α‐carboxy‐2‐nitrobenzyl) carbamoylcholine trifluoroacetate (cage II), with different structures for comparison of the 1680‐1540 cm−1 region sensitive for protein conformation, yielded consistent results. A preliminary interpretation in terms of substrate binding and local conformational changes of the receptor upon carbamoylcholine binding is provided, in analogy to the binding of acetylcholine, activation, and subsequent deactivation taking place during signal transduction.
Inhibition of hormone‐sensitive lipase by intermediary lipid metabolites
Authors: Jepson Catherine A. , Yeaman Stephen J.
Published: July 2002

Hormone‐sensitive lipase (HSL) is inhibited in a non‐competitive manner by oleoyl CoA, oleic acid and 2‐monopalmitoylglycerol, 50% inhibition being observed at concentrations of approx. 0.1 μM, 0.5 μM and 500 μM, respectively. HSL is a key enzyme in lipid metabolism, mobilising triacylglycerol and cholesterol ester stores in several tissues. Feedback inhibition of HSL by oleoyl CoA and oleic acid may therefore prevent accumulation of free fatty acids and cholesterol in the cell, whereas 2‐monoacylglycerol may act as a feedback inhibitor if the capacity of monoacylglycerol lipase is exceeded.
Autocatalytic modification of human carbonyl reductase by 2‐oxocarboxylic acids
Authors: Wermuth Bendicht , Bohren Kurt M. , Ernst Elsbeth
Published: July 2002

Carbonyl reductase occurs in multiple molecular forms. Sequence analysis has yielded a carboxyethyllysine residue in one of the enzyme forms, suggesting that pyruvate has been incorporated in a posttranslational enzymatic reaction [Krook, M., Ghosh, D., Strömberg, R., Carlquist, M. and Jörnvall, H. (1993) Proc. Natl. Acad. Sci. USA 90,502‐506]. Using highly purified carbonyl reductase from human brain we show that pyruvate and other 2‐oxocarboxylic acids are bound to the enzyme in an autocatalytic reaction. The resulting enzyme forms were indistinguishable from the native enzyme forms by electrophoresis and isoelectric focusing.
In vivo affinity label of a protein expressed in Escherichia coli
Authors: Odaka Masafumi , Kiribuchi Kyoko , Allison William S. , Yoshida Masasuke
Published: July 2002

When Tyr‐307 of the β subunit of f1 ‐ATPase from a thennophilic Bacillus strain PS3 is replaced by cysteine and expressed in Escherichia coli cells, about a half population of the mutant β subunit are labeled by Coenzyme A at Cys‐307 through a disulfide bond which is cleavable by reducing treatment. The mutant β subunit can be reconstituted into the α3β3, complex of which ATPase activity is stimulated two‐fold by reducing treatment either prior or after reconstitution. Since Tyr‐307 has been supposed to be located at one of subdomains which form the ATP binding site of the β subunit, Coenzyme A binds to the mutant β subunit as an AT(D)P analogue in E. coli cells and then covalently attaches to Cys‐307.
Phospholipid asymmetry of the outer membrane of rat liver mitochondria
Authors: Hovius Ruud , Thijssen José , van der Linden Peter , Nicolay Klaas , de Kruijff Ben
Published: July 2002

The phospholipid topology of the outer membrane of intact rat liver mitochondria and derived outer membrane vesicles was investigated by determining the accessible pool of the various phospholipid classes towards phospholipase A2, a phosphatidylcholine‐specific transfer protein and by chemical labeling using trinitrobenzene sulfonic acid. The outer membrane vesicles are sealed and have a right‐side‐out topology with a proposed localization of 55%, 77%, 100%, and at least 30% of the phosphatidylcholine, phosphatidylethanolamine, cardiolipin, and phosphatidylinositol plus phosphatidylserine in the outer leaflet, respectively. The outer membrane in intact mitochondria appears to have a similar phospholipid distribution.
Structure, function and regulation of plasma membrane H+‐ATPase
Authors: Serrano Ramón
Published: July 2002

Most antigenic determinants of yeast ATPase are located within its N‐terminal part. Amino acids 24–56, required for insertion at the plasma membrane, are highly accessible. The C‐terminus behaves as a modulable auto‐inhibitory domain in both yeast and plant ATPases. The expression of functional plant enzyme in yeast allows its mutational analysis. Plant tissues involved in active transport, such as the stomata guard cells, phloem, root epidermis and endodermis, are enriched in ATPase. One isoform is phloem‐specific. The fact that auxin induces the synthesis of ATPase in corn coleoptiles provides molecular support to the ‘Acid growth’ theory.
A d‐antifreeze polypeptide displays the same activity as its natural l‐enantiomer
Authors: Wen Dingyi , Laursen Richard A.
Published: July 2002

The d‐ and l‐forms of an α‐helical antifreeze polypeptide (AFP) have been chemically synthesized. Circular dichroism spectra of the molecules show equal and opposite ellipticities. The d‐ and the l‐enantiomers alone, and a 50:50 mixture of the two, all show identical antifreeze activity, but the enantiomeric forms are predicted to bind to the ice surface with different orientations. It is suggested that symmetry properties of certain ice surfaces permit the asymmetric binding of AFPs, and thus that AFPs are analogous to enzymes that act upon prochiral substrates.
Involvement of protein kinase in environmental stress‐induced activation of human multidrug resistance 1 (MDR1) gene promoter
Authors: Uchiumi Takeshi , Kohno Kimitoshi , Tanimura Hideyuki , Hidaka Katsuhiko , Asakuno Keizo , Abe Hiroko , Uchida Yuzo , Kuwano Michihiko
Published: July 2002

The human MDR1 gene can be induced in response to various environmental stimuli. To examine whether such stress‐induced activation of the MDR1 gene can be modulated by protein kinase, we employed a stable human cancer KB cell line which contained the bacterial chloramphenicol acetyltransferase (CAT) gene directed by the MDR1 gene promoter. H‐7, a protein kinase C inhibitor, at more than 40 μM inhibited activation of the MDR1 promoter that was induced by ethylmethane sulfonate, 5‐fluorouracil or UV irradiation. DNA binding activity of nuclear factors recognizing the MDR1 promoter was augmented in KB cells treated with UV, but decreased in cells treated concomitantly with H‐7. Okadaic acid alone was able to induce the promoter activation, and this induction was dependent on specific promoter sequences. Okadaic acid also enhanced the DNA binding activity of nuclear factors recognizing the MDR1 promoter. The phosphorylation of transacting factors may modulate the MDR1 gene promoter activity.
Alteration of the erythrocyte glutathione redox balance by N‐acetylcysteine, captopril and exogenous glutathione
Authors: Russell Jim , Spickett Corinne M. , Reglinski John , Smith W.Ewen , McMurray John , Abdullah Ibrahim B.
Published: July 2002

The effect of the thiol containing compounds N‐acetylcysteine and captopril on glutathione metabolism in human erythrocytes has been investigated non‐invasively using 1H spin echo NMR. N‐Acetylcysteine was found to increase the reduced form of glutathione while captopril increased the oxidized form, but no changes in the total glutathione concentration were observed. Incubation of the cells with buthionine sulphoximine to inhibit de novo glutathione synthesis did not affect the response. The results show that these compounds act by altering the redox balance of glutathione rather than by stimulating its synthesis, and that their mechanisms of action are different, and not simply due to the presence of the thiol group.
A membrane‐bound HIPIP type center in the thermohalophile Rhodothermus marinus
Authors: Pereira Manuela M. , Antunes Aida M. , Nunes Olga C. , da Costa Milton S. , Teixeira Miguel
Published: July 2002

A HIPIP‐type center was discovered in intact membranes of the thermohalophilic aerobe Rhodothermus marinus. In both the membrane‐bound state and after detergent solubilization and partial purification, this center exhibits an almost axial EPR spectrum, with g‐values at 2.13 and 2.03, similar to those of soluble HIPIP proteins isolated from purple bacteria. It has a high reduction potential, of 260 mV at pH 7.5. Rhodothermus HIPIP is involved in the main membrane‐bound electron‐transfer pathway, being reduced by NADH or succinate only in the presence of cyanide. The possible physiological function of this novel HIPIP‐type center is discussed.
Chloroplast import and sequential maturation of pea carbonic anhydrase: the roles of various parts of the transit peptide
Authors: Forsman Cecilia , Pilon Marinus
Published: July 2002

Chloroplast pea carbonic anhydrase is synthesised in the cytosol with an unusually long bipartite N‐terminal extension of the mature sequence previously proposed to serve as a transit peptide. Studies of import into pea chloroplasts show that the N‐terminal 69 amino acids of the previously proposed transit peptide is sufficient for translocation and localisation to the stroma, while the acidic C‐terminal part does not seem to have any function in these processes. Processing of the in vitro imported precursors is shown to be at a new cleavage site located in the middle of the actual transit peptide. The results indicate that maturation occurs in more than one step. The time‐course does not seem to be dependent on the age of the chloroplast but on the age of the translocated precursor.
Linkage between phosphorylated cystatin α and filaggrin by epidermal transglutaminase as a model of cornified envelope and inhibition of cathepsin L activity by cornified envelope and the conjugated cystatin α
Authors: Takahashi Masae , Tezuka Tadashi , Kakegawa Hisao , Katunuma Nobuhiko
Published: July 2002

Lysine‐rich phosphorylated cystatin α (P‐cystatin α) from newborn rat epidermis is a good substrate for epidermal transglutaminase (TGase) and also one of the component proteins of cornified envelope in the stratum corneum. Since the filaggrin linker segment peptide was efficiently conjugated with P‐cystatin α and was mediated by epidermal TGase in the presence of Ca2+ ions, filaggrin is a candidate for the glutamine‐rich linkage protein to conjugate with lysine‐rich P‐cystatin α. A conjugated protein was formed by epidermal TGase in the activated condition with Ca2+ ions and dithiothreitol. In contrast, the conjugated protein was not formed under chelated conditions with EDTA. The conjugated protein reacted positively with anti‐P‐cystatin α polyclonal antibody (PoAb). The conjugated protein and purified cornified envelope showed an inhibitory effect against papain and cathepsin L, but cathepsin B and H were not inhibited by these P‐cystatin α conjugates. Although the component protein, P‐cystatin α itself, inhibited cathepsin H strongly, these conjugated proteins inhibited specifically the cathepsin L family. The amino acid composition of cornified envelope protein and the conjugated protein of P‐cystatin α and filaggrin linker segment peptide was not completely the same. The conjugated protein of P‐cystatin α and filaggrin linker segment peptide showed the same inhibitory properties against cysteine proteinases as the cornified envelope. These findings suggest that the linkage protein between P‐cystatin α and filaggrin linker segment peptide may be considered a model of cornified envelope, although skin cornified envelope may be conjugated with some additional proteins.
Ion permeability induced in artificial membranes by the ATP/ADP antiporter
Authors: Tikhonova I.M. , Andreyev A.Yu. , Antonenko Yu.N. , Kaulen A.D. , Komrakov A.Yu. , Skulachev V.P.
Published: July 2002

The hypothesis on the additional function of the ATP/ADP antiporter (ANT) as uncoupling protein has been tested in proteoliposomes and planar bilayer phospholipid membranes (BLM). It is found that dissipation of the light‐induced ΔpH in the dark is very much faster in ANT‐bacteriorhodopsin proteoliposomes than in proteoliposomes containing baeteriorhodopsin as the only protein. Mersalyl treatment of ANT‐bacteriorhodopsin proteoliposomes causes further increase in the ΔpH dissipation rate due to formation of a high conductance pore. The properties of this pore are studied on ANT incorporated to BLM. They proved to be similar to those of so‐called multiple conductance channel or permeability transition pore of inner mitochondrial membrane. The conductance of the single channel is as high as 2.2 nS. The channel fails to discriminate between K+, Na+, H+ and Cl−. Thus the obtained data are consistent with the assumption that native and modified ANT might function as an H+‐specific conductor and as a permeability transition pore, respectively.
Differential effects of NaCl concentration on the constitutive activity of the thyrotropin and the luteinizing hormone/chorionic gonadotropin receptors
Authors: Cetani Filomena , Tonacchera Massimo , Vassart Gilbert
Published: July 2002

The TSH receptor (TSHR) and the receptor (LHR) are members of the family of G protein‐coupled receptors. Recently, point mutations conferring constitutive activity to the TSHR and LHR have been observed as a cause of toxic adenoma and familial/sporadic male pseudo‐precocious puberty, respectively. When evaluated by transfection in COS‐7 cells the wildtype (wt) TSHR displays definite constitutive activity towards Gs‐dependent adenylylcyclase stimulation, while available evidence shows that the LHR does not. In order to compare the constitutive activity of both receptors, we performed functional studies in COS‐7 cells using different assay conditions. Human TSHR and LHR cDNAs subcloned in the expression vector pSVL were transiently expressed in COS‐7 cells and cAMP production was determined following incubation in a medium containing physiological concentration of NaCl [isotonic (NaCl)] or in the same medium without NaCl [hypotonic (NaCl−)] or where NaCl was replaced by an isoosmolar concentration of sucrose [isotonic (sucrose)]. Cells transfected with the TSHR showed higher basal cAMP levels over cells transfected with pSVL in all conditions tested. The effect was stronger when cells were incubated in isotonic (sucrose) buffer. Cells expressing LHR exhibited a minimal increase of cAMP levels over cells transfected with pSVL in isotonic (NaCl) buffer; however, a marked increase in basal cAMP levels was observed when cells were assayed in hypotonic (NaCl−) or isotonic (sucrose) buffers. Varying the pH or incubation temperature was without effect on the results obtained with both receptors. Our data show that despite extensive sequence similarity, the LH and TSH receptors differ markedly in their basal activity. The differential sensitivity of both receptors to low NaCl concentrations, suggests that the unliganded TSH receptor is less constrained than its LH homolog and may be more susceptible to activation by a wide spectrum of mutations.
Identification of a melanoma antigen, PRAME, as a BCR/ABL‐inducible gene
Authors: Watari Kiyoshi , Tojo Arinobu , Nagamura-Inoue Tokiko , Nagamura Fumitaka , Takeshita Akihiro , Fukushima Toshihiro , Motoji Toshiko , Tani Kenzaburo , Asano Shigetaka
Published: July 2002

In order to elucidate molecular events in BCR/ABL‐induced transformation, we adopted a polymerase chain reaction (PCR)‐based technique of differential display and compared mRNA expression in human factor‐dependent cells, TF‐1, with that in factor‐independent cells, ID‐1, which were established from TF‐1 cells by transfection of BCR/ABL. Cloning and sequencing of a gene which was upregulated in ID‐1 cells revealed that the gene was identical to a melanoma antigen, PRAME. Our present study demonstrated that PRAME was markedly expressed in primary leukemic cells with chronic myeloid leukemia (CML) in blastic crisis and Philadelphia (Ph)+‐acute lymphoblastic leukemia (ALL), in which BCR/ABL played an important role as a pathogenic gene. Moreover, comparison of PRAME expression among CD34+ cells with CML in blastic, accelerated, and chronic phases revealed a higher expression in CML in advanced phases. Thus PRAME was considered to be a good candidate for a marker of Ph+‐leukemic blast cells as well as a new target antigen of leukemic blast cells that cytotoxic T cells can recognize.
Endocytosis of FcγRI is regulated by two distinct signalling pathways
Authors: Norman J.C. , Allen J.M.
Published: July 2002

Aggregation by immune complexes of receptors specific for the Fc region of IgG results in their internalisation and disposal by trafficking to lysosomes. We show here that internalisation of FcγRI by IFN‐γ treated U937 cells following receptor aggregation by cross‐linking antibodies requires the activation of two distinct signalling pathways. The pathways were functionally dissected in streptolysin‐O‐permeabilised cells by capitalising on their relative dependence on active GTP binding proteins. One pathway required the presence of GTP‐γS or active βγ subunits, the other did not. Use of inhibitors revealed that the βγ‐independent pathway required activation of PI 3‐kinases and was PKC‐independent In contrast, the βγ‐dependent pathway involved activation of phospholipase C‐β and PKC, but was PI 3‐kinase‐independent. Both these pathways were found to be active in intact cells and are likely to determine receptor trafficking following internalisation.
Genomic Exploration of the Hemiascomycetous Yeasts: 7. Saccharomyces servazzii
Authors: Casaregola Serge , Lépingle Andrée , Bon Elisabeth , Neuvéglise Cécile , Nguyen Huu-Vang , Artiguenave François , Wincker Patrick , Gaillardin Claude
Published: July 2002

The genome of Saccharomyces servazzii was analyzed with 2570 random sequence tags totalling 2.3 Mb. BLASTX comparisons revealed a minimum of 1420 putative open reading frames with significant homology to Saccharomyces cerevisiae (58% aa identity on average), two with Schizosaccharomyces pombe and one with a human protein, confirming that S. servazzii is closely related to S. cerevisiae. About 25% of the S. servazzii genes were identified, assuming that the gene complement is identical in both yeasts. S. servazzii carries very few transposable elements related to Ty elements in S. cerevisiae. Most of the mitochondrial genes were identified in eight contigs altogether spanning 25 kb for a predicted size of 29 kb. A significant match with the Kluyveromyces lactis linear DNA plasmid pGKL‐1 encoded RF4 killer protein suggests that a related plasmid exists in S. servazzii. The sequences have been deposited with EMBL under the accession numbers AL402279–AL404848.
Evidence for the involvement of the GTS1 gene product in the regulation of biological rhythms in the continuous culture of the yeast Saccharomyces cerevisiae
Authors: Wang Jinqing , Liu Weidong , Mitsui Kazuhiro , Tsurugi Kunio
Published: July 2002

In the yeast Saccharomyces cerevisiae, ultradian oscillations of energy metabolism have been observed in continuous cultures. Here, we found that the level of the GTS1 gene product oscillated in concert with the ultradian rhythm of energy metabolism. When GTS1 was inactivated by gene disruption, the metabolic oscillation was affected severely, mostly disappearing within a day, in the absence of synchronized stress‐response oscillations throughout the continuous culture. The disappearance of biological rhythms in the GTS1‐deleted mutant was substantially rescued by transformation with chimera plasmids carrying GTS1 under the control of GTS1's own promoter. On the other hand, this disappearance was not rescued by constitutive expression of GTS1 under the control of the triose phosphate isomerase promoter.
Analysis of sequence‐dependent curvature in matrix attachment regions
Authors: Yamamura Jun , Nomura Koji
Published: July 2002

Sequence‐dependent DNA conformations of matrix attachment regions (MARs) available in a database were calculated using the wedge model, and compared with randomly chosen genes, promoters, enhancers and transposons. The MARs had a longer bent part and higher angle/helical turn than the other regions. It is known that some MAR sequences have A‐tracts that cause DNA bending, and we also found many A‐tracts in examined MARs. Furthermore, non‐random and clustered distribution of A‐tracts shown here gave further evidence of the importance of A‐tracts for MAR conformations. These results suggest that DNAs of MARs have a characteristic conformation instead of conserved sequence.
A deteriorated triple‐helical scaffold accelerates formation of the Tetrahymena ribozyme active structure
Authors: Ohki Yasushi , Ikawa Yoshiya , Shiraishi Hideaki , Inoue Tan
Published: July 2002

The Tetrahymena group I ribozyme requires a hierarchical folding process to form its correct three‐dimensional structure. Ribozyme activity depends on the catalytic core consisting of two domains, P4–P6 and P3–P7, connected by a triple‐helical scaffold. The folding proceeds in the following order: (i) fast folding of the P4–P6 domain, (ii) slow folding of the P3–P7 domain, and (iii) structure rearrangement to form the active ribozyme structure. The third step is believed to directly determine the conformation of the active catalytic domain, but as yet the precise mechanisms remain to be elucidated. To investigate the folding kinetics of this step, we analyzed mutant ribozymes having base substitution(s) in the triple‐helical scaffold and found that disruption of the scaffold at A105G results in modest slowing of the P3–P7 folding (1.9‐fold) and acceleration of step (iii) by 5.9‐fold. These results suggest that disruption or destabilization of the scaffold is a normal component in the formation process of the active structure of the wild type ribozyme.
Myotonic dystrophy protein kinase phosphorylates the myosin phosphatase targeting subunit and inhibits myosin phosphatase activity
Authors: Murányi Andrea , Zhang Rongxin , Liu Feizhou , Hirano Katsuya , Ito Masaaki , Epstein Henry F. , Hartshorne David J.
Published: July 2002

Myotonic dystrophy protein kinase (DMPK) and Rho‐kinase are related. An important function of Rho‐kinase is to phosphorylate the myosin‐binding subunit of myosin phosphatase (MYPT1) and inhibit phosphatase activity. Experiments were carried out to determine if DMPK could function similarly. MYPT1 was phosphorylated by DMPK. The phosphorylation site(s) was in the C‐terminal part of the molecule. DMPK was not inhibited by the Rho‐kinase inhibitors, Y‐27632 and HA‐1077. Several approaches were taken to determine that a major site of phosphorylation was T654. Phosphorylation at T654 inhibited phosphatase activity. Thus both DMPK and Rho‐kinase may regulate myosin II phosphorylation.
Diacylglycerol effects on phosphatidylinositol‐specific phospholipase C activity and vesicle fusion
Authors: Villar Ana V , Goñi Félix M , Alonso Alicia
Published: July 2002

Diacylglycerol increased the hydrolytic activity of phosphatidylinositol‐specific phospholipase C on large unilamellar vesicles containing 5–40% phosphatidylinositol. Moreover, diacylglycerol increased the rate and extent of vesicle fusion (contents mixing) induced by the enzyme. Kinetic studies of intervesicular lipid mixing revealed that fusion was limited by the frequency of contacts involving two diacylglycerol‐rich domains.
Skeletal and cardiac ryanodine receptors bind to the Ca2+‐sensor region of dihydropyridine receptor α1C subunit
Authors: Mouton Jérôme , Ronjat Michel , Jona Istvan , Villaz Michel , Feltz Anne , Maulet Yves
Published: July 2002

In striated muscles, excitation–contraction coupling is mediated by the functional interplay between dihydropyridine receptor L‐type calcium channels (DHPR) and ryanodine receptor calcium‐release channel (RyR). Although significantly different molecular mechanisms are involved in skeletal and cardiac muscles, bidirectional cross‐talk between the two channels has been described in both tissues. In the present study using surface plasmon resonance spectroscopy, we demonstrate that both RyR1 and RyR2 can bind to structural elements of the C‐terminal cytoplasmic domain of α1C. The interaction is restricted to the CB and IQ motifs involved in the calmodulin‐mediated Ca2+‐dependent inactivation of the DHPR, suggesting functional interactions between the two channels.