bluuebunny/crossref_metadata_2025 huggingface dataset:
Phosphorylation of a 40S ribosomal subunit protein in Tetrahymena
Authors: SRIPATI Conjeevaram E. , CUNY Marguerite
Published: March 2005

In Tetrahymena the small ribosomal subunit protein S7, which appears to be the equivalent of S6 of higher eukaryotes, undergoes reversible phosphorylation under a set of defined conditions. In an attempt to understand the physiological role of such reversible phosphorylation, we examined the status of ribosomal protein S7 in growing cells and growth‐arrested cells, starving either non‐specifically for nutrients or specifically for a single essential amino acid. These experiments allowed us to dissociate S7 phosphorylation from changes in the translational activity and the stability of ribosomes. The results revealed complete lack of correlation between phosphorylation of S7 and both the growth status of the cells and the in vivo stability of ribosomes. Taken together with the observation that phosphorylation of S7 occurs only when the cells are starved in buffers containing sodium chloride or high concentrations of Tris, non‐essential ions for normal growth, our data suggest that this protein modification is required to maintain the functional integrity of the ribosomes in an altered electrostatic environment, induced by changes in the extracellular ionic conditions.
Urea synthesis from ammonia in periportal and pericentral regions of the liver lobule
Authors: KARI Frank W. , YOSHIHARA Harumasa , THURMAN Ronald G.
Published: March 2005

Rates of urea synthesis were determined in periportal and pericentral regions of the liver lobule in perfused liver from fed, phenobarbital‐treated rats by measuring the extra O2 consumed upon infusion of NH4Cl with miniature O2 electrodes and from decreases in NADPH fluorescence detected with micro‐light‐guides.

Urea synthesis by the perfused rat liver supplemented with lactate (5 mM), ornithine (2 mM) and methionine sulfoximine (0.15 mM), an inhibitor of glutamine synthetase, was stimulated by stepwise infusion of NH4Cl at doses ranging from 0.24 mM to 3.0 mM. A good correlation (r= 0.92) between decreases in NADPH fluorescence and urea production was observed when the NH4Cl concentration was increased.

Sublobular rates of O2 uptake were determined by placing miniature oxygen electrodes on periportal or pericentral regions of the lobule on the liver surface, stopping the flow and measuring decreases in oxygen tension. From such measurements local rates of O2 uptake were calculated in the presence and absence of NH4Cl and local rates of urea synthesis were calculated from the extra O2 consumed in the presence of NH4Cl and the stoichiometry between O2 uptake and urea formation. Rates of urea synthesis were also estimated from the fractional decrease in NADPH fluorescence, caused by NH Cl infusion in each region, measured with micro‐light‐guides and the rate of urea synthesis by the whole organ.

When perfusion was in the anterograde direction, maximal rates of urea synthesis, calculated from changes in fluorescence, were 177 ± 31 μmol g−1 h−1 and 61 ± 24 μmol g−1 h−1 in periportal and pericentral regions, respectively. When perfusion was in the retrograde direction, however, rates were 76 ± 23 μmol g−1 h−1 in periportal areas and 152 ± 19 μmol g−1 h−1 in pericentral regions.

During perfusion in the anterograde direction, urea synthesis, calculated by changes in O2 uptake, was 307 ± 76 μmol g−1 h−1 and 72 ± 34 μmol g−1 h−1 in periportal and pericentral regions, respectively. When perfusion was in the retrograde direction, urea was synthesized at rates of 54 ± 17 μmol g−1 h−1 and 387 ± 99 μmol g−1 h−1 in periportal and pericentral regions, respectively. Thus, maximal rates of urea synthesis were dependent upon the direction of perfusion. In addition, rates of urea synthesis were elevated dramatically in periportal regions when the flow rate per gram liver was increased (e.g. 307 versus 177 μmol g−1 h−1).

These results demonstrate that periportal regions of the liver lobule have a greater maximal capacity to synthesize urea when flow is in the physiological anterograde direction. In contrast, the primary site of urea synthesis can be shifted rapidly to pericentral regions when the direction of flow is reversed. Thus, maximal rates of urea synthesis in the perfused liver are observed in regions of the liver lobule with the highest O2 tensions.
The oxygenation‐linked binding of neutral red to spiny lobster hemocyanin
Authors: MAKINO Nobuo
Published: March 2005

The structural change of lobster hemocyanin in cooperative O2 binding was studied by the dye‐binding method. It was found that neutral red shows an O2‐linked binding to hemocyanin with a higher affinity for the oxy form. The number of the dye‐binding sites was estimated to be three in the hexameric molecule of oxyhemocyanin.

The course of the structural change in the partially oxygenated hemocyanin was examined using the absorbance change of the bound dye as a measure. It was found that the fractional change in the dye binding was considerably greater than the degree of O2 saturation of hemocyanin.

The three‐state allosteric model, which was proposed for explanation of the O2 binding properties of lobster hemocyanin [N. Makino (1986) Eur. J. Biochem. 154, 49–55], was also consistent with the effects of the dye on the O2 binding to the native hemocyanin. On the basis of this model, the dye binding to partially oxygenated hemocyanin could be connected with the populations of the affinity states. It was inferred that the binding of neutral red reflects the quaternary structure of the protein.

In contrast, O2 binding to the stripped (EDTA‐treated) hemocyanin showed a considerable decrease in the cooperativity in the presence of the dye. The O2‐binding isotherms could not be explained by the three‐state model. It is suggested that the subunit interaction is partially blocked by the dye in the absence of divalent cations.
Distinct patterns of glycosylation of colligin, a collagen‐binding glycoprotein, and SPARC (osteonectin), a secreted Ca2+‐binding glycoprotein
Authors: HUGHES R. Colin , TAYLOR Amanda , SAGE Helene , HOGAN Brigid L. M.
Published: March 2005

Mouse parietal endoderm PYS cells were labelled with [2‐3H]mannose for 16–24 h. Colligin, an Mr‐47 000 collagen‐binding protein, and SPARC, a Mr‐43 000 protein, highly homologous to the Ca2+‐binding protein osteonectin, were isolated from labelled cell extracts and culture medium respectively. Glycopeptides obtained by exhaustive digestion with pronase were analysed by lectin‐affinity, ion‐exchange, and gel‐filtration chromatography and by paper chromatography of high‐mannose oligosaccharides after endo H release. The results show that the N‐linked carbohydrate chains of colligin are exclusively the high‐mannose type, of which (Man)8(GlcNAc)2 and (Man)9(GlcNAc)2 make up 77%. This carbohydrate structure provides strong evidence that colligin is a component of the endoplasmic reticulum, and argues against a role in cell‐surface interactions. By contrast to colligin, SPARC secreted by PYS cells contains predominantly a diantennary complex type of chain containing a variable number of sialic acid and core‐substituted fucose residues. Similar glycosylation patterns to those discussed above were seen in colligin isolated from primary mouse embryonic parietal endoderm cells and the murine 3T3 cell line, and in SPARC secreted by bovine corneal endothelial cells. Unlike the type‐IV‐collagen‐binding glycoprotein studied by Dennis, J., Waller, C. and Schirrmacher, V. [J. Cell Biol. 99, 1416–1423 (1984)], removal of N‐linked oligosaccharides from colligin had no effect on its binding to native type IV collagen.
Purification and characterization of cystathionine γ‐synthase type II from Bacillus sphaericus
Authors: KANZAKI Hiroshi , KOBAYASHI Michihiko , NAGASAWA Toru , YAMADA Hideaki
Published: March 2005

Cystathionine γ‐synthase type II, which catalyzes l‐cystathionine synthesis from O‐acetyl‐l‐homoserine and l‐cysteine was purified from Bacillus sphaericus (IFO 3536) in seven steps. The purified enzyme appeared to be homogeneous by the results of polyacrylamide electrophoresis and ampholyte electrofocusing. The enzyme is a typical pyridoxal‐P dependent enzyme, has a molecular mass of 165 kDa and consists of four subunits identical in molecular mass. The enzyme catalyzed the γ‐replacement reaction and the elimination reaction was hardly detected even when a large amount of enzyme was added. In the replacement reaction, O‐acetyl‐l‐homoserine and the following thiol compounds: L and d‐cysteine, L and d‐homocysteine, sodium sulfide, various alkyl and aryl mercaptans, acted as the most suitable substrate to produce l‐cystathionine and the corresponding S‐substituted l‐homocysteine derivatives.
Studies on the functional topography of Escherichia coli RNA polymerase
Authors: GRACHEV Mikhail A. , KOLOCHEVA Tatyana I. , LUKHTANOV Evgeny A. , MUSTAEV Arkady A.
Published: March 2005

RNA polymerase was treated in the presence of promoter‐containing templates with 16 affinity reagents, derivatives on NMPs, NDPs and NTPs with reactive substituents at the terminal phosphate. This treatment was followed by addition of a pyrimidine [α‐32P]NTP. Due to ‘catalytic competence’ of some of the residues of the affinity reagents bound covalently near the active center at the first stage, active‐center‐catalyzed synthesis of a phosphodiester bond occurred, and radioactive residues with the general formula ‐pNpN (where = radioactive phosphate) appeared covalently attached to the enzyme. Such affinity labelling was super‐selective because affinity reagent residues bound outside the active center were not elongated and thus remained non‐radioactive.

Labelling took place only when the combination of the reagent and [α‐32P]NTP corresponded to the sequence of nucleotides of the promoter. With reagents having short ‘arms’, only the β subunit was labelled; the targets were His and/or Lys residues. With reagents having longer ‘arms’, the σ subunit was also labelled.
Synthesis of β‐glucans in Prototheca zopfii
Authors: RIVAS Liliana A. , LEZICA Rafael PONT
Published: March 2005

Membrane preparations from the non‐photosynthetic alga Prototheca zopfii incorporate glucose from UDP‐[3H]glucose into the trichloroacetic‐acid‐insoluble fraction and the polysaccharides insoluble in hot alkali. Time course and pulse‐chase experiments indicate that the acid‐insoluble fraction was a precursor of the alkali‐insoluble fraction. Isolation of 3H‐labeled membrane or soluble fraction showed that only membrane fractions were able to transfer radioactivity into polysaccharides. Treatment of glucosylated membranes with trypsin or cellulase only partially affect their transfer ability, indicating that the precursor was internalized in vesicles.

Analysis of the in vitro synthesized polysaccharides by enzymatic and acid hydrolysis showed that glucose and cellobiose were present as radioactive sugars. Permethylation of the polysaccharide indicates that 80% of the glucose was β‐1,4‐bonded with 20% in β‐1,3‐linkagés. This polysaccharide was found to be identical with the cell‐wall β‐glucan obtained in vivo [Rivas, L. A. & Pont Lezica, R. (1978) Planta (Berl.) 165, 348–353].
Synthesis of β‐glucans in Prototheca zopfii
Authors: RIVAS Liliana A. , PONT LEZICA Rafael
Published: March 2005

When Prototheca zopfii cells were pulse‐labeled with 14C‐containing amino acids and homogenized, 14C‐labeled membranes were obtained. In vitro incubations with the previously labeled membranes and UDP‐[3H]Glc produced a trichloroacetic‐acid‐insoluble fraction having both isotopes. A double‐labeled glucoprotein was isolated and characterized. It has a relative molecular mass of 28 000–30 000 and a carbohydrate content of 10%. The oligosaccharide chain is linked to the protein through an O‐glycosidic bond between hydroxyproline and glucose. The oligosaccharide has a polymerization degree ranging over 10–20 hexose units. Glucose is the only monosaccharide found; most of the glucose residues are β‐1,4‐linked (90%) but some are β‐1,3‐linked (10%).
Autotrophic synthesis of activated acetic acid from CO2 in Methanobacterium thermoautotrophicum
Authors: LÄNGE Siegfried , FUCHS Georg
Published: March 2005

The synthesis of acetyl‐CoA from CO2, H2, and various C1 compounds was studied in vitro with extracts and with protein fractions of Methanobacterium thermoautotrophicum. Acetyl‐CoA synthesis from CO2 and H2 by extracts required CO2 reduction to CH4 to proceed. Both processes were highly stimulated by formaldehyde which served as the carbon precursor of both CH4 and the CH3 group of acetate. Carbon monoxide in combination with formaldehyde dramatically stimulated the acetyl‐CoA synthesis up to 150‐fold. In this system, which did not require CO2 reduction to the formaldehyde and CO level, acetyl‐CoA synthesis was no longer dependent on CH4 formation. The soluble (100 000 × g supernatant) cell protein was resolved into a protein fraction [45–60% (NH4)2SO4‐fraction] which catalyzed acetyl‐CoA synthesis at a specific rate of 15 nmol · min−1· (equivalent of mg cell protein)−1 (60°C). This oxygen‐sensitive enzyme reaction required dithioerythritol for activity and was strictly dependent on (a) coenzyme A, (b) CO, and (c) N5,N10‐methylene tetrahydromethanopterin, N5‐methyl tetrahydromethanopterin or formaldehyde plus tetrahydromethanopterin. The incorporation of formaldehyde is explained by the spontaneous formation of methylene tetrahydromethanopterin. The product of the reaction, acetyl‐CoA, was quantitatively derived from CO (carboxyl of acetate) and a C1 derivative of tetrahydromethanopterin (methyl of acetate). The C1 derivative of tetrahydromethanopterin could not be replaced by a C1 derivative of tetrahydrofolate or by methyl‐coenzyme M; ATP was not required. The active protein fraction contained CO dehydrogenase and at least one corrinoid protein. These results provide strong biochemical arguments for the proposed mechanism of autotrophic acetyl‐CoA synthesis in Methanobacterium.
Complete amino‐acid sequence of the natural ATPase inhibitor from the mitochondria of the yeast Candida utilis
Authors: DIANOUX Anne Christine , HOPPE Jürgen
Published: March 2005

The complete alignment of the 63 residues of the mitochondrial ATPase inhibitor from the yeast Candida utilis has been determined. The sequence study was carried out mainly by automatic (liquid and solid‐phase) methods. Peptides were obtained by enzymatic digestion with clostripain and purified by reverse‐phase high‐performance liquid chromatography. The ATPase inhibitor contains three sets of repetitive sequences and eight clusters of charged residues, as also found in the inhibitor of Saccharomyces cerevisiae, with which it shares 58.7% homology of conserved residues. When the two yeast ATPase inhibitor sequences were compared to that of beef heart, 20 residues remained common to the three alignments, although the latter protein contained a long histidine‐rich insertion, only found in this inhibitor. Most of the homologous residues were clustered near the center of the protein, which by partial proteolytic digestion of the beef heart ATPase inhibitor [Dianoux, A. C. et al. (1982) FEBS Lett. 140, 223–228] has already been shown to be involved in the biological function.
Chlorophyll‐protein complexes of barley photosystem I
Authors: BASSI Roberto , SIMPSON David
Published: March 2005

Photosystem I (PSI) preparations with a chlorophyll a/b ratio of 6.0 were isolated from barley thylakoids using two different methods. The high‐molecular‐mass complex (CP1a*) which is resolved by non‐denaturing gel electrophoresis had the same properties as a PSI preparation (PSI‐200) isolated by Triton X‐100 solubilisation of thylakoids followed by sucrose gradient ultracentrifugation. This material had a chlorophyll:P700 ratio of 208:1 and was composed of three different chlorophyll‐protein complexes which could be separated from each other by solubilising the PSI preparation in dodecyl maltoside followed by sucrose gradient ultracentrifugation. Approximately half of the chlorophyll, including all the chlorophyll b, was located in two antenna complexes designated LHCI‐680 and LHCI‐730, which were identified by their characteristic low‐temperature fluorescence emission spectra. The rest of the chlorophyll a was associated with the PSI reaction centre, P700 Chla‐P1, which fluoresced at 720 nm. Each chlorophyll‐protein complex had a unique polypeptide composition and characteristic circular dichroic and absorption spectra. The use of dodecyl maltoside instead of dodecyl sulphate resulted in a less denatured form of LHCI‐680, which fluoresced at 690 nm at 77 K. One of the sucrose gradient fractions contained a complex consisting of only LHCI‐730 and P700 Chla‐P1 which fluoresced at 731 nm, indicating that LHCI‐730 is structurally associated with P700 Chla‐P1 and quenches its fluorescence. Approximately threequarters of the light‐harvesting antenna chlorophyll was in LHCI‐730, but only about one‐quarter of the normal complement of LHCI‐730 was required to quench the reaction centre.

By reducing the amount of Triton relative to the chlorophyll concentration, a PSI preparation (chlorophyll a/b ratio of 3.5) with a chlorophyll:P700 ratio of 300:1 was isolated. It contained no photosystem II, but a significant amount of LHCII which was functionally connected to the PSI reaction centre. Reconstitution studies demonstrated that excitation energy transfer from LHCII to PSI requires the presence of LHCI‐680, and we propose that, in PSI, the following linear excitation energy transfer sequence occurs: LHCII → LHCI‐680 → LHCI‐730 → P700 Chla‐P1.
Phenobarbital‐mediated modulation of gene expression in rat liver
Authors: LECHNER Maria Celeste , SINOGAS Carlos , OSÓRIO‐ALMEIDA Maria Leonor , FREIRE Maria Teresa , CHAUMET‐RIFFAUD Philippe , FRAIN Monique , SALA‐TREPAT José M.
Published: March 2005

Phenobarbital evoques a pleiotypic response in the liver characterized by cell hypertrophy and mono‐oxygenase induction. These phenomena arise through complex modulation mechanisms changing the pattern of protein synthesis, distinct from those triggered by other well known inducers, like steroid hormones or polycyclic hydrocarbons.

To investigate the mechanisms involved in regulating the expression of the phenobarbital‐inducible tissue‐specific genes, we constructed two libraries of recombinant bacterial plasmids pBR322 in Escherichia coli. Each library contains cDNA copies of polysomal poly(A)‐rich RNA obtained from control and 16‐h phenobarbital‐induced rat liver. A thousand cloned sequences from each library were screened by double‐cross colony hybridization using [32P]cDNA prepared from homologous and heterologous poly(A)‐rich RNAs as the probes. The statistical analysis of the results revealed that phenobarbital treatment significantly changes the relative abundance of different polysomal mRNA classes in rat liver.

Clones corresponding to mRNAs clearly induced following phenobarbital treatment have been further selected by plasmid DNA dot hybridization, and used as probes for measuring the changes in each mRNA concentration in the whole cell and in the polysomal RNAs from rat livers, at different times after phenobarbital treatment.

The fact that changes in the concentration of each specific mRNA in the polysomes does not parallel the variation of its total concentration in the cell indicates that the induced modulation of protein synthesis in the liver is brought about by mechanisms involving both transcriptional and translational regulation, since besides the increases in whole cellular mRNA concentration a marked mobilization of mRNA into active polysomes could be demonstrated during the onset of the adaptive response to phenobarbital.
Structural analysis of 5S rRNA, 5S rRNA‐protein complexes and ribosomes employing RNase H and d(GTTCGG)
Authors: LORENZ Siegfried , HARTMANN Roland K. , PIEL Norbert , ULBRICH Norbert , ERDMANN Volker A.
Published: March 2005

The hybridization of d(GTTCGG) to eubacterial 5S rRNAs, 5S rRNA‐protein complexes, 70S ribosomes and 50S and 30S ribosomal subunits was investigated. This oligonucleotide, which may be considered to be an analogue of the TΨCG loop of tRNAs, was chosen in order to investigate a possible interaction between tRNAs with ribosomal components during protein synthesis. The hybridization was analysed by RNase H hydrolysis studies and, in the case of the ribosomes and ribosomal subunits, in addition with the radioactively labelled oligodeoxyribonucleotide in binding studies.

The results obtained lead to the conclusion that nucleotides in loop c, i.e. positions 42–47, are available for oligonucleotide interaction in free Escherichia coli and Bacillus stearothermophilus 5S rRNAs and not available in the corresponding 5S rRNA‐protein complexes. The 70S ribosomes and ribosomal subunits did not interact with the oligonucleotide. Under the assumption that d(GTTCGG) is an analogue of the TΨCG loop of tRNAs and in view of the results obtained, we conclude that in the unprogrammed ribosomes the TΨCG loop of tRNAs does not interact via standard Watson‐Crick base pairs with the ribosomal 5S, 16S or 23S RNAs.
Further studies on the structure of the glycogen‐bound form of protein phosphatase‐1 from rabbit skeletal muscle
Authors: HIRAGA Akira , KEMP Bruce E. , COHEN Philip
Published: March 2005

We have reported previously that the glycogen‐bound form of protein phosphatase‐1 (termed PP‐1G) is a heterodimer, composed of the 37‐kDa catalytic (C) subunit complexed to a 103‐kDa G‐subunit that anchors the enzyme to glycogen [Strålfors, P., Hiraga, A. and Cohen, P. (1985) Eur. J. Biochem. 149, 295–303]. An antibody raised against a synthetic peptide corresponding to the phosphorylation site on the G‐subunit was found to immunoprecipitate PP‐1G specifically. No precipitation occurred if the antibody was preincubated with the synthetic peptide, or if PP‐1G was replaced by either the isolated C‐subunit or protein phosphatase‐2A. The results confirm by a new and independent method that the G‐subunit is complexed to the C‐subunit, and that it is not a contaminant.

The G‐subunit is remarkably sensitive to proteolysis. At the final stage of purification, PP‐1G was eluted as a broad peak of activity. The leading fractions contained the 37‐kDa C‐subunit and 103‐kDa G‐subunit, while the central and trailing fractions comprised the 37‐kDa C‐subunit plus a number of bands with molecular masses ranging over 40–80 kDa. The 40–80‐kDa bands were phosphorylated by cyclic‐AMP‐dependent protein kinase and tryptic digestion generated the identical phosphopeptides obtained by trypsinisation of the 103‐kDa G‐subunit. Furthermore, antibody to the G‐subunit immunoprecipitated protein phosphatase activity quantitatively in the leading, central and trailing fractions. The results demonstrate that the 40–80‐kDa polypeptides are fragments of the G‐subunit, and that fragments as small as 40 kDa retain the sites of phosphorylation as well as the ability to interact with the C‐subunit and with glycogen.

Khatra [J. Biol. Chem. (1986) 261, 8944–8952] reported that the glycogen‐bound form of protein phosphatase‐1 did not contain a G‐subunit and that it was a dimer composed of two identical C‐subunits. The present work has shown that this proposal is incorrect, and that proteolysis of the G‐subunit to fragments that stain very poorly with Coomassie blue can explain why this error was made.
Purification and characterization of a novel metalloendopeptidase from Streptococcus cremoris H61
Authors: YAN Tsong‐Rong , AZUMA Norihiro , KAMINOGAWA Shuichi , YAMAUCHI Kunio
Published: March 2005

An endopeptidase (LEP‐II), which has a unique substrate specificity, was purified to homogeneity by conventional chromatographic techniques from Streptococcus cremoris H61. The enzyme was a metalloendopeptidase since it was inhibited by EDTA and 1,10‐phenanthroline; the metal‐depleted enzyme could be fully reactivated by micromolar levels of Zn2+ and was not inhibited by specific inhibitors for serine or thiol protease. The molecular mass of the enzyme was estimated to be 80 kDa by Sephacryl S‐300 gel filtration and high‐performance liquid chromatography with a TSK‐G3000SW column. The enzyme consisted of two identical subunits and the N‐terminal sequence of LEP‐II was determined up to the 19th residue. Although the enzyme had a broad substrate specificity it specifically hydrolyzed the peptide bonds involving the amino groups of hydrophobic amino acid residues. Various small polypeptides, such as αs1‐CN(f1–23), αs1‐CN(f91–100). oxidized insulin B chain, glucagon and some biologically active peptides were hydrolyzed. However, a variety of larger polypeptides or proteins, such as αs1‐CN(f1–54), αs1‐CN(f61–123), αs1‐CN(f136–196), αs1‐casein, β‐casein, and K‐casein were not hydrolzyed. LEP‐II recognized the size of its substrates, which were limited below a molecular mass of about 3.5 kDa.
Influence of N6‐methylation of residue A(5) on the conformational behaviour of d(C‐C‐G‐A‐A‐T‐T‐C‐G‐G) in solution studied by 1H‐NMR spectroscopy
Authors: RINKEL Lambertus J. , van der MAREL Gijs A. , van BOOM Jacques H. , ALTONA Cornelis
Published: March 2005

One‐ and two‐dimensional NMR studies at 300 MHz and 500 MHz were carried out on the two oligonucleotides d(C‐C‐G‐A‐A‐T‐T‐C‐G‐G) and d(C‐C‐G‐A‐m6A‐T‐T‐C‐G‐G) in aqueous solution. NMR spectra were observed at 10 mM sample concentration over the temperature range 273–368 K. Assignments are given of the base, H1′, H2′, H2″, H3′ and of some H4′ resonances, based upon a combination of two‐dimensional correlation spectra (COSY) and two‐dimensional nuclear Overhauser effect spectra (NOESY); imino‐proton resonances were assigned with the aid of a two‐dimensional NOE experiment. Chemical shift vs temperature profiles were constructed in order to gain insight into the influence of N6‐methylation of residue A(5) on the temperature‐dependent conformational behaviour of the decamer and to determine thermodynamic parameters for the duplex‐to‐coil transition. The NOESY spectra, the imino‐proton spectra and the shift profiles of the two compounds, under conditions where each forms a B‐DNA‐type duplex, are very similar. This is taken to indicate that the influence of N6‐methylation of residue A(5) on the local structure of the duplex must be small. However, the temperature dependence of the (non‐)exchangeable proton resonances of the two compounds reveals that methylation slows down the duplex – single‐strand exchange. Furthermore, a thermodynamic analysis of the two compounds indicates that N6‐methylation slightly decreases the stability of the duplex relative to the monomeric forms (Tm is reduced from 332 K down to 325 K at 10 mM sample concentration). Proton‐proton couplings were obtained by means of one‐dimensional and two‐dimensional NMR experiments and were used in a conformational analysis of the sugar ring of each residue of the two compounds in the duplex form. The analysis indicated that all sugar rings display conformational flexibility in the intact duplex: population S‐type sugar conformation ranges from 70% to 100%. A more refined analysis of the sugar rings of the parent compound revealed a sequence‐dependent variation of the sugar geometry. This variation does not follow well the trend predicted by the Calladine/Dickerson Σ3‐sum rule [Dickerson, R. E. (1983) J. Mol. Biol. 166, 419–441; Calladine, C. R. (1982) J. Mol. Biol. 161, 343–352]; moreover the actual variations appear to be smaller in solution than those expected on the basis of known X‐ray structures.
Influence of N6‐methylation of residue A(5) on the conformational behaviour of d(C‐C‐G‐A‐A‐T‐T‐C‐G‐G) in solution studied by 1H‐NMR spectroscopy
Authors: RINKEL Lambertus J. , van der MAREL Gijs A. , van BOOM Jacques H. , ALTONA Cornelis
Published: March 2005

The solution conformations of the oligonucleotides d(C‐C‐G‐A‐A‐T‐T‐C‐G‐G) and d(C‐C‐G‐A‐m6A‐T‐T‐C‐G‐G) as a function of temperature and sample concentration were investigated by means of 1H‐NMR spectroscopy. The NMR spectra revealed that, at certain combinations of temperature and low sample and salt concentration, both compounds exist as a B‐DNA‐type duplex slowly (on the 1H‐NMR time scale) interconverting with a monomeric species. From chemical shift data and imino‐proton spectra, it is concluded that the monomeric species consists of a mixture of a hairpin form in rapid equilibrium with the random‐coil form. The double‐helical stem of the hairpin is formed by the six terminal cytidine and guanine residues, whereas the four core residues, ‐A‐(m6)A‐T‐T‐, partake in the loop. Thermodynamic analysis of the chemical shift of the resonances of the monomeric species vs temperature profiles of the two decamers and mutual comparison of these profiles indicate the following: (a) the influence of N6‐methylation of residue A(5) upon the local structure of the hairpin must be small; (b) methylation decreases the stability of the duplex relative to the monomeric species: the temperature at which the fraction duplex equals 0.5 was found to be 312 K for the parent compound and 305 K for the methylated decamer at 2 mM sample concentration; (c) methylation does not significantly alter the stability of the hairpin form relative to the random coil form: the Tm of the hairpin ⇌ random‐coil equilibrium is 308 K for the parent compound and 306 K for the methylated decamer. A higher fraction hairpin‐like structure for the N6‐methylated compound is observed under identical conditions of temperature and sample concentration: at 300 K, 2 mM sample concentration, the fraction hairpin form is 0.12 for d(C‐C‐G‐A‐A‐T‐T‐C‐G‐G) and 0.20 for d(C‐C‐G‐A‐m6A‐T‐T‐C‐G‐G). This finding appears to be a consequence of the reduced stability of the methylated dimeric species relative to the monomeric species, and to depend upon the sodium‐ion concentration: it becomes more pronounced under low‐salt conditions.
Recombinant‐DNA‐derived bovine growth hormone from Escherichia coli
Authors: LANGLEY Keith E. , LAI Por‐Hsiung , Wypych Jette , EVERETT Richard R. , BERG Thor F. , KRABILL L. F. , DAVIS Janice M. , SOUZA Lawrence M.
Published: March 2005

Bacterially synthesized, recombinant‐DNA‐derived bovine growth hormone (r‐bGH), prepared as described in the preceding paper in this journal, has been characterized in comparison with pituitary bovine growth hormone (pit‐bGH). The characterization criteria include sodium dodecyl sulfate/polyacrylamide gel electrophoresis, automated N‐terminal sequence analysis, amino acid composition, isoelectric focusing, reverse‐phase high‐performance liquid chromatography, ultraviolet absorbance, analysis for free protein thiol, sizing by gel filtration, circular dichroism, radioimmunoassay and biological activity in the hypophysectomized rat weight‐gain assay. In every respect the r‐bGH appears to be virtually identical to pit‐bGH.
Murein biosynthesis in synchronized cells of Proteus mirabilis
Authors: GMEINER Jobst , SARNOW Elke
Published: March 2005

Cells of Proteus mirabilis, synchronized by sucrose density gradient centrifugation, were grown in complex medium containing radioactive N‐acetylglucosamine. At various times, labelled murein sacculi were isolated and digested with endo‐N,O‐acetylmuramidase from Chalaropsis. The murein fragments thus obtained were separated into disaccharide peptides as the monomeric subunits and into peptide‐cross‐linked subunits by gel filtration. The subunits were further differentiated into O‐acetylated and non‐O‐acetylated species, and into subunits containing anhydro‐N‐acetylmuramic acid which were glycan chain terminators in the native sacculi. Quantification of the subunit species gave the following results.

1. At specific times during the cell cycle, murein subunits were lost from the polymer and a transient decrease in cross‐linkage was observed.

2. The overall degree of cross‐linkage in mature murein, i.e. the ratio of peptide‐cross‐linked subunits versus uncross‐linked subunits, was 1.15 as determined by regression analysis.

3. Anhydro‐N‐acetylmuramic‐acid‐containing murein subunits representing glycan chain terminators were found either peptide‐cross‐linked or uncross‐linked as monomers. Since these two subunit species were recovered in a defined ratio of 1.6, mature murein consisted of at least two different types of glycan chains. On average, each chain contained 15.4 murein subunits.

4. About 60% of the murein subunits in mature murein were O‐acetylated and showed a higher degree of cross‐linkage than the non‐O‐acetylated portion.

Finally, following the composition of the sacculus during the cell cycle revealed a complex precursor‐product relationship between non‐O‐acetylated and O‐acetylated subunits during murein maturation. The data allowed us to deduce several features of the assembly process of murein sacculi.
Structural characterization of gangliosides from murine T lymphocytes
Authors: MÜTHING Johannes , EGGE Heinz , KNIEP Bernhard , MÜHLRADT Peter F.
Published: March 2005

Mouse spleen cells were prepared from CBA/J mice, and T lymphocytes were selectively stimulated with the T cell mitogen concanavalin A and further propagated in the presence of the T cell growth factor interleukin‐2. The T cells were metabolically labeled with D‐[1‐14C]galactose and D[1‐14C]glucosamine, and the gangliosides were extracted and purified by DEAE‐Sepharose column chromatography. Carbohydrate backbone structures of the asialogangliosides, prepared by mild acid hydrolysis, were determined by (1) high‐performance liquid chromatography, (2) treatment with exoglycosidases and (3) immunostaining. Monosialylated gangliosides were isolated by gradient elution from DEAE‐Sepharose and further separated by preparative high‐performance thin‐layer chromatography in two solvent systems. Isolated fractions were characterized by (1) preparation of asialogangliosides by mild acid hydrolysis, (2) the action of Vibrio cholerae neuraminidase, and (3) fast‐atom‐bombardment mass spectrometry. The following structures were identified: IVNeuAc‐GgOse4Cer; IVNeuG‐GgOse4Cer; IVNeuAc‐GgOse5Cer; and IVNeu‐Gc‐GgOse5Cer. The latter two gangliosides were not detected on B lymphoblasts and may be T‐cell‐specific structures. All gangliosides were heterogeneous in their ceramide moieties, being substituted with C16:0, C24:0, and C24:1 fatty acids, A preliminary study of several other mouse strains showed no strain‐specific genetic variations in the T cell gangliosides. The possible role of these gangliosides is discussed.
Examination of the structural requirements for proliferation of peroxisomes and mitochondria in mouse liver by hypolipidemic agents, with special emphasis on structural analogues of 2‐ethylhexanoic acid
Authors: LUNDGREN Bo , MEIJER Johan , DePIERRE Joseph W.
Published: March 2005

We have found here that there are clear structural requirements for peroxisome proliferation (monitored as increases in carnitine acetyltransferase activity, cyanide‐insensitive palmitoyl‐CoA oxidation, catalase and increases in the protein designated PPA 80) in mouse liver. From the investigation of ten structural analogues of 2‐ethylhexanoic acid, it could be concluded that the most effective proliferators all have an ethyl group as the substituent on carbon 2 of the main chain, which consists of six carbons. The further observation from this group of compounds that a charged group is required for effective proliferation leads us to speculate that such a group is involved in the molecular mechanism as well.

Many, but not all, of the effective peroxisome proliferators in a second group of compounds contain a phenoxy group, often with a substituted α carbon. Interestingly, the 2,4‐dichlorophenoxyacetic and 2,4,5‐trichlorophenoxyacetic acids are both effective peroxisome proliferators, but the closely related p‐chlorophenoxyacetic acid is inactive in this respect, indicating that the chlorine atom at position 2 must be essential to the process in these cases.

The results presented here also indicate that the structural requirements for proliferation of mitochondria are similar to those for proliferation of peroxisomes. Certainly, the most effective peroxisome proliferators also cause large increases in ‘mitochondrial’ protein and cytochrome oxidase activity, i.e. there is an obvious qualitative correlation.
Fate of injected 125I‐labeled cholera toxin taken up by rat liver in vivo
Authors: JANICOT Michel , DESBUQUOIS Bernard
Published: March 2005

Subcellular fractionation techniques have been used to assess the localization of injected 125I‐labeled cholera toxin (125I‐CT) taken up by rat liver in vivo, and to determine whether internalization of the toxin is required for the generation of the active A1 peptide.

The uptake of injected 125I‐CT into the liver is maximal at 5 min (about 10% injected dose/g). At this time the radioactivity is for the most part recovered in the microsomal (P) fraction, but later on it progressively associates with the mitochondrial‐lysosomal (ML) and supernatant fractions. The radioactivity is enriched 7‐fold in plasma membranes at 5–15 min, and 15–60‐fold in Golgi‐endosome (GE) fractions at 15–60 min.

On analytical sucrose gradients the radioactivity associated with the P fraction is progressively displaced from the region of 5′‐nucleotidase (a plasma membrane marker) to that of galactosyltransferase (a Golgi marker). On Percoll gradients, however, it is displaced towards acid phosphatase (a lysosomal marker). Density‐shift experiments, using Triton WR 1339, suggest that some radioactivity associated with the P fraction (at 30 min) and all the radioactivity present in the ML fraction (at 2 h) is intrinsic to ácid‐phosphatase‐containing structures, presumably lysosomes. Comparable experiments using 3,3′‐diaminobenzidine cytochemistry indicate that the radioactivity present in GE fractions is separable from galactosyltransferase, and thus is presumably associated with endosomes.

The fate of injected 125I‐labeled cholera toxin B subunit differs from that of the whole toxin by a more rapid uptake (and/or clearance) of the ligand into subcellular fractions, and a greater accumulation of ligand in the ML fraction.

Analysis of GE fractions by SDS/polyacrylamide gel electrophoresis shows that, up to 10 min after injection of 125I‐CT, about 80% of the radioactivity is recovered as A subunit and 20% as B subunit, similarly to control toxin. Later on there is a time‐dependent decrease in the amount of A subunit and, at least with the intermediate GE fraction, a concomitant appearance of A1 peptide (about 15% of the total at 60 min). In contrast the radioactivity associated with plasma membranes remains indistinguishable from unused toxin.

It is concluded that, upon interaction with hepatocytes, 125I‐CT (both subunits A and B) sequentially associates with plasma membranes, endosomes and lysosomes, and that endosomes may represent the major subcellular site at which the A1 peptide is generated.
Transcription termination and processing of transcripts from tRNA‐related Xenopus satellite DNA sequences
Authors: MEYERHOF Wolfgang , WITTIG Burghardt , TAPPESER Beatrix , KNÖCHEL Walter
Published: March 2005

Nucleotide sequence homologies have been found between the transcriptional unit of a Xenopus laevis satellite DNA containing 1037‐bp monomeric repeats and tRNA‐coding genes. Since the tRNA‐homologous region forms only part of the transcriptional unit, we have analyzed the size of transcripts being made after injection of cloned DNA into Xenopus oocyte nuclei. Incubation for 6 h mainly results in three RNA species comprising 130, 183 and 196 nucleotides, which originate at the same initiation site but terminate at different T stretches. Longer incubation reveals a complex pattern of smaller RNAs most likely resulting from 3′‐end processing of primary transcripts. All these RNAs are related to tRNA, since they contain the tRNA‐homologous region which is close to the 5′ end of the transcription unit.
Purification and characterization of a ribonuclease specific for poly(U) and poly(C) from the larvae of Ceratitis capitata
Authors: SIDERIS Diamantis C. , FRAGOULIS Emmanuel G.
Published: March 2005

A specific ribonuclease was detected and purified to homogeneity from six‐day‐old larvae of the insect Ceratitis capitata and its homogeneity was checked by analysis in polyacrylamide gels in the presence of sodium dodecyl sulfate.

The nuclease specifically degrades poly(U) and poly(C) whilst it fails to do so with other single‐stranded homopolyribonucleotides. The enzyme has a pH optimum in the region 7–9 and relative molecular mass of about 25000.

The effect of this ribonuclease on the integrity of RNAs isolated from six‐day‐old larvae or rat liver was also studied.
The effect of factor Va on lipid dynamics in mixed phospholipid vesicles as detected by steady‐state and time‐resolved fluorescence depolarization of diphenylhexatriene
Authors: van de WARRT Piet , VISSER Antonie J. W. G. , HEMKER H. Coenraad , LINDHOUT Theo
Published: March 2005

We have monitored the thermotropic behavior of mixed dimyristoylglycerophosphoserine (Myr2GroPSer)/dimyristoylglycerophosphocholine (Myr2GroPCho) and Myr2GroPSer/dipalmitoylglycerophosphocholine (Pam2GroPCho) vesicles in the presence of blood‐clotting factor Va, using 1,6‐diphenyl‐1,3,5‐hexatriene as a lipid probe. The Ca2+‐independent interaction of factor Va with these vesicles caused a small increase (1–2°C) in the phase transition temperature, regardless of whether Myr2GroPChe was the lower or higher‐melting component of the mixed vesicles. The major effect of factor Va was to increase the polarization of diphenylhexatriene when the mixed vesicles were in the liquid crystalline phase. The protein did not change the anisotropy in the bilayer gel state. The increase in the polarization value above the transition temperature closely correlated with the amount of phospholipid‐bound factor Va, as verified by a direct binding technique. In addition, we found that the affinity of factor Va for Myr2GroPSer/Myr2GroPCho and Myr2GroPSer/Pam2GroPCho greatly increased at temperatures above the transition temperatures. Time‐dependent fluorescence anisotropy measurements of diphenylhexatriene embedded in vesicles in the liquid crystalline state give fluorescence decay curves which can best be fitted by two exponential functions with two rotational correlation times and a constant term. Vesicles composed of Myr2GroPSer exhibit more ordering than Myr2GroPCho vesicles. However, the order parameter of mixed vesicles composed of 40% Myr2GroPSer and 60% Myr2GroPCho (mol/mol) approached that of Myr2GroPCho. Factor Va dramatically increased the longer rotational correlation time of diphenylhexatriene embedded in mixed vesicles in the liquid crystalline state from 3.7 ns to about 17 ns. The second rank‐order parameter increased only slightly, but the calculated steady‐state anisotropy increased by twofold. These results indicate that the acidic phospholipid‐dependent binding of factor Va to mixed vesicles has an ordering effect on the acyl chains of the acidic phospholipids in the outer layer, but leaves the bulk of the phospholipids, mainly phosphatidylcholine, unaltered. None of the factor‐Va‐induced alterations in the anisotropy parameters point to the occurrence of lateral phase separation.
Identification and sequencing of cDNA clones for the rodent negative acute‐phase protein α1‐inhibitor 3
Authors: SCHWEIZER Michael , TAKABAYASHI Kenji , GEIGER Thomas , LAUX Thomas , BIERMANN Gereon , BUHLER Jean‐Marie , GAUTHIER Francis , ROBERTS Lilian M. , HEINRICH Peter C.
Published: March 2005

Rat α1‐inhibitor 3 clones were isolated by immunological screening of a λ gt11 cDNA library prepared from rat liver poly(A)‐rich RNA. The recombinant cDNA clones were identified by the absence of their immunoprecipitable products following hybrid‐arrested in vitro translation. The size of the cognate poly(A)‐rich RNA was estimated to be roughly 5000 residues. Approximately 16 h after induction of inflammation the amount of α1‐inhibitor 3 poly(A)‐rich RNA decreases as shown by dot‐blot hybridization and Northern analyses. The response of this negative acute‐phase plasma protein to inflammation may therefore be considered to be at the pretranslational level.

The characterized DNA constitutes an open reading frame of 225 amino acids followed by a canonical eucaryotic polyadenylation signal and a poly(A) tail. Sequence microheterogeneity, particularly in the 3′‐flanking region was observed. An amino acid homology of 70% for α1‐inhibitor 3 with human and rodent α2‐macroglobulin emphasizes the evolutionary relationship of the macroglobulins.
Unfolding of the trp repressor from Escherichia coli monitored by fluorescence, circular dichroism and nuclear magnetic resonance
Authors: LANE Andrew N. , JARDETZKY Oleg
Published: March 2005

The denaturation of the trp repressor from Escherichia coli has been studied by fluorescence, circular dichroism and proton magnetic resonance spectroscopy. The dependences of the fluorescence emission of the two tryptophan residues on the concentration of urea are not identical. The dependence of the quenching of tryptophan fluorescence by iodide as a function of urea concentration also rules out a two‐state transition. The circular dichroism at 222 nm decreases in two phases as urea is added. Normalised curves for different residues observed by 1H NMR also do not coincide, and require the presence of at least one stable intermediate. Analysis of the dependence of the denaturation curves on the concentration of protein indicate that the first transition is a partial unfolding of the dimeric repressor, resulting in a loss of about 25% of the helical content The second transition is the dissociation and unfolding of the partially unfolded dimer. At high concentrations of protein (500 μM) about 73% of the repressor exists as the intermediate in 4 M urea. The apparent dissociation constant is about 10−4 M; the subunits are probably strongly stabilised by the subunit interaction. The native repressor is stable up to at least 70°C, whereas the intermediate formed at 4 M urea can be denatured reversibly by heating (melting temperature ∼60°C, ΔH∼ 230 kJ/mol).
The site of inhibition of the chloroplast electron‐transport system by 2,3‐dithiopropan‐1‐ol (BAL)
Authors: SHAHAK Yosepha , HIND Geoffrey , PADAN Etana
Published: March 2005

BAL (2,3‐dithiopropan‐1‐ol) treatment of chloroplasts has previously been reported to induce a block in electron transport from water to NADP+ at a site preceding plastocyanin [Belkin et al. (1980) Biochim. Biophys. Acta 766, 563–569]. In the present work the block was further characterized. The following properties of BAL treatment are described.

a) Inhibition of electron transport from water to lipophilic acceptors but not to silicomolybdate.

b) Inhibition of the slow, sigmoidal phase of chlorophyll a fluorescence induction.

c) Inability of N, N, N′,N′,‐tetramethyl‐p‐phenylenediamine to bypass the inhibition of NADP+ photoreduction with water as the electron donor.

d) Inhibition of electron transport from externally added quinols to NADP+

e) Inhibition of cytochrome f reduction by photosystem II, but not its oxidation by photosystem I.

f) Inhibition of cytochrome b6 turnover and cytochrome f rereduction after single‐turnover flash illumination under cyclic electron‐flow conditions.

The BAL‐induced block is therefore located between the secondary quinone acceptor (QB) and the cytochrome b6f complex. It was further found that (a) the isolated cytochrome complex is not inhibited after BAL treatment; (b) BAL‐reacted plastoquinone‐1 inhibits electron transport in chloroplasts; (c) BAL does not inhibit electron transport in chromatophores of Rhodospirilum rubrum or Rhodopseudomonas capsulata. It is suggested that the inhibition of electron transport in chloroplasts results from specific reaction of BAL with the endogenous plastoquinone.