bluuebunny/crossref_metadata_2025_split huggingface dataset:
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.
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.
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.
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.
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.
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
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.
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.
Accounting for the ATP‐consuming processes in rabbit reticulocytes
Authors: SIEMS Werner , DUBIEL Wolfgang , DUMDEY Renate , MÜLLER Marianne , RAPOPORT Samuel Mitja 
Published: March 2005

The report deals with a detailed balance of ATP production and consumption of the rabbit reticulocyte.1. The sum‐total of ATP produced amounts to 135mmol · l−1· h−1.2. About 70% of the ATP consumption has been accounted for by specific processes. The main contributing processes are the globin synthesis with about 28%, the Na+, K+‐ATPase with 23% and the proteolysis with more than 15%. 30% of ATP consumption has not been accounted for.3. Cycloheximide (20 μM) leads to a dissociation between synthesis and degradation of proteins, which argues against any obligatory connection between these processes.4. More than 90% of the lysine liberated from mitochondria by proteolysis were reutilized for the globin synthesis demonstrating the high nitrogen economy of reticulocytes.5. Each of the ATP‐consuming processes studied appears to control ATP production in an independent manner without competition with each other.
Raman spectroscopy and normal vibrations of peptides
Authors: LAGANT Philippe , VERGOTEN Gérard , FLEURY Guy , LOUCHEUX‐LEFEBVRE Marie‐H. 
Published: March 2005

Raman and infrared spectra have been recorded on the peptide Gly‐lPro‐Gly‐Gly in its zwitterionic form and on its protected homolog Z‐Gly‐lPro‐Gly‐Gly‐OMe (Z=N‐benzyloxycarbonyl and OMe = methyl ester). Both peptides adopt a type‐II β‐turn conformation into appropriate solvents, that is in dimethylsulfoxide and chloroform respectively.A normal coordinate treatment has been performed on Gly‐lPro‐Gly‐Gly using a modified Urey‐Bradley force field and the characteristic normal modes of vibrations. Their corresponding frequencies have been extracted from experimental investigations.
Acyltransferase‐catalyzed cleavage of arachidonic acid from phospholipids and transfer to lysophosphatides in macrophages derived from bone marrow Comparison of different donor‐ and acceptor substrate combinations
Authors: FLESCH Inge , ECKER Barbara , FERBER Ernst 
Published: March 2005

In a previous paper it was shown that in prelabeled murine thymocytes a direct CoA‐mediated transfer of arachidonic acid from phosphatidylcholine to lysophosphatidylethanolamine occurs which does not involve the intermediate formation of free fatty acid. The transfer is ATP‐independent and is catalyzed by the acyl‐CoA: lysophosphatide acyltransferase operating in reverse. In prelabeled thymocytes phosphatidylcholine was the only arachidonoyl donor and lysophosphatidylethanolamine the only lysoacceptor.In murine bone‐marrow‐derived macrophages a series of CoA‐mediated transfer reactions were detected leading to a redistribution of arachidonic acid between phospholipids. Using exogenous substrates a bidirectional transfer from 1‐acyl‐2‐arachidonoylglycerophosphocholine to lysophosphatidylethanolamine occurs. An unidirectional transfer from l‐acyl‐2‐arachidonoylglycerophosphoinositol to lysophosphatidylcholine and from 1‐acyl‐2‐arachidonoylglycerophosphoinositol to lysophosphatidylethanolamine was observed. Plasmalogenic lysoacceptors generally have a weaker acceptor capacity than the correspondent acyllysophospholipid. In macrophages the CoA‐mediated transfer of arachidonoyl moieties is independent of ATP and Mg2+ and is totally inhibited by sodium cholate, indicating that it is catalyzed by the acyl‐CoA: lysophosphatide acyltransferase.
Ribosomal RNA‐protein cross‐links, induced by γ‐irradiation of 40S and 60S ribosomal subunits of L cells
Authors: CAZILLIS Michèle , GIOCANTI Nicole , HOUSSAIS Jean‐François , EKERT Bernard 
Published: March 2005

The 40S and 60S ribosomal subunits of L cells are γ‐irradiated in the absence of oxygen at low radiation doses to keep the integrity of the ribosomal structure. We show that under these experimental conditions, specific cross‐links are induced in situ between rRNA and ribosomal proteins due to close contact between their reactive groups.We found that about 15 proteins are cross‐linked to the 28S RNA. Most of them belong to the core proteins of the 60S ribosomal subunits. A few high‐molecular mass proteins which might be factors are also bound to 28S RNA.Between 8 and 11 proteins are covalently linked to 18S RNA; 8 of these have been previously described as transferable proteins from one subunit to the other. Only 3 are core proteins of the small subunit.The contribution of these results to the study of the three‐dimensional ribosomal structure is also discussed.
Comparison of the tryptic digestion pattern of subfragments 1 from V1 and V3 rat cardiac isomyosins
Authors: LOMPRÉ Anne‐Marie , HAN Kia‐Ki , BOUVERET Pierre , RICHARD Claude , SCHWARTZ Ketty 
Published: March 2005

The limited tryptic digestion patterns of the chymotryptic subfragment 1 (S1) of the two rat ventricular isomyosins V1 and V3, were compared under several conditions. Pure S1V1 was obtained from 3‐week‐old rats and pure S1V3 from adult rats 6 weeks after hypophysectomy. To localize the sites of trypsin susceptibility and to determine the distribution of the peptides along the S1 molecule, we used, as a probe, antibodies raised against a pig cardiac 29‐kDa peptide. We demonstrate that this peptide contains the N‐acetyl group located on the N‐terminal part of the cardiac myosin molecule.In S1V1 we observed two major sites of proteolysis, independently of the digestion conditions: they are located at 27kDa and 80kDa from the N terminus as in skeletal muscle S1. S1V3 appears much more sensitive to the proteolysis conditions: at least two additional sites of cleavage are present in the 50‐kDa peptide when digested at pH 8.0. Decrease in the pH from 8.0 to 7.0 or the presence of Mg‐ATP have no effect on the digestion of S1V1 while these ambient factors protect the 50‐kDa peptide of S1V3 from breakdown. We conclude that the 50‐kDa peptide is a variable portion of the myosin molecule, the conformation of which is sensitive to ambient factors such as the pH or the presence of nucleotides.
Use of a monoclonal antibody to distinguish between precursor and mature forms of human lysosomal α‐glucosidase
Authors: OUDE ELFERINK Ronald P. J. , STRIJLAND Anneke , SURYA Inge , BROUWER‐KELDER Elisabeth M. , KROOS Marian , HILKENS John , HILGERS Jo , REUSER Arnold J. J. , TAGER Joseph M. 
Published: March 2005

The maturation of lysosomal α‐glucosidase in cultured human skin fibroblasts was studied using a monoclonal antibody that distinguishes between the precursor and mature forms of the enzyme.1. Monoclonal antibodies against α‐glucosidase isolated from placenta were produced by the hybridoma technique [Hilkens et al. (1981) Biochim. Biophys. Acta 678, 7–11]. One of these monoclonal antibodies, that synthesized by clone 43G8, reacts with the mature forms, but not with the precursor form of α‐glucosidase isolated from urine.2. By means of pulse‐labelling studies, it could be shown that monoclonal antibody 43G8 does not react with either the intracellular or the secreted precursor of α‐glucosidase from cultured human skin fibroblasts. However, the antibody does react with the intermediate and mature forms of α‐glucosidase.3. Endocytosis of the precursor of α‐glucosidase from urine by fibroblasts is followed by its conversion to a form with lower molecular mass.4. After endocytosis urinary precursor α‐glucosidase is converted to a form that binds to monoclonal antibody 43G8. The t1/2 for this conversion is 2 h. The conversion is inhibited by addition of leupeptin to the culture medium.5. It is concluded that a thiol proteinase is involved in the maturation of α‐glucosidase in fibroblasts and the appearance of the antigenic determinant for 43G8.