bluuebunny/crossref_metadata_2025 huggingface dataset:
The E2 protein of human papillomavirus type 16
Authors: LEES Emma M. , DRIESSEN Huub P. C. , CRAWFORD Lionel V. , CLARKE Anthony R.
Published: March 2005

The E2 open reading frame of human papillomavirus type 16 was inserted into the Escherichia coli vector pKK223‐3, and expressed to greater than 15% of total cellular protein when induced with isopropyl β‐d‐thiogalactopyranoside. The highest expressing clone was grown in bulk and the E2 protein purified to homogeneity by the following procedure: (a) isolation of the insoluble protein fraction; (b) extraction with urea; (c) quaternary amino‐ethyl‐Sepharose ion‐exchange chromatography and (d) renaturation and chromatography on dextran sulphate. That the purified protein was fully functionally active was confirmed by its specific DNA‐binding properties and its ability to activate gene transcription by over two orders of magnitude in an in vivo assay.
A novel peroxisomal nonspecific lipid‐transfer protein from Candida tropicalis
Authors: TAN Hironobu , OKAZAKI Koei , KUBOTA Ichiro , KAMIRYO Tatsuyuki , UTIYAMA Hiroyasu
Published: March 2005

We have sequenced the nucleotides of the gene POX18 that encodes PXP‐18, a major peroxisomal polypeptide inducible by oleic acid in the yeast Candida tropicalis. POX18 had a single open reading frame of 127 amino acids. Some 33% of the amino acid sequence of the predicted basic polypeptide (13 805 Da), was identical to that of the nonspecific lipid‐transfer protein (sterol carrier protein 2) from rat liver. PXP‐18, purified to near homogeneity from isolated peroxisomes, had an amino‐terminal sequence identical to that of the predicted polypeptide except for the initiator methionine, and had nonspecific lipid‐transfer activity comparable to that of its mammalian equivalents. Unexpectedly, PXP‐18 lacked the cysteine residue thought to be essential for the activity of this protein in mammals. RNA blot analysis showed that the POX18 gene was expressed exclusively in cells grown on oleic acid, suggesting that PXP‐18 has a role in the β‐oxidation of long‐chain fatty acids. PXP‐18 modulated acylcoenzyme A oxidase activity at low pH.
Use of the biotin‐avidin system for labelling, isolation and characterization of neural cell‐surface proteins
Authors: VON BOXBERG Ysander , WÜTZ Renate , SCHWARZ Uli
Published: March 2005

We describe a method for the selective labelling, isolation and electrophoretic analysis of cell‐surface molecules and extracellular matrix components. Intact tissues are reacted with activated esters of biotin and the labelled surface molecules identified on Western blots with horseradish‐peroxidase‐coupled or 35S‐labelled streptavidin. Alternatively, the biotinylated proteins can be purified from tissue homogenates by affinity chromatography on an avidin‐agarose column. Evidence is presented to show that this method is indeed specific for membrane and matrix components. Its practical application to embryonic neural tissues is demonstrated.
Purification and characterization of hydroxypyruvate reductase from a serine‐producing methylotroph, Hyphomicrobium methylovorum GM2
Authors: IZUMI Yoshikazu , YOSHIDA Toyokazu , KANZAKI Hiroshi , TOKI Shin‐ichiro , MIYAZAKI Silvia Susana , YAMADA Hideaki
Published: March 2005

Hydroxypyruvate reductase of a serine‐producing methylotroph, Hyphomicrobium methylovorum GM2, was purified to complete homogeneity, crystallized and characterized, the first time for an enzyme from a methylotroph. The enzyme was found to be a dimer composed of identical subunits (38 kDa), the molecular mass of the enzyme being about 70 kDa. The enzyme was stable against heating at 25°C for 10 min at pH values between 5 and 9. Optimal activity was observed at pH 6.8 and around 45°C. The enzyme catalyzed the reduction of hydroxypyruvate with the oxidation of only NADH. Other than hydroxypyruvate, only glyoxylate served as a substrate. The Km values were found to be 0.175 mM for hydroxypyruvate and 10.8 mM for glyoxylate. Taking advantage of the high substrate specificity of this enzyme, a means of enzymatic determination of hydroxypyruvate was established.
Complete amino acid sequence of tenebrosin‐C, a cardiac stimulatory and haemolytic protein from the sea anemone Actinia tenebrosa
Authors: SIMPSON Richard J. , REID Gavin E. , MORITZ Robert L. , MORTON Craig , NORTON Raymond S.
Published: March 2005

The complete amino acid sequence of the cardiac stimulatory and haemolytic protein tenebrosin‐C, from the Australian sea anemone Actinia tenebrosa, has been determined by Edman degradation of the intact molecule and fragments produced by treatment of the polypeptide chain with cyanogen bromide and enzymatic cleavage with endoproteinase Asp‐N, thermolysin and trypsin.

The molecule is a single‐chain polypeptide consisting of 179 amino acid residues with a calculated molecular mass of 19 797 Da. Tenebrosin‐C shows a high degree of amino acid sequence similarity (63%) with Stoichactis helianthus cytolysin III [Blumenthal, K. M. and Kem, W. R. (1983) J. Biol. Chem. 258, 5574–5581] and is identical to a partial sequence (90 residues) reported for equinatoxin, a cardiostimulatory and haemolytic protein isolated from the European sea anemone Actinia equina [Ferlan, I. and Jackson, K. (1983) Toxicon Suppl. 3, 141–144]. No amino acid sequence similarity was detected between tenebrosin‐C and other protein sequences stored in available databases. The predicted secondary structure of tenebrosin‐C suggests that it is a compact, highly structured molecule.
Phosphorylation of yeast hexokinases
Authors: VOJTEK Anne B. , FRAENKEL Dan G.
Published: March 2005

We show by the use of 32P‐labeling in vivo that hexokinase 2 and hexokinase 1 in Saccharomyces cerevisiae are phosphoproteins. The highest labeling was after incubation in medium with a low concentration of glucose, when labeling appears to be predominant even without use of immunoprecipitation. The nature of the modification is not known, but it has properties consistent with a phosphomonoester of serine or threonine. The cAMP‐dependent protein kinase plays a negative role in hexokinase phosphorylation, in that there was reduced labeling in strains (bcyl) lacking a regulatory subunit, and increased labeling during growth with high concentrations of glucose in a strain attenuated in the catalytic subunit (tpklw1). The function of the modification is not known, but there was a correlation between the extent of labeling and the expression of kinase‐dependent high‐affinity glucose uptake.
Molecular characterization of three rat liver serine‐protease inhibitors affected by inflammation and hypophysectomy
Authors: PAGES Gilles , ROUAYRENC Jean François , LE CAM Ginette , MARILLER Marcel , LE CAM Alphonse
Published: March 2005

Rat hepatocytes have the potential to secrete three similar acidic glycoproteins, serine protease inhibitors 1, 2 and 3 (SPI‐1, SPI‐2, SPI‐3), recognized by the same antibodies. They were synthesized as precursors of comparable sizes (45 kDa), which were post‐translationally modified by N‐glycosylation at three (SPI‐3) or four (SPI‐1 and SPI‐2) sites. This appeared to account for the size difference of mature proteins. The mRNA sequences, derived from cDNA clones, displayed a high degree of similarity (70–90%), except the sequence of the antiprotease‐reactive centers which were completely divergent. SPI‐1 and SPI‐2 mRNAs were of similar sizes (1.8 kb), and were smaller than that of SPI‐3 (2.2 kb); the difference corresponded to a longer, 3′‐end untranslated sequence. Production of SPI‐1 and SPI‐2, which was constitutive in the normal animal, could be abolished by hypophysectomy and was strongly decreased during acute inflammation. In contrast, production of SPI‐3, which was barely detectable in normal rats, was transiently induced during inflammation.
Preliminary crystallographic data for protease Ω
Authors: PICKERSGILL Richard W. , SUMNER Ian G. , GOODENOUGH Peter W.
Published: March 2005

Protease Ω from Carica papaya L. has been purified and crystallized. The crystals are trigonal, space group P3112 (or P3212), with a= 7.42 ± 0.02 nm, c= 7.79 ± 0.02 nm with one molecule in the asymmetric unit. The crystals diffract to 0.19‐nm resolution using synchrotron radiation.
Prolyl aminopeptidase from rat brain and kidney
Authors: TURZYNSKI Andreas , MENTLEIN Rolf
Published: March 2005

Based on the liberation of proline from ProLeuGlyNH2 (MIF‐1, melanostatin) manganese‐activated prolyl aminopeptidase activities were purified from rat brain and kidney cytosolic fractions. They were distinguished from other di‐ and tripeptidases and an arylamidase liberating N‐terminal proline. Purified prolyl aminopeptidase from both sources had identical molecular properties (native Mr 300000, subunit Mr 54000) and very similar catalytic properties. The action of the purified enzymes was not restricted to proline. Other, in particular lipophilic, amino acids were cleaved from di‐, tri‐ and oligopeptides with even higher velocities. Peptides with N‐terminal penultimate proline residues were not degraded. From a comparison of molecular data, action on peptides, influence of pH values, inhibitors and activators, it is concluded that the activity is identical with leucyl aminopeptidase (EC 3.4.11.1) and that a separate prolyl aminopeptidase (EC 3.4.11.5) does not exist in rats.
Purification and characterization of a DNA polymerase from the archaebacterium Thermoplasma acidophilum
Authors: HAMAL Abdellah , FORTERRE Patrick , ELIE Christiane
Published: March 2005

A thermophilic DNA polymerase has been purified to near homogeneity from the archaebacterium Thermoplasma acidophilum. Analysis of the purified enzyme by sodium dodecyl sulfate gel electrophoresis revealed a single polypeptide of 88 kDa which co‐sediments with the DNA polymerase activity on sucrose gradients. Combination of sedimentation and gel filtration analyses indicates that this DNA polymerase is an 88‐kDa monomeric enzyme in its native form. The DNA polymerase is resistant to aphidicolin, slightly sensitive to 2′,3′‐dideoxyribosylthymine triphosphate and inhibited by N‐ethylmaleimide when preincubation with this reagent is performed at 65°C. We find that a 3′→ 5′ exonuclease activity is associated with the purified DNA polymerase; the two activities of the enzyme are optimal at 65°C but the exonuclease activity is active in a broader range of lower temperatures and is more thermostable than the DNA polymerase activity.
Thermodynamic nonideality in macromolecular solutions
Authors: SHEARWIN Keith E. , WINZOR Donald J.
Published: March 2005

Second virial coefficients and hence covolumes for self‐interaction of five proteins, viz. ribonuclease, ovalbumin, bovine serum albumin, catalase and α‐crystallin, have been determined by analyzing the concentration dependence of the partition coefficient obtained from frontal chromatographic studies on either Fractogel TSK HW55 or porous glass beads. The resulting estimates of the effective radii essentially duplicate their Stokes counterparts and thereby provide further justification for assuming the approximate identity of the thermodynamic and hydrodynamic radii of hydrated globular proteins. Gel chromatographic evaluation of second virial coefficients for protein/dextran systems has led to elimination of the sphere/sphere model as a valid thermodynamic description of the space‐filling effects in protein/polymer mixtures, since it does not predict the observed independence of covolume, expressed per unit mass of polymer, upon size of the polymer. This requirement is met by the sphere/rod model [Edmond, E. & Ogston, A. G. (1968) Biochem. J. 109, 569–576] and also by the sphere/flexiblesegment model [Hermans, J. (1982) J. Chem. Phys. 77, 2193–2203]. Furthermore, similar studies of the effect of solute radius on covolume for interaction with dextran T70 attest to the adequacy of either model for predicting the thermodynamic nonideality arising from the inclusion of dextrans in protein solutions, and also provide the relevant calibration of the model.
Structure of the human gene for α‐enolase
Authors: GIALLONGO Agata , OLIVA Daniele , CALÌ Larissa , BARBA Giovanna , BARBIERI Giovanna , FEO Salvatore
Published: March 2005

In mammals there are at least three isoforms of the glycolytic enzyme enolase encoded by three similar genes: α, β and γ. In this report we describe the isolation and characterization of the human α‐enolase locus. The gene appears to exist as a single copy in the haploid genome and is composed of 12 exons distributed over more than 18000 bases. The structure of this gene has a high degree of similarity to that of the human and rat γ‐enolase genes, with identical positions for all the intron regions. Primer extension and S1 nuclease protection experiments indicate that transcription is initiated at multiple sites. The putative promoter region, like that of other housekeeping genes, lacks canonical TATA and CAAT boxes, is extremely G + C‐rich and contains several potential SP1 binding sites. Furthermore, various sequences similar to known regulatory elements were detected.
The control of lipase‐catalysed transesterification and esterification reaction rates
Authors: GOLDBERG Michel , THOMAS Daniel , LEGOY Marie‐Dominique
Published: March 2005

The reaction rate of two lipase‐catalysed reactions, esterification and transesterification, were studied in a liquid/solid two‐phase system in order to investigate the effect of water partition between the enzyme preparation and the liquid phase composed of only the reactants, i.e. without the conventional solvents.

Lipase from Candida cylindracea was used for these studies. The enzyme was inactive in dehydrated systems. In the case of monoester synthesis, the reaction rate increased with increasing water activity. The reaction rates of the non‐specific C. cylindracea lipase‐catalysed reactions were very sensitive to the nature of the substrates in this unusual system. For instance, the transesterification reaction rate of ethyl propionate was 48 times higher with nonanol than heptanol in the case of dehydrated substrates, but only 2.2 times higher in the case of watersaturated substrates. The results presented here demonstrate the absolute necessity to consider the polarity of every substrate, because of its ability to modify the water partition between the solid phase (enzyme preparation) and the liquid phase (substrate and product), which results in drastic changes in enzyme activity.

Contrary to esterification, which is known to be activated by the water produced, the rate of transesterification remained constant at the beginning of the reaction. However, when transesterification and esterification were carried out in the same liquid phase, the transesterification reaction rate was controlled by the water produced by the concomitant esterification. Activation effects of the water molecules produced during the enzymatic reaction were of exactly the same order of magnitude for both reactions.
Osmotic stress and abscisic acid induce expression of the wheat Em genes
Authors: MORRIS Peter C. , KUMAR Amar , BOWLES Dianna J. , CUMING Andrew C.
Published: March 2005

The early‐methionine‐labelled (Em) polypeptide is the single most abundant cytosolic protein of dry wheat embryos. It is encoded by messenger RNA which accumulates during the later (maturation) stages of embryogenesis. The accumulation of Em mRNA can be induced in isolated developing embryos, in culture, by the application of the plant growth regulator, abscisic acid, which prevents precocious germination.

Precocious germination is also inhibited by the culture of embryos under conditions of osmotic stress when accumulation of Em mRNA is induced. This induction occurs in the absence of any significant increase in the endogenous levels of embryonic abscisic acid although there is a requirement for the continued presence of the growth regulator. Additionally, expression of Em genes can be repeated during early germination, if imbibing embryos are subjected to osmotic stress. Induction of Em‐gene expression by osmotic stress is consistent with the proposed role of the Em polypeptide in mediating the remarkable tolerance of cereal embryos to the programmed desiccation undergone during their maturation.
Recent advances in the molecular analysis of inherited disease
Authors: MALCOLM Susan
Published: March 2005

Many important human genes have been cloned during the last ten years. In some cases, using reverse genetic techniques [Orkin, S. H. (1986) Cell 47, 845–850], disease‐causing genes have been isolated whose product was previously unknown. Important examples include the dystrophin protein which, when mutated, gives rise to either Duchenne or Becker muscular dystrophy [Koenig, M., Hoffman, E. P., Bertelson, C. J., Monaco, A. P., Feener, C. and Kunkel, L. M. (1987) Cell 50, 509–517; Monaco, A. P., Bertelson, C. J., Liechti‐Gallati, S. & Kunkel, L. M. (1988) Genomics 2, 90–95; Koenig, M., Monaco, A. P. & Kunkel, L. M. (1988) Cell 53, 219–228] and the cystic fibrosis transmembrane conductance regulator (CFTR) [Riordan, J. R., Rommens, J. M., Kerem, B.‐S., Alon, N., Rozmahel, R., Grzelczak, Z., Zielenski, J., Lok, S., Plavsic, N., Chou, J.‐L., Drumm, M. L., Ianuzzi, M. C., Collins, F. S. & Tsui, L.‐C. (1989) Science 245, 1066–1073]. Recently the technology for systematically detecting single base‐pair changes by chemical methods, enzymatic methods or direct DNA sequencing has greatly expanded and simplified. In addition to providing structural information about these clinically important genes and information on disease‐causing mutations, these studies have led to an increased understanding of mechanisms of mutation, to the discovery of novel genetic mechanisms and to important clinical applications of carrier detection and pre‐natal diagnosis. The recent rapid progress has been made possible by the development of DNA amplification using the polymerase chain reaction (pcr) invented by Saiki and colleagues [Saiki, R. K., Chang, C‐A., Levenson, C. H., Warren, T. C., Boehm, C. D., Kazazian, H. H. & Ehrlich, H.A. (1988) N. Engl. J. Med. 319, 537–541].
Structural characterization of the human DNA topoisomerase I gene promoter
Authors: KUNZE Norbert , KLEIN Michael , RICHTER Arndt , KNIPPERS Rolf
Published: March 2005

We have isolated a genomic DNA fragment from HeLa cells containing the promoter region and the first two exons of the human gene encoding DNA topoisomerase I (hTOP1). Transcription of hTOP1 mRNA initiates at multiple sites which are clustered 247 nucleotides and 210 nucleotides upstream of the translation‐initiation site of the protein coding region. The nucleotide sequence of the region preceding the transcription‐initiation sites is G/C rich and contains sequence motifs which are known binding sites of the transcription factors Oct1 (octameric transcription factor 1), Sp1 and AP2 (activator protein 2). Furthermore, one cAMP‐responsive element is present 50 nucleotides upstream of the transcription‐initiation site nearest the 5′ end. Neither TATA nor CAAT boxes were found in the promoter region of the hTOP1 gene. A 918‐bp fragment containing the sequence elements described above drives the transient expression of a chloramphenicol acetyl transferase (CAT) gene sequence in transfected HeLa and 293 cells. In addition we analyzed a 10‐kb fragment containing the promoter and exons 1 and 2 for regions of DNase I hypersensitivity. We detected one prominent DNase‐I‐hypersensitive region in the promoter close to the putative transcription‐factor‐binding sites and several weaker regions in intron 2.
A systematic approach towards the complete assignment of 13C resonances for horse ferrocytochrome c
Authors: GAO Yuan , BOYD Jonathan , WILLIAMS Robert J. P.
Published: March 2005

The complete 1H‐NMR assignments for horse ferrocytochrome c have been reported by Wand and colleagues and by our group at Oxford. Using these 1H assignments, we now report chemical shift assignment for 205 13C resonances arising from horse ferrocytochrome c. This is from a total of 437 13C nuclei with covalently attached protons. These chemical shift assignments have been achieved using 1H‐detected two‐dimensional heteronuclear 1H‐13C correlation techniques. The data have been collected from samples of horse ferrocytochrome c without isotopic enrichment. The complete 13C assignments for all carbons with covalently linked protons are reported for the amino acids Ala, Thr, Val and Gly. Specific assignments are tabulated for all 49 methyl groups, for 52 of 92 α‐carbon resonances, for 10 resonances associated with the heme group, for all aromatic side‐chain 13C resonances which have covalent protons and give rise to observable cross peaks under the experimental conditions used, as well as for a number of other side chains of aliphatic amino acids.
Cloning and DNA sequencing of the fbc operon encoding the cytochrome bc1 complex from Rhodobacter sphaeroides
Authors: YUN Chang‐Hyon , BECI Rose , CROFTS Antony R. , KAPLAN Samuel , GENNIS Robert B.
Published: March 2005

The ubiquinol: cytochrome‐c oxidoreductase (cytochrome bc1 complex) is a central component of the mitochondrial respiratory chain as well as the respiratory and/or photosynthetic systems of numerous prokaryotic organisms. In Rhodobacter sphaeroides, the bc1 complex has a dual function. When the cells are grown photosynthetically, the bc1 complex is present in the intracytoplasmic membrane and is a critical component of the cyclic electron transport system. When the cells are grown in the dark in the presence of oxygen, the same bc1 complex is a necessary component of the cytochrome‐c2‐dependent respiratory chain. The fact that the bc1 complex from R. sphaeroides has been extensively studied, plus the ability to manipulate this organism genetically, makes this an ideal system for using site‐directed mutagenesis to address questions relating to the structure and function of the bc1 complex.

In the current work, the cloning and complete sequence of the fbc operon from R. sphaeroides is reported. As in other bacteria, this operon contains three genes, encoding the Rieske 2Fe–2S subunit, the cytochrome b subunit, and the cytochrome c1 subunit. Recombination techniques were used to delete the entire fbc operon from the chromosome. The resulting strain cannot grow photosynthetically, but can grow aerobically utilizing a quinol oxidase. Photosynthetic growth is restored by providing fbc operon on a plasmid, and the reappearance of the protein subunits and the spectroscopic features due to the bc1 complex are also demonstrated. Finally, a mutation is introduced within the gene encoding the cytochrome b subunit which is predicted to confer resistance to the inhibitor myxothiazol. It is shown that the resulting strain contains a functional bc1 complex which, as expected, is resistant to the inhibitor.

Hence, this system is suitable for the detailed characterization of the bc1 complex, combining site‐directed mutagenesis with the biochemical and biophysical techniques which have been previously developed for the study of photosynthetic bacteria.
Structure/activity relationships of C‐terminal neuropeptide Y peptide segments and analogues composed of sequence 1–4 linked to 25–36
Authors: BECK‐SICKINGER Annette G. , JUNG Günther , GAIDA Wolfram , KÖPPEN Herbert , SCHNORRENBERG Gerd , LANG Rudolf
Published: March 2005

C‐terminal analogues of neuropeptide Y have been synthesized. The influence of chain length, single‐amino‐acid substitutions and segment substitutions on receptor binding, biological activity and conformational properties has been investigated. Receptor binding and in vivo assays revealed biological activity already for amino acids 28–36 of neuropeptide Y [neuropeptide Y‐(Ac‐28–36)‐peptide] which increased with increasing chain length. Replacement of Arg25 in neuropeptide Y‐(Ac‐25–36)‐peptide had no influence on binding, whereas Arg33 and Arg35 cannot be replaced by lysine or ornithine without considerable decrease in receptor binding. The introduction of conformational constraints by the 2‐aminoisobutyric acid residue (Aib) in position 30 and replacing the amino acids 28–32 by Ala‐Aib‐Ala‐Aib‐Ala decreased receptor binding. However, the corresponding Aib‐Ala‐Aib‐Ala‐Aib‐substituted analogue and a more flexible analogue with Gly5 at position 28–32 exhibited considerable affinity for the receptor. All these substitutions led to a decrease in postsynaptic activity. Strong agonistic activities could be detected in a series of 10 discontinuous analogues, which are constructs of N‐terminal parts linked via different spacer molecules to C‐terminal segments. One of the most active molecules was neuropeptide Y amino acids 1–4 linked to amino acids 25–36 through aminohexanoic acid (Ahx) [neuropeptide Y‐(1–4‐Ahx‐25–36)‐peptide].
Thermodynamics of information transfer between subunits in oligomeric enzymes and kinetic cooperativity
Authors: RICARD Jacques , GIUDICI‐ORTICONI Marie‐Thérèse , BUC Jean
Published: March 2005

The strength of quatenary constraints between two subunits of a polymeric enzyme depends upon the number of neighboring subunits and upon whether these subunits are liganded or not. These quaternary constraints between two subunits of a complex polymeric enzyme may be expressed, however, in terms of quaternary constraints that exist within ideal dimers. The influence of quaternary constraints on the reaction rate of a complex polymeric enzyme may thus be expressed in terms of the intersubunit strain that exists within dimers. This conclusion, that was far from evident, appears to be the consequence of the postulates of structural kinetics, and derive as well from usual thermodynamic principles.

The structural steady‐state equations may be expressed in terms of partition and sub‐partition functions. As applied to structural kinetic models, a partition function expresses how, during the steady state, the energy of a population of enzyme molecules is distributed over n states. Similarly a sub‐partition function describes how, during the steady state, the energy of these enzyme molecules is partitioned among only n–k of these states. Although the concept of partition function was initially formulated for equilibrium processes, it may be extended without any loss of generality to non‐equilibrium processes. Moreover it is reminiscent of the concept of binding polynomial presented some years ago by Wyman for the equilibrium binding of a ligand to a protein. With this formalism derived from statistical mechanics, a structural rate equation may be derived from the ratio of a subpartition function of degree n–1 and of a partition function of degree n. Again these properties are the consequence of the postulates of structural kinetics associated with simple ideas derived from statistical thermodynamics.
Characteristic binding of human plasma apolipoprotein B to gangliotetraosylceramide and gangliotriaosylceramide
Authors: LEDVINOVA Jana , IWAMORI Masao , NAGAI Yoshitaka
Published: March 2005

The binding of human plasma low‐density lipoproteins (LDL), freshly prepared by discontinuous ultracentrifugation, to several neutral and acidic glycosphingolipids was examined by TLC immunostaining with the anti‐[apolipoprotein B (apoB)] antibody. ApoB was found to bind characteristically to the asialogangliosides, gangliotetraosylceramide (Gg4Cer) and gangliotriaosylceramide (Gg3Cer), the former being a more potent receptor than the latter, indicating that the sequences Galβ1‐3GalNAcβ1‐4Gal and GalNAcβ1‐4Gal are involved in the binding of apoB. A weak positive reaction with fucosylgangliotetraosylceramide (IV2Fuc‐Gg4Cer), which has the same internal recognition sequence, was also observed (the binding ability was only 1/7 of that in the case of Gg4Cer). No binding to other neutral glycosphingolipids, or glycosphingolipid sulfates (I3‐SO3‐GalCer) and gangliosides, was detected, and therefore substitution of the receptor glycolipid with sialic acid was thought to inhibit the binding.

The results indicate that, along with the binding of apoB to the LDL‐binding domain of the receptor glycoprotein, interaction with some carbohydrate chains in the receptor, or with glycolipids coexisting on the plasma membrane, may be important for the binding of apoB to cells.
Human poly(ADP‐ribose) polymerase gene
Authors: YOKOYAMA Yuichi , KAWAMOTO Takeshi , MITSUUCHI Yasuhiro , KUROSAKI Tomohiro , TODA Katsumi , USHIRO Hiroshi , TERASHIMA Masako , SUMIMOTO Hidetoshi , KURIBAYASHI Isao , YAMAMOTO Yasutake , MAEDA Tomoho , IKEDA Hisao , SAGARA Yusuke , SHIZUTA Yutaka
Published: March 2005

The promoter region of the poly(ADP‐ribose) polymerase gene has been isolated using a Sau3AI genomic library derived from human leukocyte. It lacks typical transcriptional regulatory elements such as TATA and CAAT boxes, but it contains two potential Sp1 binding sites and three putative AP‐2 binding elements. The region up to nucleotide position – 99 in relation to the predominant transcriptional initiation site exhibits promoter activity as judged by chloramphenicol acetyltransferase assay and the activity is enhanced both by cAMP and by phorbol ester. Northern blot and Western blot analyses have revealed that expression of the polymerase gene is also stimulated by both of these compounds in cultured HeLa cells. Southern blot hybridization of genomic DNA separately digested with various endonucleases gives a discrete single band in each case when the 5′‐untranslated region of the polymerase cDNA is used as a probe. These results indicate that poly(ADP‐ribose) polymerase is encoded by a unique gene whose expression is regulable by cAMP and by phorbol ester.
Activation and inhibition of the calcium‐release channel of isolated skeletal muscle heavy sarcoplasmic reticulum
Authors: WYSKOVSKY Wolfgang , HOHENEGGER Martin , PLANK Brigitte , HELLMANN Gertrude , KLEIN Sabine , SUKO Josef
Published: March 2005

Calcium‐independent calcium efflux from heavy sarcoplasmic reticulum (HSR) of skeletal muscle was found to be biphasic, with half‐times of 2–6 s and 200–400 s for the first and second phase, respectively. Calcium‐, AMP‐ and caffeine‐induced calcium efflux was triphasic, with half‐times of 0.05–0.2 s, 1–5 s and 100–400 s for the first, second and third phases, respectively. This very fast first phase is certainly due to calcium efflux via the calcium‐release channel of HSR vesicles. Both ruthenium red and neomycin inhibited the first phase of the calcium‐independent calcium efflux and the first phase of the calcium‐, AMP‐ or caffeine‐induced calcium efflux completely, whilst the second phase was fully inhibited by ruthenium red only and partially inhibited by neomycin at high concentrations, indicating that the second phase of calcium release also occurs via the calcium‐release channel.

Various models for calcium efflux through the release channel have been tested by simulation. Activation and inhibition of the channel‐mediated calcium efflux from HSR cannot be explained by two states of the calcium‐release channel (open or closed), but requires the existence of at least three states. A channel with one open state and two closed states, resulting in a rapid inactivation, is the most simple model compatible with the experimental data. According to this model, activation is assumed to reduce inactivation of the channel, whilst inhibition assumes an acceleration of channel inactivation. This mechanism most likely applies to neomycin. An additional open‐blocked state has to be assumed for inhibition by ruthenium red.
Direct repeats in the non‐coding region of rabbit mitochondrial DNA
Authors: MIGNOTTE Françoise , GUERIDE Monique , CHAMPAGNE Anne‐Marie , MOUNOLOU Jean‐Claude
Published: March 2005

In rabbit we observed heteroplasmy at an exceptionally high level, the heterogeneity occurring within the non‐coding region of the DNA. Mitochondrial DNA (mt DNA) was cloned in pBR322 and the nucleotide sequence analysis of an EcoRI–Hind III fragment encompassing the non‐coding region revealed that although there are common features with other mammalian mtDNAs (termed large central‐conserved‐sequence block, conserved‐sequence blocks 1, 2 and 3 and termination‐associated elements) the non‐coding region shows an unusual organization; two stretches of tandem repeats of 20 bp and 153 bp are present in a part containing the origin of H‐strand replication (OH) and probably the promoters for transcription as judged from other vertebrates. The long repeats are located between tRNAPhe and conserved sequence block 3 and the short repeats are located between conserved sequence blocks 1 and 2. When cloned in Escherichia coli (recA or recBC sbcb) DNA fragments containing the short repeats show length differences corresponding to various copy numbers of repeats. Electrophoretic analysis of the appropriate restriction fragments of rabbit mtDNA reveals extended intra‐ and inter‐individual length heterogeneity. Both sets of repeats are involved in the generation of heterogeneity and are present in variable copy numbers from one mtDNA molecule to another. Moreover, rearrangement of the motives of the short repeat are observed to different extents in the mtDNA from one animal to another. The occurrence, maintenance and possible involvement of these repeated sequences, capable of forming stable secondary structures, are discussed in relation to their location in the region of control signals.
Isolation and characterization of an inhibitor of ribosome‐dependent GTP hydrolysis by elongation factor G
Authors: VOIGT Jürgen , NAGEL Klaus
Published: March 2005

Two inhibitors of ribosome‐dependent GTP hydrolysis by elongation factor (EF)G were found in the ribosome wash of Escherichia coli strain B. One of these inhibitors was purified to homogeneity and characterized. The isolated inhibitor was found to consist of two polypeptide subunits with apparent molecular masses of 23 kDa and 10 kDa. Inhibition of EF‐G GTPase could not be overcome by increasing amounts of the elongation factor or high concentrations of GTP, but was reversed by large amounts of ribosomes. The effect of the inhibitor was reduced by increasing concentrations of either 30S or 50S ribosomal subunits. EF‐G‐dependent GTPase of 50S ribosomal subunits was not affected by the inhibitor. These findings clearly show that the inhibitor interferes with the modulation of EF‐G GTPase activity by the interactions between 30S and 50S ribosomal subunits. Under conditions, where 30S CsCl core particles are able to associate with 50S subunits and to stimulate EF‐G GTPase, the effect of the inhibitor was considerably reduced when intact 30S ribosomal subunits were substituted by 30S CsCl core particles. This finding indicates that 30S CsCl split proteins are important for the action of the inhibitor and that the inhibitor does not affect the EF‐G GTPase merely by interfering with the association of ribosomal subunits. Furthermore, poly(U)‐dependent poly(phenylalanine) synthesis was considerably less sensitive to the inhibitor than EF‐G GTPase. When ribosomes were preincubated with poly(U) and Phe‐tRNAPhe, poly(phenylalanine) synthesis was considerably less affected by the inhibitor, whereas EF‐G GTPase was still sensitive.
Changes of the oligomeric structure of legumin from pea (Pisum sativum L.) after succinylation
Authors: SCHWENKE Klaus Dieter , ZIRWER Dietrich , GAST Klaus , GÖRNITZ Eckhard , LINOW Karl‐Joachim , GUEGUEN Jacques
Published: March 2005

The influence of various levels of succinylation on the structure of the legumin from pea seed has been studied by the techniques of sedimentation velocity, viscometry, fluorescence and circular dichroism spectroscopy, as well as dynamic light scattering. The protein dissociates gradually into the 3S subunit forming a 7S intermediate. At a level of 75–80% succinylation, sudden unfolding of the protein occurs characterized by drastic changes in viscometric and spectroscopic properties. The fluorescence spectra point to the formation of a novel organized structure at a moderate degree of modification before the molecular unfolding takes place.

The succinylated subunit was shown to have a sedimentation coefficient of 3.2S, a diffusion coefficient of 5.03 × 10−7 cm2· s−1 a Stokes' radius of 4.24 nm, a partial specific volume of 0.703 ml/g, an intrinsic viscosity of 0.13 dl/g, a molar mass of 52.2 kDa and a frictional ratio of 1.74.
Evidence that enzymes of a novel aerobic 2‐amino‐benzoate metabolism in denitrifying Pseudomonas are coded on a small plasmid
Authors: ALTENSCHMIDT Uwe , ECKERSKORN Christoph , FUCHS Georg
Published: March 2005

A new pathway for aerobic metabolism of 2‐aminobenzoate which proceeds via anthranoyl‐CoA has recently been revealed in a Pseudomonas strain KB740. This bacterial strain was found to contain a small 8.1‐kbp plasmid pKB740 which appears to harbour the genes encoding for two key enzymes catalyzing the initial reactions of the pathway, 2‐aminobenzoate coenzyme A ligase and 2‐aminobenzoyl‐coenzyme A monooxygenase/reductase. The evidence is as follows:

The plasmid content of the culture varied by a factor of ten depending on the growth substrates; it was highest when cells were grown aerobically on 2‐aminobenzoate.

The plasmid pKB740 could be introduced into Escherichia coli strain JM83 by transformation. Wild‐type E. coli and E. coli JM83 are unable to metabolize 2‐aminobenzoate whereas the transformed E. coli JM83 cells could grow with this aromatic compound as sole organic substrate and oxidize it completely to CO2. The plasmid recovered from E. coli had the same restriction map as the original plasmid, but was dimerized.

The two key enzyme activities were demonstrated in the transformed E. coli in sufficiently high amounts to explain growth. They appear to be regulated on the transcription level by induction; they were formed only during aerobic growth in the presence of 2‐aminobenzoate, as in the parent Pseudomonas.

The N‐terminal amino acid sequence of 2‐aminobenzoyl‐CoA monooxygenase/reductase was similar to the consensus sequence of the FAD binding site of different flavoenzymes. The data also prove that the enzyme with two flavin functions is an α2 homodimer.

Southern blotting of digested chromosomal and plasmid DNA and hybridization against a labelled 15‐base oligonucleotide derived from the N‐terminal amino acid sequence of 2‐aminobenzoyl‐CoA monooxygenase/ reductase revealed that the gene for this enzyme is coded on the plasmid rather than on the chromosome. The gene was localized on a 3.2‐kbp restriction fragment.

The formation of 2‐aminobenzoyl‐CoA monooxygenase/reductase protein in transformed E. coli was demonstrated by Western blotting of proteins of cell extracts separated by SDS/PAGE. The enzyme protein band, which was stained by a procedure based on antibodies against 2‐aminobenzoyl‐CoA monooxygenase/reductase, was demonstrated in transformed E. coli.
Distances between functional sites of the Ca2++ Mg2+‐ATPase from sarcoplasmic reticulum using Co2+ as a spectroscopic ruler
Authors: CUENDA Ana , HENAO Fernando , GUTIERREZ‐MERINO Carlos
Published: March 2005

Cobalt ion inhibits the Ca2++ Mg2+‐ATPase activity of sealed sarcoplasmic reticulum vesicles, of solubilized membranes and of the purified enzyme. To use Co appropriately as a spectroscopic ruler to map functional sites of the Co2++ Mg2+ ‐ATPase, we have carried out studies to obtain the kinetic parameters needed to define the experimental conditions to conduct the fluorimetric studies.

The apparent K0.5 values of inhibition of this ATPase are 1.4 mM, 4.8 mM and 9.5 mM total Co2+ at pH 8.0, 7.0 and 6.0, respectively. The inhibition by Co2+ is likely to be due to free Co binding to the enzyme. Millimolar Ca2+ can fully reverse this inhibition, and also reverses the quenching of the fluorescence of fluoresceinlabeled sacroplasmic reticulum membranes due to Co2+ binding to the Ca2++ Mg2+‐ATPase. Therefore, we conclude that Co2+ interacts with Co2+ binding sites.

Co2+· ATP can be used as a substrate by this enzyme with Vmax of 2.4 ± 0.2 p.mol ATP hydrolyzed min−1 (mg protein)−1 at 20–22°C and pH 8.0, and with a K0.5 of 0.4–0.5 mM.

Co2+ partially quenches, about 10 ± 2%, the fluorescence of fluorescein‐labeled sarcoplasmic reticulum Ca2+ Mg2+ATPase upon binding to this enzyme at pH 8.0. From the fluorescence data we have estimated an average distance between Co2+ and fluorescein in the ATPase of 1.1–1.8 nm or 1.3–2.1 run for one or two equidistant Co2+ binding sites, respectively.

Co2+· ATP quenches about 20‐25% of the fluorescence of fluorescein‐labeled Ca2++ Mg2+‐ATPase, from which we obtain a distance of 1.1–1.9 nm between Co2+ and fluorescein located at neighbouring catalytic sites.
The tricarboxylate carrier from rat liver mitochondria
Authors: GLERUM D. Moira , CLAEYS Dirk , MERTENS Wim , AZZI Angelo
Published: March 2005

The tricarboxylate carrier from rat liver mitochondria has been purified and reconstituted into phospholipid vesicles. Its activity has been characterized by both a radioactive citrate uptake assay and a coupled enzymatic assay. A Km of 40 1tM and a Vmax of 1.56 p.mol × min−1× mg−1 have been determined for the carrier. Cholesterol levels of between 5–10% of total lipid content are shown to cause a decrease in carrier activity.
Tooth ‘enamelins’ identified mainly as serum proteins
Authors: STRAWICH Elsa , GLIMCHER Melvin J.
Published: March 2005

The major enamelin protein component present in EDTA or EDTA/guanidine hydrochloride extracts of developing bovine enamel has a molecular mass of about 67 kDa; it has an amino acid composition similar to that of bovine serum albumin and reacts with polyclonal and monoclonal antibodies to albumin. Two‐dimensional separation of the components in the enamelin extract by isoelectric focusing and SDS/PAGE reveal that the major ∼ 67‐kDa component and almost all of the minor Coomassie‐staining protein components of 7ap; 67 kDa, as well as many of the other minor components with different molecular masses, also react with polyclonal and monoclonal antialbumin. The ∼ 67‐kDa band eluted after SDS/PAGE, as well as the major ∼ 67‐kDa spots eluted after two‐dimensional separation, were found to have N‐terminal amino acid sequences identical to that of bovine serum albumin. Albumin accounted for at least 70–80% of the total protein content of the enamelin extract and was essentially the only protein in the ∼ 67‐kDa component. The serum proteins α‐2 HS glycoprotein, γ‐globulin and fetuin, and the proline‐rich salivary protein termed P‐B were also identified in the enamelin extract. The serum proteins and the salivary protein account for > 95% of the proteins in the enamelin extracts. Of the remaining very small amounts of non‐serum or salivary protein isolated from the enamelin extracts, three minor components were isolated which had N‐terminal amino acid sequences which were not similar to any known protein in the protein sequence data base and could therefore conceivably be true ‘enamelins’ synthesized by ameloblasts. One additional protein had the first five N‐terminal amino acids and residue 8 of amelogenin, residues 6 and 7 being different from those of amelogenin. Two other very minor protein components had amino acid compositions distinct from the amelogenins and the serum proteins, but were N‐terminally blocked on attempted sequencing. None of the components in the neutral soluble low‐ionic‐strength extract or in the 4 M guanidine hydrochloride extract, both of which consist principally of amelogenins, immunoreacted with anti‐albumin or with any of the antibodies to other serum proteins and fetuin, despite the fact that the amelogenin extracts also contain non‐amelogenin proteins.

On the basis of the data presented, studies employing antibodies to the so‐called enamelin proteins and hypotheses as to their molecular conformation, their roles as evolutionary markers, or their positive role in mineralization should be reconsidered and reviewed.
Purification and characterization of an (ADP‐ribose)n glycohydrolase from human erythrocytes
Authors: TANUMA Sei‐ichi , ENDO Hiroyoshi
Published: March 2005

An (ADP‐ribose)n, glycohydrolase from human erythrocytes was purified approximately 13000‐fold and characterized. On sodium dodecyl sulfate/polyacrylamide gel the purified enzyme appeared homogeneous and had an estimated relative molecular mass (Mr) of 59000. Amino acid analysis showed that the enzyme had a relatively high content of acidic amino acid residues and low content of basic amino acid residues. Isoelectrofocusing showed that the enzyme was an acidic protein with pI value of 5.9. The mode of hydrolysis of (ADP‐ribose)n by this enzyme was exoglycosidic, yielding ADP‐ribose as the final product. The Km value for (ADP‐ribose)n, (average chain length, n = 15) was 5.8 μM. and the maximal velocity of its hydrolysis was 21 μmol · min−1. mg protein−1. The optimum pH for enzyme activity was 7.4. KCI was more inhibitory than NaCl. The enzyme activity was inhibited by ADP‐ribose and cAMP but not the dibutyryl‐derivative (Bt2‐cAMP), cGMP or AMP. These physical and catalytic properties are similar to those of cytosolic (ADP‐ribose)n, glycohydrolase II, but not to those of nuclear (ADP‐ribose)n, glycohydrolase I purified from guinea pig liver [Tanuma, S., Kawashima, K. & Endo, H. (1986)J. Biol. Chem. 261, 965–969]. Thus, human erythrocytes contain (ADP‐ribose)n, glycohydrolase II.

The kinetics of degradation of poly(ADP‐ribose) bound to histone H1 by purified erythrocyte (ADP‐ribose)n, glycohydrolase was essentially the same as that of the corresponding free poly(ADP‐ribose). In contrast, the glycohydrolase showed appreciable activity of free oligo(ADP‐ribose), much less activity on the corresponding oligo(ADP‐ribose) bound to histone H1. The enzyme had more activity on oligo(ADP‐ribose) bound to mitochon‐drial and cytosolic free mRNA ribonucleoprotein particle (mRNP) proteins than on oligo(ADP‐ribose) bound to Histone HI. It did not degrade mono(ADP‐ribosyl)‐stimulatory guanine‐nucleotide‐binding protein (Gs) and ‐inhibitory guanine‐nucleotide‐binding protein (Gi) prepared with cholera and pertussis toxins, respectively. These results suggest that cytosolic (ADP‐ribose)n, glycohydrolase II may be involved in extranuclear de(ADP‐ribosyl)n, ation, but not in membrane de‐mono(ADP‐ribosyl)ation.
Effect of metabolic alterations on the density and the contents of cathepsins B, H and L of lysosomes in rat macrophages
Authors: MUNO Daisaku , SUTOH Noriko , WATANABE Tsuyosi , UCHIYAMA Yasuo , KOMINAMI Eiki
Published: March 2005

Crude lysosomal preparations from non‐cultured peritoneal rat macrophages were shown to separate into high‐density fractions rich in cathepsin B and H and low‐density fractions rich in cathepsin L when layered on Percoll density gradients. Morphologically, the heavy lysosome fractions were found to consist mainly of lysosomes labeled with gold particles for anti‐(cathepsin B, H and L). The light lysosome fractions contained lysosomes labeled with anti‐(cathepsin B, H and L) and many other contaminants. In addition, small vesicles labeled by anti‐(cathepsin L) were detected in these fractions. Addition of calf serum to the cultured macrophages induced an increase in the density of lysosomes in both dose‐dependent and time‐dependent fashions. Cathepsins B, H and L all shifted to the heavy lysosome fractions following the addition of serum. Progressive increase in fluorescence‐labeled calf IgG in the heavy lysosome fractions after its addition suggests that the continuous entrance of excess proteins to lysosomes causes an increase in their density. This idea is supported by the fact that the density of lysosomes increased in parallel with the accumulation of horseradish peroxidase taken up in the heavy lysosome fractions. Increase in the density of lysosomes after treatment with ethyl(2S.3S)‐3[(S)‐3‐methyl‐1‐(3‐methyl‐butylcarbamoyl)]oxirane‐2‐carboxylate (E‐64‐d) was marked in the cells cultured with serum‐containing medium but slight in serum‐deprived cells. However, the level of pyruvate kinase, an autophagic sequestration marker in heavy autolysosomes from E‐64‐d‐treated cells, was much higher in serum‐deprived cells, indicating that the contribution of heterophagic sequestration towards an increase in the density of lysosomes is much greater than that of autophagy.