bluuebunny/crossref_metadata_2025 huggingface dataset:
Kinetics of binding of lucensomycin to natural and artificial membranes
Authors: CAPUOZZO Elisabetta , SALERNO Costantino , STROM Roberto , CRIFÒ Carlo
Published: March 2005

The binding of the polyenic antibiotic lucensomycin to native or modified human erythrocyte ghosts and to model membranes has been studied by monitoring the absorbance variations of the polyene at 320 nm. The nonsteroidal components of the membranes (such as proteins and phospholipids) seem to affect the rate of the individual reaction steps leading to the formation of cholesterol‐lucensomycin complexes rather than the ratio among these heterologous aggregates at equilibrium.
Crystallization of and X‐ray investigations on glucose dehydrogenase from Bacillus megaterium
Authors: PAL Gour Pada , JANY Klaus‐Dieter , SAENGER Wolfram
Published: March 2005

The tetrameric glucose dehydrogenase from Bacillus megaterium M1286 belongs to the ‘short' family of dehydrogenases with 262 amino acids per subunit (Mr∼ 30000), and does not require Zn2+ for enzymatic action. It was crystallized as complex with its coenzyme NAD from a 1–2% protein solution by the batch method using ammonium sulfate as precipitant at pH 6.5. Crystals appeared within two days as clusters of large plates with maximum dimensions of 2 mm, which diffract X‐rays to a resolution of at least 0.2 nm. The space group is orthorhombic P212121, unit‐cell dimensions are a= 15.03 nm, b= 10.42 nm and c= 6.74 nm. Assuming one molecule (∼ 120 kDa) per asymmetric unit the VM value is 0.0022 nm3/Da and the solvent content of the crystals is 45% based on a partial specific volume for the protein of 0.723 ml/g. The crystallization was further improved by using the microdialysis technique where instead of clusters, single crystals appeared within 7 days.
Structure of the major triglycosyl phenol‐phthiocerol of Mycobacterium tuberculosis (strain Canetti)
Authors: DAFFÉ Mamadou , LACAVE Charlotte , LANÉELLE Marie‐Antoinette , LANÉELLE Gilbert
Published: March 2005

Phenol‐phthiocerol glycolipids have been found previously in Mycobacterium leprae, M. kansasii, M. bovis and M. marinum, but not in M. tuberculosis. A search for such glycolipids in this latter species showed that the Canetti strains of M. tuberculosis synthesize a major triglycosyl phenol‐phthiocerol, accompanied by minor amounts of other glycolipids with a similar aglycone moiety.

The triglycoside moiety has the following structure: 2,3,4‐tri‐O‐methyl L‐fucopyranosyl(α1→3)L‐rhamno‐pyranosyl(α1→3)2‐O‐methyl L‐rhamnopyranosyl(α1 –.

The aglycone moiety consists in phenol‐phthiocerol (two homologs). Its two secondary alcohol functions are esterified by mycocerosic acids (homologs with 26–32 carbon atoms and with 2–4 methyl branches). The proposed structure differs on several points from the M. leprae glycolipids, but presents some analogy with the major glycolipid of M. kansasii.

A minor monoglycosyl phenol‐phthiocerol was also studied. Its overall structure is very similar to that of M. bovis, with 2‐O‐methyl rhamnose as sugar moiety.
Multiple mRNA species code for the catalytic subunit of the cAMP‐dependent protein kinase from LLC‐PK1 cells
Authors: ADAVANI S. Rao , SCHWARZ Manuela , SHOWERS Mark O. , MAURER Richard A. , HEMMINGS Brian A.
Published: March 2005

We present evidence for the existence of two forms of the catalytic (C) subunit of the cAMP‐dependent protein kinase. A λgt‐11 cDNA library constructed from poly(A)‐rich RNA from the porcine kidney cell line, LLC‐PK1, was screened using a 1.5‐kb EcoRI fragment from a bovine cDNA for the C subunit. Two independent classes of cDNAs were identified on the basis of partial restriction map and sequence data. These two cDNAs, λCAT4 and λCAT3, apparently encode two forms of C subunit designated Cα and Cβ, respectively. The nucleotide sequence of the Cα and Cβ cDNAs revealed differences in the coding region and particularly in the 3′ untranslated region. However, the deduced amino acid sequences of Cα and Cβ subunits were 96% homologous to the sequences so far determined. Specific probes from the 3′ coding region of the two cDNA species were used to investigate C subunit mRNA expression in LLC‐PK1 cells. Northern analysis showed a major mRNA species of 2.8 kb with the Cα probe while the Cβ probe detected two mRNA species of 5.0 kb and 3.8 kb. These data were supported by genomic blot analysis which showed distinct hybridization patterns with either the Cα or Cβ probes. All the available evidence suggests that at least two distinct genes encode the C subunit which are expressed in LLC‐PK1 cells.
Accessibility of the globular domain of histones H1 and H5 to antibodies upon folding of chromatin
Authors: RUSSANOVA Valya R. , DIMITROV Stephan I. , MAKAROV Vladimir L. , PASHEV Iliya G.
Published: March 2005

Antibodies to the globular domain of histones H1 and H5 were purified by affinity chromatography and used to study the accessibility of this region of H1 and H5 in folded and unfolded rat liver and hen erythrocyte chromatin respectively. The different conformations of the chromatin filament were induced by varying the ionic strength from 1 mM to 80 mM NaCl and maintained by fixation with glutaraldehyde. Treatment with glutaraldehyde at a given salt concentration affected neither the orientation of nucleosomes relative to the fiber axis nor the compactness of chromatin. Solid‐phase immunoassay and inhibition experiments showed no binding of the antibody against the globular domain of H1 to chromatin at the entire range of salt concentrations, while the antibody to the whole H1 molecule reacted with chromatin at low salt. On the other hand, the antibody to the globular region of H5 reacted with hen erythrocyte chromatin independently of the extent of chromatin condensation. These results indicate that the antigenic determinants of the globular domain of H5 are accessible to the antibody both in folded and unfolded chromatin, while those of the same region of H1 are masked, probably by interaction with DNA or proteins.
Ion transport by lasalocid A across red‐blood‐cell membranes
Authors: FERNANDEZ Erik , GRANDJEAN Jean , LASZLO Pierre
Published: March 2005

Na+ and K+ fluxes mediated by lasalocid A across erythrocyte membranes have been determined from 23Na‐NMR peak areas and chemical shifts, respectively. In similar experiments, Cl− transport has been monitored by NMR signal intensities. Taking into account the external pH variations, the results are readily explainable in terms of charge‐balance conservation. The effect of disodium 4,4′‐diisothiocyanostilbene‐2,2′‐disulfonate, an anion‐exchange inhibitor, has also been studied.
Substrate specificity of GM2 and GD3 synthase of Golgi vesicles derived from rat liver
Authors: KLEIN Dieter , POHLENTZ Gottfried , SCHWARZMANN Günter , SANDHOFF Konrad
Published: March 2005

Several GM3 derivatives have been synthesized. Among them were lyso‐GM3 derivatives and GM3 analogues with modifications in the sialic acid moiety. They were used as glycolipid acceptors in assays for GM2 and GD3 synthase of rat liver Golgi. Analysis of the resulting enzyme activities and of the reaction products revealed different substrate specificities for GM2 and GD3 synthase although the normal glycolipid acceptor for both transferases is ganglioside GM3. Specificity of GD3 synthase is strongly determined by the substrate's negative charge and the acyl residue in amide bond to the amino group of neuraminic acid, while GM2 synthase reacts quite indifferently to these changes in the sialic moiety of the substrate. Both enzymes seem to be sensitive to the spatial extension at the neuraminic acid's carboxylic group.
A novel archaebacterial NAD+‐dependent alcohol dehydrogenase
Authors: RELLA Rocco , RAIA Carlo A. , PENSA Marcella , PISANI Francesca M. , GAMBACORTA Agata , DE ROSA Mario , ROSSI Mosè
Published: March 2005

An NAD+‐dependent alcohol dehydrogenase (alcohol:NAD+ oxidoreductase, EC 1.1.1.1) was detected in cellular extracts of the extreme thermophilic archaebacterium Sulfolobus solfataricus. The enzyme was purified to homogeneity and shown to be a dimer with a native molecular mass of 71 kDa by sucrose gradient centrifugation and SDS electrophoresis.

The enzyme has a broad substrate specificity that includes linear and branched primary alcohols, linear and cyclic secondary alcohols, linear and cyclic ketones and anisaldehyde.

The enzyme has an extraordinary thermophilicity and a remarkable thermostability, and appears to have some properties and a structure different from those previously described for thermophilic alcohol dehydrogenases.
Translational control at the level of initiation between mRNAs for pre‐existing proteins and a 22‐kDa heat‐shock protein of Chlamydomonas by small cytosolic RNAs
Authors: KLOPPSTECH Klaus , LORBERBOUM Haya , DE GROOT Nathan , HOCHBERG Abraham A.
Published: March 2005

Small cytosolic RNAs (scRNAs) from human placenta inhibit translation of poly(A)‐rich RNA from Chlamydomonas in the wheat germ cell‐free system. The major exception is the mRNA for a nuclear‐coded 22‐kDa chloroplast heat‐shock protein whose translation is much less affected. Evidence is presented which suggests that scRNAs do not directly interact with the mRNAs but with a factor of the wheat germ system instead. It has been found that run‐off translation of polyribosomes is not impaired by scRNAs whereas the formation of initiation complexes in vitro, again with the exception of those of the mRNA for the 22‐kDa heat‐shock protein, is heavily affected. From this evidence we conclude that scRNAs interfere with the action of one or more of the wheat germ initiation factors and that the translation of the mRNA for the 22‐kDa heat‐shock protein is much less dependent upon this (these) factor(s).
Frequency domain fluorescence studies of yeast phosphoglycerate kinase and its ternary complex
Authors: WASYLEWSKI Zygmunt , EFTINK Maurice R.
Published: March 2005

A frequency domain fluorescence study of yeast phosphoglycerate kinase has been performed to observe the effect of substrates on the structure and dynamics of the enzyme. At 20°C and pH 7.2, a biexponential decay is observed for tryptophanyl emission. The short fluorescence lifetime (0.4 ns) component is associated with a spectrum having a 329‐nm maximum and a 18.4‐kJ/mol activation energy, Ea, for thermal quenching. The long‐lifetime (3.5 ns) component has a 338‐nm maximum and an Ea of only 7.9 kJ/mol. Tentatively we assign the short and long‐lifetime components to Trp‐333 and Trp‐308. Binding of the substrates ATP and 3‐phosphoglycerate leads to a significant increase in the fluorescence lifetime, the red shift of the emission spectrum and in the decrease in the Ea for both components. Acrylamide‐quenching studies indicate that the two tryptophan residues have about the same degree of kinetic exposure to the quencher and that the binding of the substrates causes a very slight change in the quenching pattern. These fluorescence studies indicate that the binding of the substrates to phosphoglycerate kinase may influence the conformational dynamics around the two tryptophan residues located on one of the protein's domains.
Conformational properties of azurin in solution as determined from resolution‐enhanced Fourier‐transform infrared spectra
Authors: SUREWICZ Witold K. , SZABO Art. G. , MANTSCH Henry H.
Published: March 2005

Infrared spectra of the blue copper protein azurin and of apoazurin from P. fluorescens were obtained in aqueous solution. Using resolution enhancement procedures, a number of component bands were identified in the region of the amide I mode, and these bands were assigned to various components of protein secondary structure. A quantitative analysis of these infrared spectra indicates that the secondary structure of P. fluorescens azurin in solution is very similar to those determined previously by X‐ray diffraction for the crystals of azurins from other bacterial species. The major components of this structure are β strands and turns. Infrared spectra also evince a remarkable thermal stability of the native azurin. A significant unfolding of the protein could only be detected at temperatures above approximately 76°C. While the secondary structure of apoazurin is practically indistinguishable from that of the native protein at room temperature, the thermal stability of the apo form is significantly reduced.
Three different proteins exhibiting NAD‐dependent acetaldehyde dehydrogenase activity from Alcaligenes eutrophus
Authors: JENDROSSEK Dieter , STEINBÜCHEL Alexander , SCHLEGEL Hans G.
Published: March 2005

The existence of three different proteins exhibiting NAD‐dependent acetaldehyde dehydrogenase activity was confirmed in Alicaligenes eutrophus. The fermentative alcohol dehydrogenase, which also exhibits acetaldehyde dehydrogenase activity, is one of these proteins. The other two proteins were purified from A. eutrophus N9A mutant AS4 grown on ethanol applying chromatography on DEAE‐Sephacel and triazine‐dye affinity media. Acetaldehyde dehydrogenase II, which amounts to about 14% of the total soluble protein in cells grown on ethanol, was purified to homogeneity. The relative molecular masses of the native enzyme and of the subunits were 195 000 or 56 000, respectively. This enzyme exhibits a high affinity for acetaldehyde (Km= 4 μM). Acetaldehyde dehydrogenase I amounts only to less than 1% of the total soluble protein. The relative molecular masses of the native enzyme and of the subunits were 185 000 and 52 000, respectively. This enzyme exhibits a low affinity for acetaldehyde (Km= 2.6 mM). Antibodies raised against acetaldehyde dehydrogenase II did not react with acetaldehyde dehydrogenase I.

Two different strains, A. eutrophus N9A mutant AS1, which represents a different mutant type and can utilize both ethanol or 2,3‐butanediol, and the type strain of A. eutrophus (TF93), which can utilize ethanol, form two acetaldehyde dehydrogenases during growth on ethanol, too. As in AS4, one of these enzymes from each strain amounted to a substantial portion of the total soluble protein in the cells. These major acetaldehyde dehydrogenases were purified from both strains; they resemble acetaldehyde dehydrogenase II isolated from AS4 in all relevant properties. Antibodies against the enzyme isolated from AS4 gave identical cross‐reactions with the enzymes isolated from AS1 and TF93.
Somatic antigens of Pseudomonas aeruginosa
Authors: KNIREL Yuriy A. , PARAMONOV Nikolay A. , VINOGRADOV Evgeny V. , SHASHKOV Alexander S. , DMITRIEV Boris A. , KOCHETKOV Nikolay K. , KHOLODKOVA Elena V. , STANISLAVSKY Evgeny S.
Published: March 2005

O‐specific polysaccharides, obtained on mild acid degradation of lipopolysacchrides of the serologically related strains Pseudomonas aeruginosa O3 (Lányi classification), O25 (Wokatsch classification) and immunotypes 3 and 7 (Fisher classification), are built up of trisaccharide repeating units involving 2‐acetamido‐2,6‐dideoxy‐D‐galac‐tose (N‐acetyl‐D‐fucosamine), 2,3‐diacetamido‐2,3‐dideoxy‐D‐mannuronic acid or 2,3‐diacetamido‐2,3‐dideoxy‐L‐guluronic acid and 3‐acetamidino‐2‐acetamido‐2,3‐dideoxy‐D‐mannuronic acid or 3‐acetamidino‐2‐acetamido‐2,3‐dideoxy‐L‐guluronic acid. Lányi O3(a),3d,3f and Wokatsch O25 polysaccharides contain also O‐acetyl groups.

On the basis of solvolysis with anhydrous hydrogen fluoride, resulting in trisaccharide fragments with N‐acetylfucosamine residue at the reducing terminus, chemical modifications of the acetamidino group (alkaline hydrolysis to the acetamido group or reductive deamination to the ethylamino group), as well as analysis by 1H‐NMR (including nuclear Overhauser effect experiments) and 13C‐NMR spectroscopy, and fast‐atom bombardment mass spectrometry, it was concluded that the repeating units of the polysaccharides have the following structures:

image
where HexNAcAmA →α‐L‐GulNAcAmA (∼70%) or β‐D‐ManNAcAmA (∼30%).

Lányi O3(a),3d,3f polysaccharide involves two types of repeating units, which differ from each other only in the configuration at C‐5 of the 3‐acetamidino‐2‐acetamido‐2,3‐dideoxyuronic acid residue. Lányi O3(a),3c, O3a,3d,3e and Fisher immunotypes 3 and 7 polysaccharides contain, together with the major repeating units shown above, a small proportion of units in which the derivative of α‐L‐guluronic acid is replaced by the corresponding β‐D‐manno isomer.

The data obtained provide the opportunity to substantiate the serological interrelations between these strains of P. aeruginosa by the presence in the O‐specific polysaccharides of common monosaccharides or disaccharide fragments. The distinctions between them stem from the presence or absence of the O‐acetyl group, a different configuration of the glycosidic linkage of the N‐acetylfucosamine residue and/or a different configuration at C‐5 of one or both derivatives of diaminouronic acids.
Nomenclature and symbols for folic acid and related compounds
Authors: BLAKLEY Raymond L.
Published: March 2005

The folates are a group of heterocyclic compounds based on the 4‐[(pteridin‐6‐ylmethyl)amino]benzoic acid skeleton conjugated with one or more l‐glutamate units. The document is a revision of recommendations prepared by the IUPAC‐IUB Commission on Biochemical Nomenclature in 1964. The main changes are a consequence of the much greater information that now exists about the stereochemistry of folates. Recommendations on the nomenclature of reduced compounds and substituents are included, as are proposed symbols and abbreviations.
Isolation and structural characterization of an 8‐O‐(4‐amino‐4‐deoxy‐ β‐l‐arabinopyranosyl)‐3‐deoxy‐d‐manno‐octulosonic acid disaccharide in the lipopolysaccharide of a Proteus mirabilis deep rough mutant
Authors: SIDORCZYK Zygmunt , KACA Wieslaw , BRADE Helmut , RIETSCHEL Ernst Th. , SINNWELL Volker , ZÄHRINGER Ulrich
Published: March 2005

On short treatment of the lipopolysaccharide of a deep rough mutant of Proteus mirabilis (strain R4 5) with hydrochloric acid (0.01 M, 10 min, 100°C) a compound was released which, on high‐voltage paper electrophoresis, migrated slightly to the cathode (MGlcN= 0.19) and which stained with ninhydrin, alkaline silver nitrate and in the thiobarbituric acid assay. After purification and derivatization, its chemical structure was identified by combined gas‐liquid chromatography/mass spectrometry and two‐dimensional shift‐correlated proton nuclear magnetic resonance spectroscopy as 8‐O‐(4‐amino‐4‐deoxy‐β‐l‐arabinopyranosyl)‐3‐deoxy‐d‐manno‐octulosonic acid. This disaccharide was also present in a wild‐type strain of P. mirabilis.
Use of protein blotting to study the DNA‐binding properties of histone H1 and H1 variants
Authors: WRIGHT Jonathan M. , WIERSMA Paul A. , DIXON Gordon H.
Published: March 2005

Sub‐types of histone H1 have been observed in a variety of tissues from several organisms. One of the best characterized H1 variants is H5 from avian erythrocytes. Several lines of evidence suggest that H5 has a greater affinity for DNA than H1 and is thus thought to account, in part, for the highly condensed and transcriptionally repressed state of avian erythrocyte chromatin. In trout there is an analogous erythrocyte‐specific H1 variant, previously termed ‘H5’ [B. L. A. Miki and J. M. Neelin (1975) Can. J. Biochem. 53 1158–1169). Using a sensitive and rapid protein‐blotting procedure which is specific amongst the histones for histone H1 and its variants, we compared DNA‐binding properties of the trout erythrocyte histone ‘H5’ and chicken H5. By increasing the NaCl concentration of the binding buffer, a gradual decrease in the amount of DNA that bound to chicken H1, trout H1 and trout erythrocyte ‘H5’ variant was observed, such that at concentrations above 0.37 M, negligible amounts of DNA were bound. By contrast, chicken H5 bound a significantly greater amount of DNA even at a concentration of 0.4 M NaCl. Based on the DNA‐binding, properties, we conclude that the trout erythrocyte variant ‘H5’ is more closely related to H1 than to H5. By assaying the DNA‐binding affinity of calf thymus H1 peptide fragments, generated by protease and chemical cleavage, and the sperm‐specific H1 variants of the annelid, Platynereis dumerilii, which posses greatly shortened C‐terminal tails, we conclude that a domain that includes a very small portion of the C‐terminal tail and part of the globular domain is sufficient for the binding of H1 to DNA.
A kinetic model for lipoxygenases based on experimental data with the lipoxygenase of reticulocytes
Authors: LUDWIG Peter , HOLZHÜTTER Hermann‐Georg , COLOSIMO Alfredo , SILVESTRINI Maria Chiara , SCHEWE Tankred , RAPOPORT Samuel M.
Published: March 2005

A comprehensive kinetic model for lipoxygenase catalysis is proposed which includes the simultaneous occurrence of dioxygenase and hydroperoxidase activities and is based on the assumption of a single binding site for substrate fatty acid and product.

The aerobic reaction of purified lipoxygenase from rabbit reticulocytes with 9,12(Z,Z)‐octadecadienoic acid (linoleic acid) as substrate was studied.

The rate constants and the dissociation constants of this enzyme were calculated for the model from progress curves; the model describes correctly the experimental data.

The following kinetic features of the reticulocyte enzyme are assumed to apply generally to lipoxygenases. (a) The enzyme shows autoactivation by its product. (b) The rate‐limiting step is the hydrogen abstraction. (c) Both substrate fatty acid and its product are competitive inhibitors of the lipoxygenase. (d) Lowering the oxygen concentration enhances the degree of substrate inhibition, whereas product inhibition is not influenced. (e) If substrate is in excess the oxygen concentration determines the share of dioxygenase and hydroperoxidase activities of the enzyme.

As predicted from the model it was found that at low concentrations of oxygen the regio– and stereo‐specificities of the dioxygenation are diminished.

During the autoactivation phase the steady‐state approximation does not hold.
Biochemical properties and hormonal regulation of barley nuclease
Authors: BROWN Peter H. , HO Tuan‐hua David
Published: March 2005

The amino acid composition and NH2‐terminal amino acid sequence of barley nuclease (EC 3.1.30.2) were determined. The amino acid composition is similar to that of mung bean nuclease, and therefore the biochemical properties of barley nuclease were characterized and compared with those of mung bean and other plant nucleases. The 3′‐nucleotidase activity of barley nuclease is greater for purine than for pyrimidine ribonucleotides. The enzyme has little activity towards ribonucleoside 2′ and 5′‐monophosphates, and deoxyribonucleoside 3′ and 5′‐monophosphates, and is also inactive towards the 3′‐phosphoester linkage of nucleoside cyclic 2′,3′ and 3′,5′‐monophosphates. The enzyme hydrolyzes dinucleoside monophosphates, showing strong preference for purine nucleosides as the 5′ residues. Barley nuclease shows significant base preference for homoribonucleic acids, catalyzing the hydrolysis of polycytidylic acid > polyuridylic acid > polyadenylic acid ≫ polyguanylic acid. The enzyme also has preference for single‐stranded nucleic acids. Hydrolysis of nucleic acids is primarily endonucleolytic, whereas the products of digestion possess 5′‐phosphomonoester groups. Nuclease activity is inhibited by ethylenediaminetetraacetic acid and zinc is required for reactivation. Secretion of nuclease from barley aleurone layers is dependent on the hormone gibberellic acid [Brown, P. H. and Ho, T.‐h. D. (1986) Plant Physiol. 82, 801–806]. Consistent with these results, gibberellic acid induces up to an eightfold increase in the de novo synthesis of nuclease in aleurone layers. The secreted enzyme is a glycoprotein having an apparent molecular mass of 35 kDa. It consists of a single polypeptide having an asparagine‐linked, high‐mannose cligosaccharide. The protein portion of the molecule has a molecular mass of 33 kDa.
Mutants affecting tRNAPhe from Escherichia coli Studies of the suppression of thermosensitive phenylalanyl‐tRNA synthetase
Authors: DELAMARCHE Christian , VACHER Jean , BUCKINGHAM Richard H.
Published: March 2005

Four mutants of pheV, a gene coding for tRNAPhe in Escherichia coli, share the characteristic that when carried in the plasmid pBR322, they lose the capacity of wild‐type pheV to complement the thermosensitive defect in a mutant of phenylalanyl‐tRNA synthetase. One of these mutants, leading to the change C2→U2 in tRNAPhe, is expressed about 10‐fold lower in transformed cells than wild‐type pheV. This mutant, unlike the remaining three (G15→A15, G44→A44, m7G46→A46), can recover the capacity to complement thermosensitivity when carried in a plasmid of higher copy number. The other three mutants, even when expressed at a similar level, remain unable to complement thermosensitivity. A study of charging kinetics suggests that the loss of complementation associated with these mutants is due to an altered interaction with phenylalanyl‐tRNA synthetase. The mutant gene pheV(U2), when carried in pBR322, can also recover the capacity to complement thermosensitivity through a second‐site mutation outside the tRNA structural gene, in the discriminator region. This mutation, C(‐6)→T(‐6), restores expression of the mutant U2 to about the level of wild‐type tRNAPhe.
Studies on the sequence and structure of the Escherichia coli K‐12 nupG gene, encoding a nucleoside‐transport system
Authors: WESTH HANSEN Svend Erik , JENSEN Nina , MUNCH‐PETERSEN Agnete
Published: March 2005

The nupG gene, encoding one of the two active nucleoside‐transport systems in Escherichia coli K‐12, has been cloned on the multicopy plasmid pBR322 and derivatives thereof. The recombinant plasmids complemented a chromosomal nupG mutation.

A genetic map was determined by digestion with restriction endonucleases and the nucleotide sequence of a 3‐kb stretch of DNA has been determined on fragments cloned into M13 phages. An open reading frame of 1254 bp, encoding a protein with a calculated molecular mass of 45.333 kDa, was deduced to be the coding region of nupG.

Minicell‐forming strains carrying plasmids containing this gene were shown to produce a hydrophobic, membrane‐bound polypeptide with an apparent molecular mass of approximately 43 kDa.
Biochemical and immunological evidence for a second nitrate reductase in Escherichia coli K12
Authors: IOBBI Chantal , SANTINI Claire‐Lise , BONNEFOY Violaine , GIORDANO Gerard
Published: March 2005

Genes different from those of the narGHI operon and encoding a nitrate reductase activity have been cloned by Bonnefoy et al. (unpublished results). We have shown by the use of well‐known assay methods that the encoded enzyme activity is catalyzed by a true nitrate reductase and not by trimethylamine‐N‐oxide reductase or formate dehydrogenase. The biochemical and immunological study, employing anti‐(nitrate reductase) serum raised against the known enzyme, revealed that Escherichia coli contains a second nitrate reductase (nitrate reductase Z) which shares some similarities as well as differences with the known enzyme. By using a strain with a deletion of the narGHI operon and carrying a multicopy plasmid having the nitrate reductase Z genes, we have shown that nitrate reductase Z is a membrane‐bound molybdoenzyme able to couple formate oxidation with nitrate reduction. Like the known nitrate reductase, this enzyme has chlorate reductase activity.

The molecular mass and pH and temperature dependence of enzyme Z are similar to these of the known enzyme. On the other hand the two enzymes have significant differences in their electrophoretic mobility on polyacrylamide gels. Unlike the known enzyme, enzyme Z is synthesized in small amounts; the expression of its structural genes does not seem to be induced by nitrate, repressed by oxygen or activated by the product of the fnr gene.

The immunological comparison of the two enzymes was performed by rocket immunoelectrophoresis, double diffusion on agar plates and immunoblots. These techniques disclosed a difference between the two enzymes in their recognition by the antiserum and showed that E. coli has two types of nitrate reductase.
Influence of diet on the transcriptional and post‐transcriptional regulation of malic enzyme induction in the rat liver
Authors: KATSURADA Akihiko , IRITANI Nobuko , FUKUDA Hitomi , NOGUCHI Tamio , TANAKA Takehiko
Published: March 2005

Fasted rats were refed a carbohydrate/protein diet, a carbohydrate diet (without protein) or a protein diet (without carbohydrate) to investigate, using a cDNA cloned in our laboratory, the regulatory mechanisms involved in hepatic malic enzyme induction. In the carbohydrate/protein diet, although the enzyme activity and the mRNA concentration of malic enzyme were increased about 7‐fold above the levels in the fasted rat, the rate of transcription was increased only 2‐fold. In the carbohydrate diet group the rate of transcription and the concentration of mRNA were increased to the levels in the carbohydrate/protein diet group, whereas the enzyme activity increased only to 60% of those levels. Protein appears to contribute to an increase in the translation of malic enzyme mRNA. In the protein diet group the transcriptional rate was not low, but the mRNA concentration was about half in comparison with the level of the carbohydrate/protein diet group. Further, dietary fat did not reduce the transcriptional activity, but reduced the mRNA concentration and the enzyme activity to half of the basal levels. Therefore, it is suggested that fat stimulates the degradation of the mRNA in liver cytosol, whereas carbohydrate tends to stabilize the mRNA. On the other hand 3,5,3′‐triiodothyronine treatment increased the transcriptional activity by 1.5–2‐fold above the basal values on all the diets and even on fasting. Thus, it is suggested that 3,5,3′‐triiodothyronine increases the transcriptional activity of malic enzyme independently from nutritional regulation, while the nutrients are predominantly involved in the post‐transcriptional steps.
A comparison of the local environment of Cu(II) in native and half‐Cu‐depleted bovine serum amine oxidase
Authors: MONDOVI Bruno , MORPURGO Laura , AGOSTINELLI Enzo , BEFANI Olivia , McCRACKEN John , PEISACH Jack
Published: March 2005

Electron spin‐echo envelope modulation spectroscopy has been used to compare the local environment of Cu(II) in native bovine serum amine oxidase, containing two copper atoms/dimer molecule, and in a protein preparation, half depleted of copper, which has little enzymatic activity. For each preparation, two different populations of coordinated imidazoles with inequivalent magnetic coupling to copper could be recognized. In addition, water was shown to be a ligand to copper. No differences in coordinated ligand structures between the native and half‐Cu‐depleted preparations could be seen. In addition, the amount of ambient, non‐coordinated water detected for native and half‐Cu‐depleted proteins was found to be nearly equivalent. However, the addition of phenylhydrazine, an inhibitor that binds to the pyrroloquinoline quinone cofactor but not to Cu(II) in the native enzyme, displaces ambient water near copper.
Integration of porin synthesized in vitro into outer mitochondrial membranes
Authors: ONO Hideyu , TUBOI Syozo
Published: March 2005

Porin, an intrinsic protein of outer mitochondrial membranes of rat liver, was synthesized in vitro in a cell‐free translation system with rat liver RNA. The apparent molecular mass of porin synthesized in vitro was the same as that of its mature form (34 kDa). This porin was post‐translationally integrated into the outer membrane of rat liver mitochondria when the cell‐free translation products were incubated with mitochonadria at 30°C even in the presence of a protonophore (carbonyl cyanide m‐chlorophenylhydrazone). Therefore, the integration of porin seemed to proceed energy‐independently as reported by Freitag et al. [(1982) Eur. J. Biochem. 126, 197–202]. Its integration seemed, however, to require the participation of the inner membrane, since porin was not integrated when isolated outer mitochondrial membranes alone were incubated with the translation products. Porin in the cell‐free translation products bound to the outside of the outer mitochondrial membrane when incubated with intact mitochondria at 0°C for 5 min. When the incubation period at 0°C was prolonged to 60 min, this porin was found in the inner membrane fraction, which contained monoamine oxidase, suggesting that porin might bind to a specific site on the outer membrane in contact or fused with the inner membrane (a so‐called OM‐IM site). This porin bound to the OM‐IM site was integrated into the outer membrane when the membrane fraction was incubated at 30°C for 60 min. These observations suggest that porin bound to the outside of the outer mitochondrial membrane is integrated into the outer membrane at the OM‐IM site by some temperature‐dependent process(es).
Quantitative measurement of the error in the cryptic stereospecificity of methylmalonyl‐CoA mutase
Authors: MICHENFELDER Martina , HULL William E. , RÉTEY János
Published: March 2005

Samples of methylmalonyl‐CoA and (2H3)methylmalonyl‐CoA were prepared by a combination of chemical and enzymic methods. After ion‐exchange chromatography the unlabelled methylmalonyl‐CoA was pure, the deuterated substance contained 11 – 12% dephospho‐CoA derivative.

The sample of unlabelled methylmalonyl‐CoA was incubated in deuterated buffer with catalytic amounts of methylmalonyl‐CoA mutase, epimerase, and coenzyme B1 2. The progress of the reaction was monitored directly by 1H‐NMR spectroscopy at 500 MHz. After equilibrium was established, a slow mutase‐catalysed deuterium incorporation into migratable positions of succinyl‐CoA was observed.

The sample of (2H3)methylmalonyl‐CoA was incubated in unlabelled buffer with a mixture of methylmalonyl‐CoA mutase, epimerase and coenzyme B1 2. In withdrawn aliquots, the reaction was interrupted by acidification and the lyophilised samples were examined by 1H‐NMR spectroscopy in deuterium oxide. Both rearrangement and protium incorporation into migratable positions of succinyl‐CoA were monitored.

At comparable methylmalonyl‐CoA to succinyl‐CoA conversion rates, deuterium loss from migratable positions was 4–6 times faster than the corresponding protium loss. It is confirmed that the stereochemical error of the mutase is amplified by isotope discrimination when deuterium is in migratable positions, whereas it is diminished when protium is in migratable positions.
Characterization of N‐linked gluco‐oligomannose type of carbohydrate chains of glycoproteins from the ovary of the starfish Asterias rubens (L.)
Authors: DE WAARD Pieter , KAMERLING Johannis P. , VAN HALBEEK Herman , VLIEGENTHART Johannes F. G. , BROERTJES Jan J. S.
Published: March 2005

Glycoproteins were isolated from the ovary of the starfish Asterias rubens (L.). After delipidation, sugar analysis revealed the presence of mannose, glucose and N‐acetylglucosamine in a molar ratio of 9.0:1.3:2.3. Subsequently, hydrazinolysis, re‐N‐acetylation, reduction and high‐voltage paper electrophoresis were carried out, resulting in a mixture of neutral oligosaccharide alditols which was fractionated on Bio‐Gel P‐4. The alditols, investigated by 500‐MHz 1H‐NMR spectroscopy, turned out to be of the oligomannose type or of the gluco‐oligomannose type containing 9 mannose and 1–3 glucose residues. The most abundant compounds were established to be:

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Subunit composition of the H+‐ATPase complex from anaerobic bacterium Lactobacillus casei
Authors: MILEYKOVSKAYA Evgenia I. , ABULADZE Anna N. , OSTROVSKY Dmitrii N.
Published: March 2005

The H+‐ATPase complex has been isolated from the membranes of the anaerobic bacterium Lactobacillus casei by two independent methods.

The crossed‐immunoelectrophoresis of the 14C‐labelled ATPase complex against antibodies to a highly purified soluble ATPase has been used. The subunit composition of the complex has been established by autoradiography. The soluble part of L. casei ATPase, in contrast to coupling factor F1‐ATPases of aerobic bacteria, chloroplasts and mitochondria which include two kinds of large subunit (α and β), consists of one kind of large subunit with a molecular mass of 43 kDa. Moreover, a minor polypeptide of 25 kDa has been found in the soluble ATPase. Factor Fo of L. casei ATPase complex consists of a 16‐kDa subunit and two subunits with molecular masses less than 14 kDa.

A dicyclohexylcarbodiimide‐sensitive ATPase complex has been isolated from L. casei membranes by treating them with a mixture of octyl glucoside and sodium cholate. The complex, purified by centrifugation on a sucrose density gradient, contains the main subunits with molecular masses of 43 kDa, 25 kDa and 16 kDa and a dicyclohexylcarbodiimide‐binding subunit with a molecular mass less than 14 kDa.
Regulation of the ontogeny of rat liver metallothionein mRNA by zinc
Authors: ANDREWS Glen K. , GALLANT Karen R. , CHERIAN M. George
Published: March 2005

To investigate the role of metals in the regulation of the ontogenic expression of rat liver metallothionein (MT) mRNA, the concentrations of zinc, MT and MT mRNA were determined in lives of fetal and newborn rats from dams which were fed with a control or zinc‐deficient or copper‐deficient or iron‐deficient diet from day 12 of gestation. The liver samples were analyzed for MT‐mRNA levels using a mouse MT‐I cRNA probe. Although the newborn hepatic levels of each metal (zinc or copper or iron) was specifically reduced corresponding to the respective mineral deficiencies, the hepatic concentrations of total MT and MT‐I mRNA were significantly decreased only in pups born from zinc‐deficient dams. Injection of the zinc‐deficient newborn pups with 20 mg Zn as ZnSO4/kg restored with MT‐I mRNA levels to slightly above control values within 5 h of injection. The hepatic zinc, MT and MT‐I mRNA levels were observed to increase significantly in control fetal rat liver on days 17–21 of gestation but there were little changes in either zinc or MT in fetal livers from zinc‐deficient dams during the late gestational period. The MT‐I mRNA level also did not show an increase on days 18 and 20 of gestation in zinc‐deficient fetal liver as compared to controls. These results demonstrate a direct role of zinc in hepatic MT gene expression in rat liver during late gestation. Immunohistochemical localization of MT using a specific antibody to rat liver MT showed that the staining for MT in zinc‐deficient pup liver was mainly in the cytosol in contrast to the significant nuclear MT staining observed in control newborn rat liver. The results suggest that maternal zinc deficiency has a marked effect not only in decreasing the levels of hepatic MT and MT‐I mRNA but also in the localization of MT in newborn rat liver.
The role of thiols in nucleotide uptake into synaptic vesicles from Torpedo marmorata
Authors: LEE Deborah Anne , WITZEMANN Veit
Published: March 2005

We have employed sulfhydryl group reagents in an attempt to determine the mechanism by which the transport of nucleotides into synaptic vesicles is controlled. Transport proved to be sensitive to N‐ethylmaleimide; radiolabelled N‐ethylmaleimide was used to locate the sulfhydryl group to the translocase‐associated molecule previously identified as a polypeptide of Mr 34000 [Lee and Witzemann (1983) Biochemistry 22, 6123–6130]. The nucleotide uptake was 75% inhibited by the mercurials p‐hydroxymercuribenzoate and p‐chloromercuriphenylsulfonate. Uptake was also sensitive to the reagents phenylarsine oxide and iodosobenzoic acid, which are specific for dithiols. These results indicate that a readily accessible dithiol is critical for nucleotide transport. Using the lipophilic oxidants iodosobenzoic acid and plumbagin, we demonstrated that nucleotide uptake was inhibited upon oxidation of the dithiol but that this did not involve an alteration in the affinity of the translocase for its substrate.
Spectral and potentiometric analysis of cytochromes from Bacillus subtilis
Authors: de VRIJ Wim , van den BURG Bertus , KONINGS Wil N.
Published: March 2005

Bacillus subtilis cytoplasmic membranes contain several cytochromes which are linked to the respiratory chain. At least six different cytochromes have been separated and identified by ammonium sulphate fractionation and ion‐exchange chromatography. They include two terminal oxidases with CO‐binding properties and cyanide sensitivity. One of these is an aa3‐type cytochrome c oxidase which has characteristic absorption maxima in the reduced—oxidized difference spectrum at 601 nm in the α‐band and at 443 nm in the Soret band regions. In the α‐band two separate electron transitions with Em=+205 mV and Em=+335 mV can be discriminated by redox potentiometric titration. The other CO‐binding cytochrome c oxidase contains two cytochrome b components with α‐band maxima at 556 nm and 559 nm. Cytochrome b556 can be reduced by ascorbate and has an Em++ 215 mV, whereas cytochrome b559 has an Em=+140 mV. Furthermore a complex consisting of a cytochrome b564 (Em=+140 mV) associated with a cytochrome c554 (Em=+250 mV) was found. This cytochrome c554, which can be reduced by ascorbate, appears to have an asymmetrical α‐peak and stains for heme‐catalyzed peroxidase activity on SDS‐containing polyacrylamide gels. A protein with a molecular mass of about 30 kDa is responsible for tis activity. A cytochrome b559 (Em=+ 65 mV) appears to be an essential part of succinate dehydrogenase. Finally a cytochrome c550 component with an apparent mid‐point potential of Em=+ 195 mV has been detected.
Studies of the functional topography of Escherichia coli RNA polymerase
Authors: GODOVIKOVA Tatyana S. , GRACHEV Mikhail A. , KUTYAVIN Igor V. , TSAREV Igor G. , ZARYTOVA Valentina F. , ZAYCHIKOV Evgeny F.
Published: March 2005

Amidation of the 5′‐phosphate group of the heptanucleotide pdApdApdApdTpdCpdGprC and of its derivatives of the general formula (pdN)npdGprC (n= 0–5) with imidazole, N‐methylimidazole, and 4‐dimethylaminopyridine afforded a series of phosphorylating affinity reagents. The parent oligonucleotides of this series complementary to promoter A2 of T7 phage over the region (− 5 to + 2) are known to be efficient primers of the synthesis of RNA by Escherichia coli RNA polymerase with promoter A2 as template. Treatment of the complex RNA‐polymecase · promoter‐A2 with affinity reagents followed by addition of [α‐32P]UTP resulted in labelling of RNA polymerase by the residues ‐(pdN)npdGprCrU (+ radioactive phosphate). This affinity labelling was highly selective because elongation of the covalently bound residues (pdN)npdGprC by rU residues was catalyzed by the active center of RNA polymerase. The most efficient reagents were N‐methylimidazolides. A dramatic change of the pattern of labelling of the subunits β, β′, and σ took place with changing n. Maximum labelling of the β subunit occurred at n= 1 and of the σ subunit at n= 5. The targets in both the subunits were His residues. The α subunit was not specifically labelled.
Characterization of an arginine‐specific protein kinase tightly bound to rat liver DNA
Authors: LEVY‐FAVATIER Florence , DELPECH Marc , KRUH Jacques
Published: March 2005

A new protein kinase has been characterized among the proteins tightly bound to rat liver DNA and released by DNase I and RNase A treatment. This enzyme was separated by gel filtration from this released material. Its apparent molecular mass was found to be 34 kDa and it is made of a single unit.

The main characteristic of this protein kinase is that it is arginine‐specific. Isolation of phosphoarginine required the use of proteolytic enzymes at alkaline pH since the phosphate bond is highly acid‐labile. This protein kinase is able to autophosphorylate and to phosphorylate a single chromosomal protein of 11 kDa also tightly bound to DNA. It uses ATP and dATP as phosphate donors and is cAMP‐independent. Its optimal activity requires Mn2+ ions. Vanadate, spermine and heparins have no effect on its activity.
The role of iron in the activation of mannoni and altronic acid hydratases, two Fe‐requiring hydro‐lyases
Authors: DREYER Jean‐Luc
Published: March 2005

d‐Altronate hydratase and d‐mannonate hydratase belong to a class of Fe2+‐requiring enzymes, but the function of iron in these enzymes is largely unknown. Methods are described for the convenient preparation of both these hydratases from Escherichia coli and studies related to metal activation are presented. The enzymes are inactive in the absence of a bivalent metal and a reducing agent such as dithiothreitol. Fe2+ at low concentrations activates the enzymes efficiently, but inhibits them over 2 mM. Furthermore Mn2+ is also capable of activating aldonic acid hydratases and appears to be a constituent of the enzyme active center. A marked synergistic activation is observed in the presence of both ions, raising the possibility that the enzyme has two binding sites for ions. Upon activation, the two aldonic acid hydratases incorporate a single Fe atom and contain no Fe‐S core, in contrast to other characterized Fe‐hydratases, such as aconitase or maleic acid hydratase. The incorporated iron is losely bound (with Kd about 4.5 mM and 20 mM for mannonate and altronate hydratase, respectively) and can be readily removed with EDTA. The enzymes exhibit no requirement for sulphide ions and are insensitive to thiol reagents. A first‐order inhibition is observed with iron chelators and can be removed by competition with excess metal ions. No change in the absorption spectra is observed upon oxidation‐reduction or activation with metals. The activated enzymes exhibit no electron paramagnetic (EPR) spectrum under anaerobic conditions; in the presence of oxygen, an intense EPR spectrum develops in Fe2+‐activated samples with signal at g= 1.98, which upon reaction of the enzyme with the substrate moves into a species with signals at g= 4.15 and g= 9.07, with EPR parameters very similar to those of oxidized rubredoxins.
Highly acidic glycans from sea cucumbers
Authors: MOURÃO Paulo A. S. , BASTOS Izaias G.
Published: March 2005

The body wall of the sea cucumber contains high amounts of sulfated glycans, which differ in structure from glycosaminoglycans of animal tissues and also from the fucose‐rich sulfated polysaccharides isolated from marine algae and from the jelly coat of sea urchin eggs. In Ludwigothurea grisea, glycans can be separated into three fractions which differ in molecular mass and chemical composition. The fraction containing a high‐molecular‐mass component has a high proportion of fucose and small amounts of amino sugars, whereas another fraction contains primarily a sulfated fucan. The third fraction, which represents the major portion of the sea cucumber polysaccharides, contains besides fucose, approximately equimolar proportions of glucuronic acid and amino sugars, and has a sulfate content higher than that in the other two fractions. Both D and L‐isomers of fucose are found in these polysaccharides, and the sulfate is linked to the O‐3 position of the fucose residues. The attachment position of the sulfate groups to the glucuronic acid units and amino sugars is still undetermined. It is possible that these compounds are involved in maintaining the integrity of the sea cucumber's body wall, in analogy with the role of other macromolecules in the vertebrate connective tissue.
Identification of the 38‐kDa subunit of rabbit skeletal muscle glycogen synthase as glycogenin
Authors: PITCHER Julie , SMYTHE Carl , CAMPBELL David G. , COHEN Philip
Published: March 2005

Glycogen synthase from rabbit skeletal muscle has been shown to be a complex of two types of subunit which have apparent molecular masses of 86 kDa and 38 kDa and are present in a 1:1 molar ratio. The 38‐kDa component was separatéd from the 86‐kDa catalytic subunit by gel filtration in the presence of 2 M LiBr, and a number of chymotryptic peptides were sequenced. This demonstrated that the 38‐kDa subunit was glycogenin, the protein that is bound covalently to glycogen and believed to be the ‘primer' involved in the initiation of de novo glycogen synthesis.