bluuebunny/crossref_metadata_2025 huggingface dataset:
Structural analysis of the sequence coding for an inducible 26‐kDa protein in human fibroblasts
Authors: HAEGEMAN Guy , CONTENT Jean , VOLCKAERT Guido , DERYNCK Rik , TAVERNIER Jan , FIERS Walter
Published: March 2005

When human fibroblast cells were stimulated with poly(I) · poly(C) in the presence of cycloheximide for the production of interferon‐β (IFN‐β), a 26‐kDa protein could be immunoprecipitated by antiserum raised against partially purified human IFN‐β [Content, J., De Wit, L., Pierard, D., Derynck, R., De Clercq, E. & Fiers, W. (1982) Proc. Natl Acad. Sci. USA 79, 2768–2772]. In our hands this 26‐kDa protein showed no antiviral activity. Other investigators have, however, reported the presence in the same conditions of a second type of IFN, a socalled β2 species [Weissenbach, J., Chernajovsky, Y., Zeevi, M., Shulman, L., Soreq, H., Nir, U., Wallach, D., Perricaudet, M., Tiollais, P. & Revel, M. (1980) Proc. Natl Acad. Sci. USA 77, 7152–7156] of which the mRNA structure and protein characteristics strongly suggests identity with the 26‐kDa product.

In this paper we describe the nucleotide sequence of the 26‐kDa cDNA and part of the corresponding genomic clone. The cDNA clones were isolated from a library made with mRNA from induced human fibroblasts. As, however, the information thus obtained was still incomplete, genomic clones were isolated from a total human DNA library. In this way, the entire region coding for the 26‐kDa protein was established, as well as the neighbouring sequences including the inducible promoter area. From the deduced polypeptide sequence a number of characteristics of the 26‐kDa protein can be explained. It turns out that the 26‐kDa protein gene and the socalled ‘IFN‐β2’ gene are identical. However, extensive homology searches indicate that the 26‐kDa protein does not show statistically significant sequence homology with any known interferon species. Hence, the question of whether the 26‐kDa product represents a novel IFN species remains open.
Biological activity of synthetic heptaacyl lipid A representing a component of Salmonella minnesota R595 lipid A
Authors: GALANOS Chris , LÜDERITZ Otto , FREUDENBERG Marina , BRADE Lore , SCHADE Ulrich , RIETSCHEL Ernst Th. , KUSUMOTO Shoichi , SHIBA Tetsuo
Published: March 2005

A synthetic lipid A (preparation 516), containing seven acyl groups and representing one component of natural free lipid A of Salmonella minnesota R595, has been investigated for biological activity in a number of endotoxin test systems. It was found that the synthetic preparation was, in typical in vivo endotoxin tests (lethality, pyrogenicity, Shwartzman reactivity) as well as in its antigenicity and macrophage activation capacity, significantly less active than natural Salmonella lipid A. However, in other in vitro assay systems (B‐cell mitogenicity, complement activation, Limulus amoebocyte lyzate gelation) it expressed similar activity as Salmonella lipid A.
A cell‐free system from Rhizobium meliloti to study the specific expression of nodulation genes
Authors: DUSHA Ilona , SCHRÖDER Joachim , PUTNOKY Péter , BÁNFALVI Zsófia , KONDOROSI Adam
Published: March 2005

An in vitro transcription‐translation system was developed using cell‐free extracts from the symbiotic nitrogen‐fixing bacterium Rhizobium meliloti strain 41. Conditions for preparation of the 30000xg supernatant extract and for measurement of protein‐synthesizing activity were determined and compared to the activity of an Eschericha coli cell‐free system. Genes expressed in the free‐living or in the symbiotic state were studied. The product of a recA‐like gene (41‐kDa protein) was synthesized both in R. meliloti and E. coli extracts, although less efficiently in the heterologous system. In agreement with earlier results obtained in E. coli minicells, three proteins (44, 28.5 and 23 kDa) were synthesized from a cloned 3.3 × 103‐base DNA region carrying genes for nodulation (nod). However, differences in the transcription‐translation of nod and host specificity (hsn) genes were observed when protein expression was compared in R. meliloti and E. coli cell‐free extracts, and the possible explanations of these findings are discussed.
3‐Azi‐1‐methoxybutyl D‐maltooligosaccharides specifically bind to the maltose/maltooligosaccharide‐binding protein of Escherichia coli and can be used as photoaffinity labels
Authors: THIEME Roland , LAY Helga , OSER Andreas , LEHMANN Jochen , WRISSENBERG Sabine , BOOS Winfried
Published: March 2005

Maltooligosaccharides with two to six (α1–4)‐linked glucose residues, carrying at their reducing end a 3‐azi‐1‐methoxybutyl group in either α or in β glycosidic linkage, were synthesized. These maltooligosaccharide analogues inhibit maltose uptake via the maltose‐binding‐protein‐dependent transport system in Escherichia coli. The concentration of half‐maximal inhibition of maltose transport, at 15 nM concentration, decreases with increasing chain length of the analogue, levelling off at 40 μM after a chain length of four glucose residues in the α series and at 350 μM after a chain length of three glucose residues in the β series. The inhibition of maltose transport occurs at the level of the periplasmic maltose‐binding protein. 3‐Azi‐1‐methoxybutyl α‐D‐[3H]maltotrioside was bound by the maltose‐binding protein with a Kd of 0.18 mM. Irradiation at 350 nm of purified maltose‐binding protein in the presence of 4 μM of this substrate labeled the protein covalently; labeling was prevented by 1 mM maltose. Using a crude preparation of periplasmic proteins two proteins were labeled, the maltose‐binding protein and α‐amylase.

Thus, 3‐azi‐1‐methoxybutyl α‐D‐maltooligosaccharides are potent photoaffinity labels for proteins with maltooligosaccharides‐binding sites.
Porcine submaxillary mucin contains α2→3‐ and α2→6‐linked N‐acetyl‐ and N‐glycolyl‐neuraminic acid
Authors: SAVAGE Angela V. , KOPPEN Piet L. , SCHIPHORST Wietske E. C. M. , TRIPPELVITZ Leo A. W. , VAN HALBEEK Herman , VLIEGENTHART Johannes F. G. , VAN DEN EIJNDEN Dirk H.
Published: March 2005

Four acidic trisaccharides have been obtained by alkaline borohydride reductive cleavage (β‐elimination) of a fraction of porcine submaxillary mucin precipitating at 60 – 75% ethanol. Their structures have been investigated using the techniques of methylation analysis involving gas‐liquid chromatography/mass spectrometry along with high‐resolution 1H‐NMR analysis. Two of the four oligosaccharides, B1 – B4, contain N‐acetylneuraminic acid (NeuAc) while two contain N‐glycolylneuraminic acid (NeuGc). The following structures are proposed for the acidic trisaccharide fraction: (B1) NeuAcα(2→3)Galβ(1→3)GalNAcol, (B2) NeuGcα(2→3)Galβ(1→3)GalNAcol, (B3)Galβ(1→3)[NeuAcα(2→6)]GalNAcol and (B4) Galβ(1→3)[NeuGcα(2→6)]GalNAcol, (GalNAcol = reduced N‐acetylgalactosamine.

These oligosaccharides were present in a molar ratio of 69:22:4:5. Although oligosaccharide B4 has previously been found in porcine submaxillary mucin B1, B2 and B3 have not. Furthermore, oligosaccharide B2 is a novel structure.
The B and Z forms of the d(m5C‐G)3 and d(br5C‐G)3 hexamers in solution
Authors: ORBONS Leonard P. M. , VAN DER MAREL Gijs A. , VAN BOOM Jacques H. , ALTONA Cornelis
Published: March 2005

The non‐exchangeable proton resonances of the hexadeoxynucleoside pentakisphosphates d(m5C‐G)3 and d(br5C‐G)3 in the B form as well as in the Z form were assigned by means of two‐dimensional correlated spectroscopy and two‐dimensional nuclear Overhauser enhancement spectroscopy. The complete proton NMR spectrum of the B form of the methylated compound was assigned in a pure 2H2O solution as well as in a 2H2O/C2H3O2H mixed solvent, containing 5 mM MgCl2. In the latter solvent the B form occurs in slow equilibrium (on the NMR time scale) with the Z form, the resonances of which also were fully assigned. The proton resonances of the B and Z forms of the brominated fragment were assigned in a 2H2O/C2H3O2H solution containing 5 mM MgCl2. A new and general method is described for the sequential assignment of the non‐exchangeable proton resonances of oligonucleotides in the Z form.
Conformational analysis of the B and Z forms of the d(m5C‐G)3 and d(br5C‐G)3 hexamers in solution
Authors: ORBONS Leonard P. M. , ALTONA Cornelis
Published: March 2005

The B and the Z forms of the DNA hexamers d(m5C‐G)3 and d(br5C‐G)3 were investigated by means of NMR spectroscopy. It is demonstrated that the low‐salt form of d(m5C‐G)3 is a B DNA structure. The form, which becomes increasingly predominant when increasing amounts of MgCl2 and/or methanol are added to the solution, has Z DNA characteristics. It is shown that the major geometrical features of the Z form of d(m5C‐G)3 in the crystal structure are maintained in solution, with the dC residues S sugar conformation, γ+ and the base in the anti orientation and the dG residues N (except the 3′‐terminal residue), γt and syn. Neither the Z form of the methylated nor that of the brominated compound resembles the Z′ form, in which the deoxy guanosine sugar rings adopt a C1′‐exo conformation. Substitution of m5C by br5C causes no perceptible conformational changes in either the B or in the Z forms.
Pseudomonas aeruginosa cytochrome oxidase
Authors: BLATT Yoav , PECHT Israel
Published: March 2005

The steady‐state kinetics of Pseudomonas aeruginosa cytochrome oxidase were studied. Reduced cytochrome c551 and azurin from the same bacteria were used as the electron‐donating substrates, while dioxygen served as the electron acceptor. Oxidized cytochrome c551 and azurin exhibited product inhibition of the reaction. However, apo‐azurin and azurin derivatives in which the copper was substituted by the redox‐inert ions Ni2+, Co2+, Cd2+ and Zn2+, did not show any effect on the kinetics. These observations implied that complex formation between the substrates or the products and the enzyme is not a rate‐limiting step and is not the cause for product inhibition. The integrated rate law for a reaction scheme in which we assumed that complex formation was not rate limiting was fitted to the complete reaction traces. The results suggested that it is the low thermodynamic driving force, expressed in the small differences in redox potential between the substrates and heme c of the enzyme, which cause the observed product inhibition.
ATP synthase complex from beef heart mitochondria
Authors: FREISLEBEN Hans‐Joachim , ZIMMER Guido
Published: March 2005

ATPase/ATP synthase preparations originally contain protein bands in the 28–31‐kDa region. The present study demonstrates separation of the band at 29 kDa (adenine nucleotide translocator) from a band at approximately 31 kDa. In cholate/ammonium sulfate‐extracted ATP synthases removal of the 31‐kDa band results in decrease of ATP–Pi exchange and oligomycin sensitivity of the ATPase activity.

It is suggested that the protein band at 31 kDa is heterogeneous, containing diverse activities, the identities of which are yet to be determined.
Simultaneous synthesis and hydrolysis of ATP regulated by the inhibitor protein in submitochondrial particles
Authors: BELTRÁN Carmen , TUENA DE GÓMEZ‐PUYOU Marietta , DARSZON Alberto , GÓMEZ‐PUYOU Armando
Published: March 2005

Coupled submitochondrial particles from bovine heart with ATP synthases devoid of control by the inhibitor protein of Pullman and Monroy [J. Biol. Chem. 238, 3762–3769 (1963)] can be prepared by incubation of Mg‐ATP particles in 50 mM phosphate, 250 mM sucrose, and > 95% D2O (pD 7.8) at 38°C. As monitored with oxonol, the respiring particles build up and maintain a ΔΦ about 5–10% lower than that of the starting preparation. With oligomycin ΔΦ of the two preparations is the same. In the presence of an ATP trap (hexokinase and glucose), the two types of particles carry out oxidative phosphorylation at comparable rates. Low concentrations of oligomycin induce a small enhancement of the rate of ATP synthesis in non‐controlled particles. In the absence of an ATP trap, net accumulation of ATP, as driven by electron transport in particles without control by the inhibitor protein, is low. Apparently this is due to lack of control by the inhibitor protein of ATP hydrolysis that occurs during oxidative phosphorylation.
The Na+/H+ exchange system in glial cell lines
Authors: JEAN Thierry , FRELIN Christian , VIGNE Paul , LAZDUNSKI Michel
Published: March 2005

1. The properties of the Na+/H+ exchange system in the glial cell lines C6 and NN were studied from 22Na+ uptake experiments and measurements of the internal pH (pHi) using intracellularly trapped biscarboxyethylcarboxyfluorescein. In both cell types, the Na+/H+ exchanger is the major mechanism by which cells recover their pHi after an intracellular acidification. The exchanger is inhibited by amiloride and its derivatives. The pharmacological profile (ethylisopropylamiloride > amiloride > benzamil) is identical for the two cell lines. Both Na+ and Li+ can be exchanged for H+. Increasing the external pH increases the activity of the exchanger in the two cell lines. In NN cells the external pH dependence of the exchanger is independent of the pHi. In contrast, in C6 cells, changing the pHi value from 7.0 to 6.5 produces a pH shift of 0.6 pH units in the external pH dependence of the exchanger in the acidic range. Decreasing pHi activates the Na+/H+ exchanger in both cell lines.

2. Increasing the osmolarity of the external medium with mannitol produces an activation of the exchanger in C6 cells, which leads to a cell alkalinization. Mannitol action on 22Na+ uptake and the pHi were not observed in the presence of amiloride derivatives. Mannitol produces a modification of the properties of interaction of the antiport with both internal and external H+. It shifts the pHi dependence of the system to the alkaline range and the external pH (pHo) dependence to the acidic range. It also suppresses the interdependence of pHi and pHo controls of the exchanger's activity. NN cells that possess an Na+/H+ exchange system with different properties do not respond to mannitol by an increased activity of the Na+/H+ exchanger. The action of mannitol on C6 cells is unlikely to be mediated by an activation of protein kinase C.
Association of messenger RNA with the cytoskeletal framework in rat L6 myogenic cells
Authors: PRAMANIK Saroj K. , WALSH Richard W. , BAG Jnanankur
Published: March 2005

The distribution of mRNA between the detergent‐soluble and insoluble (cytoskeleton) fractions in rat L6 myoblast and myotube cells was examined. Approximately 85% of cytoplasmic mRNA in both myoblasts and myotubes was found associated with the cytoskeletal framework. The cytoskeleton‐bound mRNA was present as polysomes. In contrast, the mRNA of the detergent‐soluble fraction was not associated with ribosomes and was thus considered to be the repressed population. The association of mRNA with the cytoskeletal framework was not affected by treatments leading to dissociation of polysomes. Differential distribution of mRNA between the soluble and cytoskeleton‐bound fractions was analyzed by in vitro translation. The mRNAs coding for polypeptides of molecular masses 40 kDa and 60 kDa were preferentially enriched in the soluble fraction.

The nature of binding between mRNA and the cytoskeletal framework was examined following in vivo cross‐linking of RNA and protein by irradiating muscle cells with ultraviolet light. It was observed that this treatment covalently linked RNA and the neighbouring protein moieties without any detectable damage to the cytoskeletal framework, as measured by the distribution of RNAs and proteins between the cytoskeleton and soluble fractions. Analysis of the polypeptide moieties cross‐linked to the mRNA have shown that a large number of polypeptides of molecular masses between 15–220 kDa were associated with both cytoskeleton‐bound and soluble mRNAs. The polypeptide moieties of these mRNA‐protein complexes were not only similar in the cytoskeleton and soluble mRNA‐protein complexes but also were similar between myoblasts and myotubes. However, one polypeptide of 165 kDa was preferentially associated with the cytoskeleton‐bound mRNA‐protein complexes. Interestingly this 165‐kDa polypeptide was also preferentially enriched in the total proteins from the cytoskeleton fraction. This result suggests a possible role of the 165‐kDa polypeptide in association between mRNA and the cytoskeletal framework. To examine the mechanism of interaction between mRNA and the cytoskeletal framework we have reported here a ghost monolayer transcription system from myotubes. This transcription system was able to synthesize rRNA and mRNA. The mRNA transcribed in vitro was preferentially associated with the cytoskeleton structure present in the ghost monolayer system.
Nucleotide sequence of the pbpA gene and characteristics of the deduced amino acid sequence of penicillin‐binding protein 2 of Escherichia coli K12
Authors: ASOH Sadamitsu , MATSUZAWA Hiroshi , ISHINO Fumitoshi , STROMINGER Jack L. , MATSUHASHI Michio , OHTA Takahisa
Published: March 2005

We have determined the nucleotide sequence of the pbpA gene encoding penicillin‐binding protein (PBP) 2 of Escherichia coli. The coding region for PBP 2 was 1899 base pairs in length and was preceded by a possible promoter sequence and two open reading frames. The primary structure of PBP 2, deduced from the nucleotide sequence, comprised 633 amino acid residues. The relative molecular mass was calculated to be 70867. The deduced sequence agreed with the NH2‐terminal sequence of PBP 2 purified from membranes, suggesting that PBP 2 has no signal peptide. The hydropathy profile suggested that the NH2‐terminal hydrophobic region (a stretch of 25 non‐ionic amino acids) may anchor PBP 2 in the cytoplasmic membrane as an ectoprotein. There were nine homologous segments in the amino acid sequence of PBP 2 when compared with PBP 3 of E. coli. The active‐site serine residue of PBP 2 was predicted to be Ser‐330. Around this putative active‐site serine residue was found the conserved sequence of Ser‐Xaa‐Xaa‐Lys, which has been identified in all of the other E. coli PBPs so far studied (PBPs 1A, 1B, 3, 5 and 6) and class A and class C β‐lactamases. In the higher‐molecular‐mass PBPs 1A, 1B, 2 and 3, Ser‐Xaa‐Xaa‐Lys‐Pro was conserved. In the putative peptidoglycan transpeptidase domain there were six amino acid residues, which are common only in the PBPs of higher molecular mass.
Immunofractionation of chromatin regions associated with histone H1°
Authors: MENDELSON Ella , LANDSMAN David , DRUCKMANN Shulamit , BUSTIN Michael
Published: March 2005

Two monoclonal antibodies, which were elicited against histone H5, bind to purified rat liver chromatin and to rat liver H1° but not to rat liver H1. The monoclonal antibodies were immobilized on CNBr‐Sepharose and the resulting immunoaffinity column was used to fractionate rat liver oligonucleosomes. Enzyme‐linked immunoabsorbant assay (ELISA) and immunoblotting experiments indicate that the nucleosomes bound to the column were tenfold enriched in their content of H1°. Oligonucleosomes, prepared from the livers of either untreated or 3‐methylcholanthrene‐treated adult rats, were fractionated on the anti‐H1° affinity column. The DNA purified from the unfractionated nucleosomes, from the unbound nucleosomes and from the nucleosomes which were bound to the column was examined with various 32P‐labeled probes. A slight enrichment in H1° was detected in the coding region of the rat albumin gene. In contrast DNA which was bound to the column was significantly depleted in sequences hybridizing with total cellular RNA (which contains mostly ribosomal RNA) and with sequences hybridizing to the 3′‐terminal region of a cytochrome P‐450 gene, which is inducible by the chemical carcinogen 3‐methylcholanthrene, regardless of whether isolated from control or from carcinogentreated rat livers. Our experiments clearly demonstrate that chromatin can be efficiently immunofractionated. The results suggest that the H1° content of chromatin regions containing genes which are constitutively transcribed is not necessarily different from that of regions containing non‐transcribed genes and that highly inducible genes may be segregated into chromatin regions which are depleted of H1°.
Relevance of the lariat model to the splicing of the H‐2 gene family
Authors: BOUGUELERET Lydie , CLAVERIE Jean‐Michel
Published: March 2005

We examined the splicing of the H‐2 gene family, taking the H‐2Kd as a prototype, in the framework of the lariat model. We mainly investigated the mechanism described by Konarska et al. [Nature (Lond.) 313, 552–557 (1985)] who propose a direct interaction between the 5′ splicing site and the branching region. We also checked each of the H‐2 introns for the presence of patterns resembling the published consensus for the branching region. The known splicing events in the H‐2 gene family are not always consistent with the current models, and our results indicate that slightly different mechanisms govern the splicing of different introns. A tentative explanation of the alternative splicing of the first and last intron, previously described, is given. The removal of the large third intron is not easily rationalized unless new rules for an additional multistep processing are postulated.
Pyrophosphate‐caused inhibition of the aminoacylation of tRNA by the leucyl‐tRNA synthetase from Neurospora crassa
Authors: AIRAS R. Kalervo , CRAMER Friedrich
Published: March 2005

Inorganic pyrophosphate inhibits the aminoacylation of tRNALeu by the leucyl‐tRNA synthetase from Neurospora crassa giving very low Ki, PPiapp. values of 3–20 μM. The inhibition by pyrophosphate, together with earlier kinetic data, suggest a reaction mechanism where leucine, ATP and tRNA are bound to the enzyme in almost random order, and pyrophosphate is dissociated before the rate‐limiting step. A kinetic analysis of this mechanism shows that the measured Kiapp. values do not give the real dissociation constant but it is about 0.4 mM. Other dissociation constants are 90 μM for leucine, 2.2 mM for ATP and 1 μM for tRNALeu. At the approximate conditions of the living cell (2 mM ATP, 100 μM leucine and 150 μM PPi) the leucyl‐tRNA synthetase is about 85% inhibited by pyrophosphate.
Control of ribosome biosynthesis in plant cell cultures under heat‐shock conditions
Authors: NOVER Lutz , MUNSCHE Dieter , NEUMANN Dieter , OHME Karin , SCHARF Klaus‐Dieter
Published: March 2005

The immediate block of ribosome biosynthesis in heat‐shocked tomato cell cultures is primarily caused by the complete inhibition of pre‐rRNP processing. Depending on the heat‐shock conditions synthesis of pre‐rRNP goes on, though at a reduced level. Synthesis and/or preservation of pre‐rRNP during heat shock as well as its efficient processing in the recovery period are thoroughly improved by preconditioning of cells to the hyperthermic treatment. Such preinduced cultures are characterized by their content of preformed heat‐shock proteins, whose dominant representative (hsp 70) becomes highly enriched in the characteristic granular rRNP material observed in nucleoli of heat‐shocked cells. This is shown by immune fluorescence staining and microautoradiography.
Nucleotide sequence of a cDNA clone encoding Scylliorhinus caniculus protamine Z2
Authors: BERLOT‐PICARD Frederique , VODJDANI Guilane , DOLY Janine
Published: March 2005

A cDNA library was constructed from a protamine‐enriched fraction of dogfish (Scylliorhinus caniculus) mRNA. The nucleotide sequence of a 440‐bp insert was determined, and its produced protein sequence confirmed its identification as a cysteine‐rich protamine Z2 [Martinage, A., Gusse, M., Belaiche, D., Sautiere, P. and Chevaillier, P. (1985) Biochim. Biophys. Acta 831, 172–178]. The frequency of utilization of the different triplets coding for arginine, which represents 30–70% of the total amino acid residues for trout, mouse and dogfish protamines, is discussed. An alternative repetitive sequence of CGC‐AGG was found in the N terminus of the protein. Analysis of the 3′ flanking region after the mRNA‐terminating TAA codon identified an inverted repeat sequence and an ACCA sequence, which may be possible vestiges of a histone‐like termination signal.
Purification, characterization and origin of rat gastric peroxidase
Authors: DE Swapan K. , BANERJEE Ranajit K.
Published: March 2005

A membrane‐bound peroxidase (EC 1.11.1.7) from rat stomach has been solubilized by 0.2% cetyl‐trimethylammonium bromide in the presence of 1.2 M NH4Cl. The enzyme was purified 3355‐fold to apparent homogeneity as judged by acid polyacrylamide gel electrophoresis and appears to be a cationic protein. In sodium dodecyl sulfate gel electrophoresis, the enzyme shows single polypeptide band of Mr 45000. In gel permeation, the Mr has been estimated as 47000. Spectral properties indicate the presence of Soret band at 412 nm which shifts to 425 nm on complexation with CN− and to 430 nm on reduction with dithionite. The velocity constant, k1 for the reaction of the proxidase with H2O2 is 1.38 × 107 M−1 s−1 and Km for H2O2 is 0.1 mM. The enzyme contains active sulphydryl groups and is inhibited by sulphydryl reagents of which p‐hydroxymercuribenzoate is more reactive than mersalyl or N‐ethylmaleimide. The enzyme is very resistant to thermal denaturation up to 65°C and also to chaotropic reagents at least up to 2 M above which it is inactivated. The enzyme shows similarity with the intestinal eosinophil peroxidase as regards the molecular mass, spectral, kinetic and some of the catalytic properties. However, they differ significantly in terms of their interaction with fluoride ion, sulphydryl reagents, chaotropic reagent and also with the antiserum against the gastric peroxidase. Histochemically, the gastric peroxidase is shown to be localised in the gastric gland proper of the fundic stomach, rich in parietal and chief cells.
Catalysis by hog‐kidney aminoacylase does not involve a covalent intermediate
Authors: RÖHM Klaus H. , VAN ETTEN Robert L.
Published: March 2005

Hog kidney aminoacylase (N‐acylamino acid amidohydrolase; acylase I) is shown to catalyze the exchange of acetate oxygens with water at a significant rate only when alanine is present simultaneously. These studies, conducted using the 18O‐isotope induced shift on 13C‐NMR spectra, provide evidence in favor of a linear kinetic mechanism as opposed to a ‘ping‐pong’ double‐displacement mechanism. At pH values above neutrality, aminoacylase I also catalyzes the exchange of alanine oxygens with those of water. Ionic strength and pH effects on the kinetics of aminoacylase I are examined and the results are interpreted in terms of a model of the enzyme active site.
The nifH gene product is required for the synthesis or stability of the iron‐molybdenum cofactor of nitrogenase from Klebsiella pneumoniae
Authors: FILLER Wendy A. , KEMP Richard M. , NG Jasmine C. , HAWKES Timothy R. , DIXON Ray A. , SMITH Barry E.
Published: March 2005

The MoFe protein of nitrogenase from Klebsiella pneumoniae contains an iron‐molybdenum cofactor, FeMoco, the synthesis or processing of which involves the products of at least five genes, nifQ, nifB, nifN, nifE and nifV. We have detected FeMoco activity in extracts of strains which synthesise neither of the MoFe protein subunits, indicating that FeMoco can be synthesised prior to combination with the MoFe protein polypeptides. Expression of the nifH gene (or a large part of it), was essential for FeMoco activity to be observed either in the presence or in the absence of the MoFe protein subunits. The nifH gene product was not involved in the control of the transcription of other nif gene products known to be involved in FeMoco synthesis or processing, nor was it essential for the stability of performed FeMoco before its combination with the MoFe protein polypeptides.
Nucleotide sequence and characteristics of the gene for l‐lactate dehydrogenase of Thermus caldophilus GK24 and the deduced amino‐acid sequence of the enzyme
Authors: KUNAI Kenji , MACHIDA Masayuki , MATSUZAWA Hiroshi , OHTA Takahisa
Published: March 2005

The gene for L‐lactate dehydrogenase (LDH) (EC 1.1.1.27) of Thermus caldophilus GK24 was cloned in Escherichia coli using synthetic oligonucleotides as hybridization probes. The nucleotide sequence of the cloned DNA was determined. The primary structure of the LDH was deduced from the nucleotide sequence. The deduced amino acid sequence agreed with the NH2‐terminal and COOH‐terminal sequences previously reported and the determined amino acid sequences of the peptides obtained from trypsin‐digested T. caldophilus LDH. The LDH comprised 310 amino acid residues and its molecular mass was determined to be 32808. On alignment of the whole amino acid sequences, the T. caldophilus LDH showed about 40% identity with the Bacillus stearothermophilus, Lactobacillus casei and dogfish muscle LDHs. The T. caldophilus LDH gene was expressed with the E. coli lac promoter in E. coli, which resulted in the production of the thermophilic LDH. The gene for the T. caldophilus LDH showed more than 40% identity with those for the human and mouse muscle LDHs on alignment of the whole nucleotide sequences.

The G + C content of the coding region for the T. caldophilus LDH was 74.1%, which was higher than that of the chromosomal DNA (67.2%). The G + C contents in the first, second and third positions of the codons used were 77.7%, 48.1% and 95.5% respectively. The high G + C content in the third base caused extremely non‐random codon usage in the LDH gene. About half (48.7%) the codons in the LDH gene started with G, and hence there were relatively high contents of Val, Ala, Glu and Gly in the LDH. The contents of Pro, Arg, Ala and Gly, which have high G + C contents in their codons, were also high. Rare codons with U or A as the third base were sometimes used to avoid the TCGA sequence, the recognition site for the restriction endonuclease, TaqI. Two TCGA sequences were found only in the sequence of CTCGAG (XhoI site) in the sequenced region of the T. caldophilus DNA. There were three segments with similar sequences in the two 5′ non‐coding regions, probably the promoter and ribosome‐binding regions, of the genes for the T. caldophilus LDH and the Thermus thermophilus 3‐isopropylmalate dehydrogenase.
Substitution of uridine in vivo by the intrinsic photoactivable probe 4‐thiouridine in Escherichia coli RNA
Authors: FAVRE Alain , BEZERRA Roberto , HAJNSDORF Eliane , DUBREUIL Yolande LEMAIGRE , EXPERT‐BEZANÇON Alain
Published: March 2005

In vivo incorporation of the uridine‐photoactivable analogue, 4‐thiouridine, into the ribosomal RNA of an Escherichia coli pyrD strain has been demonstrated. It is highly dependent on the exogeneous uridine and 4‐thiouridine concentrations as well as on temperature. We have defined conditions allowing the substitution of 13 ± 2% of the uridine residues in bulk RNA by 4‐thiouridine. On a high‐Mg2+ sucrose gradient, 33 ± 3% of ribonucleic particles sediment as 70S ribosomes, the remaining being in the form of non‐associated 50S and 30S particles containing immature rRNA. The thiolated 70S ribosomes tolerate a 4–5% substitution level (40 thiouridine molecules/particle).

Surprisingly, 3–4% of ribosomal proteins, about two protein molecules/particle, were spontaneously covalently bound to 4‐thiouridine‐substituted rRNA. Specific 366‐nm photoactivation increased this proportion to 10–12%, i.e. up to six or seven ribosomal protein molecules/particle. The photochemical cross‐linking proceeds with aparent first‐order kinetics with a quantum yield close to 5 × 10–3. Although extensive photodynamic breakage of rRNA occurs under aerobic conditions, both the kinetics and yield of ribosomal protein cross‐linking were independent of oxygenation conditions. The thiolated (4.5%) 70S ribosomes allowed the poly(U)‐directed poly(Phe)synthesis at 48% the control rate. Photoactivation decreased this activity to 28% and 10% when performed under nitrogen and in aerated conditions, respectively.
Regions necessary for pH‐dependent assembly of gizzard myosin rod
Authors: KUMON Akira , KUBA Masako , MURAKAMI Noriko , YASUDA Seiji , TAKASHIMA Toshinobu , MATSUMURA Sueo , SUEZAKI Yukio
Published: March 2005

Aggregated and disaggregated forms of gizzard myosin rod and its fragments in various concentrations of NaCl (0–0.30 M) at various pH (7.4–8.6) were distinguished from each other by their permeability through a Sepharose 4B column.

The rod existed in three forms, namely: large aggregates impermeable to the column, small aggregates eluted at the void volume of the column and a disaggregated monomer which penetrated the column. The relative proportions of the three forms varied depending on the salt concentration and pH. The monomeric rod was detected in NaCl solutions above 0.20 M and its relative proportion at 0.25 M NaCl was larger than those of the small and large aggregates. The small aggregates of the rod were predominant at below 0.05 M NaCl and, upon decrease in pH from 8.6 to 7.4, these small aggregates in NaCl solutions between 0.10 M and 0.15 M were replaced by the large aggregates.

Light meromyosin, which corresponded to the C‐terminal two‐thirds of the rod, existed exclusively as large aggregates in NaCl solutions below 0.15 M; increase of NaCl concentration to above 0.20 M resulted in the formation of its monomer, instead of the large aggregates. In contrast to the rod, no small aggregated form of the light meromyosin was detected.

Truncated light meromyosin which had lost a small segment from either the C‐terminal or N‐terminal of light meromyosin was eluted only as a monomer in any NaCl concentration at any pH.

It may be deduced from the above results that a small segment in the light meromyosin is requisite for the assembly of both rod and light meromyosin in the NaCl solutions below 0.15 M and that the relative proportion of small and large aggregates of the rod is determined in a pH‐dependent manner by the subfragment 2 segment, the N‐terminal third of the rod.
Isoforms of myosin and actin in human, monkey and rat myometrium
Authors: CAVAILLÉ Françoise , JANMOT Chantal , ROPERT Sylvie , d'ALBIS Anne
Published: March 2005

Using several electrophoretic procedures, we have compared the forms of myosin and actin in pregnant and non‐pregnant uterus of woman, monkey (Macaca fascicularis) and rat. On non‐dissociating gels, native myosin of the three species migrates as a single band, of identical mobility independently of the physiological state. Remigration of this band in dissociating conditions shows that it is constituted of two heavy chains of respectively 201 kDa and 205 kDa; the relative proportions of these two bands are different for the three animal species but do not vary during pregnancy. Using two‐dimensional gel electrophoresis, we found that the 17‐kDa light chain of purified uterus myosin exists under two isoelectric forms, the more acidic one becoming progressively predominant at the end of pregnancy in the human as in the monkey uterus, while we observed no changes in the rat. In two‐dimensional gel electrophoresis, actin of human, monkey and rat uterus is present under three isoforms, the most basic one (the γ form) increasing early in pregnancy in the two primate species but being always the most abundant form in the rat. The ATPase activity of human uterus myosin was found to be similar for the protein extracted from both pregnant and non‐pregnant uterus. The changes observed in the 17‐kDa light chain and in the actin isoforms might nevertheless participate in the modifications of contractility of the uterus during pregnancy of the primates.
On the oxidation pathways of the mitochondrial bc1 complex from beef heart
Authors: ESPOSTI Mauro DEGLI , TSAI Ah‐Lim , PALMER Graham , LENAZ Giorgio
Published: March 2005

We have investigated the oxidation of the reduced ubiquinol: cytochrome c (bc1 complex) isolated from beef heart mitochondria. The oxidation of cytochrome c1 by both potassium ferricyanide and cytochrome c in the ascorbate‐reduced bc1 complex is not a first‐order process. This is taken as evidence that cytochrome c1 is in rapid equilibrium with the Rieske iron‐sulphur center. Among the several inhibitors tested, only 5‐n‐undecyl‐6‐hydroxy‐4,7‐dioxobenzothiazole and stigmatellin are seen to affect this redox equilibrium between the high‐potential centers of the beef heart bc1 complex.

The oxidation of cytochrome b by cytochrome c in both the succinate‐reduced and the fully reduced bc1 complex is blocked by all the inhibitors tested. This inhibition occurs simultaneoulsy with an acceleration in the oxidation of cytochrome c1, even after extraction of the endogenous ubiquinone which is present in the bc1 preparation. Almost complete extraction of ubiquinone from the bc1 complex has no effect upon the rapid phase of cytochrome b oxidation, nor does it alter the inhibition of cytochrome b oxidation by the various inhibitors.

The oxidation of cytochrome b by exogenous ubiquinones is stimualted by myxothiazol and partially inhibited by antimycin. However, the addition of both these inhibitors together completely blocks the oxidation of cytochrome b by quinones. In contrast, the simultaneous addition of antimycin and myxothiazol has no such synergistic effect upon the oxidation of cytochrome b by cytochrome c.

Our data show that intramolecular electron transfer from cytochrome(s) b to the Rieske iron‐sulphur center can take place in the bc1 complex without involvement of endogenous ubiquinone‐10. This electron pathway is sensitive to all the inhibitors of the enzyme.
Energy dependence of protein translocation into chloroplasts
Authors: FLÜGGE U. Ingo , HINZ Giselbert
Published: March 2005

The translocation of in vitro synthesized precursor proteins into intact spinach chloroplasts was investigated with respect to its energy requirement. It was demonstrated that MgATP itself, and not a transmembrane electrochemical gradient across the envelope membrane, promotes protein import. By manipulating the external and the stromal level of MgATP, we provided evidence that MgATP energized the protein import not within the chloroplast but at the outside of the envelope membrane. It is postulated that an MgATP‐dependent phosphorylation/dephosphorylation cycle at the outer membrane face was involved in the course of protein translocation into the chloroplast.
Structure and regulation of the sheep metallothionein‐Ia gene
Authors: PETERSON M. Gregory , MERCER Julian F. B.
Published: March 2005

Screening of a sheep genomic λ library with a sheep metallothionein‐I cDNA clone resulted in the isolation of a 13,200‐base‐pair fragment containing a metallothionein gene which DNA sequence analysis identified as the gene encoding the cloned cDNA. The two introns occur at identical positions to those in other mammalian metallothioneins but are considerably larger. The first intron contains a DNA element that is present in a related but not identical form in many places in the sheep genome. Comparison of the promoter sequences of this gene (sMT‐Ia) with the promoters of metallothionein genes from other species identified a number of conserved regions which may be important in the regulation of this gene by heavy metals, glucocorticoids and α‐interferon. In sheep fibroblasts, the levels of sMT‐Ia mRNA was found to be maximally elevated (95‐fold) in the presence of zinc or cadmium and elevated 30‐fold in the presence of copper. Dexamethasone had no effect upon mRNA levels. Thus this gene shows a pattern of regulation similar to the human MT‐If gene, but distinct from the other human and mouse metallothionein genes so far reported.
High‐affinity binding sites for human Glu‐plasminogen unveiled by limited plasmic degradation of human fibrin
Authors: TRAN‐THANG Chiê , KRUITHOF Egbert K. O. , ATKINSON Jeffrey , BACHMANN Fedor
Published: March 2005

The binding of human 125I‐Glu‐plasminogen to human plasmin‐degraded fibrin was studied. Treatment of preformed and polymerized fibrin with 0.01 IU plasmin/ml resulted in an increased binding of 125I‐Glu‐plasminogen depending upon the length of time of preincubation of fibrin with plasmin. Binding reached a plateau of 30% of total added radioactivity after 60 min. At this time, less than 10% of fibrin had been digested. Polyacrylamide/urea/acetic acid gel electrophoresis revealed that the radioiodinated plasminogen bound to plasmin‐degraded fibrin was of the Glu form. Computerized non‐linear regression analysis of the binding experiments revealed that limited plasmic degradation of fibrin progressively generates high‐affinity binding sites (Kd∼ 0.3 μM) for Glu‐plasminogen. At the time of maximal Glu‐plasminogen binding approximately 5 high‐affinity binding sites per 100 molecules of fibrin had been generated. The low‐affinity type of binding sites were also identified. These observations describe a new mechanism which exquisitely modulates the plasmic breakdown of fibrin by a continuous renewal of high‐affinity binding sites for Glu‐plasminogen on the surface of the fibrin gel during the fibrinolytic process.
The proton channel, CF0, in thylakoid membranes
Authors: LILL Holger , ENGELBRECHT Siegfried , SCHÖNKNECHT Gerald , JUNGE Wolfgang
Published: March 2005

We investigated the conductance of pea thylakoid membranes and their capacity for photophosphorylation as function of the extraction of chloroplast coupling factor CF1. The degree of extraction was varied via the incubation time in EDTA‐containing hypo‐osmolar medium and was measured by rocket electroimmunodiffusion. The conductance of thylakoid membranes was measured by flash kinetic spectrophotometry.

The time course of extraction followed the time course of thylakoid swelling. Contrary to expectation increasing loss of CF1 did not primarily increase the velocity of proton efflux from each vesicle. Instead proton‐tight vesicles were converted to leaky ones, which lost phosphorylating activity. Two subpopulations occurred, although both types of vesicles, leaky and proton‐tight ones, were CF1‐depleted to a similar degree. This implied that only a small fraction of CF1‐lacking CF0 was functional as a proton channel. Tight vesicles had no functional channels while leaky ones had at least one. We determined the proportion of tight vesicles in three independent ways: via the residual phosphorylation activity, via measurements of proton efflux and via measurements of the electric relaxation across the membrane. The results obtained were identical. A statistical evaluation of the data led us to the following conclusions. (a) EDTA treatment produced vesicles containing approximately 105 chlorophyll molecules, equivalent to a total of approximately 100 CF0CF1 per vesicle. (b) Even at the highest degree of extraction (75% of total CF1 extracted) only 2.5 out of 75 exposed CF0 per vesicle were proton‐conducting. (c) The unit conductance of one open CF0 channel was 169 ± 18 fS at pH 7.5 and room temperature. At an electrical driving force of 100 mV this was equivalent to the passage of approximately 105 protons/s.

The most important consequence of this relatively high unit conductance was that a single open CF0 channel was capable of dissipating the protonmotive force of one vesicle, thereby deactivating the whole remaining catalytic capacity of this vesicle.
Reconstitution of CF1‐depleted thylakoid membranes with complete and fragmented chloroplast ATPase
Authors: ENGELBRECHT Siegfried , LILL Holger , JUNGE Wolfgang
Published: March 2005

Chloroplast ATPase (CF1) was isolated from spinach, pea and maize thylakoids by EDTA extraction followed by anion‐exchange chromatography. CF1 was purified and resolved by HPLC into integral CF1, and CF1 lacking the δ & ɛ subunits: CF1(–δ) and CF1(–ɛ). Washing Mono‐Q‐bound CF1 with alcohol‐containing buffers followed by elution without alcohol produced the β subunit and in separate peaks CF1(–δ) and CF1(–ɛ). Elution from Mono Q in the presence of tenside yielded a βδ fragment, CF1(–δ) and CF1(–δɛ).

Chloroplasts were CF1‐depleted by EDTA extraction. Reconstitution of photophosphorylation in these ‘EDTA vesicles’ was obtained by addition of CF1 and its fragments. CF1, CF1(–δ) and CF1(–δɛ) were active with cross‐reactivity between spinach, pea and maize. δ‐containing CF1 always reconstituted higher activities than δ‐deficient CF1. The βδ fragment and dicyclohexylcarbodiimide (DCCD)‐inhibited CF1 also were reconstitutively active while β and DCCD‐inhibited CF1(–δ) were not.

These results support the notion that subunit δ can function as a stopcock to the CF0 proton channel as proposed by Junge, W., Hong, Y. Q., Qian, L. P. and Viale, A. [(1984) Proc. Natl Acad. Sci. USA 81, 3078–3082].
Protein synthesis in yeast Saccharomyces cerevisiae
Authors: NASRIN Nargis , AHMAD Mir Firoz , NAG Mihir K. , TARBURTON Philip , GUPTA Naba K.
Published: March 2005

Antibodies prepared against a homogeneous preparation of Co‐eIF‐2A20 [Ahmad et al. (1985) J. Biol. Chem. 260, 6955–6959] reacted with several polypeptides including an 80‐kDa polypeptide present in a crude yeast ribosomal salt wash. This 80‐kDa polypeptide, containing Co‐eIF‐2A (Co‐eIF‐2A80) activity, has been extensively purified using a two‐step purification procedure involving an immunoaffinity column chromatograph prepared using antibodies against Co‐eIF‐2A20 (fraction II) and hydroxyapatite chromatography (fraction III). The factors, eIF‐2 + homogeneous Co‐eIF‐2A80 (fraction III) promoted Met‐tRNAf· 40S complex formation with an AUG codon but not with a physiological mRNA or a polyribonucleotide messenger poly(U,G) whereas eIF‐2 + a partially purified Co‐eIF‐2A80 preparation (fraction II) promoted Met‐tRNAf· 40S complex formation with an AUG codon as well as with globin mRNA and poly(U,G) messenger. This factor‐promoted Met‐tRNAf binding to 40S ribosomes depends absolutely on the presence of a polyribonucleotide messenger containing an initiation codon (such as AUG or GUG). Other polyribonucleotide messengers tested, such as poly(U), poly(A) and poly(A,C), were completely ineffective in this binding reaction. This result indicates that the Met‐tRNAf· 40S · mRNA complex is formed by a direct interaction between Met‐tRNAf, 40S ribosomes and the initiation site in mRNA. A mechanism has been proposed for Met‐tRNAf· 40S · mRNA complex formation in yeast.
Effect of deletions within the leader peptide of pre‐ornithine transcarbamylase on mitochondrial import
Authors: LINGELBACH Klaus R. , GRAF Lynda J. , DUNN Ashley R. , HOOGENRAAD Nicholas J.
Published: March 2005

The uptake of the cytoplasmically synthesized mammalian enzyme, ornithine transcarbamylase, into mitochondria is directed by an N‐terminal peptide of 32 amino acids. We have investigated some of the structural requirements for the import of the enzyme from rat liver into isolated mitochondria and into mitochondria of COS cells transfected with cDNA encoding the precursor form of ornithine transcarbamylase. Deletion of 21 amino acids from the N terminus of the leader peptide blocked the import of the precursor; deletion of 5 amino acids at positions 15–19 from the N terminus of the leader peptide had no deleterious effect on the import of the enzyme, nor on the processing and assembly of subunits in mitochondria. The region deleted contained three of eight basic residues in the leader peptide suggesting that specific structural elements containing basic residues, rather than the general basic nature of the leader, may be involved in mitochondrial import.
A 1H‐NMR study of human interleukin‐1β
Authors: GRONENBORN Angela M. , CLORE G. Marius , SCHMEISSNER Ursula , WINGFIELD Paul
Published: March 2005

Complete identification of spin systems in the aromatic region of recombinant human interleukin‐1β has been achieved using two‐dimensional homonuclear Hartmann‐Hahn spectroscopy. In addition, sequence‐specific assignments for the four tyrosine residues have been carried out with the help of a series of mutant proteins, obtained by site‐directed mutagenesis of the cloned gene. It is shown that, for the mutant proteins investigated, either none or only local structural changes occur. The use of NMR spectroscopy to determine the structural identity of site‐directed mutant proteins with respect to the wild‐type protein is discussed.