bluuebunny/crossref_metadata_2025 huggingface dataset:
Analysis of the DNA topoisomerase‐II‐mediated cleavage of the long terminal repeat of Drosophila 1731 retrotransposon
Authors: NAHON Evelyne , BEST‐BELPOMME Martin , SAUCIER Jean‐Marie
Published: March 2005

The interaction of DNA topoisomerase II with the long terminal repeat (LTR) of the Drosophila melanogaster 1731 retrotransposon was studied. The covalent binding of topoisomerase II to the LTR was strongly stimulated by different inhibitors of the enzyme 4′‐demethylepipodophyllotoxin‐9‐(4,6‐O‐2‐ethylidene‐β‐>‐glucopyranoside (VP‐16), 4′‐(9‐acridinylamino)methanesulfon‐m‐anisidine) (m‐AMSA) and an ellipticine derivative. Enzyme‐mediated DNA cleavage could be observed in the absence of inhibitors and was stimulated in their presence. Cleavage occurred predominantly at sites located within or at the boundary of alternating purine/pyrimidine tracts in agreement with previous observations [Spitzner, J. R., Chung, I. K. & Muller, M. T. (1990) Eukaryotic topoisomerase II preferentially cleaves alternating purine‐pyrimidine repeats, Nucleic Acids Res. 18, 1–11]. In addition, all of the cleavage sites observed in the absence of inhibitor were located in the U3 region of the LTR. The site specificity of drug‐induced cleavage was studied and the conformity of the cleavage sites with previously established consensus sequences was examined. Our results suggest that DNA topoisomerase II, through its ability to alter the degree of DNA supercoiling, might be involved in the control of different functions of the LTR.
Investigation of the oxygenation of phospholipids by the porcine leukocyte and human platelet arachidonate 12‐lipoxygenases
Authors: TAKAHASHI Yoshitaka , GLASGOW Wayne C. , SUZUKI Hiroshi , TAKETANI Yutaka , YAMAMOTO Shozo , ANTON Monika , KÜHN Hartmut , BRASH Alan R.
Published: March 2005

When arachidonate 12‐lipoxygenase purified from porcine leukocytes was incubated aerobically with 1‐palmitoyl‐2‐arachidonoyl‐sn‐glycero‐3‐phosphocholine, the phospholipid reacted at up to 30% of the rate of a free fatty acid substrate; the esterified arachidonic acid was oxygenated predominantly to the (12S)‐12‐hydroperoxy product. The porcine leukocyte enzyme was also capable of metabolizing phosphatidylcholine containing esterified (15S)‐15‐hydroperoxy‐5,8,11,13‐eicosatetraenoic acid; oxygenation occurred predominantly at the 14R position. Reaction with mitochondrial and endoplasmic membranes of rat liver produced esterified (12S)‐12‐hydroperoxy‐5,8,10,14‐eicosatetraenoic acid and (13S)‐13‐hydroperoxy‐9,11‐octadecadienoic acid as major oxygenation products. Thus, porcine leukocyte 12‐lipoxygenase is capable of oxygenating not only free polyenoic fatty acids but also more complex substrates such as phospholipids and biomembranes. In contrast, the human platelet 12‐lipoxygenase is almost inactive with these esterified polyenoic fatty acids. In regard to the function of these enzymes, the leukocyte‐type of 12‐lipoxygenase has similar catalytic activities to the mammalian 15‐lipoxygenase and its physiological function may include the structural modification of membrane lipids.
Nucleotide sequence and promoter‐specific effect of a negative regulatory region located upstream of the mouse proliferating cell nuclear antigen gene
Authors: MATSUOKA Shuhei , YAMAGUCHI Masamitsu , HAYASHI Yuko , MATSUKAGE Akio
Published: March 2005

Different portions of the 5′‐upstream region of the mouse proliferating cell‐nuclear‐antigen (PCNA) gene were combined with the bacterial chloramphenicol acetyltransferase (CAT) gene of a CAT vector. A transient expression assay of CAT activity in mouse neuroblastoma N18TG2 cells transfected with these recombinant plasmids and RNase protection analysis have revealed the existence of a negative regulatory region between nucleotides –1231 and –624 (+1 denotes the transcription initiation site). The CAT expression levels were gradually increased, depending on the extent of deletion from the 5′‐terminus in this region, suggesting that the negative regulatory region consists of multiple elements with rather weak repressing activities.

Significant sequence similarity was found between the negative regulatory region of the PCNA gene and those of the several reported genes. A 752‐bp segment containing this negative regulatory region repressed the function of the PCNA gene promoter in an orientation‐independent and position‐independent manner. However, the negative regulatory region showed almost no repressing effect on the functions of the heterologous gene promoters such as the simian virus 40 enhancer promoter, the enhancer promoter in the Rous sarcoma virus long‐terminal repeat and the mouse DNA polymerase β gene promoter. These results suggest that the negative regulatory region of the mouse PCNA gene functions specifically to its own promoter. This unique property is discussed in comparison with that of the negative regulatory elements of the mouse DNA polymerase β gene.
Anomalous interaction of Sp1 and specific binding of an E‐box‐binding protein with the regulatory elements of the Na,K‐ATPase α2 subunit gene promoter
Authors: IKEDA Keiko , NAGANO Kei , KAWAKAMI Kiyoshi
Published: March 2005

We identified cis elements in the 5′‐flanking region of rat Na,K‐ATPase α2 subunit gene (Atp1 a2) using transient transfection assays in L6 rat skeletal muscle myoblast cells. By 5′‐deletion mutation analysis, the region between nucleotide positions –175 and –108 was identified as a positive regulatory region. In the region, the distal E box (nucleotides –144 to –139) acts as a negative regulatory element, and the Sp1 consensus sequence (nucleotides –123 to –118) and the GGGAGG sequence (nucleotides –114 to –109) act as positive regulatory elements. Gel‐retardation analysis revealed that binding factors are an E‐box‐binding protein and Sp1. DNase I foot‐printing and methylation‐interference analyses revealed that Sp1 binds to the region from nucleotides –122 to –101 and the E‐box‐binding protein to the region from nucleotides –144 to –136. T4 DNA polymerase footprinting revealed that there are three Sp1‐binding sites in the region and that Sp1 binds to one of the three sites in a mutually exclusive manner. The mechanism by which Sp1 activates the Atp1a2 promoter is discussed.
Recombinant coho salmon insulin‐like growth factor I
Authors: MORIYAMA Shunsuke , DUGUAY Stephen J. , CONLON J. Michael , DUAN Cunming , DICKHOFF Walton W. , PLISETSKAYA Erika M.
Published: March 2005

Recombinant coho salmon insulin‐like growth factor I (rsIGF‐I) was produced in Escherichia coli, purified and characterized. The rsIGF‐I expression vector was constructed by polymerase chain reaction and cloning into a plasmid containing a phage T7 RNA polymerase promoter. The rsIGF‐I was recovered from bacterial inclusion bodies, solubilized under reducing conditions, immediately refolded, then fractionated by a two‐step ion‐exchange chromatography on DEAE‐52 and Mono‐S columns. It was further purified by HPLC on a reverse‐phase Asahi‐Pak C4P‐50 C4 column. Purification of rsIGF‐I was monitored by SDS/PAGE and immunoblot with anti‐[human somatomedin C (SM C)/IGF‐I] serum. The rsIGF‐I appeared as a single band with molecular mass of 7 kDa, the same size as recombinant human IGF‐I (rhIGF‐I) and cross‐reacted with anti‐(human SM C/IGF‐I) serum. The amino acid sequence of rsIGF‐I contained an NH2‐terminal methionine residue followed by the sequence predicted for mature sIGF‐I. At concentrations in the range 3.9–250 ng/ml, rsIGF‐I significantly stimulated sulfate uptake by the cultured branchial cartilage of coho salmon. The stimulatory effect of rsIGF‐I was concentration dependent and slightly more potent than that of rhIGF‐I at the highest concentration tested.
Reconstitution of holotransketolase is by a thiamin‐diphosphate‐magnesium complex
Authors: BOOTH Christine K. , NIXON Peter F.
Published: March 2005

When human erythrocyte apo‐transketolase is mixed with cofactors and substrates, the progress curve exhibits a lag phase. Elimination of the lag phase requires the presence of saturating concentrations of cofactors, thiamin diphosphate and Mg2+. The most simple explanation of the observed hysteretic transition is that the slow binding of a Mg2+‐thiamin‐diphosphate species precedes slow isomerisation of the enzyme to the active form. Although the hysteretic transition involves more than one process, it does not involve the dissociation–association of enzyme subunits. The best estimate of the apparent Km, 1.59 ± 0.23 μM, for the binding of Mg2+‐thiamin diphosphate to transketolase was obtained in the presence of a high non‐inhibitory concentration of magnesium and varied concentrations of thiamin diphosphate. Thus the reconstitution of the human enzyme differs from the yeast enzyme, which undergoes a rate‐limiting dimerisation during reconstitution.
Overexpression of Desulfovibrio vulgaris Hildenborough cytochrome c553 in Desulfovibrio desulfuricans G200
Authors: BLANCHARD Laurence , MARION Dominique , POLLOCK Brent , VOORDOUW Gerrit , WALL Judy , BRUSCHI Mireille , GUERLESQUIN Françoise
Published: March 2005

Plasmid pRC41, containing the cyf gene encoding cytochrome c553 from Desulfovibrio vulgaris Hildenborough, was transferred by conjugation from Escherichia coli to Desulfovibrio desulfuricans G200. The structural properties of the purified protein were studied by one‐dimensional and two‐dimensional NMR. A heterogeneity in the folding of the cytochrome isolated from D. vulgaris Hildenborough and from D. desulfuricans G200 was observed for the oxidized form. Temperature, pH and salt‐dependence studies indicated that the heterogeneity does not result from an intermediate in the protein unfolding process, but derives from two conformations which are not in dynamic equilibrium.
Conformation of thymosin β4 in water determined by NMR spectroscopy
Authors: CZISCH Michael , SCHLEICHER Michael , HÖRGER Susanne , VOELTER Wolfgang , HOLAK Tad A.
Published: March 2005

The conformational preferences of a 43‐amino‐acid G‐actin‐binding peptide, thymosin β4, in water at 1, 4 and 14°C, and at pH 3.0 and 6.5 were studied by NMR. NMR showed that thymosin β4 lacks a uniquely folded conformation in water. However, some preferential α‐helical conformations of thymosin β4 can be observed in aqueous solutions. The segment at residues 5–16 showed characteristic interactions for conformations in both the β‐strand and α‐helical regions of the ø‐Ψ space, based on strong CαH(i)‐NH(i+1) interactions and NH‐NH, CαH(i)‐NH(i+3), and CαH(i)‐CβH(i+3) interactions, respectively. At 1–4°C, another segment at residues 31–37 also shows both β and α conformations, forming however a less well‐defined helix than the segment at residues 5–16. At 14°C, the conformational population of the helix at positions 5–16 is shifted more towards the random and turn‐like structures, whereas the segment at positions 31–37 becomes exclusively a random coil.
Induction of translational thermotolerance in liver of thermally stressed rats
Authors: DE MAIO Antonio , BECK Stephen C. , BUCHMAN Timothy G.
Published: March 2005

Heat‐shock gene expression in cultures of single cell types has been well characterized but little is known about the heat‐shock response of intact organs in vivo. In this study, the kinetics of hepatic heat‐shock gene expression and the induction of thermotolerance were characterized in rats. Animals were subjected to a defined, reversible stress by increasing the core body temperature to 41°C or 42°C for 30 min. New synthesis of the inducible form of the heat shock‐70 family of proteins (hsp‐72) peaked simultaneously with the maximal level of hsp‐72 transcripts at both temperatures. These data are consistent with previous observations in cultures of hepatoblastoma cells after thermal stress [De Maio, A., Beck, S. C. & Buchman, T. G. (1993) Circ. Shock 40, 177–186].

The incorporation of radioactive amino acids into polypeptides by the liver was blocked during the first hour of recovery after heat shock at 42°C. This inhibition of protein synthesis by thermal stress could be prevented by prestressing rats at 42°C for 30 min and allowing the rats to recover for 24 h at normal body temperature (37°C). This phenomenon, previously defined as ‘translational thermotolerance’, correlates with the hepatic content of hsp‐72; maximal protection occurs 24 h after a 42°C thermal stress when hsp‐72 (protein) is also maximum and decreases with the clearance of hsp‐72 from the liver. These data suggest that the presence of hsp‐72 within the liver may modulate the organ response to subsequent stresses and may be important to organ and animal survival after repeated insults.
Interferon‐γ up‐regulates a unique set of proteins in human keratinocytes
Authors: HONORÉ Bent , LEFFERS Henrik , MADSEN Peder , CELIS Julio E.
Published: March 2005

Treatment of proliferating and quiescent primary human keratinocytes with interferon‐γ (IFN‐γ) (100 U/ml, 23.5 h) followed by two‐dimensional gel analysis revealed three proteins, IGUP I‐3421 (Mr= 48 200, pI= 6.06); IGUP I‐3524 (Mr= 56 900, pI= 5.92), a protein homologous to peptidechain‐release factor and tryptophanyl‐tRNA syntetase; and IGUP I‐5111 (Mr= 30400, pI= 5.76) recorded in the keratinocyte protein database (Celis et al., 1991, 1992) that are highly and specifically up‐regulated by IFN‐γ among several agents tested including 14 other cytokines, second messengers [dibutyryl cAMP (Bt2cAMP), dibutyryl cGMP (Bt2cGMP)] and compounds known to affect keratinocytes [4β‐phorbol 12‐myristate 13‐acetate (PMA), retinoic acid, Ca2+, dexamethasone, lipopolysaccharides, foetal calf serum]. Protein IGUP I‐5111 was selected for further studies as its level is affected by simian‐virus‐40 transformation and because peptide sequences were available in the microsequence database. The cDNA was cloned from a fibroblast cDNA library using degenerate oligodeoxyribonucleotides and expressed in AMA cells using the vaccinia virus expression system. Database searches indicated that the predicted protein, which migrated with the AMA variant of keratinocyte protein IEF SSP 5111, is novel although it exhibits weak similarity to cytoskeletal proteins. IGUP I‐5111 contains the RGD sequence found in many extracellular glycoprotein ligands of the integrin receptor family and it is found at least partially in the culture supernatant.

Considering the presence of IFN‐γ in psoriatic plaques as well as its putative involvement in the pathophysiology of the disease it was of interest to determine whether the set of proteins was up‐regulated in these cells. Two‐dimensional gel analysis of the protein phenotype of non‐cultured, unfractionated psoriatic keratinocytes failed to reveal up‐regulation of any of the three IFN‐γ‐induced proteins suggesting that the effect of IFN‐γin vivo may be modulated by the activity of other cytokine(s) or growth factor(s). Psoriatic keratinocytes were equally sensitive to IFN‐γ as their normal counterparts.
Human progelatinase A can be activated by autolysis at a rate that is concentration‐dependent and enhanced by heparin bound to the C‐terminal domain
Authors: CRABBE Thomas , IOANNOU Chris , DOCHERTY Andrew J. P.
Published: March 2005

Activation of the latent precursor of human gelatinase A (progelatinase A) was investigated using recombinant proenzyme purified from culture medium conditioned by transfected mouse myeloma cells. A 4.0 μM progelatinase A solution was activated to a maximum of 48% of the activity produced by 4‐aminophenylmercuric acetate (APMA) simply by its incubation at 37°C for 12 h, though at lower starting concentrations the rate and extent of activation were reduced. Activation was shown to be the result of a single autolytic cleavage at the Asn80‐Tyr81 peptide bond that removes the propeptide and converts the Mr= 72000 proenzyme into the Mr= 66000 active species also produced by APMA activation. It is proposed that this cleavage is a bimolecular event catalysed by previously activated gelatinase A. The addition of heparin increased by approximately eightfold the initial rate of progelatinase A autolytic activation but did not affect the activation of a deletion mutant that lacked the C‐terminal domain [des‐(418–631)progelatinase A]. The inference that this increase resulted from an interaction between heparin and the C‐terminal domain was supported by the finding that, unlike des‐(418–631)gelatinase A, both full‐length gelatinase A and the isolated C‐terminal domain were able to bind to heparin–Sepharose CL‐6B and that, at NaCl concentrations sufficient to abolish this binding, heparin had no effect. We conclude that heparin is able to enhance autolytic activation by acting as a template that approximates active → latent gelatinase A and suggest that a similar mechanism may account for the cell‐surface activation of this enzyme.
Photoaffinity labeling of the human red‐blood‐cell urea‐transporter polypeptide components
Authors: NEAU Philippe , DEGEILH Françoise , LAMOTTE Hervé , ROUSSEAU Bernard , RIPOCHE Pierre
Published: March 2005

The tritiated urea analogue 1‐(3‐azido‐4‐chlorophenyl)‐3methyl‐2‐thiourea ([3H]MeACPTU) was used as a probe to photolabel the human red‐blood‐cell membrane facilitated urea transporter. On irradiation, [3H]MeACPTU incorporated irreversibly into white ghost membranes. SDS/gel electrophoresis of membranes revealed radioactive incorporation in five major bands of 200, 110, 60, 40 and 14 kDa. The labeling of the 40‐kDa and 60‐kDa bands was partly prevented by the presence of a high concentration of other urea analogues such as thiourea and 1‐(3,4‐dichlorophenyl) 2‐thiourea (DCPTU). The photolabeling pattern obtained with white ghosts of the Kidd blood‐group type Jk(a−,b−) showed no labeling of the 40‐kDa polypeptide. Protecting experiments carried out with anti‐Jka, anti‐Jkb and anti‐Jk3 sera prevented radioactive incorporation in the 60‐kDa band and in the 110‐kDa band. Urea permeability of pink ghosts of blood type Jk(a+,b+) measured in the presence of Jk3 antibodies was 19% lower than the control values. However, urea permeability of frog urinary bladder epithelial cells was not affected by the presence of Jk‐reactive antibodies.

These results support the hypothesis that the Kidd antigen and the facilitated urea transporter are the same protein. Our estimation of the number of copies in each cell is close to that of the previously published value of 14000.
Big endothelin‐1 structure important for specific processing by endothelin‐converting enzyme of bovine endothelial cells
Authors: OKADA Kenji , ARAI Yukiko , HATA Mikiko , MATSUYAMA Kenji , YANO Mitsuo
Published: March 2005

Phosphoramidon‐sensitive endothelin‐converting enzyme of bovine endothelial cells showed substrate selectivity for big endothelin‐1 (ET‐1) when compared to big ET‐1(1–38), big ET‐2(1–37), big ET‐2(1–38) and big ET‐3(1–41). To investigate the big ET‐1 structure important for specific conversion by the endothelin‐converting enzyme, we synthesized a series of truncated analogues of big ET‐1, measured the hydrolysis of their Trp21–Val22 bonds, and found that a 16‐residue peptide, big ET‐1(19–34), is the minimal peptide sequence. This suggests that an unusually long carboxy‐terminal sequence is required for big ET‐1 conversion. Alanine substitution for individual amino acids in the carboxy‐terminal region of big ET‐1(19–34) demonstrated that His27, Val29, Pro30, Tyr31, Gly32, Leu33 and Gly34 are more important than Asn23, Thr24, Pro25, Glu26 and Val28 for eliciting efficient hydrolysis of the Trp21–Val22 bond, even though the former residues are located at more distant positions from the cleavage sites than are the latter. These results, together with the fact that big ET‐2 and big ET‐3 show heterogeneity in the big ET‐1 residues His27, Val28, Val29 and Gly34, suggest that the His27‐Val‐Val‐Pro‐Tyr‐Gly‐Leu‐Gly34 sequence in the carboxy‐terminal region of big ET‐1 plays the most important role in selective conversion by endothelin converting enzyme.
Structural investigation of transglutaminase by Fourier transform infrared spectroscopy
Authors: TANFANI Fabio , BERTOLI Enrico , SIGNORINI Marco , BERGAMINI Carlo M.
Published: March 2005

The secondary structure of transglutaminase was investigated by Fourier tranform infrared spectroscopy. Spectra of the protein in both H2O and 2H2O were analyzed by deconvolution and second derivative methods in order to observe the overlapping components of the amide‐I band. The quantitative analysis of the amide‐I‐band components was made by a curve‐fitting procedure. The protein was studied in the absence and in the presence of 1 mM GTP, 1 mM Ca2+ and 1 mM GTP/1 mM Ca2+. The quantitative analysis of infrared spectra revealed that no remarkable changes in the secondary structure of the enzyme are induced by GTP, Ca2+ or Ca2+/GTP. Major changes, however were observed in the thermal‐denaturation behavior of the protein. The protein showed maximum of denaturation at temperatures over 50–55°C in the absence or in the presence of 1 mM Ca2+ and over 55–60° in the presence of 1 mM GTP or 1 mM Ca2+/1 mM GTP. The results obtained indicate that GTP induces a stabilization of the tertiary structure of the enzyme, even in the presence of 1 mM Ca2+. The thermal denaturation patterns of the protein suggest the occurrence of Ca2+‐dependent aggregation.
Synergistic binding of hydrophobic probes and zinc ligands to thermolysin
Authors: PFUETZNER Richard A. , CHAN William W.‐C.
Published: March 2005

The strong synergism previously observed in the binding of inhibitors to two Zn‐proteases, has also been found for thermolysin. As in earlier cases, the effects are produced by a small Zn‐ligand (e.g. a hydroxamate) in the presence of another compound which contains the key structural features of specific substrates (a specificity probe). For thermolysin, the most effective specificity probes are hydrophobic derivatives of amines and amino acids (e.g. carbobenzyloxy‐L‐alaninol). Even the simple combination of benzyl alcohol and formohydroxamate displays considerable synergism. The above effects are temperature dependent and correlate well with a thermally induced conformational isomerization reported recently for this enzyme.

Our results seem to be related to previous observations of substrate synergism in the reverse reaction and to superactivation by chemical modification of this enzyme. All these effects are consistent with a change in the environment of the catalytically important zinc atom upon binding of the hydrophobic side chain of the substrate. With the inclusion of thermolysin, binding synergism is now known to occur in an endopeptidase as well as in exopeptidases of diverse specificity. The general occurrence of this phenomenon in zinc proteases and its possible significance are discussed in an accompanying study.
Preferential sites of proteolytic cleavage of bovine, human and rat thyroglobulin
Authors: GENTILE Fabrizio , SALVATORE Gaetano
Published: March 2005

The sites and the sequence of the proteolytic cleavages of bovine, human and rat thyroglobulin, during the limited proteolysis with thermolysin and trypsin, were determined by sequencing the NH2 termini of the peptides produced and comparing them to the cDNA‐derived sequences of bovine, human and rat thyroglobulin. Major cleavage sites of bovine thyroglobulin included residues 240, 502, 993, 1218, 1784 with thermolysin, and 240, 520, 1142, 1783, 2515 with trypsin. Cleavage sites of human thyroglobulin included residues 503, 982, 990, 1405, 1831 with thermolysin, and 522, 1627, 2513 with trypsin. Those of rat thyroglobulin included residues 501, 1776, 1784 with thermolysin, and 522, 1771, 1825, 2515 with trypsin (numbered as in bovine thyroglobulin). Thus, thyroglobulin from various species presents well localized and conserved regions particularly sensitive to proteolysis. The most sensitive region extended for 30 residues after residue 500. Another major cluster of cleavages was centered around residue 1800; this region was only partially sensitive in human thyroglobulin. A conserved tryptic site lay at the COOH terminus of the molecule. Most cleavage sites occurred within the inserted sequences that disrupt the Cys‐rich, tandem repeats of thyroglobulin and either contain or are located near exon–intron junctions. Several cleavage sites lay in proximity of early iodinated or hormonogenic tyrosyl residues or of putative N‐linked glycosylation sites. While a predominantly β‐type secondary structure and a rigid three‐dimensional structure were predicted for the Cys‐rich repeats, stretches of predicted α‐helices, β‐strands and irregular structure were interspersed in the regions surrounding the cleavage sites. These data demonstrate the existence of conserved regions of thyroglobulin inherently sensitive to proteolysis, which most likely represent solvent‐exposed regions of the primary structure, possibly forming loops at the surface of thyroglobulin.
Characteristics of lysophospholipase activity expressed by cytosolic phospholipase A2
Authors: FUJIMORI Yumi , KUDO Ichiro , FUJITA Ken‐ichi , INOUE Keizo
Published: March 2005

Evidence has accumulated to suggest that a wide variety of mammalian cells and tissues express a cytosolic phospholipase A2 with arachidonoyl preference (cPLA2). Purified rabbit platelet‐derived cPLA2, as well as the human recombinant enzyme originally identified in the monocytic leukemic cell line U937, exhibit significant lysophospholipase activity. Several series of experiments indicated that a single protein mediated both activities. Treatment of the purified enzyme with p‐bromophena‐cylbromide or an anti‐(rabbit platelet cPLA2) monoclonal antibody, RHY‐5, suppressed the activity of phospholipase A2 without any appreciable effect on lysophospholipase activity, suggesting that the domain(s) required for phospholipase A2 activity may be located separately from that for lysophospholipase activity. Lysophospholipase activity was appreciably detected above the critical micellar concentration of the substrate. Lysophosphatidylcholine was also hydrolyzed efficiently when it was incorporated into liposomes made of dialkylphosphatidylcholine. The hydrolysis of lysophospholipid was dependent on the fatty acid bound at the sn1 position; the relative rates of hydrolysis of 1‐oleoyllysophosphatidylcholine, 1‐palmitoyllysophosphatidylcholine, and 1‐stearoyllysophosphatidylcholine were 23, 8, and 1, respectively. A similar order of reactivity was observed with lysophospholipid incorporated into dialkylphosphatidylcholine liposomes. cPLA2 may function not only as an arachidonate liberation enzyme but also as an enzyme responsible for degradation of certain molecular species of lysophospholipids formed in membranes.
Cloning and characterization of the poly(hydroxyalkanoic acid)‐depolymerase gene locus, phaZ1, of Pseudomonas lemoignei and its gene product
Authors: JENDROSSEK Dieter , MÜLLER Beate , SCHLEGEL Hans Günter
Published: March 2005

Four different DNA fragments each coding for poly(hydroxyalkanoic acid) depolymerase (phaZ1–phaZ4) were isolated in pUC plasmids from a genomic library of Pseudomonas lemoignei in Escherichia coli. All recombinant strains secreted a highly active poly(3‐hydroxybutyric acid) depolymerase and produced large translucent halos on an opaque medium containing poly(3‐hydroxybutyric acid) granules. One DNA region (phaZ1) was present in seven independently isolated clones. Three other cloned DNA fragments were different from phaZ1 and from each other (phaZ2–phaZ4). In phaZ1, an open‐reading frame of 1245 bp was identified from the nucleotide sequence of a 5435‐bp MboI fragment (57 mol G+C/100 mol) of this region and encoded a novel poly(hydroxyalkanoic acid) depolymerase of P. lemoignei, poly(3‐hydroxybutyric acid) depolymerase C. A leader‐sequence peptidase‐cleavage site was predicted from the deduced amino acid sequence between Ala37 and Leu38. The calculated relative molecular masses of the precursor and the putative mature protein were 43468 and 39581, respectively. The polypeptide contains a lipase consensus sequence (Gly‐Xaa‐Ser‐Xaa‐Gly) and an unusually high proportion of threonine residues (22 of 36 amino acids) near the C‐terminus. The N‐terminus of the deduced amino acid sequence of PhaZ1 differed from that of the purified poly(3‐hydroxybutyric acid) depolymerases A, B and the poly(3‐hydroxyvaleric acid) depolymerase of P. lemoignei.

The phaZ1 gene product, poly(3‐hydroxybutyric acid) depolymerase C, was partially purified from recombinant E. coli (pUC91 ::phaZ1). The purified protein was specific for poly(hydroxyalkanoic acid) consisting of monomers of four or five carbon atoms and for p‐nithrophenylbutyrate as substrates. The polymer‐hydrolyzing activity, but not the p‐nitrophenylate esterase activity, was inhibited by complex media such as Luria‐Bertani medium and by soluble E. coli proteins. The enzyme protein did not cross‐react with antibodies raised against purified poly(3‐hydroxyvaleric acid) depolymerase of P. lemoignei.
Mutants of Methylobacterium extorquens and Paracoccus denitrificans deficient in c‐type cytochrome biogenesis synthesise the methylamine‐dehydrogenase polypeptides but cannot assemble the tryptophan‐tryptophylquinone group
Authors: PAGE M. Dudley , FERGUSON Stuart J.
Published: March 2005

Five mutants of Methylobacterium extorquens and four mutants of Paracoccus denitrificans that have a general defect in c‐type cytochrome synthesis also failed to assemble an active methylamine dehydrogenase. In all cases methanol dehydrogenase, another periplasmic enzyme, was fully active. All nine mutant strains accumulated both the heavy and light subunits of methylamine dehydrogenase to essentially wild‐type levels. In all nine mutants, the heavy‐subunit and light‐subunit polypeptides were proteolytically processed, suggesting that translocation to the periplasm had occurred; in the case of the P. denitrificans mutants, a periplasmic location for the heavy and light subunits was confirmed experimentally. While specific quinone staining of the methylamine dehydrogenase light subunit in wild‐type M. extorquens and P. denitrificans strains could readily be demonstrated, the light subunit polypeptides accumulated by the mutants did not quinone stain, indicating that the methylamine dehydrogenase prosthetic group, tryptophan tryptophylquinone, is not assembled in the absence of functional c‐type cytochromes.
Characterization of two glycosylated boar spermadhesins
Authors: CALVETE Juan José , SOLÍs Dolores , SANZ Libia , DÍAZ‐MAURIÑO Teresa , SCHÄFER Wolfram , Mann Karlheinz , TÖPFER‐PETERSEN Edda
Published: March 2005

Boar spermadhesins AQN‐1, AQN‐3 and AWN form a recently described protein family, synthesized by the sexual accessory glands, and become associated with the sperm head upon ejaculation. They contain 109–133 amino acid residues, two conserved disulphide bridges, are not glycosylated, and have 40–60% primary structure identity. These boar polypeptides are multifunctional proteins, which possess heparin‐, serine‐protease‐inhibitor‐ and/or zona‐pellucida‐glycoprotein‐binding capability and have, therefore, been implicated in sperm capacitation and sperm‐oocyte attachment. AQN‐2 (18–20 kDa), however, is unique among boar spermadhesins in that it is the only member of the family which is known to be glycosylated and which possesses weak zona‐pellucida‐binding but not seminal‐plasma‐inhibitor‐binding ability. In this study we report the structural and functional characterization of the two glycoproteins contained in the AQN‐2 fraction. One component is identical with PSP‐I, a major porcine seminal plasma protein whose function has not yet been identified, while the second protein is a glycosylated isoform of AQN‐3. Here we show that the inability of the glycosylated boar spermadhesins to bind seminal‐plasma protease inhibitors as well as the weak binding of glycosylated AQN‐3 to zona pellucida glycoproteins is due to the presence of the oligosacharide chain on a conserved asparagine residue. This indicates that modification of a spermadhesin polypeptide framework may serve to modulate its ligand‐binding capabilities.
Evidence for the functional importance of Cys298 in D‐amino acid oxidase from Trigonopsis variabilis
Authors: SCHRÄDER Thomas , ANDREESEN Jan R.
Published: March 2005

D‐Amino acid oxidase from Trigonopsis variabilis was purified to homogeneity by a combination of freeze/thawing, isoelectric precipitation and chromatography on Mono Q. This purification procedure required very little working effort. The homogeneous enzyme exhibited a ratio A280/A450 of about 6.5 and was obtained in high yield (63%) and a good stability. Using D‐methionine as a substrate, a specific activity of 120 U/mg was determined colorimetrically at 26°C, corresponding to 185 U/mg polarographically at 37°C. Polyclonal antibodies were raised against the homogeneous protein and Western immunoblot analysis showed that the 39‐kDa subunit can undergo defined cleavages at the carboxy terminus of amino acid positions 104, 106 and 108, leading to 27‐kDa and 12‐kDa fragments as revealed by SDS/PAGE, which are still enzymically active in their native form. The enzyme was inactivated by all sulfhydryl‐modifying reagents tested. Inactivation by 5,5′‐dithio‐bis(‐2‐nitrobenzoate) was correlated with a modification of up to 2 mol/mol protein of the six cysteine residues present in the monomer. Identification of the most reactive cysteine was achieved by inactivation of the enzyme with the fluorescent, sulfhydryl‐modifying reagent monobromobimane. In the presence of a substrate amino acid, under anaerobic conditions, the protein could be protected from modification and, thus, inactivation by this reagent. Peptide mapping by reverse phase chromatography of endoproteinase Glu‐C‐digested monobromobimane‐labeled enzyme revealed one major fluorescence peak which was not obtained when the protein was modified in the presence of a substrate amino acid under anaerobic conditions. Isolation and sequencing of the labeled peptide led to the identification of Cys298 as the reactive cysteine residue.
ATP synthesis in mitochondria
Authors: HATEFI Youssef
Published: March 2005

CONTENTS Structure and composition of the ATP synthase complex – Mechanism of ATP hydrolysis and synthesis–ATP hydrolysis – ATP synthesis– Unisite ATP synthesis – Multisite ATP synthesis –Energy transduction by the ATP synthase complex–Interaction of(Proton electrochemical potential) with F0F1– Energy transduction and transfer within the ATP synthase complex
Participation in cellular prostaglandin synthesis of type‐II phospholipase A2 secreted and anchored on cell‐surface heparan sulfate proteoglycan
Authors: SUGA Hinako , MURAKAMI Makoto , KUDO Ichiro , INOUE Keizo
Published: March 2005

Rat‐liver‐derived BRL‐3A cells, which express both type‐II phospholipase A2 (PLA2) and cytosolic PLA2 (cPLA2), generated prostaglandin E2 (PGE2) in the presence of fetal calf serum. When the cells were treated with tumor necrosis factor (TNF), PGE2 generation was greatly stimulated. The production of PGE2 observed in both cases was suppressed by a type‐II PLA2‐specific inhibitor, thielocin A1. Appreciable amounts of type‐II PLA2 were released into the medium from the TNF‐stimulated cells when heparin was added extracellularly. The release of type‐II PLA2 from TNF‐stimulated cells was also found in the presence of heparan sulfate or dextran sulfate, whereas other glycosaminoglycans showed no effects under the same conditions. These findings suggest that type‐II PLA2 expressed in BRL‐3A cells mostly associates with the cell surface by binding to cellular heparan sulfate proteoglycan. Removal of cell‐surface‐associated type‐II PLA2, by either extracellular addition of heparin or by prior treatment of the BRL‐3A cells with heparitinases, resulted in marked reduction of PGE2 synthesis in the cells. Exposure of BRL‐3A cells to thrombin also induced the apparent secretion of type‐II PLA2, and thrombin‐stimulated PGE2 generation was suppressed by heparin effectively. Type‐II PLA2 secreted and attached to heparan sulfate on the cell surface may therefore play an essential role in PGE2 synthesis by BRL‐3A cells.
Two states of the nucleotide‐binding site of sarcoplasmic reticulum adenosine triphosphatase detected by the calcium‐dependent reaction with adenosine 5′‐[γ‐imidazolidate]triphosphate and adenosine 5′‐[β‐imidazolidate]diphosphate
Authors: GUTOWSKI‐ECKEL Zeynep , BÄUMERT Hans G.
Published: March 2005

The Ca2+‐ATPase from sarcoplasmic reticulum can be inhibited by adenosine 5′‐[γ‐imidazolidate]triphosphate through the formation of an intramolecular cross‐link at the active site which is dependent on the presence of Ca2+ [Bill, E., Gutowski, Z. & Bäumert, H. G. (1988). Calciumdependent inactivation of the Ca2+‐ATPase from sarcoplasmic reticulum by chemically reactive adenosine triphosphate, Eur. J. Biochem. 176, 119–124]. In the present study we show that adenosine 5′[β‐imidazolidate]diphosphate is likewise an inhibitor of the Ca2+‐ATPase effecting a similar inhibition pattern on phosphate release and Ca2+ transport. The overall reaction is Ca2+ dependent and produces a protein band that in SDS/PAGE is indistinguishable from that seen with ATP[imidazolidate]. This shows that the side chain of Asp351 which is claimed to be involved in the crosslinking reaction must be in reach of both the β and the γ phosphate moiety of the respective nucleotides. The cross‐linked product is formed by a two‐step reaction. The first step is the fast reaction of nucleotide imidazolidate presumably at the phosphorylation site (Asp351) underformation of a mixed anhydride that covalently links nucleotide and protein. Subsequently, the nucleotide is released by a substitution reaction with a second amino acid side chain. This cross‐linking reaction is strictly Ca2+ dependent and, remarkably, requires Ca2+ to be added before addition of the inhibitor. It proceeds at two rates and suggests that there are two states of the nucleotide‐binding site. This is also supported by the fact that in the absence of Ca2+, ATP[imidazolidate] reacts only in approximately 50% of the calculated ATP‐binding site (based on 80–90% ATPase of total sarcoplasmic reticulum protein) with no subsequent cross‐linking reaction.
A high‐resolution solid‐state 13C‐NMR study on [1‐13C]Ala and [3‐13C]Ala and [1‐13C]Leu and Val‐labelled bacteriorhodopsin
Authors: TUZI Satoru , NAITO Akira , SAITÔ Hazime
Published: March 2005

We have recorded 100.7‐MHz high‐resolution solid‐state 13C‐NMR spectra of [3‐13C]Ala, [1‐13C]Ala‐labelled, Leu‐labelled and Val‐labelled bacteriorhodopsin (bR), to analyze the conformation and dynamics of transmembrane α helices and hydration‐induced conformational changes. We assigned the 13C‐NMR signals of these 13C‐labelled amino acid residues to portions of the transmembrane α helices, loops and N‐terminus and C‐terminus, based on the conformation‐dependent 13C chemical shift. The assignment of peaks to the transmembrane α helices is straightforward in view of the characteristic 13C chemical shifts of the Cβ and carbonyl carbons, referred to the data of the model system. The signals of the transmembrane α helices were further divided into three or four peaks which are ascribed to either a dispersion of torsion angles of the α helices or variation of environments around the helices. In addition, we found that conformation of the N‐terminus and C‐terminus of bR is virtually the random‐coil form which undergoes rapid reorientational motion in the hydrated system. Further, we show that the higher‐order (secondary and/or tertiary) structure of bR is influenced by the hydration/dehydration process of the purple membrane, as viewed from the peak profile of the Ala Cβ13C signals. This change of structure occurs between relative humidities of 2% and 4%, consistent with the shift of the absorption maxima of retinal, which arise from the protonation/deprotonation processes of the Schiff base. In contrast, no such change of the carbonyl signals occurs for the Leu, Val and Ala residues. For the latter two residues, some spectral changes were noted at the stage of full hydration.
Evidence that the reaction of the UDP‐N‐acetylglucosamine 1‐carboxyvinyltransferase proceeds through the O‐phosphothioketal of pyruvic acid bound to Cys115 of the enzyme
Authors: WANKE Christoph , AMRHEIN Nikolaus
Published: March 2005

The enzyme UDP–N‐acetylglucosamine 1‐carboxyvinyltransferase (enolpyruvyltransferase, EC 2.5.1.7) catalyses the transfer of the intact 1‐carboxyvinyl moiety of phosphoenolpyruvate to the 3′‐hydroxyl group of the glucosamine moiety of UDP–(2′)‐N‐acetylglucosamine with the concomitant release of inorganic phosphate, the first committed step in the biosynthesis of the bacterial cell wall peptidoglycan. Overexpression of the enzyme from Enterobacter cloacae in Escherichia coli allowed the isolation of large amounts of purified enzyme (approx. 900 mg/20 g fresh mass bacteria). By incubating the enzyme with 14C‐labelled phosphoenolpyruvate, 32P‐labelled orthophosphate and unlabelled UDP–(2′)‐N‐acetyl‐(3′)‐1‐carboxyvinylglucosamine, we were able to isolate and characterise a reaction intermediate, covalently bound to the protein. It contains stoichiometric quantities of the C3 moiety (0.98 mol/mol) as well as of the phosphate moiety (0.95 mol/mol) of phosphoenolpyruvate relative to protein. The rapid turnover of this protein‐bound intermediate in the presence of UDP–(2′)‐N‐acetylglucosamine towards the product UDP–(2′)‐N‐acetyl‐(3′)‐1‐carboxyvinylglucosamine suggests that the intermediate is kinetically competent. We also present evidence that the intermediate is bound as the O‐phosphothioketal of pyruvic acid to Cys115 of the enzyme. This is the same Cys residue to which phosphomycin, an irreversible inhibitor of the UDP–GlcNAc carboxyvinyltransferase, binds covalently. Exchange of Cys115 for a Ser residue resulted in an inactive enzyme, demonstrating the essential role of Cys115 for the reaction.

The only other enzyme known to catalyse the transfer of the intact 1‐carboxyvinyl moiety of phosphoenolpyruvate to a substrate is the 3‐phosphoshikimate 1‐carboxyvinyltransferase (EC 2.5.1.19), the sixth enzyme of the shikimate pathway. The reaction of this synthase is known to proceed through a single, tightly but not covalently bound, tetrahedral intermediate. Even though the two enzymes share similarities in their primary amino acid sequences, their reaction mechanisms appear to be substatially different.
A plasma membrane ‘vacuum cleaner’ for daunorubicin in non‐P‐glycoprotein multidrug‐resistant SW‐1573 human non‐small cell lung carcinoma cells.
Authors: MÜLDER H. Spiko , van GRONDELLE Rienk , WESTERHOFF Hans V. , LANKELMA Jan
Published: March 2005

A multidrug resistant (MDR) human non‐small cell lung carcinoma cell line, SW‐1573/2R120 (2R120), not containing the drug‐efflux pump P‐glycoprotein (PgP), has been studied for the transport of daunorubicin (DN) across the cellular plasma membrane. Earlier, reduced initial DN‐uptake rates and lower cellular DN steady‐state concentrations were found for this cell line, when it was compared to the SW‐1573 wild‐type cell line. This finding was an indication for the presence of another cellular drug‐efflux pump. However, we found similar DN‐efflux rates in drug‐free medium for the two cell lines, while for Pgp‐containing MDR SW‐1573/2R160 (2R160) cells the efflux rate was increased compared to wild‐type cells. In order to elucidate differences in DN transport across the cellular plasma membrane, the association of DN with plasma membranes of intact cells was investigated, using fluorescence‐resonance‐energy transfer. For this purpose, the plasma‐membrane probe 1‐(4‐trimethyl‐ammoniumphenyl)‐6‐phenyl‐1,3,5‐hexatriene (TMA‐DPH) was chosen since, because of the overlap between the emission spectrum of TMA‐DPH and the excitation spectrum of DN, transfer of energy can be achieved from TMA‐DPH to DN. Cells were loaded with TMA‐DPH and, after addition of 10 μM DN, the TMA‐DPH fluorescence was quenched. Rapid initial quenching proved to be similar in the MDR 2R160 (Pgp‐containing) cells and in the SW‐1573 wild‐type cells (21±1% and 20±2%, respectively), but was less in the MDR 2R120 cells not containing Pgp (14±1%). This finding correlated with a lowered amount of DN dissolved in the plasma membrane of 2R120 cells. We interpret these data to be the result of a ‘vacuum‐cleaner’ pumping system other than Pgp which removes DN from a plasma membrane compartment and equilibrates relatively slowly with the interior of the cell.
Insulin‐like growth factor‐1 modulates steroid hormone effects on osteocalcin synthesis in human MG‐63 osteosarcoma cells
Authors: PIRSKANEN Asta , JÄÄSKELÄINEN Tiina , MÄENPÄÄ Pekka H.
Published: March 2005

The 1,25‐dihydroxyvitamin‐D3‐(calcitriol)‐induced medium osteocalcin and cellular osteocalcin mRNA concentrations are increased in a dose‐dependent and time‐dependent manner by prior treatment of the human MG‐63 osteosarcoma cells with insulin‐like growth factor‐I (IGF‐I). In addition, IGF‐I reverses the inhibitory effects of dexamethasone and enhances the effects of retinoic acid on osteocalcin synthesis. The stimulatory effect of IGF‐I on osteocalcin synthesis is accompanied by stabilization of osteocalcin mRNA and a decrease of AP‐1 binding to the osteocalcin promoter. The binding of the vitamin‐D receptor (VDR) to its cognate response element is not affected by IGF‐I. In contrast to its effects on osteocalcin synthesis, both baseline and calcitriol‐stimulated alkaline phosphatase activities are decreased by IGF‐I treatment. Furthermore, IGF‐I has mitogenic effects on MG‐63 cells. The proliferation of the cells and the levels of c‐jun mRNA are greatly increased during IGF‐I treatment. Calcitriol reduces or eliminates both these effects. The low concentrations of IGF‐I used in this study suggest that IGF‐I is an important normal regulator of the synthesis of osteocalcin, a bone calcium‐binding protein participating in bone mineralization, by modulating the effects of steroid hormones on its synthesis.
Hexadecylpalmitoyglycerol or ceramide is linked to similar glycophosphoinositol anchor‐like structures in Trypanosoma cruzi
Authors: de LEDERKREMER Rosa M. , LIMA Carlos E. , RAMIREZ Maria I. , GONÇALVEZ Marinei F. , COLLI Walter
Published: March 2005

The lipopeptidophosphoglycan from Trypanosoma cruzi is a glycosylated inositol‐phosphoceramide isolated from epimastigotes at the stationary phase of growth (4–5 days). We have now purified two similar glycoinositolphospholipids (glycoinositolphospholipid A and glycoinositolphospholipid B) from epimastigotes after the second day of culture growth. [3H]Palmitic acid was incorporated into 1‐O‐hexadecyl‐2‐O‐palmitoylglycerol in glycoinositolphospholipid A and into ceramide in glycoinositolphospholipid B. The lipids were released by incubation with glycosylphosphatidylinositol‐specific phospholipase C from Bacillus thuringiensis or by chemical methods. After alkaline hydrolysis, the lipids were analysed by GLC/MS. In glycoinositolphospholipid A the resulting lipids corresponded to 1‐O‐hexadecylglycerol and palmitic acid. The ceramide components in glycoinositolphospholipid B are sphinganine, palmitic acid and lignoceric acid. The oligosaccharides could be degraded by nitrous acid and further enzymic treatment showed that the two glycoinositolphospholipids isolated from T. cruzi share the common core structure of the glycosylphosphatidylinositol membrane anchors. The microheterogeneity was determined, as well as the substitution by galactose, and was mainly in the furanose configuration as was previously described for lipopeptidophosphoglycan. However, methylation analysis indicated that 20% of the galactose is in the pyranose from. Both glycoinositolphospholipids mainly differ in the lipid moiety.
The expression of human plasma cholesteryl‐ester‐transfer protein in HepG2 cells in induced by sodium butyrate
Authors: SPERKER Bernhard , MARK Michael , BUDZINSKI Ralph‐M.
Published: March 2005

Although human plasma cholesteryl‐ester‐transfer protein (CETP) is primarily synthesized in the liver, its expression in a number of transformed liver cell lines is very low. However the use of the human hepatoma cell line HepG2 as a model system for the regulation of CETP on mRNA level is facilitated by a quantitative reverse‐transcribed polymerase chain reaction. We demonstrate a time‐dependent and concentration‐dependent 3–4‐fold induction of CETP mRNA by sodium butyrate. CETP mass in the medium is also augmented; however, the effect on protein level is less pronounced.