bluuebunny/crossref_metadata_2025 huggingface dataset:
Characterization of reconstituted ATPase complex proteoliposomes prepared from the thermophilic cyanobacterium Synechococcus 6716
Authors: VAN WALRAVEN Hendrika S. , LUBBERDING Henk J. , MARVIN Hans J. P. , KRAAYENHOF Ruud
Published: March 2005

The preparation and some properties are described of proteoliposomes consisting of the ATPase complex and lipids from the thermophilic cyanobacterium Synechococcus 6716. In the proteoliposomes (about 200 nm in diameter) only a low amount of protein can be incorporated (protein/lipid ratio of 0.01 w/w) and they show very few protein particles on freeze‐fracture replicas. The octyl glucoside and cholate dialysis method of reconstitution yielded stable proteoliposomes with a relatively low proton permeability. ATP hydrolysis and 32Pi/ATP exchange activities were about 400 and 120nmol · min−1· mg protein−1, respectively; the former was strongly stimulated by an uncoupler. ATP hydrolysis induces membrane energization as monitored by membrane‐potential‐ and surface‐potential‐indicating probes and by different pH indicators trapped inside the vesicles. The probes used were a membrane‐bound fluorescent aminoacridine, which monitors surface charge‐density changes, the native carotenoids and added oxonol VI for monitoring electrical potential in the membrane and the pH indicators neutral red and cresol red. The different rise kinetics of these probes indicate that proton accumulation upon ATP hydrolysis involves at least two steps: a membrane‐localized potential charge and proton transfer followed by a much slower acidification of the bulk intravesicular space. Internal neutral red and cresol red seem to discriminate between proton translocation to the internal interface and bulk space, respectively.
The lipopolysaccharide of a chloridazon‐degrading bacterium
Authors: WEISSHAAR Rüdiger , LINGENS Franz
Published: March 2005

Lipopolysaccharide of a chloridazon‐degrading bacterium was obtained by a two‐stage extraction procedure with phenol/EDTA in a yield of 0.3% of dried bacteria. The carbohydrate moiety consisted of heptose, 3‐deoxyoctulosonic acid and d‐glucose in a molar ratio of 1:2:2 · 3. Lipid A was composed of 1 mol 2,3‐diamino‐2,3‐dideoxy‐d‐glucose, 2 mol amide‐bound and 2.6 mol ester‐bound fatty acids/mol. Amide‐bound fatty acids were 3‐hydroxydodecanoic acid and 3‐hydroxyhexadecanoic acid; dodecanoic acid and R‐(‐)‐3‐hydroxydodec‐5‐cis‐enoic acid were found to be present in ester linkage. Under conditions of acidic hydrolysis, the latter was converted into the cis and trans isomers of 5‐hexyltetrahydrofuran‐2‐acetic acid. Dodecanoic acid was demonstrated to be linked with the hydroxy groups of the amide‐bound fatty acids. The taxonomic significance of these results, especially the demonstration of 2,3‐diamino‐2,3‐dideoxy‐d‐glucose, is discussed.
Study of the respiratory chain in Micrococcus luteus (lysodeikticus) by electron‐spin‐resonance spectroscopy
Authors: CROWE Brian A. , OWEN Peter , CAMMACK Richard
Published: March 2005

Low‐temperature electron spin resonance spectroscopy was used to investigate the redox centres of Micrococcus luteus membranes. Three different types of iron‐sulphur centres were distinguished. Two of these, a [4Fe‐4S]3+‐type cluster giving rise to a signal at g= 2.01 in the oxidized state and a [2Fe–2S] cluster with a spectrum at g= 2.03 and 1.93 in the reduced state, were attributable to succinate dehydrogenase. Another, generating signals in the reduced state at g= 2.027, 1.90 and 1.78 was identified as a ‘Rieske’ iron‐sulphur centre. This latter cluster had a mid‐point potential (pH 7.0) of + 130 mV. In addition, signals characteristic of high‐spin ferric haem (g= 6.20), low‐spin ferric haem (g= 3.67, 3.36 and 3.01) and Cu2+ (g= 2.18 and 2.02) were also detected. The ferric‐haem features, together with the Cu2+ and ‘Rieske’ centres, were enriched in membrane residues insoluble in Triton X‐100, which are known from difference spectroscopy to contain cytochromes b‐560, c‐550 and a‐601 (aa3 oxidase). The signals demonstrated by electron spin resonance for M. luteus membranes showed marked similarities to those documented for the complexes II, III, and IV of mitochondria. However, signals analogous to complex I (NADH‐ubiquinone reductase) could not be demonstrated for M. luteus membranes.
Characterization of succinate dehydrogenase from Micrococcus luteus (lysodeikticus) by electron‐spin‐resonance spectroscopy
Authors: CROWE Brian A. , OWEN Peter , PATIL Daulat S. , CAMMACK Richard
Published: March 2005

Low‐temperature electron spin resonance spectroscopy has been used to study the biophysical properties of succinate dehydrogenase from the gram‐positive bacterium Micrococcus luteus. The paramagnetic redox centres of the enzyme were identified in a succinate‐dehydrogenase–antigen complex, which had been purified with the aid of monospecific serum from membranes solubilized with Triton X‐100. The centres were characterized in further detail using the membrane‐bound and Triton‐solubilized forms of the enzyme. These studies distinguished two types of iron‐sulphur centres, viz. a [4Fe‐4S]3+ cluster displaying a narrow signal at g= 2.01 in the oxidized state (conventionally termed centre S‐3) and a [2Fe–2S] cluster with an axial signal at g= 2.03 and 1.93 in the reduced state (conventionally termed centre S‐1).

Centre S‐3 had a mid‐point redox potential of + 10 mV, a comparatively low value for this type of cluster. The behaviour of the g= 1.93 signal of centre S‐1 was a complex function of the redox potential, microwave power and temperature of measurement. When measured at low power (i.e. non‐saturating conditions), the intensities observed for the g= 1.93 signal poised at various critical potentials in the redox titration were similar. However, the corresponding intensities differed markedly at high power, where conditions were saturating. It is proposed that under saturating conditions the spin‐lattice relaxation of the [2Fe–2S] cluster S‐1 (mid‐point potential +70 mV) is enhanced by centre S‐3 between the potential range +10 –+ 70 mV and by an ESR‐silent centre, termed centre S‐2, with a mid‐point potential of −295 mV.
The primary structure of turkey muscle acylphosphatase
Authors: CAMICI Guido , MANAO Giampaolo , CAPPUGI Gianni , BERTI Andrea , STEFANI Massimo , LIGURI Gianfranco , RAMPONI Giampietro
Published: March 2005

The complete primary structure of turkey muscle acylphosphatase has been determined. The sequence was derived from peptides obtained by digestion of the carboxymethylated protein with pepsin and thermolysin and by subdigestion of some of the cyanogen bromide fragments with trypsin and Staphylococcus aureus protease.

Peptides were purified by preparative finger prints and/or preparative high‐performance liquid chromatography. Sequencing of the various peptides was achieved by manual Edman degradation and by time‐course analysis of amino acids released by carboxypeptidases. The amino‐terminal blocking group (acetyl) was determined by fast atom bombardment mass spectrometry.

This sequence was compared with that of horse muscle enzyme determined previously.
Purification and characterization of various esterases from rat liver
Authors: ROBBI Mariette , BEAUFAY Henri
Published: March 2005

The major rat liver microsomal esterases acting on o‐nitrophenylacetate with isoelectric points 5.0, 5.5, 6.1 and 6.4 were resolved by isoelectric focusing. Molecular weights were determined by sedimentation analysis in isokinetic gradients of sucrose and, after purification, in sodium dodecyl sulphate polyacrylamide gel electrophoresis. Their subunit molecular weights were between 57000 and 60 000. They behaved as monomers except the pI‐6.1 enzyme which behaved as a trimer. Esterases of pI 5.0, pI 6.1 and pI 6.4 behaved like glycoproteins of the polymannose type in the presence of 125I‐labelled concanavalin A. Preparations of the pI‐5.0 enzyme contained two esterases of highly homologous structure. Antibodies directed against this preparation did not inhibit but precipitated pI‐5.0 esterase activity quantitatively. They did not react with the pI‐6.1 and pI‐6.4 esterases but precipitated several non‐immunologically related esterases. Two of these enzymes were inducible by phenobarbital. Total activity was very low in 3‐day‐old animals. Individual esterase activities rose at different rates during development; the enzyme focusing near pI 5.0 was about three times more active in adult females than in males. All microsomal esterases are located on the luminal side of the endoplasmic reticulum.
Binding sites for monoclonal antibodies and for mRNPs on SV40 large T‐antigen determined with a cleavage map
Authors: SCHWYZER Martin , TAI Yung , STUDER Erwin , MICHEL Marcel R.
Published: March 2005

Immune complexes of simian virus 40 large T‐antigen with monoclonal papovavirus protein antibodies PAb 416, PAb 402, or PAb 423 were bound to protein‐A – Sepharose and then cleaved into discrete fragments by limited tryptic proteolysis. PAb 402 protected a specific cleavage site, located approximately within amino acid residues 450–500, from tryptic proteolysis; PAb 423 protected another site within residues 675–699. As shown by immunoblotting, 125I‐labeled PAb 416 was bound to a 17‐kDa N‐terminal fragment of large T‐antigen (amino acid residues 1‐130), and PAb 423 was bound to several overlapping fragments derived from the C terminus of large T‐antigen. These monoclonal antibodies were then used as accessibility probes to study the interaction of mRNPs with cytoplasmic large T‐antigen. Whereas small T‐antigen and nuclear large T‐antigen were fully immunoreactive, cytoplasmic large T‐antigen reacted poorly with PAb 402 or polyclonal antibodies unless the mRNP moiety was removed by treatment with EDTA/RNase A. In contrast, mRNP/T‐antigen complexes were fully immunoreactive with PAb 416 or PAb 423 and did not require treatment with EDTA/RNase A. The results suggest that the binding site of PAb 402 is blocked due to the interaction with mRNPs whereas the N‐terminal binding site of PAb 416 and the C‐terminal binding site of PAb 423 remain accessible to antibodies.
The role of proteolytic enzymes derived from crude bacterial collagenase in the liberation of hepatocytes from rat liver
Authors: HATTON Mark W. C. , BERRY Leslie R. , KRESTYNSKI Fred , SWEENEY George D. , REGOECZI Erwin
Published: March 2005

Crude bacterial collagenase was chromatographed on DEAE‐cellulose to yield three peaks with proteolytic activity: an arginine esterase (DEAE‐1), gelatinase (DEAE‐2) and a caseinolytic activity (DEAE‐3). The arginine esterase and gelatinase activity fractions were slightly contaminated with each other but neither possessed caseinolytic activity; the caseinolytic fraction was devoid of arginine esterase and gelatinase activities. In addition, crude collagenase was fractionated by ZnII‐affinity chromatography to produce a gelatinase peak (ZnII peak 1), which was free from arginine esterase and caseinolytic activities. The four fractions were compared to crude collagenase in their ability to liberate rat hepatocytes by using either liver slices or a standard perfusion technique. Compared to crude collagenase (0.05–0.1% w/v), which produced 70–80% liver digestion with approximately 80% cell viability, digestion with equivalent quantities of the isolated enzymic activities was relatively poor. Gelatinase activity (ZnII peak 1) was wholly ineffective and DEAE‐1 and DEAE‐2 each possessed only slight digestive properties. Hepatocyte liberation by the caseinolytic activity, DEAE‐3, was partially successful (30–40% digestion, 25–30% viability) but only a portion of liver tissue was digested regardless of the quantity of DEAE‐3 used. However, by mixing certain fractions before perfusion two gelatinase‐dependent, cell‐releasing mechanisms were identified: (a) DEAE‐3 with ZnII peak 1 and (b) DEAE‐1 mixed with either DEAE‐2 or ZnII peak 1. Each system compared creditably with the digestive properties of an equivalent activity of crude collagenase. At present we are attempting to determine any differences between hepatocytes produced by the two enzymic mechanisms.
Selective transcription of a cloned cauliflower mosaic virus DNA fragment in vitro by soybean RNA polymerase II in the presence of dinucleotide primers
Authors: COOKE Richard M. , PENON Paul , GOT Claude , MIASSOD Raymond
Published: March 2005

Transcription of a cloned cauliflower mosaic virus (CaMV) DNA fragment (plasmid pCa8) was studied at a low enzyme: DNA ratio. Preincubation with purine nucleoside triphosphates leads to essentially random transcription, while in the presence of a dinucleoside monophosphate and a purine nucleoside triphosphate in the preincubation medium certain combinations prime preferential transcription of the eucaryotic moiety of the chimeric plasmid. Characterisation of transcription primed by the most efficient combination, ApG + ATP, shows that a low enzyme: DNA ratio is absolutely essential for selective initiation. Interestingly the presence of the eucaryotic insertion is essential for the transcription of vector sequences. Analysis of RNA primed by ApG + ATP and of short chains synthesised in the presence of the GTP analogue 3′‐OMeGTP shows a high degree of selectivity of transcription initiation sites. Hybridisation of primed RNA to restriction fragments of pCa 8 shows that initiation occurs within a limited region of the inserted CaMV fragment.
o‐Phthalaldehyde, a fluorescence probe of aldolase active site
Authors: PALCZEWSKI Krzysztof , HARGRAVE Paul A. , KOCHMAN Marian
Published: March 2005

Conditions were determined in which approximately one mole of o‐phthalaldehyde reacts with one mole of aldolase subunit yielding a stable fluorescent isoindole derivative. During this chemical modification, a linear relationship was observed between the enzyme inactivation and absorbance change (337 nm) or fluorescence change (λem 420 nm, and λex 338 nm) characteristic for isoindole ring formation. The reaction follows second‐order kinetics, k= 1.1 × 103 M−1 s−1, in 50 mM borate buffer, pH 8.4 at 25°C. The modification of aldolase results in loss of approximately one ‐SH group per protein subunit. The enzyme is protected against modification by substrates and competitive inhibitors. Essentially no isoindole derivative is formed when the glycerol‐1‐phosphate‐lysyl derivative of aldolase is used for modification studies. It is concluded that aldolase modification occurs at the activesite region. Isolation of cross‐linked peptides suggests that Lys‐227 and Cys‐336 are involved in formation of the isoindole derivative. This result supports Cys‐336 as the active‐site cysteine necessary for aldolase catalytic activity.

Fluorescence studies have shown that the isoindole group linked to aldolase has its λmax,em markedly shifted toward shorter wavelength in comparison to the fluorescence of free isoindole derivatives in aqueous solution. In model studies a linear relationship between λmax, em of 1‐(β‐hydroxyethylthio)‐2−‐β‐hydroxyethylisoindole and the solvent polarity or acidity was observed. The results of the studies suggest that the microenvironment of the cleft in aldolase which binds isoindole appears to be of low acidity and low polarity. The apparent low polarity experienced by the isoindole probe may be due to its location in an actual low‐polarity portion of the active site, or may be due to non‐relaxing surroundings of the probe.
Nidogen: a new, self‐aggregating basement membrane protein
Authors: TIMPL Rupert , DZIADEK Marie , FUJIWARA Sakuhei , NOWACK Hans , WICK Georg
Published: March 2005

Nidogen was purified from a mouse tumor basement membrane where it accounted for 2–3 % of the total proteins. It was isolated as two forms (A and B) of a monomer (Mr=80000) each consisting of a single polypeptide chain folded into a globular head connected to a small tail. The B form of the monomer was shown to be capable of aggregating into a nest‐like structure (Mr250000). A smaller form (Mr=45000) was observed in some of the extracts. The amino acid composition of nidogen was different to that of other basement membrane proteins. It contained about 10% carbohydrate, with N‐linked and O‐linked oligosaccharide chains in similar proportions. Isoelectrofocussing demonstrated a limited heterogeneity of nidogen with pI in the range 6.5–7. Monomeric nidogen failed to interact with other basement membrane components and heparin. Aggregation could be induced by limited proteolysis and was reversed by detergents or high salt concentrations. Together with the observation that most of the nidogen could be solubilized only after destroying the collagenous matrix, the data indicate that aggregation of nidogen reflects an activity involved in matrix assembly.

Specific antibodies raised against nidogen did not distinguish between the monomeric and aggregated form of the protein but showed that the fragment was antigenically deficient. These antibodies did not cross‐react with collagen type IV, laminin, entactin and heparansulfate proteoglycan. Immunofluorescence staining and absorption studies demonstrated that nidogen is a common component of authentic basement membranes. Larger forms of nidogen (Mr about 100000 and 150000) were found in organ cultures of Reichert's membrane suggesting that it is synthesized in precursor forms.
Role of phosphate in the regulation of the Pasteur effect in Saccharomyces cerevisiae
Authors: LAGUNAS Rosario , GANCEDO Carlos
Published: March 2005

The occurrence of the Pasteur effect in Saccharomyces cerevisiae in several conditions has been examined. In these conditions measurements of a series of metabolites potentially involved in the regulation of the effect were performed. These included, among others, adenine nucleotides, citrate, fructose 2,6‐bisphosphate and phosphate. Only phosphate changed in a consistent way, increasing in anaerobiosis when the Pasteur effect occurred. It is concluded that, with the available data, only phosphate may be considered as a regulator of the Pasteur effect in this microorganism.
Purification and properties of a double‐stranded ribonuclease from the yeast Saccharomyces cerevisiae
Authors: MEAD David J. , OLIVER Stephen G.
Published: March 2005

A double‐stranded ribonuclease has been purified more than 90‐fold to near homogeneity from the yeast, Saccharomyces cerevisiae. The enzyme shows a high specificity for double‐stranded RNA as its substrate. It has a molecular weight of 27000 as determined by sodium dodecyl sulphate/polyacrylamide gel electrophoresis. The enzyme degrades dsRNA optimally at 30°C; it is stimulated by KCl and by the –SH reagent, dithiothreitol. In contrast to RNase III from Escherichia coli, the yeast enzyme is inhibited by divalent cations. Physiological studies have demonstrated that in vivo levels of the enzyme activity fell during the latter part of the exponential growth phase but rose during stationary phase. The specific activity of the enzyme in nitrogen‐starved yeast cells was 2–3‐fold higher than in non‐starved cells. The enzyme could be detected in yeast strains containing both, one or none of the species of cytoplasmic dsRNA (L and MdsRNAs) and may, therefore, have some wider role.
Proton‐NMR studies of the solution conformations of vitamin‐D‐induced bovine intestinal calcium‐binding protein
Authors: DALGARNO David C. , LEVINE Barry A. , WILLIAMS Robert J. P. , FULLMER Curtis S. , WASSERMAN Robert H.
Published: March 2005

1H NMR is used to study the solution structure of vitamin‐D‐induced bovine intestinal calcium‐binding protein. The study of the native protein is aided by the recently published crystal structure; it is shown that the conformations of the molecule in the crystal and in solution are very similar. The effect of pH and temperature on the native structure is described. The structure of the apo protein is then described, and the effect of pH and temperature on its fold is outlined. A comparison between apo and native protein folds is made which indicates that the folds are very similar. The two folds are related by a calcium titration, which indicates that the protein binds two calcium ions sequentially. Both steps in the Ca2+ titration occur under conditions of slow exchange (kex 80s−1). The effect of binding Ca2+ ions is to cause twisting motions of helices, with the helices acting as rods, relaying the conformational change induced by Ca2+ binding to the linker regions of the protein.
On the interactions of adenosine 3′,5′‐monophosphate with the components of protein kinase I
Authors: SWILLENS Stéphane
Published: March 2005

Cyclic‐AMP‐dependent protein kinase is activated through the dissociation of active catalytic subunits from a regulatory dimer. The regulatory subunit consists of four cyclic‐AMP‐binding sites and two binding sites for catalytic subunits.

Under well defined experimental conditions, protein kinase activation obeys apparent positive cooperativity and is linearly coupled to cyclic AMP binding.

The simulation of theoretical models is used for testing working hypotheses. Here we demonstrate that the proposed stoichiometry of protein kinase activation may account for the experimentally observed properties of the system. The restrictive conditions under which theory and experimental observations are compatible are: (1) functional dependence between the two monomers of the regulatory dimer, (2) the only complexes which can accumulate at equilibrium in the considered conditions are R2C2, (cyclic AMP)2R2C and (cyclic AMP)4R2 (where R and C are the regulatory and catalytic subunits of protein kinase).

An experimental procedure is proposed in order to check the validity of the theoretical predictions.

The determination of the sequence of events leading to activation or inactivation of protein kinase is discussed.
Purification and properties of a thiol protease from rat liver nuclei
Authors: MOTIZUKI Mituyoshi , TSURUGI Kunio , OGATA Kikuo
Published: March 2005

A thiol protease was purified about 800‐fold from the chromatin fraction of rat liver by employing Sepharose 6B gel filtration, chromatofocusing and Sephadex G‐100 gel filtration. It was nearly homogeneous on sodium dodecyl sulfate/polyacrylamide gel electrophoresis and its molecular weight was about 29000. The isoelectric point of the enzyme was 7.1. The pH optimum for degradation of 3H‐labelled ribosomal proteins was 4.5. It is noticeable that the maximal activity was shifted to pH 5.5 by DNA, and that 30–40 % of the maximal activity was observed at neutral pH in the presence of DNA. The activity was increased about twice by 2–4 mM dithiothreitol. The protease may be specific for the nuclei because it is different from all lysosomal thiol proteases ever known.
Deuterium isotope effects on the rates of steroid – glucocorticoid receptor interactions
Authors: ARÁNYI Péter
Published: March 2005

Rate constants of association and dissociation of several steroids to and from glucocorticoid receptor protein of chick thymus cytosol were determined in water and deuterium oxide. Substitution of deuterium for hydrogen did not influence association rate constants. Dissociation rate constants decreased about twofold in deuterium oxide in case of steroids containing an 11‐β‐hydroxyl group but remained unchanged if the steroid had no hydroxyl or had an α‐hydroxyl group at position 11. Presence of molybdate ions decreased but did not abolish the deuterium isotope effect. These findings suggest that the 11‐β‐hydroxyl group, known to be present in every optimal glucocorticoid agonist molecule, participates in a kinetically relevant hydrogen bond, and that this hydrogen bond may have a role in glucocorticoid action.
Adenosine(5′)tetraphospho(5′)adenosine‐binding protein of calf thymus
Authors: RAPAPORT Eliezer , FELDMAN Laurie
Published: March 2005

1. An adenosine(5′)tetraphospho(5′)adenosine (Ap4A) binding protein has been purified from calf thymus. The protein is comprised of a single polypeptide of Mr 54000 and is capable of high‐affinity (Kd= 13 μM) binding of Ap4A with great substrate specificity.

2. The Ap4A binding protein has been isolated in two forms: a “free”, or non‐polymerase‐bound, form which predominates, and a similar form which copurifies with DNA polymerase α, but which can be resolved from it.

3. The free form of Ap4A binding protein contains associated adenosine(5′)tetraphospho(5′)adenosine phosphohydrolase (Ap4Aase) activity, while the form resolved from DNA polymerase α contains no such activity.

4. The Ap4Aase activity, which catalyzes the phosphohydrolysis of Ap4A to ATP and AMP, is strongly inhibited by low levels (50–100 μM) of Zn2+ without any effect on the Ap4A binding protein activity.

5. This difference in associated Ap4Aase activity between free and polymerase‐bound forms of the protein, plus the copurification mentioned above, indicate a specific association between Ap4A binding protein and DNA polymerase α.
Incorporation of 1,N6‐ethenoadenosine into the 3′ terminus of tRNA using T4 RNA ligase
Authors: PAULSEN Harald , WINTERMEYER Wolfgang
Published: March 2005

A fluorescent derivative of tRNAfMet from Escherichia coli has been prepared which contains 1,N6‐ethenoadenosine (ɛA) in the place of adenosine 73, the fourth residue from the 3′ end. The labeled tRNA, tRNAfɛA73Met, is fully active with respect to aminoacylation, formylation and formylmethionyl transfer to puromycin. The preparation procedure entails the chemical removal of four nucleotides from the 3′ end of tRNAfMet, ligation of the truncated molecule with ɛA 3′,5′‐bisphosphate by use of T4 RNA ligase and repair of the C‐C‐A end with nucleotidyl transferase. The fluorescence of fMet‐tRNAfɛA73Met has been exploited for studying tRNA‐ribosome complexes. Upon binding the tRNA into the ribosomal P site, the fluorophor experiences a change of its molecular environment as indicated by an increased fluorescence intensity. On the other hand, iodide quenching experiments indicate that, in the complex, the fluorophor is not shielded against solvent access. The results suggest that (a) adenosine 73 is not involved in direct contacts with the ribosome and (b) the stacking of the 3′‐terminal A‐C‐C‐A sequence is changed upon binding to the ribosome.
Identification of the titrating group in the heme cavity of myoglobin
Authors: KRISHNAMOORTHI Ramaswamy , La MAR Gerd N.
Published: March 2005

The pH dependence of the proton NMR chemical shifts of met‐cyano and deoxy forms of native and reconstituted myoglobins reflects a structural transition in the heme pocket modulated by a single proton with pK 5.1–5.6. Comparison of this pH dependence of sperm whale and elephant myoglobin and that of the former protein reconstituted with esterified hemin eliminates both the distal histidine as well as the heme propionates as the titrating residue. Reconstitution of sperm whale met‐cyano myoglobin with hemin modified at the 2,4‐positions leads to a systematic variation in the pK for the structural transition, thus indicating the presence of a coupling between the titrating group and the heme π system. The results are consistent with histidine FG3 (His‐FG3) being the titrating group, and a donor‐acceptor π‐π interaction between its imidazole and the heme is proposed.
Evidence for an incomplete dolichyl‐phosphate pathway of lipoglycan formation in Volvox carteri f. nagariensis
Authors: MÜLLER Thomas , BAUSE Ernst , JAENICKE Lothar
Published: March 2005

Crude membrane fractions from Volvox carteri in the presence of detergent and metal complexing agent catalyze the transfer of glucose from dolichyl phosphate glucose to branched dolichyl diphosphate chitobiosyl penta‐mannoside Dol‐PP‐(GlcNAc)2‐(Man)5, a known intermediate of the lipid‐mediated pathway of N‐glycosylation of proteins, resulting in the formation of Dol‐PP‐(GlcNAc)2‐(Man)5‐(Glc)1. Under the various conditions tested, neither Dol‐P‐Man nor other known mannosyl donors of the nucleoside‐activated or lipid‐activated type can serve as donor molecules for the elongation of the lipid‐linked heptasaccharide. On the other hand, calf liver microsomes in similar experiments mannosylated the heptasaccharide further with Dol‐P‐Man up to a nonamannoside, Dol‐PP‐(GlcNAc)2‐(Man)9. A direct glucosylation of the acceptor, however, with Dol‐P‐Glc failed in this system.

The (GlcNAc)2‐(Man)5‐(Glc)1, obtained after mild acid hydrolysis of the above glycolipid is not significantly split by an unspecific α‐glucosidase from yeast. However, Volvox microsomes liberated most of the glucose indicating a specific glucosidase in the membranes of the alga. This enzyme does not act on (GlcNAc)2‐(Man)9‐(Glc)1, the usual protein‐linked carbohydrate intermediate of trimming processes of N‐glycosidic glycoproteins.

The data on glycolipid formation let us postulate that in Volvox the normal N‐glycosylation pathway differs from that found in higher plants and animals either by a lack of evolution or by mutation in the genes coding for the mannosyl transferases involved.
The biosynthesis of the ubiquinol‐cytochrome c reductase complex in yeast
Authors: DE HAAN Muus , VAN LOON Adolphus P. G. M. , KREIKE Jan , VAESSEN Ruud T. M. J. , GRIVELL Leslie A.
Published: March 2005

The nuclear gene coding for the imported 14‐kDa subunit of the ubiquinol–cytochrome c reductase of yeast mitochondria has been sequenced in an attempt to define regulatory and protein topogenic elements. The gene has a length of 381 base pairs and is potentially capable of encoding a polypeptide of 14561 Da. It is transcribed into a single low‐abundance RNA of 680 nucleotides whose 5′ and 3′ termini map, respectively, 30–35 nucleotides upstream and 180–190 nucleotides downstream of the initiator and termination codons. Consistent with the estimated low level of the mRNA, codon usage in the gene is not strongly biased and other features, characteristic of highly expressed genes in yeast, are absent. The 14‐kDa protein is predicted to be a predominantly hydrophilic protein, with only a single, short hydrophobic stretch located between positions 19–38. Comparison with other imported mitochondrial proteins so far sequenced has failed to reveal unifying features that might serve as targeting elements. Steady‐state levels of the 14‐kDa and 11‐kDa subunits are reduced in mit− mutants which synthesize truncated forms of apocytochrome b and in these, newly synthesized subunits exhibit a specifically increased turnover rate. We suggest that association of these two subunits with the complex may be mediated or enhanced by interaction with other subunits, in particular cytochrome b.
Studies on the dynamic syn‐anti equilibrium in purine nucleosides and nucleotides with the aid of 1H and 13C NMR spectroscopy
Authors: STOLARSKI Ryszard , HAGBERG Curt‐Eric , SHUGAR David
Published: March 2005

Analyses of 1H and 13C NMR spectra have been utilized to extend studies on the dynamic equilibrium syn–anti about the glycosidic bond of purine nucleosides and nucleotides. With the aid of chemically synthesized model analogous in fixed syn and anti conformations, and the introduction of appropriate corrections for the conformation of the exocyclic chain of the sugar moiety, it is possible to evaluate quantitatively the relative populations of the syn and anti conformers from the experimentally observed chemical shifts of H(2′) and C(2′). The resulting agreement between the data based on H(2′) chemical shifts with those deduced from C(2′) chemical shifts extends the validity of this procedure, and furnishes more accurate results than those previously based on uncorrected H(2′) chemical shifts alone. The overall findings are briefly compared with those derived from measurements of proton relaxation times and the Overhauser effect, as well as by X‐ray diffraction in the solid state. Attention is drawn to the potential utility of the results, including chemical shift data, in studies on interactions of nucleosides and nucleotides with appropriate enzyme systems.
Proteins from the prokaryotic nucleoid
Authors: PACI Maurizio , PON Cynthia L. , LOSSO M. Adele , GUALERZI Claudio
Published: March 2005

The 1H‐NMR spectra of the two Escherichia coli basic, low‐Mr (∼ 9000) DNA‐binding proteins NS1 and NS2 and of their native complex NS were studied at 400 MHz and a number of resonances and resonance peaks were assigned. As in the case of some eukaryotic histones, the presence of a large number of high‐field perturbed Phe resonances, several shielded and deshielded methyl resonances and backbone NH protons quite inaccessible to the solvent clearly indicate the existence of extensive tertiary and, even more so, quaternary structures involving hydrophobic interactions. These structures are lost upon heating, but readily reform upon cooling. Spectral differences between NS1, NS2 and NS and the greater thermal stability of NS indicate that molecules of the heterologous subunits (NS1 and NS2) aggregate (dimerize) preferentially in comparison to the self‐aggregation of the homologous subunits. Unlike those of the eukaryotic histones, the tertiary and quaternary structures of NS are insensitive to extensive variations of the ionic strength.
Induction of surface IgG receptors in cytomegalovirus‐infected human fibroblasts
Authors: FREY Jürgen , EINSFELDER Birgitt
Published: March 2005

Binding studies on diploid human fibroblasts with human immunoglobulin G (IgG) demonstrate the existence of a specific receptor for this class of immunoglobulin. The receptor preferentially binds aggregated human IgG and recognizes these complexes via the Fc portion of the molecule. Cytomegalovirus infection of diploid human fibroblasts results in a more than 100‐fold increase in the number of IgG‐receptors present on the cell surface. The binding of aggregated IgG by these newly expressed receptors exhibits the characteristics of the binding mediated by the receptors detectable in uninfected cells.
Isolation of two β‐xylosidase genes ofBacillus pumilusand comparison of their gene products
Authors: PANBANGRED Watanalai , KAWAGUCHI Osamu , TOMITA Tetsushi , SHINMYO Atsuhiko , OKADA Hirosuke
Published: March 2005

The chromosomal DNA fragments ofBacillus pumilusIPO, a potent xylan‐hydrolyzing bacterium, were ligated to a vector plasmid, pBR322, and used to transformEscherichia coliC600 cells. Two hybrid plasmids, pOXD28 and pOXN29, were found to enable the transformants to produce β‐xylosidase. The former was found to contain a 2.6‐MDaBglII fragment and the latter, a 7.7‐MDaPstI fragment, both coding β‐xylosidase, but xylanase is coded only on the latter hybrid plasmid. The DNAs inserted in both plasmids originated from theB. pumiluschromosome, but from different regions, as shown by Southern hybridization and the analysis of restriction fragments. β‐Xylosidases I and II, coded on pOXN29 and pOXD28 respectively, were purified to homogeneous preparations and compared. Both were dimer enzymes consisting of 65000–70000‐Da subunits. Specific activity and theKmvalue of β‐xylosidase I top‐nitrophenyl β‐D‐xyloside as substrate were respectively 100 and 1/40 times those of β‐xylosidase II. The mobilities of β‐xylosidases I and II on polyacrylamide gel electrophoresis were also different.

β‐Xylosidase I, the gene of which is located near the xylanase gene on pOXN29, can convert xylooligosaccharides to xylose, but β‐xylosidase II had little activity on xylobiose. These results suggest that β‐xylosidase I is the main enzyme for xylan hydrolysis inB. pumilus
1H NMR studies of calmodulin
Authors: DALGARNO David C. , KLEVIT Rachel E. , LEVINE Barry A. , WILLIAMS Robert J. P. , DOBROWOLSKI Zbigniew , DRABIKOWSKI Witold
Published: March 2005

Two tryptic fragments of the Ca2+‐binding protein calmodulin have been studied by high‐resolution 1H NMR. TR1C (residues 1–77) spans the first two domains of the protein and TR2C (residues 78–148) spans the second two domains. The spectra indicate that each of the two‐domain peptides assumes a conformation which is very close to that in the native protein. This characteristic holds both in the presence and in the absence of Ca2+ ions. Therefore, the resonance assignments obtained for the relatively simpler fragment spectra can be used to assign the spectrum of whole calmodulin. Analysis of the chemical shift patterns and nuclear Overhauser enhancement effects of several assigned resonances indicates that each half of calmodulin can be modelled after the two EF‐hand Ca2+‐binding proteins for which crystal structures are available, namely parvalbumin and intestinal Ca2+‐binding protein.
Brain pyridoxine‐5‐phosphate oxidase
Authors: CHURCHICH Jorge E.
Published: March 2005

Pyridoxine‐5‐phosphate oxidase has been purified 2250‐fold from pig brain by affinity chromatography. The enzyme of 60000 molecular weight is made up of two identical size subunits and binds 1 mol of FMN/mol dimer. 1 mol of the fluorescent inhibitor phospho‐pyridoxal oxime interacts with 1 mol of the dimeric protein to yield a dissociation constant KD= 0.2 μM.

Resolution of the holoenzyme into apoprotein and free FMN does not induce dissociation of the dimeric structure. The oxidase catalyzes the oxidation of the natural substrates pyridoxine 5‐phosphate and pyridoxamine 5‐phosphate, but phospho‐pyridoxyl derivatives of the aromatic carboxylic acids, p‐aminobenzoate and m‐amino‐benzoate, are also excellent substrates of the enzyme. Introduction of electron‐withdrawing substituents into the structure of benzene increases the Kcat values. A comparison of the Kcat values obtained with several synthetic substrates suggests that electron‐withdrawing substituents tend to stabilize carbanionic intermediates formed in the earlier stages of catalysis.
Enzyme‐linked immunosorbent assay for the neuropeptide ‘head activator’
Authors: SCHALLER H. Chica , BODENMÜLLER Heinz , ZACHMANN Beate , SCHILLING Elke
Published: March 2005

By exposing different sites of the ‘head activator’, different sets of antibodies were designed and produced which recognised either the amino or the carboxy terminus of the free ‘head activator’, which reacted with ‘head activator’ in a tissue‐fixed conformation, or which bound to the ‘head‐activator’ sequence, if it was part of a larger precursor‐like molecule. An enzyme‐linked immunosorbent assay (ELISA) was developed to characterise the antibodies and also to assay minute amounts of ‘head activator’ or ‘head‐activator’‐like immunoreactivities in animal or tissue extracts. A competitive ELISA is described which uses biotin‐avidin for enhancement. The assay is sensitive with an antibody specific for the amino terminus in the range of 0.5–50 fmol, with an antibody specific for the carboxy terminus in the range of 20–400 fmol. The ELISA specific for the amino terminus is 10‐times more sensitive than a radio‐immunoassay with tritiated ‘head activator’ [H. Bodenmüller and B. Zachmann (1983) FEBS Lett. 159, 237–240]. Previously no radioimmunoassay existed with specificity for the carboxy terminus.
Metabolic imbalance in a Saccharomyces cerevisiae mutant unable to grow on fermentable hexoses
Authors: ALONSO Alberto , PASCUAL Carlos , HERRERA Luis , GANCEDO Juana M. , GANCEDO Carlos
Published: March 2005

A mutant of Saccharomyces cerevisiae unable to grow on fermentable hexoses has been studied. The mutant grew normally on galactose or maltose. It was also able to grow on a medium containing glucose or fructose with a 25‐fold excess of d‐xylose. Assay of the glycolytic enzymes in vitro did not show differences between the parental and the mutant strains. Upon addition of fructose, metabolites up to triose phosphates accumulated and the ATP dropped to low levels. It is proposed that an imbalance between the initial and final segments of glycolysis that depletes the cell of ATP produces the observed phenotype.
Modification of the intramolecular turnover of terminal carbohydrates of dipeptidylaminopeptidase IV isolated from rat‐liver plasma membrane during liver regeneration
Authors: KREISEL Wolfgang , REUTTER Werner , GEROK Wolfgang
Published: March 2005

An intramolecular turnover of the terminal carbohydrates l‐fucose, N‐acetylneuraminic acid and d‐galactose is a characteristic property of several liver plasma membrane glycoproteins, first demonstrated for dipeptidylaminopeptidase IV (EC 3.4.14.5., DPP IV). The core carbohydrates d‐mannose and N‐acetyl‐d‐glucosamine turn over like the polypeptide chain. The ratio of apparent half‐lives of l‐fucose and l‐methionine of DPP IV is shifted from 0.17 in normal liver to 0.60 in regenerating liver. The ratio of half‐lives of N‐acetylneuraminic acid and l‐methionine is only slightly changed from 0.43 in normal liver to 0.61 in regenerating liver. The ratio of apparent half‐lives of d‐mannose and l‐methionine amounts to 0.80 in normal liver and 0.71 after partial hepatectomy. From this a drastic reduction of the intramolecular turnover of l‐fucose on plasma membrane DPP IV in regenerating liver can be derived. The intramolecular N‐acetylneuraminic acid turnover is affected to only a minor extent. d‐Mannose turns over like the polypeptide in both normal and regenerating liver. The intramolecular l‐fucose turnover may be involved in membrane glycoprotein recycling, which presumably is altered in regenerating liver. Additionally, l‐fucose could regulate the rate of degradation of DPP IV, since core‐fucosylated glycoproteins appear to be resistant to mammalian endo‐N‐acetylglucosaminidase.
Characterization of peroxisomal and mitochondrial carnitine acetyltransferases purified from alkane‐grown Candida tropicalis
Authors: UEDA Mitsuyoshi , TANAKA Atsuo , FUKUI Saburo
Published: March 2005

Properties of peroxisomal and mitochondrial carnitine acetyltransferases purified from an alkane‐grown yeast, Candida tropicalis, were compared each other. The molecular weight of both enzymes was estimated to be about 420000 by analytical ultracentrifugation and gel filtration chromatography with Sepharose 6B. However, each enzyme gave two subunits on the polyacrylamide slab gel electrophoresis in the presence of sodium dodecyl sulfate: the peroxisomal enzyme (64000 and 57000) and the mitochondrial enzyme (64000 and 52000). The subcellularly distinct enzymes gave a similar amino acid composition except for the contents of some amino acids: glycine, valine, glutamic acid and aspartic acid. Their isoelectric point was somewhat different: 5.11 for the peroxisomal enzyme and 5.22 for the mitochondrial enzyme. Both enzymes had the same amino‐terminal residue (glutamic acid or glutamine) and the heat stability, and was indistinguishable immunochemically. These results suggest that peroxisomal and mitochondrial carnitine acetyltransferases of C. tropicalis cells may be products of the same nuclear gene. Differences in the molecular weight of the subunits of the enzymes would result from modification or processing of the common protein in the step of distribution to the respective organelles, that is, so‐called post‐translational modification.
Oxidation of glutathione during hydroperoxide metabolism
Authors: EKLÖW Lena , MOLDÉUS Peter , ORRENIUS Sten
Published: March 2005

In the present study freshly isolated rat hepatocytes treated with the glutathione reductase inhibitor BCNU (1,3‐bis(2‐chloroethyl)‐1‐nitrosourea) were used to investigate the metabolism of tert‐butyl hydroperoxide and of hydrogen peroxide formed in different intracellular compartments. Glycolate, benzylamine and hexobarbital were used to stimulate H2O2 production in the peroxisomal, mitochondrial and endoplasmic reticular/cytosolic compartments, respectively. Our results support previous findings that catabolism of H2O2 formed in the mitochondrial and cytosolic compartments occurs predominantly by the glutathione peroxidase system, whereas H2O2 generated within the peroxisomes is metabolized by catalase. They further reveal that the capacity of uninhibited glutathione reductase to reduce glutathione disulfide, formed during hydroperoxide metabolism by glutathione peroxidase, is high and that a decreased NADPH/NADP+ redox level, rather than insufficient reductase activity, is responsible for the accumulation and subsequent excretion of cellular glutathione disulfide observed during hydroperoxide metabolism. Finally, our results demonstrate that H2O2 generated during cytochrome P‐450‐mediated drug oxidation is metabolized primarily by the glutathione peroxidase system.
Proteolytic degradation of vimentin and glial fibrillary acidic protein in rat astrocytes in primary culture
Authors: CIESIELSKI‐TRESKA Jaroslava , GOETSCHY Jean‐François , AUNIS Dominique
Published: March 2005

Vimentin and glial fibrillary acidic (GFA) protein in rat astrocytes in primary culture were identified with their respective specific antisera on electroblots of one‐dimensional and two‐dimensional polyacrylamide gels. Each antiserum revealed respectively vimentin (58 kDa, pI 5.3), GFA protein (50 kDa, pI 6–5.8) and several more acidic, lower molecular mass vimentin‐immunoreactive and GFA protein‐immunoreactive polypeptides. These were more apparent when astrocyte proteins were extracted in the absence of EGTA and in the presence of Ca2+ ions suggesting that the lower immunoreactive polypeptides result from a Ca2+‐sensitive proteolytic degradation of vimentin and GFA protein. The extent of proteolysis of GFA protein was higher when the differentiation of astrocytes was induced with 1,N6‐dibutyryl‐adenosine 3′,5′‐monophosphate (Bt2cAMP). In contrast, the proteolysis of vimentin was not affected by the state of differentiation of astrocytes. The results indicate the existence of at least two distinct degradative pathways for intermediate filament proteins in astrocytes in primary culture and suggest that in differentiated astrocytes the activity of proteinase(s) involved in the degradation of GFA protein increases.
Elucidation of the structure of methanopterin, a coenzyme from Methanobacterium thermoautotrophicum, using two‐dimensional nuclear‐magnetic‐resonance techniques
Authors: VAN BEELEN Patrick , STASSEN Alphons P. M. , BOSCH Johannes W. G. , VOGELS Godfried D. , GUIJT Willem , HAASNOOT Cornelis A. G.
Published: March 2005

Methanopterin is a coenzyme involved in methanogenesis. From 2 kg wet cells of Methanobacterium thermoautotrophicum about 35 μmol methanopterin were isolated. The structure of this compound was elucidated by various two‐dimensional nuclear‐magnetic‐resonance techniques. Methanopterin was identified as N‐[1′‐(2″‐amino‐4″‐hydroxy‐7″‐ methyl‐6″‐ pteridinyl)ethyl]‐4‐[2′,3′,4′,5′‐ tetrahydroxypent‐1′‐ yl (5′→1″) O‐α‐ribofuranosyl‐5″‐phosphoric acid] aniline, in which the phosphate group is esterified with α‐hydroxyglutaric acid. The molecular formula of the sodium salt of methanopterin at pH 7.0 is C30H38O16N6PNa3·xH2O (x is about 4). The anhydrous sodium salt of methanopterin has a molecular mass of 838.60 Da and the molar absorption coefficient at 342 nm is 7.4 mM−1 cm−1 at pH 7.0.
Chemical modification of the F0 part of the ATP synthase (F1F0) from Escherichia coli
Authors: STEFFENS Karl , SCHNEIDER Erwin , HERKENHOFF Brigitte , SCHMID Roland , ALTENDORF Karlheinz
Published: March 2005

The purified F0 part of the ATP synthase complex from Escherichia coli was incorporated into liposomes and chemically modified by various reagents. The modified F0‐liposomes were assayed for H+ uptake and, after reconstitution with F1, for total and dicyclohexylcarbodiimide‐sensitive ATPase activity.

The water‐soluble carbodiimide, 1‐ethyl‐3‐(‐3‐dimethylaminopropyl)carbodiimide methiodide, (1.2 mM), inhibited H+ uptake to a great extent. Binding of F1 was almost unaffected, but the hydrolysis of ATP was uncoupled from H+ transport. This is reflected by the inhibition of dicyclohexylcarbodiimide‐sensitive ATPase activity. Woodward's reagent K, N‐ethyl‐5‐phenylisoxazolium‐3′‐sulfonate, inhibited both H+ uptake and total ATPase activity.

Modification of arginine residues by phenylglyoxal (20 mM) was followed by inhibition of the F1 binding activity by 80% of the control. H+ translocation was reduced to 70%.

Diethylpyrocarbonate (3 mM) exhibited a strong inhibiting effect on H+ uptake but not on F1 binding.

Modification of tyrosine (by tetranitromethane) as well as lysine residues (by succinic anhydride) did not affect F0 functions.

From the data presented we conclude that carboxyl‐groups, different from the dicyclohexylcarbodiimide‐binding site, are involved in H+ translocation through F0 and, in part, in the functional binding of F1. Furthermore, for the latter function, also arginine residues seem to be important. The role of histidine residues remains unclear at present.