bluuebunny/crossref_metadata_2025 huggingface dataset:
CD4 gene transcription is transiently repressed during differentiation of myeloid cells to macrophage‐like cells
Authors: PIMENTEL‐MUIÑOS Felipe X. , LOPEZ‐GUERRERO José A. , FRESNO Manuel , ALONSO Miguel A.
Published: March 2005

The CD4 glycoprotein, which serves as receptor for human immunodeficiency virus (HIV), is expressed in several types of cells of hematopoietic origin, including T lymphocytes and monocytes. Triggering differentiation of peripheral blood monocytes, monocytic U‐937 or promyelocytic HL‐60 precursor cells to macrophage‐like cells by phorbol ester treatment transiently induced both a rapid reduction in surface CD4, demonstrated by flow‐cytometry analysis, and a gradual loss of CD4 mRNA, revealed by Northern‐blot analysis. Experiments in HL‐60 cells to determine the cause of the observed decay in CD4 mRNA levels suggested that the half‐life of CD4 transcripts did not diminish but increased after phorbol ester stimulation. Direct measurement of CD4 gene transcription by run‐on analysis indicated that the rate of synthesis of new CD4 mRNA molecules was reduced approximately 10‐fold after phorbol ester stimulation, whereas the rate of synthesis of c‐fos mRNA resulted in a 2.5‐fold increase. These data suggest that phorbol ester treatment specifically reduces CD4 mRNA levels by repressing CD4 gene transcription. These findings may be relevant to understand the regulation of CD4 gene expression during differentiation.
Characterisation of a thermostable α‐amylase from Bacillus brevis
Authors: STEFANOVA Miglena , EMANUILOVA Elka
Published: March 2005

Biochemical characterization of a novel heat‐stable α‐amylase, produced by a thermophilic strain of Bacillus brevis, has been made. The pattern of the enzyme action on different substrates was studied. It was found that reducing groups were rapidly liberated from amylopectin, soluble and insoluble starch compared to amylose and glycogen. B. brevisα‐amylase acted via endo‐attack producing mainly maltopentaose during the first hour of hydrolysis. The enzyme showed high activity towards maltohexaose and maltoheptaose. The α‐amylase from B. brevis had a neutral pI and was found to be a glycoprotein, containing 9.2% (by mass) neutral sugars. The enzyme protein possessed a unique high glycine content. Calcium or sodium ions in appropriate concentrations were required for enzyme thermostability.
Identification of cholecystokinin/gastrin peptides in frog and turtle
Authors: JOHNSEN Anders H. , REHFELD Jens F.
Published: March 2005

Peptides homologous to mammalian cholecystokinin (CCK) and gastrin in brain, antrum, and small intestine of an amphibian (the bullfrog, Rana catesbeiana) and a reptile (the turtle, Pseudomys scripta) were characterized. All tissues contained peptides racting with antisera specific for the carboxyamidated C‐terminal tetrapeptide common for CCK and gastrin. Extracts of all tissues, except the turtle antrum, also reacted with an antiserum specific for mammalian sulfated CCK, while no extract contained peptides reacting with an antiserum specific for mammalian gastrin. Both species contained predominantly small acidic forms in the brain and larger less acidic forms in the antrum and intestine. The antral peptides of both species were identified. The largest frog gastrin was a 47‐residue peptide: DLLASLTHEQ KQLIMSQLLP ELLSELSNAE DHI HPMRDRD YAGWMDF · NH2. The largest turtle gastrin was a 52‐residue peptide: DLLEALSQDQ KLLMAKFLPH IYAELANREG NWHEDAALRP LHDHDYPGWM DF. NH2. They display 51% similarity. Having Tyr in position 7 from the C‐terminus, both resemble structuraly mammalian CCK rather than gastrin, which suggests the CCK is phylogenetically older than gastrin. The turtle antral peptide contains a Tyr followed by Pro as in chicken gastrin. Thus, apparently at the stage of reptiles, a route different from the mammalian was in order to evolve a specific gastrin function.
Inhibition of triosephosphate isomerase from Trypanosoma brucei with cyclic hexapeptides
Authors: KUNTZ Douglas A. , OSOWSKI Rudolf , SCHUDOK Manfred , WIERENGA Rik K. , MÜLLER Klaus , KESSLER Horst , OPPERDOES Fred R.
Published: March 2005

Two series of oligopeptides have been synthesized. Their effects on the activity of purified triosephosphate isomerase from Trypanosoma brucei and various other organisms have been studied. Using detailed three‐dimensional structure information, the first series consisted of both cyclic and linear hypdrophilic peptides that were designed to mimic the β turns of the subunit interface loops of the trypanosome triosephosphate isomerase dimer. None of these exerted any inhibitory effect. The second series consisted of more hydrophobic cyclic peptides, originally designed to inhibit a hepatic transport system. Several of these were very effective in inhibiting the trypanosome triosephosphate isomerase, but not the homologous enzymes from rabbit, dog, yeast or Escherichia coli. The most active peptide, cyclo[‐Trp‐Phe‐d‐Pro‐Phe‐Phe‐Lys(Z)‐], exerted 50% inhibitory activity at a concentration of 3 μM. The nature of the inhibitory action one of these compounds cyclo[‐Trp‐Tyr(OSO3Na)‐d‐Pro‐Phe‐Thr(OSO3Na)‐Lys(Z)‐] was studied in more detail. Its inhibition was non‐competitive and reversible and more than one peptide was able to bind/active site.
Cloning, sequencing and in vivo expression of genes encoding the Fo part of the sodium‐ion‐dependent ATP synthase of Propionigenium modestum in Escherichia coli
Authors: KAIM Gerog , LUDWIG Wolfgang , DIMROTH Peter , SCHLEIFER Karl H.
Published: March 2005

A DNA fragment containing the genes encoding subunits of the Fo part of the sodium‐translocating ATPase of Propionigenium modestum was cloned in Escherichia coli and sequenced. The predicted amino acid sequences of subunits a, b and c of the P. modestum ATPase were compared with those of the corresponding subunits of proton‐translocating ATPases from other bacteria and chloroplasts. Deletion mutants of E. coli, lacking different genes for ATPase subunits, were transformed with a recombinant plasmid, containing the genes for the subunits a, c, b, δ and part of α of the ATPase of P. modestum. Functionally reconstituted ATPase activity could be demonstrated for the transformants. The identity of the vector containing P. modestum genes was verified by restriction analysis of plasmid DNA.
Lipomide dehydrogenase from Azotobacter vinelandii: site‐directed mutagenesis of the His450‐Glu455 diad
Authors: BENEN Jacques , van BERKEL Willem , DIETEREN Nicole , ARSCOTT David , WILLIAMS Charles , VEEGER Cees , de KOK Arie
Published: March 2005

Three amino acid residues in the active site of lipoamide dehydrogenase from Azotobacter vinelandii were replaced with other residues. His450, the active‐site base, was replaced with Ser, Tyr or Phe. Pro451, from X‐ray analysis found to be in cis conformation positioning the backbone carbonyl of His450 close to N3 of the flavin, was changed to Ala. Glu455, from X‐ray analysis expected to be involved in modulating the pKa of the base (His450), was replaced with Asp and Gln. The general conclusion is that mutation of the His‐Glu diad impairs intramolecular electron transfer between the disulfide/dithiol and the FADH−/FAD.

The wild‐type enzyme functions according to a ping‐pong mechanism in the physiological reaction in which the formation of NADH is rate‐limiting. Above pH 8.0 the enzyme is strongly inhibited by the product NADH. The pH dependence of the steady‐state kinetics using the NAD+ analog 3‐acetylpyridine adenine dinucleotide (AcPyAde+) reveals a pKa of 8.1 in the pKm AcPyAde+ plot indicating that this pKa is related to the deprotonation of His450 [Benen, J., Berkel van, W., Zak, Z., Visser, T., Veeger, C. & Kok de, A. (1991) Eur. J. Biochem. 202, 863–872] and to the inhibition by NADH.

The mutations considerably affect turnover. Enzymes with the mutations Pro451 → Ala, His450 → Phe and His450 → Tyr appear to be almost inactive in both directions. Enzymes His450 → Ser is minimally active, V at the pH optimum being 0.5% of wild‐type activity in the physiological reaction. Rapid reaction kinetics show that for the His450‐mutated enzymes the reductive half reaction using reducted 6,8‐thioctic acid amide [Lip(SH)2] is rate‐limiting and extremely slow when compared to the wild‐type enzyme. For enzyme Pro451 Ala it is concluded that the loss of activity is due to over‐reduction by Lip(SH)2 and NADH. The Glu455‐mutated enzymes are catalytically competent but show strong inhibition by the product NADH (enzyme Glu455 → Asp more than Glu455 → Gln). The inhibition can largely be overcome by using AcPyAde+ instead of NAD+ in the physiological reaction.

The rapid reaction kinetics obtained for enzymes Glu455 → Asp and Glu455 → Gln deviate from the wild‐type enzyme. It is concluded that this difference is due to cooperativity between the active sites in this dimeric enzyme. Rapid reaction kinetics of enzymes His450 → Ser and Glu455 → Gln show the existence of two intermediates at the two‐electron reduced level: a species with the NAD+ bound, the flavin reduced and the disulfide intact (oxidized) and a species with NAD+ bound, the disulfide reduced and the flavin oxidized. No spectral evidence is obtained for the participation of a proposed flavin C4a adduct intermediate [Thorpe, C. & Williams, C. H. Jr (1981) Biochemistry 20, 1507–1513] in the reaction mechanism of enzymes His450 → Ser and Glu455 → Gln.
The essentail active‐site lysines of clostridial glutamate dehydrogenase
Authors: LILLEY Kathryn S. , ENGEL Paul C.
Published: March 2005

Glutamate dehydrogenase (GDH) of Clostridium symbiosum, like GDH from other species, is inactivated by pyridoxal 5′‐phosphate (pyridoxal‐P). This inactivation follows a similar pattern to that for beed liver GDH, in which a non‐covalent GDH‐pyridoxal‐P complex reacts slowly to form a covalent complex in which pyridoxal‐P is in a Schiff's‐base linkage to lysine residues.

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The equilibrium constant of this first‐order reaction on the enzyme surface determines the final extent of inactivation observed [S. S. Chen and P. C. Engel (1975) Biochem. J. 147, 351–358]. For clostridial GDH, the maximal inactivaton obtained was about 70%, reached after 10 min with 7mM pyridoxal‐P pH 7. In keeping with the model,

inactivation became irreversible after reduction with NaBH4.

The NaBH4‐reudce enzyme showed a new absorption peak at 325 nm.

K
m values for NAD+ and glutamate were unalterd, although Vmax values were decreased by 70%.

Kinectic analysis of the inactivation gave values of 0.81 ± 0.34 min−1 for K3 and 3.61 ± 0.95 mM for K2/K1. Thelineat plot of 1/(1‐R) against 1/[pyridoxal‐P], where R is the limiting residual activity reached in an inactivation reaction, gave a slightly higher valus for K2/K1 of 4.8 ± 0.47 mM and K4 of 0.16 ± 0.01 min−1.

NADH, NAD+, 2‐oxoglutrate, glutarate and succinate gave partial protection against inactivation, the biggest effect being that of 40 mM succinate (68% activity compared with 33% in the control). Paired combinations of glutarate or 2‐oxoglutarate and NAD+ gave slightly better protection than the separate components, but the most effective combination was 40mM 2‐oxoglutarate with 1 mM NADH (85% activity at equilibrium). 70% inactivated enzyme showed an incorporation of 0.7 mM pyridoxal‐P/mol subunit, estimated spectrophotometrically after NaBH4 reduction, in keeping with the 1:1 stoichiometry for the inactivation. In a sample protected with 2‐oxoglutarate and NADH, however, incorporation was 0.45mol/mol, as against 0.15mol/mol expected (85% active).

Tryptic peptides of the enzyme, modified with and without protection, were purified by HPLC. Two major peaks containing phosphopyridoxyllysine were unique to the unprotected enzyme. These peaks yielded three peptide sequences clearly homologous to sequences of other GDH species. In each case, a gap at which no obvious phenythiohydantion‐amino‐acid was detected, matched a tryptic cleavage. Modification of these three lysines, corresponding to Lys92, Lys116 and Lys128 in Escherichia coli GSH, appears to be mutually exclusive and modification of any one of them abolishes activity. Comparison with the cystal structure indicated that these lysines are clustered in the activesite cleft and likely to interact with the dicarboxylate substrate.
A tungsten‐containing active formylmethanofuran dehydrogenase in the thermophilic archaeon Methanobacterium wolfei
Authors: SCHMITZ Ruth A. , RICHTER Monika , LINDER Dietmat , THAUER Rudolf K.
Published: March 2005

Methanobacterium wolfei is a thermophilic methanogenic archaeon which requires tungsten or molybdenum for growth. We have found that the orgainsm contains two formylmethanofuran dehydrogenases, one of which is a tungsten enzymes. Indirect evidence indicated that the other formylmethanofuran dehydrogenase is a molydbenum enzyme. The tungsten enzyme was purified and characterized. The native enzyme had an apparent molecular mass of 130kDa. SDS/PAGE revealed a composition of three subunits of apparent molecular mass 35, 51 and 64kDa, the N terminalamino acid sequences of two of which were determined. 0.3–0.4 mol tungsten/mol enzyme was found but no moybdenum. The pterin cofactor was identified as molybdopterin guanine dincleotide. The purified enzymes exhibited a specific activity of 8.3 μmol · min−1· mg protein−1 and an apparent km for formylmethanofuran and methylviologen of 13μM and 0.4mM, respectively. The optimum temperature for activity was 65°C. At 40–60°C, the rate increased with a Q10 of 1.9; the activation energy of the reaction was 45kJ/mol. The enzyme was found to require potassium ions for thermostability. The oxygen‐sensitive enzyme was not inactivated by cyanide.
Kinetics of daunorubicin transport by P‐glycoprotein of intact cancer cells
Authors: SPOELSTRA Ellen C. , WESTERHOFF Hans V. , DEKKER Henk , LANKELMA Jan
Published: March 2005

Drug permeation across the plasma membrane of multidrug‐resistant cells depends on the kinetics of the P‐glycoprotein‐mediated pump activity as well as well as on the passive permeation of the drug. We here demonstrate a method to characterize kinetically the pump in intact cells.

To this purpose, we examined the membrane‐transport properties of daunorubicin in various sensitive cancer cell lines and in their multidurg resistant (MDR) counterparts. First, we determined the passive permeability coefficient for daunorubicin. Then, using a flow‐through system, the drug flux into the cell was measured after inhibition of the P‐glycoprotein‐mediated efflux pump. Combining the two results allowed us to calculate the intracellular free concentration of the drug. In the steady‐state, the pump rate must equal the net rate of passive diffusion of the drug and, therefore, the same experiments gave us the pumping rate of daunorubicin. These experiments were then repeated at various extracellular durg concentrations. By plotting the pumping rate versus the intracellular durg concentration, we then characterized the P‐glycoprotein kinetically.

Four independent methods were used to measure the passive permeability coefficient for the cell line A2780. Similar values were obtained. Maximal pump rates (Vmax) showed a good correlation with the amount of P‐glycoprotein in the cell lines used. We obtained saturation curves for the variation of the pump rates the intracellular daunorubicin concentration. These curves were typical for positive cooperativity, which provides evidence that at least two binding sites for daunorubicin are present on the active transport system of daunorubicin. The apparent Km values for P‐glycoprotein‐mediated transport, the intracellular free cytosolic daunorubicin conccntrations at half‐maximal velocity for the cell lines used, were approximately 1.5μM. Except for the cell lines with the highest amount of P‐glycoprotein, the passive efflux rate of daunorubicin proved to be a substantial part of the total daunorubicin efflux rate for the cell lines used. In cell lines with relatively low levels of P‐glycoprotein, passive daunorubicin efflux was even the main route of daunorubicin transport from the cell, determining the intracellular steady‐state concentrations of daunorubicin.
The pH in reversed micelles as imposed by the dihydrogen/proton redox cuple and indicated by viologens and cytochrome c3 using hydrogenase as redox catalys
Authors: van DIJK Cees , SPRUIJT Ruud , LAANE Colja , VEEGER Cees
Published: March 2005

The pH values in reversed micelles were measured, making use of the hydrogenase enzyme as redox catalyst short‐circuiting the viologen oxidized/semiquinone redox states. The hydrogenases from Desulfovibrio vulgaris (Hildenborough) and from Megasphaera elsdenii were applled. The observed pH values in reversed micelles were not dependent onthe type of hydrogenase.

Two cationic [cetyltrimethylammonium bromide and dodecylammonium propionate (DAP)] and two anionic sodiumdodecyl sulphate, sodium di(ethylhexyl)sufosuccinate types of reversed micelles were used in combination with viologens having distiguishable valencies. It was observed that, in the cationic‐reversed micelles, the dissociation constant for the semiquinone dimer had about the same value as compared to bulk water, while this value was significantly higher in the anionic‐reversed micelles. Furthemore, the dissociation Constant was independent of the concentration of viologen semiquinone in the reversed micell, indicating that exchange kinetics are faster than the dimerisation process. With the exception of DAP, a linear relation exists, pH = a · pHrm+ b, between the pH of the bulk water and the pH as measured in the reversed micelle(pHrm). In all these case the value of a is smaller than unity, the value of b ranges between 1.6–2.7. For DAP the pHrm is always around 7. In DAP‐reversed micelles, the counter‐ion propionate probably serves as an internal buffer. Using cytochrome c3, as pH indicator in combination with N,N′‐di(3‐aminopropl)‐4,4′‐bipyridinium)4 + to take care of electron transfer, in cetyltrimethylammonium‐bromide‐reversed micelles the pHrm is about the same as indicated by the viologen; in SDS‐reversed micelles the pHrm is always lower than that incidated by N,N′‐di(3‐aminopropyl)‐4,4′‐pyridinium4 +. In contrast to cytochrome c3 from D. vulgaris, which in reversed micelles cannot become reduced directly by its D. Vularis hydrogenase, the hydrogenase of M. elsdenii is able to reduce its ferredoxin directly.
Characterization of the promoter region of the porcine opn (osteopontin, secreted phosphoprotein 1) gene
Authors: ZHANG Qi , WRANA Jeffrey L. , SODEK Jaro
Published: March 2005

Osteopontin (secreted phosphoprotein‐1, Opn) is a phosphorylated glycoprotein expressed by transformed cells, macrophages, activated T‐lymphocytes, specialized epithelial cells and bone cells that is characteristically enriched in milk and in the mineralized matrix of bone. The synthesis of Opn by bone cells is regulated by glucocorticoids and growth factors, which promote bone formation, and by the osteotropic hormone calcitriol (1,25‐dihydroxycholecalciferol) and retinoic acid, which mediate bone resorption, indicating a bifunctional role for this protein in bone remodelling. To study the transcriptional regulation of the opn gene, two genomic clones (10 and 15 kb) encoding the opn gene were isolated from a porcine liver genomic library cloned into λ phage. From the 15‐kb clone a 4‐kb EcoRI fragment containing the first two exons and 2.6 kb of the 5′ flanking region of the opn gene was sequenced, and the transcriptional start site determined by primer extension analysis and S1 nuclease mapping. To identify the opn promoter, chimeric chloramphenicol acetyltransferase constructs were prepared using fragments from the first intron and the 5′ flanking region of the opn gene. Transient transfection of porcine bone cells with these constructs showed strong promoter activity located within 74bp upstream from the transcription initiation site. Within this region a TATA sequence, TTTAAA, was identified at positions −26 to −31. However, the highest transcription rate was observed in a construct extending 180 bp upstream that included a CCGCCC Sp1 binding sequence (−63 to −68), and an AP1 site (−74 to −80). Further upstream in the 5′ flanking region and within the first intron of the opn, a number of consensus sequensus could be identified. Chimeric constructs containing a GGGTCAtatGGTTCA direct repeat consensus sequence for a vitamin D3 response element located at nucleotides –2245 to –2259 responded to the addition of 0.1μM calcitriol by a 2.5‐fold stimulation of transcription, although a > 2‐fold increase was also observed in shorter constructs – 180 to – 905 lacking such a consensus. Promoter activity was also exhibited by a region containing a TTTAAA sequence in the first intron that corresponded to the putative promoter site reported for mouse opn in macrophages (Miyazaki, Y., Setoguchi, M., Yoshida, S., Higuchi, Y., Akizuki, S. & Yamamoto, S. (1990) J. Biol. Chem. 265, 14432–14438). However, Primer extension and hybridization analysis of both porcine and monocyte/macrophage and bone mRNA failed to reveal an Opn mRNA transcribed from the alternative promoter, indicating that the same promter regulates transcription of the opn gene in monocytes and macrophages as well as in bone.
The chloroplast‐targeting domain of plastocyanin transit peptide can form a helical structure but does not have a high affinity for lipid bilayers
Authors: ENDO Toshiya , KAWAMURA Kasumi , NAKAI Masato
Published: March 2005

Conformational properties and interactions with lipid membranes were studied for the chemically synthesized peptides PC(1–37) and PC(1–43), corresponding to the N‐terminal 37 and 43 residues, respectively, of the transit peptide of the precursor to plastocyanin of Silene pratensis. PC(1–43) covers the entire chloroplast targeting domain of the transit peptide. CD spectra of PC(1–37) and PC(1–43) showed that both peptides have little ordered structure in aqueous solutions but form partially helical conformations in the presence of detergent micelles or in methanol. Vesicle disruption and direct‐binding experiments revealed, however, that neither PC(1–37) nor PC(1–43) had a high affinity for lipid membranes. Since in the intact plastocyanin transit peptide the chloroplast‐targeting domain is followed by a hydrophobic thylakoid‐transfer domain, the plastopcyanin precursor may well be transported to the chloroplast surface first with the aid of the thylakoid‐transfer domain. The chloroplast‐targeting domain may then form a helical structure in the lipid environments, and a chloroplast‐specific motif displayed on the helical structure may be recognized by a receptor protein located at the chloroplast envelope membranes.
A mutation at Gly314 of the β subunit of the Escherichia coli pyridine nucleotide transhydrogenase abolishes activity and affects the NADP(H)‐induced conformational change
Authors: AHMAD Suhail , GLAVAS Natalie A. , BRAGG Philip D.
Published: March 2005

Escherichia coli RH1 contains a mutation causing complete loss of pyridine nucleotide transhydrogenase activity. A single base change in the chromosomal DNA resulted in the replacement of Gly314 of the β subunit by a Glu residue. The mutant enzyme was partially purified and its trypsin cleavage products examined. The distinct pattern of polypeptides given by proteolysis of the normal transhydrogenase in the presence of NADP(H) was absent when the mutant enzyme was treated with trypsin. However, the β subunit of the mutant enzyme retained its ability to bind to NAD‐agarose. Further substitutions were made at Gly314 converting it to Ala, Val or Cys by the use of site‐directed mutagenesis. All substitutions for Gly314 abolished the activity completely. The enzyme containing the Gly314→Ala mutation was studied in detail and behaved exactly as the enzyme containing the Gly314→Glu mutation. It is concluded that the mutation in the β subunit abolished the NADP(H)‐induced conformational change in the mutant enzyme. This conformational change, caused by NADP(H) binding, is required to cleave the normal β subunit at Arg265 by trypsin.

The genes encoding the pyridine nucleotide transhydrogenase were completely resequenced and several corrections have been made to the previously published sequence [Clarke et al. (1986) Eur. J. Biochem. 158, 647–653].
Characterization and purification of a membrane‐bound archaebacterial pyrophosphatase from Sulfolobus acidocaldarius
Authors: MEYER Wolfgang , SCHÄFER Günter
Published: March 2005

Plasma membranes of thermoacidophilic archaebacterium Sulfolobus acidocaldarius (DSM 639) display a pyrophosphate‐hydrolyzing activity [M. Lübben & G. Schäfer (1987) Eur. J. Biochem. 164, 533–540]. In our present work, we solubilized and purified this pyrophosphatase to homogeneity. It consists of single subunit with a molecular mass of 17 – 18 kDa, forming an oligomer of 70 kDa under native conditions. Edman degradation revealed 30 amino acids of the N‐terminus. The enzyme cleaves phosphoric‐acid‐anhydride bonds independently of monovalent or divalent cations. Temperature and pH optima of 75°C and 3.5–3.7, respectively, characterize it as an ectoenzyime. Membrane lipids of Sulfolobus stimulate the activity. The dolichol‐pyrophosphate‐complexing peptide‐antibiotic bacitracin inhibited growth of Sulfolobus. A possible function of the acid pyrophosphatase is the hydrolysis of dolichol pyrophosphate in connection with glycosylation reactions of membrane proteins.
Evidence for polynuclear aggregates of ferric daunomycin
Authors: MATZANKE Berthold F. , BILL Eckhard , BUTZLAFF Christian , TRAUTWEIN Alfred X. , WINKLER Heiner , HERMES Christoph , NOLTING Hans‐Friedrich , BARBIERI Renato , RUSSO Umberto
Published: March 2005

The interaction of the antitumor agent daunomycin (DN) with ferric iron has been analysed by Mössbauer spectroscopy, EPR, extended X‐ray absorption fine structure (EXAFS), and magnetic susceptibility measurements. In contrast to literature data, at millimolar iron and anthracycline concentrations no solitary Fe(DN)3 complexes are formed in appreciable amounts. The Mössbauer spectroscopic analysis revealed severe dependencies on temperature, on the preparation procedure, the time allowed for equilibration, and on the metal/ligand ratio. The Mössbauer spectra exhibit two components: a broad magnetic sextet and a quadrupole doublet at an Fe/DN molar ratio of 1:3 and exclusively a doublet at a molar ratio of 1:20, indicating an equilibrium of these two spectral components. The EPR spectra are dominated by a signal at geff=2. Double integration of the EPR signals enabled the determination of thier spin density and a correlation between EPR and Mössbauer spectra. The Mössbauer sextet species is EPR invisible and corresponds to magnetically ordered polynuclear aggregates with high magnetic anisotropy. EXAFS and susceptibility measurements provide additional evidence for the formation of polynuclear aggregates of ferric daunomycin. The quadrupole doublet species in the Mössbauer spectra correlates with the g=2 signal in EPR. This species is also related to a magnetically ordered system, exhibiting, however, superparamagnetic behavior due to loss magnetic anisotropy. Since daunomycin forms dimers in aqueous solution at millimolar concentrations, we conclude that the cooperative phenomena observed in EPR and Mössbauer spectra are a consequence of its stacking effects.
Molecular evolution of alanine/glyoxylate aminotransferase 1 intracellular trageting
Authors: PURDUE P. Edward , LUMB Michael J. , DANPURE Christopher J.
Published: March 2005

In mammals, the subcellular distribution of alanine:glyoxylate aminotransferase 1 (AGT) is species dependent, with the proportion of AGT targeted to mitochondria varying between 0% and greater than 90%, the remainder being located in the peroxisome. In order to extend our studies on the molecular evolution of intracellular targeting of AGT, we have investigated the organization and expression the AGT genes of rabbit, which has all of its AGT located in the peroxisome, and marmoset, which has approximately 50% of its AGT located in the peroxisome and 50% in the mitochondrion. Southern‐blot analysis indicates that, in both of these species, AGT is encoded by a single‐copy gene, as has previously been shown for human (all AGT in the peroxisome) and rat (50% AGT in the peroxisome and 50% in the mitochondrion). Comparison of the cDNA sequences encoding marmoset, rabbit, human and rat AGT, Combined with transcript mapping and in vitro mitochondrial protein‐import analysis, has provided a molecular explanation for the differential trageting of AGT in these species. As in the rat, marmoset AGT is synthesized in two forms, via the use of alternative transcription and translation‐initiation sites. These two forms of AGT differ only in the presence of absence of a 22‐amino‐acid amino – terminal peptide, which acts as a cleavable mitochondrial – targeting sequence, directing the longer form of AGT to mitochondria. The shorter form of AGT, lacking the mitochondrial – targeting sequence, is presumed to be localized in the peroxisomes. In humans and rabbits, similar but distinct evolutionary mutational events within the AGT gene have resulted in exclusion of the region encoding the mitochondrial‐targeting sequence from the open reading frame, explaining the exclusive peroxisomal localization of AGT in these species. We discuss the impact of these results on our understanding of both the evolution of species dependence of AGT subcellular distribution and the recent identification of amino acid changes in human AGT which result in mistargeting of this protein to mitochondria.
The serine‐protease inhibitor of cartilage matrix is not a chondrocytic gene product
Authors: BÖHM Beate , AIGNER Thomas , KINNE Raimund , BURKHARDT Harald
Published: March 2005

Human articular cartilage contains significant amounts of antileukoprotease, a cationic low‐molecular‐mass serine‐protease inhibitor, which was originally purified from mucous secretions (synonym: secretory leukocyte proteinase inhibitor). As it was not known whether the inhibitor molecule is also synthesized locally, we investigated antileukoprotease gene expression in chondrocytes. No antileukoprotease‐specific mRNA was detected in adult or foetal human chondrocytes by in situ hybridization, Northern‐blot analysis or polymerase chain reaction. Concurrently, the chondrocytesremained unstained on immunohistology, whereas immunoreactive antileukoprotease was demonstrated in the cartilage matrix. By Northern‐blot analysis, the antileukoprotease message was detected in the promyelocytic cell line HL60, the myelomonocytic cell line U937 and even in mature polymorphonuclear leukocytes from the peripheral blood of healthy donors. Immunoperoxidase staining of polymorphonuclear leukocytes for the antileukoprotease protein indicated that this cell is likely to be the physiological source of the inhibitor in serum. The results further suggest an accumulation of the inhibitor in the cartilage matrix.
The structure of neutral protease from Bacillus cereus at 0.2‐nm resolution
Authors: STARK Wilhelm , PAUPTIT Richard A. , WILSON Keith S. , JANSONIUS Johan N.
Published: March 2005

The crystal structure of the neutral protease from Bacillus cereus has been refined to an R factor of 17.5% at 0.2‐nm resolution. The enzyme, an extracellular metalloendopeptidase, copies of two domains and binds one zinc and four calcium ions. The structure is very similar to that of thermolysin, with which the enzyme shares 73% amino‐acid sequence identity. The active‐site cleft between the two domains is wider in neutral protease than in thermolysin. This suggests the presence of a flexible hinge region between the two domains, which may assist enzyme action. The high‐resolution analysis allows detailed examination of possible causes for the difference in thermostability between neutral protease and thermolysin.
Biochemical analysis of the biogenesis and function of the Escherichia coli export factor SecY
Authors: SWIDERSKY Ulla E. , RIENHÖFER‐SCHWEER Annette , WERNER Pamela K. , ERNST Friedrich , BENSON Spencer A. , HOFFSCHULTE Hedda K. , MÜLLER Matthias
Published: March 2005

SecY is an integral plasma‐membrane protein of Escherichia coli which is essential for the export of periplasmic and outer‐membrane proteins containing cleavable signal sequences. We have synthesized SecY in vitro using an E. coli transcription/translation system. In the absence of membranes, SecY remained largely soluble but cosedimented on sucrose gradients with the membrane fraction when inside‐out plasma‐membrane vesicles (INV) had been added cotranslationally. Membrane association of SecY was unaffected if the endogenous SecY of the INV had been inactivated by either antibodies, a mutation or trypsin treatment. In contrast, inactivation of the INV SecY interfered with membrane targeting and, consequently, the processing of precursors to β‐lactamase and λ receptor. When SecY‐deprived INV were, however, first functionally reconstituted with in‐vitro‐ synthesized SecY, targeting and translocation of λ receptor were partially resorted. Thus, the assembly of SecY into INV in vitro leads to an active enzyme. In addition, we show that the prlA4 allele of the secY gene suppresses signal‐sequence mutatios of the λ receptor in vitro. Collectively, our results demonstrate that SecY, while funcitoning as a membrane‐located receptor for precursors of exported proteins, appears to be virtually independent of pre‐existing SecY for its own membrane integration.
Expression of recombinant soluble and membrane‐bound catechol O‐methyltransferase in eukaryotic cells and identification of the respective enzymes in rat brain
Authors: TILGMANN Carola , MELEN Krister , LUNDSTRÖM Kenneth , JALANKO Anu , JULKUNEN Ilkka , KALKKINEN Nisse , ULMANEN Ismo
Published: March 2005

The rat and human recombinant soluble and membrane‐bound catechol O‐methyltransferase (S‐ and MB‐COMT, respectively) were expressed using mammalian and baculovirus vectors. Law levels of rat and human S‐COMT polypeptides were detected by immunoprecipitation in K‐562 cell lines transfected with the S‐COMT vectors. From K‐562 cells transfected with the rat MB‐COMT construct, both S‐ and MB‐COMT recombinant proteins were detected by a rat COMT‐specific antiserum. Infection of lepidopteran Spodoptera frugiperda cells with recombinant S‐ or MB‐COMT baculovirus constructs yielded high amounts of enzymically active and immunoreactive S‐ or MB‐COMT proteins, respectively. Pulse/chase experiments with [35S]methionine‐labelled insect cells infected with the MB‐COMT baculovirus showed that the 30‐kDa recombinant human MB‐COMT polypeptide was not processed into the 25‐kDa S‐COMT form. Subcellular fractionations of insect cells followed by immunoblotting with COMT antiserum, showed that recombinant S‐COMT was found only in the soluble, cytoplasmic fraction, whereas MB‐COMT resided both in soluble and membrane fractions. The recombinant MB‐COMT sedimented in Percoll gradients at the density of 1.042 g/ml cosedimenting with the plasma‐membrane marker. Fractionation and immunoblotting experiments on homogenized total rat brains indicated that the rat S‐COMT (24 kDa) and some of the rat MB‐COMT (28 kDa) was recovered in soluble fractions, whereas the microsomal material having COMT activity contained the MB‐COMT polypeptide. The rat brain microsomal MB‐COMT had a density of 1.042 g/ml in Percoll gradients, cosedimenting with the plasma‐membrane and rough‐endoplasmic‐reticulum marker enzymes. The meta/para methylation ratio of dihydroxybenzoic‐acid substrate by different recombinant rat brain COMT‐containing subcellular fractions was analysed.
Primary structures of ribosomal proteins L3 and L4 from Bacillus stearothermophilus
Authors: HERWIG Susanne , KRUFT Volker , WITTMANN‐LIEBOLD Brigitte
Published: March 2005

Ribosomal proteins L3 and L4 were purified to homogeneity from total protein isolated from the 50S subunit of Bacillus stearothermophilus by reversed‐phase high‐performance liquid chromatography (RP‐HPLC). Amino acid sequences of both proteins were determined by automated N‐terminal sequence analysis and sequencing of internal peptides. Using oligonucleotides deduced from the N‐terminal region of protein L3 as hybridization probes, a DNA fragment coding for proteins L3, L4 and the N‐terminal part of protein L23 has been identified, cloned and sequenced. The organization of the genes is identical to that found in the S10 operon of Escherichia coli. Comparison of the sequences of proteins L3 and L4 with those of other organisms revealed that all proteins of the L3 family are highly conserved. On the other hand, the archaebacterial L4 proteins show no significant sequence similarity to the E. coli L4 protein whereas the L4 protein of B. stearothermophilus is significantly similar to all of the L4 proteins and thus justifies the membership of all the L4 proteins in one protein family. The results are discussed with respect to the phylogenetic relationship between eubacteria, archaebacteria and eukaryotes and possible functional domains of proteins L3 and L4.
Purification and characterization of a recombinant murine interleukin‐6
Authors: ZHANG Jian‐guo , MORITZ Robert L. , REID Gavin E. , WARD Larry D. , SIMPSON Richard J.
Published: March 2005

Murine interleukin‐6 (IL‐6), when expressed in Escherichia coli using the pUC9 vector, accumulated as insoluble aggregates or ‘inclusion bodies’. After selective urea washing of the inclusion bodies, to remove extraneous proteins, murine IL‐6 was solubilized with 8 M guanidine hydrochloride and then rapidly purified to homogeneity by gel‐permeation chromatography followed by reversed‐phase HPLC. It was demonstrated that complete disulfide bond formation in murine IL‐6 occurred during the early urea washing/guanidine hydrochloride extraction steps, so no refolding step was required. When fully reduced murine IL‐6 was dissolved in 8 M guanidine hydrochloride and allowed to airoxidize, complete disulfide bond formation, monitored by analytical reversed‐phase HPLC, was shown to occur within 13 h at 6°C.

About 25 mg pure protein was obtained from 37 g wet cells. This recombinant murine IL‐6 had a specific activity in the hybridoma growth factor assay of 2×108 U/mg, which is equivalent to that of native murine IL‐6.

During the purification procedure, a number of variant forms of murine IL‐6 were isolated and partially characterized. Two of these forms, T1 and T3, were C‐terminal deletants of murine IL‐6 lacking about 60 and 20 amino acids from the C‐terminus, respectively, while the other form, T2, was an N‐terminal deletant lacking 37 amino acids from the N‐terminus. None of these variant forms of murine IL‐6 bound to the murine IL‐6 receptor and, consequently, all were inactive in the hybridoma growth factor assay.
Precise epitope mapping of monoclonal antibodies to the cytoplasmic side of the acetylcholine receptor α subunit
Authors: TZARTOS Socrates J. , REMOUNDOS Michael S.
Published: March 2005

The epitopes for twelve monoclonal antibodies against the cytoplasmic side of the acetylcholine receptor (AChR) α subunit were precisely mapped using over 300 continuously overlapping synthetic peptides attached on poly(ethylene) rods. mAb cross‐reactive between Torpedo and human AChR generally bound to the homologous peptides from both species. Epitopes 4–10‐residues long were identified. One mAb could bind to either arm on both sides of a β‐turn structure. Five mAb bound to a very‐immunogenic cytoplasmic epitope on α373–380 (VICE‐α). Three of the mAb against VICE‐α were earlier found to cross‐react with non‐AChR protein(s), present in thymomas from myasthenia gravis patients but absent in thymomas from non‐myasthenics. Since VICE‐α has a potentially crucial pathogenic role, the antigenic role of each residue within it was subsequently studied by 55 analogues, most having single amino acid substitutions. All the mAb against VICE‐α bound similarly but not identically to the analogues, thus explaining their known binding heterogeneity. Lys373 proved indispensable for mAb binding. Ile376, Glu377, Gly378 and Lys380 were quite critical, while Ser 374, Ala375 and Val379 seemed rather inactive. These data should prove instructive in searches for VICE‐α‐like epitopes carrying autoantigens with potential involvement in myasthenia gravis and should further expand the applications of the anti‐(AChR) mAb in AChR studies.
Mapping of three unique Ca2+‐binding sites in human annexin II
Authors: JOST Matthias , THIEL Carsten , WEBER Klaus , GERKE Volker
Published: March 2005

Site‐directed mutagenesis was employed to map and characterize Ca2+‐binding sites in annexin II, a member of the annexin family of Ca2+– and phospholipid‐binding proteins which serves as a major cellular substrate for the tyrosine kinase encoded by the src oncogene. Several single amino acid substitutions were introduced in the human annexin II and the various mutant proteins were scored for their affinity towards Ca2+ in different assays. The data support our previous finding [Thiel, C., Weber, K. and Gerke V. (1991) J. Biol. Chem. 266, 14732–14739] that a Ca2+–binding site is present in the third of the four repeat segments which comprise the 33‐kDa protein core of annexin II. In addition to Gly206 and Thr207, which are localized in the highly conserved endonexin fold of the third repeat, Glu246 is involved in the formation of this site. Thus the architecture of this Ca2+–binding site in solution is very similar, if not identical, to that of Ca2+ sites identified recently in annexin V crystals [Huber, R., Schneider, M., Mayr, I., Römisch, J. and Paques, E.‐P. (1990) FEBS Lett. 275, 15–21]. In addition to the site in repeat 3, we have mapped sites of presumably similar architecture in repeats 2 and 4 of annexin II. Again, an acidic amino acid which is located 40 residues C‐terminal to the conserved glycine at position 4 of the endonexin fold is indispensable for high‐affinity Ca2+ binding: Asp161 in the second and Asp321 in the fourth repeat. In contrast, repeat 1 does not contain an acidic amino acid at a corresponding position and also shows deviations from the other repeats in the sequence surrounding the conserved glycine. These results on annexin II together with the crystallographic information on annexin V reveal that annexins can differ in the position of the Ca2+ sites. Ca2+–binding sites of similar structure are present in repeats 2, 3, and 4 of annexin II while in annexin V they occur in repeats 1, 2, and 4. We also synthesized an annexin II derivative with mutations in all three Ca2+ sites. This molecule shows a greatly reduced affinity for the divalent cation. However, it is still able to bind Ca2+, indicating the presence of (an) additional Ca2+ site(s) of presumably different architecture.
Biosynthesis of vitamin B12
Authors: LINGENS Brigitte , SCHILD Thomas A. , VOGLER Bernhard , RENZ Paul
Published: March 2005

The 5,6‐dimethylbenzimidazole moiety of vitamin B12 is formed from riboflavin in aerobic and some aerotolerant bacteria. Thereby C1′ of riboflavin is transformed into C2 of the vitamin B12 base. In the present publication a study on this transformation with riboflavin 2H‐labeled in the 1′R or 1′S position is described. This study was undertaken in order to find out if one of the two hydrogens at C1′ is transferred to C2 of 5,6‐dimethylbenzimidazole.

The 2H‐labeled riboflavin samples were synthesized starting from unlabeled or 1‐2H‐labeled ribose and 3,4‐dimethylaniline yielding N‐β‐d‐ribopyranosyl‐3,4‐dimethylaniline. The unlabeled riboside was reduced to N‐d‐ribityl‐3,4‐dimethylaniline with sodium cyanoborotrideuteride, the 2H‐labeled riboside with sodium cyanoborohydride. The ribityl derivatives were transformed into N‐d‐ribityl‐2‐phenylazo‐4,5‐dimethylaniline, and condensed with barbituric acid to riboflavin.

The reduction of the ribosyl compound to the ribityl derivtive is only partially stereospecific. Thus the riboflavin synthesized from unlabeled ribose had a 2H ratio of 3/1 (1′R/1′S), the riboflavin obtained from d‐[1‐2H1]ribose of 1/3 (1′R/1′S). The 2H content in these positions was determined from the 1H‐NMR spectra. These spectra showed also that 1 mol 2H/mol riboflavin was present in position 1′.

The deuterated riboflavin samples were incubated under aerobic conditions with broken cell preparations of Propionibacterium shermanii. The deuterium content of the 5,6‐dimethylbenzimidazole isolated was determined by mass spectrometry and by 1H NMR. These measurements revealed that the hydrogen in the pro‐S position at C1′ of riboflavin is retained during 5,6‐dimethylbenzimidazole formation, and is thus found at C2 of this base.
Inhibition of calcium‐dependent release of noradrenaline from PC12 cells by botulinum type‐A neurotoxin
Authors: SHONE Clifford C. , MELLING Jack
Published: March 2005

(a) Clostridium botulinum type‐A neurotoxin (BoNTA) inhibited the calcium‐dependent release of noradrenaline from PC12 cells in a dose‐dependent manner. Under conditions in which intact PC12 cells were incubated with BoNTA for 20 h at 37°C, a neurotoxin concentration of approximately 0.12 ± 0.03 μM was required to inhibit 50% of the calcium‐dependent noradrenaline release.

(b) PC12 cells, differentiated in the presence of nerve growth factor for 14 days, showed a similar dose‐dependent inhibition of noradrenaline release by BoNTA with unchanged sensitivity. No specific saturable binding of 125I‐labelled BoNTA was observed to either differentiated or undifferentiated PC12 cells, suggesting a lack of high‐affinity acceptors on the cell surface for the neurotoxin. It is proposed that BoNTA enters PC12 cells either by non‐specific binding to the cell membrane or via a low‐concentration low‐affinity acceptor molecule.

(c) A study of the long‐term effects of BoNTA on noradrenaline release from PC12 cells showed that the neurotoxin remains active within the growing cells for several days. Noradrenaline release from PC12 cells exposed to BoNTA (0 3 μM) for 24 h was reduced to less than 20% of control values over a subsequent 4‐day period. After 8 days, release levels were significantly lower (60–65%) than control values, despite a more than 10‐fold increase in the cell mass.

(d) Investigation of the subcellular distribution of BoNTA after incubation with PC12 cells for 96 h revealed the bulk of the toxin (94–98%) to be associated with the cell membrane fraction. Of this, 50–80% of the BoNTA was associated with the nuclear and cell debris fraction and 11–25% was recovered in the large‐granule‐vesicle fraction; the specific binding of the neurotoxin to these membrane fractions was found to be similar.

(e) Examination of the form of the cell‐associated BoNTA after incubation for 96 h with PC12 cells revealed no evidence of any significant degradation of either neurotoxin subunit. This suggests that the neurotoxin adopts a relatively stable form within the cell. On SDS/PAGE under non‐reducing conditions, no trace of protein bands corresponding to either of the BoNTA subunits were observed, suggesting that little or none of the neurotoxin subunits exists in a monomeric form within the cells. A considerable portion (50–70%) of the membrane‐associated BoNTA was found to be present in the form of large disulphide‐linked aggregates which, on SDS/PAGE in the presence of a thiol, dissociated into the bands corresponding to the neurotoxin heavy‐chain and light‐chain subunits. Examination of the neurotoxin aggregates associated with the dense‐core‐vesicle fraction showed them to contain a greater proportion of the light‐chain subunit (29.3%± 10.3) compared to the control toxin.

(f) The above observations are discussed in relation to the mode of action of BoNTA.