bluuebunny/crossref_metadata_2025 huggingface dataset:
Monoclonal antibodies directed against the cell‐surface‐exposed part of PhoE pore protein of the Escherichia coli K‐12 outer membrane
Authors: VAN DER LEY Peter , AMESZ Hans , TOMMASSEN Jan , LUGTENBERG Ben
Published: March 2005

Six monoclonal antibodies directed against the trimeric form of outer‐membrane pore protein PhoE of Escherichia coli K‐12 were isolated and characterized. All six antibodies bind to PhoE protein in intact cells and as isolated trimers, but not to dodecyl‐sulphate‐denatured monomers. Cross‐reaction with the related pore proteins OmpF protein and OmpC protein was not observed. A hybrid pore protein in which the amino‐terminal 74 amino acids of PhoE protein have been replaced by the corresponding part of OmpF protein is able to bind the six monoclonal antibodies. Five of the antibodies bind to the PhoE proteins of thirteen different Enterobacteriaceae when expressed in E. coli K‐12, whereas the other antibody recognizes PhoE protein from nine of these strais. Four monoclonal antibodies are able to block adsorbtion of the PhoE‐protein‐specific phage TC45 to its receptor on whole cells. None of the antibodies has any effect on the uptake rate of the antibiotic cefsulodin through PhoE protein pores. Five antibodies are able to direct the complement‐mediated killing of PhoE‐protein‐carrying cells. It is concluded that the six monoclonal antibodies recognize at least three distinct cell‐surface‐exposed epitopes whose specificity is determined by the carboxy‐terminal 256 amino acids of PhoE protein.
Isolation of plasma membrane from human blood monocytes
Authors: FAYLE David R. H. , SIM Pheng‐Siew , IRVINE Debra K. , DOE William F.
Published: March 2005

The isolation of plasma membrane from human peripheral blood monocytes is described. Monocytes were isolated by centrifugal elutriation, to eliminate an adherence step, thus minimizing functional and surface antigenic alterations to the cells. Monocytes were surface‐labelled with a radiolabelled monoclonal antibody, 125I‐WVH‐1, and then disrupted by nitrogen cavitation. Membranes were separated according to equilibrium buoyant density by isopycnic centrifugation on a sucrose gradient. The subcellular membranes were localized using marker enzymes for the plasma membrane, 5′‐nucleotidase and leucine 2‐naphthylamidase (leucine aminopeptidase), and for intracellular membranes: galactosyltransferase (Golgi), arylsulfatase C (endoplasmic reticulum), monoamine oxidase (mitochondria), catalase (peroxisomes), β‐hexosaminidase and β‐glucuronidase (lysosomal vesicles) and lactate dehydrogenase (cytosol). The monoclonal antibody 125I‐WVH‐1 was shown to label the plasma membrane, as judged by known markers, and represents a highly specific trace label, applicable to the use of plasma membrane as an immunogen for monoclonal antibody production. The NAD‐splitting enzyme, NAD+ nucleosidase, was detected and its presence on the plasma membrane was demonstrated. The subcellular localization of non‐specific esterase in human mononuclear phagocytes is controversial. No evidence was found for α‐naphthyl acetate esterase activity on the plasma membrane or in lysosomal vesicles. However, a membrane‐bound esterase in fractions with properties similar to the smooth endoplasmic reticulum was detected.
Free flow electrophoresis as a tool for enrichment of mutants with temperature‐dependent lethal mutations in lipid A synthesis
Authors: HANSEN‐HAGGE Thomas , LEHMANN Volker , LÜDERITZ Otto
Published: March 2005

Free flow electrophoresis was shown to be a useful tool to enrich for mutants conditionally defective in lipid A synthesis. The method was based on the observation that electrophoretic mobility of bacterial cells is dependent on the structure of lipopolysaccharides and is influenced by lesions in the synthesis of the O‐specific chains as well as by lesion in the synthesis of the complete 3‐deoxy‐D‐manno‐octulosonic acid (dOclA) lipid A region. Using this procedure a new mutant conditionally defective in dOclA‐8‐P snythesis was isolated (mutant Ts5). Following a shift to nonpermissive conditions it accumulates a mixture of at least two equally represented lipid A precursor structures. One is made up of glucosamine, phosphate and 3‐hydroxymyristic acid in a molar ratio 1.0:1.0:2.0 and lacks dOclA and the nonhydroxylated fatty acids lauric, myristic and palmitic acid (precursor Ia). The other one (precursor Ib) has the same basic composition with additional palmitic acid. The precursor preparation derived from mutant Ts5 thus differs from previously described lipid A intermediates by the relatively high substitution by palmitic acid. The implications of the above findings to the biosynthesis of lipid A are discussed.
Structural studies on the linkage unit of ribitol teichoic acid of Lactobacillus plantarum
Authors: KOJIMA Naoya , ARAKI Yoshio , ITO Eiji
Published: March 2005

Structural studies were carried out on the linkage unit which joins ribitol teichoic acid to peptidoglycan in the cell walls of Lactobacillus plantarum AHU 1413. The heating of the cell walls at pH 2.5 led to release of only 5% of ribitol teichoic acid components as water‐soluble material. In contrast, the same treatment of the cell walls after N‐acetylation led to release of about 80% of the teichoic acid moiety, giving a teichoic‐acid‐linked sugar preparation which contained about equimolar amounts of mannosamine, glucosamine and glycerol as minor components.

The teichoic‐acid‐linked sugar was hydrolyzed by mild alkaline treatment into a disaccharide, N‐acetylmannosaminyl(β1→4)N‐acetylglucosamine and ribitol teichoic acid linked to glycerol. The Smith degradation of the N‐acetylated cell walls gave a characteristic fragment, 1,2‐ethylenediol‐phospho‐glycerol‐phospho‐N‐acetylmannosaminly(β1→4)‐N‐acetylglucosamine. Furthermore, when the intact cell walls were subjected to the NaNO2 treatment followed by NaBH4 reduction, the ribitol teichoic acid moiety was recovered for the most part in the water‐soluble polymer fraction, from which a sugar, N‐acetylmannosaminyl‐2,5‐anhydromannitol, was released by mild alkaline treatment.

These results lead to the conclusion that the ribitol teichoic acid chain in the intact cell walls of this organism is linked to peptidoglycan through a unique linkage unit, glycerol‐phospho‐N‐acetylmannosaminyl(β1→4)‐glucosamine. The anomalous stability of the linkage between the teichoic acid moiety and peptidoglycan against acid hydrolysis seems to be accounted for by the involvement of the N‐unsubstituted glucosamine residue in the phosphodiester bridge that joins the two polymers.
Enzymic synthesis of the 4Fe–4S clusters of Clostridium pasteurianum ferredoxin
Authors: BONOMI Franco , PAGANI Silvia , KURTZ Donald M.
Published: March 2005

Ex novo enzymic synthesis of the two 4Fe–4S clusters of Clostridium pasteurianum ferredoxin has been achieved by incubation of the apoprotein with catalytic amounts of the sulfurtransferase rhodanese in the presence of thiosulfate, dl‐dihydrolipoate and ferric ammonium citrate. This enzymic reconstitution procedure was compared to a chemical one, in which the enzyme was replaced by sodium sulfide.

A further comparison was made with the results previously obtained in the enzymic synthesis of the 2Fe–2S cluster of spinach ferredoxin, allowing the following conclusions to be drawn. (1) The nature of the cluster to be inserted into the reconstituted iron‐sulfur protein is determined by the apoprotein itself. (2) The refolding of the structure of the iron‐sulfur proteins around the newly inserted cluster is the rate‐limiting step in both chemical and enzymic reconstitution. (3) Rhodanese appears to play a role in the recovery of the native architecture of the reconstituted iron‐sulfur protein(s).

The extension to the 4Fe–4S centers of the rhodanese‐based biosynthetic system allows this enzymic route to be proposed as a general way to the in vivo synthesis of iron‐sulfur structures.
Identification and characterization of fragments of major glycoproteins from platelet membrane after chymotrypsin treatment
Authors: McGREGOR John L. , CLEZARDIN Philippe , JAMES Elizabeth , McGREGOR Lilian , DECHAVANNE Marc , CLEMETSON Kenneth J.
Published: March 2005

Human platelets were surface‐labeled by the periodate/NaB3H4 method or by lactoperoxidase‐catalysed iodination with 125I. The labeled platelets were treated with chymotrypsin under conditions known to give platelets which aggregate with fibrinogen without stimulation with ADP. Platelets and supernatant were then analysed by various gel electrophoretic techniques including isoelectricfocusing/sodium dodecyl sulfate‐polyacrylamide gel electrophoresis under reducing or non‐reducing conditions and two‐dimensional non‐reduced/reduced sodium dodecyl sulfate polyacrylamide gel electrophoresis followed by fluorography or indirect autoradiography.

Chymotrypsin‐treatment of surface‐labeled platelets degraded the major glycoproteins Ib, IIb and IIIa but also GP4.9−5.4120, GPlc and GPV. The membrane‐bound fragments of GPIb, IIb and IIIa could be identified and also the supernatant fragments of GPIb and GPV. GPIIIa was also cleaved within a loop structure formed by disulfide bond(s).

The fact that remnants of both GPIIb and IIIa are left on chymotrypsin‐treated platelets which aggregate spontaneously with fibrinogen may indicate that a complex formed by these remnants constitutes the fibrinogen‐binding site on platelets.
A high‐affinity site for glutathione in the cytoplasm of Escherichia coli and its possible role in potassium retention
Authors: MEURY Jean , ROBIN Aline
Published: March 2005

Glutathione‐deficient mutants of Escherichia coli, unlike the wild type, exhibit a fast leak and a fast turnover of K+ at steady state.

In a medium with low K+ the growth rate is very slow when unsupplemented with glutathione; when supplemented with glutathione at a concentration as low as 0.1 μM the growth rate is similar to that of the wild type. The ability of glutathione to restore a wild‐type growth rate can be accounted for by an immediate reduction of the K+ leak.

Two analogs of glutathione also reduce the K+ leak: γ‐glutamylaminobutyrylglycine (ophthalmic acid) and αγ‐glutamylcystinylbisglycine.

Glutathione binds with high affinity (Kd 50 nM) to a cytoplasmic protein; ophthalmic acid and αγ‐glutamylcystinylbisglycine compete with glutathione for the binding site (Ki 0.1 μM and 1 μM), thereby ruling out the possibility that the thiol is involved both in reducing the K+ leak and in binding to the high affinity binding site. For each of the three peptides the Kd for binding is similar to the minimal concentration that achieves the maximal reduction of the K+ leak. The results suggest that the binding of glutathione should play a role in the retention of K+ in E. coli.
Purification and some of the functions of the products of bacteriophage T4 recombination genes, uvs X and uvs Y
Authors: YONESAKI Tetsuro , RYO Yeikoh , MINAGAWA Teiichi , TAKAHASHI Hideo
Published: March 2005

The nonessential T4 genes uvsX and uvsY are involved in DNA repair and general recombination. Using newly isolated amber mutants of these genes, we have identified the gene products (gp) by sodium dodecyl sulfate (SDS)/polyacrylamide gel electrophoresis. Their relative molecular masses are 39000 and 16000, respectively. In the normal wild‐type infection process they are produced early but not late in infection. Their synthesis continues for a longer period when DNA synthesis is blocked. We have developed procedures to isolate these gene products at a purity of more than 95% for gpuvsX and at 70% for gpuvsY, as judged by SDS/polyacrylamide gel electrophoresis and staining with Coomassie brilliant blue dye. The purification procedures suggest that these products may be membrane proteins. Using both an agarose gel assay and electron microscopy, we find that the product of the gene uvsX catalyzes the assimilation of a linear single‐stranded fd DNA fragment into superhelical double‐stranded fd DNA (RFI). The reaction requires ATP and Mg2‐ besides substrate DNAs and uvsX protein. The T4 uvsX protein therefore is similar to the Escherichia coli recA protein in molecular size and function, but differs in antigenic property.
Phosphate dependency of phosphofructokinase 2
Authors: LALOUX Monique , VAN SCHAFTINGEN Emile , FRANCOIS Jean , HERS Henri‐Géry
Published: March 2005

Experiments performed at micromolar concentrations of inorganic phosphate support the conclusion that liver phosphofructokinase 2 would be completely inactive in the absence of inorganic phosphate or arsenate. The concentration of inorganic phosphate that allowed half‐maximal activity decreased with increasing pH, being approximately 0.11 mM at pH 6.5 and 0.05 mM at pH 8. The effect of phosphate was to increase V and to decrease Km for fructose 6‐phosphate, without affecting Km for ATP. Citrate and P‐enolpyruvate inhibited the enzyme non‐competitively with fructose 6‐phosphate and independently of the concentration of inorganic phosphate. Phosphorylation of the enzyme by the catalytic subunit of cyclic‐AMP‐dependent protein kinase did not markedly modify the phosphate requirement and its effect of inactivating phosphofructokinase 2 could not be counteracted by excess phosphate. A nearly complete phosphate dependency was also observed with phosphofructokinase 2 purified from Saccharomyces cerevisiae or from spinach leaves. By contrast, the fructose 2,6‐bisphosphatase activity of the liver bifunctional enzyme was not dependent on the presence of inorganic phosphate. Phosphate increased this activity about threefold when measured in the absence of added fructose 6‐phosphate and a half‐maximal effect was reached at approximately 0.5 mM phosphate. Like glycerol phosphate, phosphate counteracted the inhibition of fructose 2,6‐bisphosphatase by fructose 6‐phosphate, but a much higher concentration of phosphate than of glycerol phosphate was required to reach this effect.
Use of tyrosine‐containing polymers to characterize the substrate specificity of insulin and other hormone‐stimulated tyrosine kinases
Authors: ZICK Yehiel , GRUNBERGER George , REES‐JONES Robert W. , COMI Richard J.
Published: March 2005

Synthetic copolymers containing tyrosine residues were used to characterize the substrate specificity of the insulin receptor kinase and compare it to tyrosine kinases stimulated by epidermal growth factor, insulin‐like growth factor‐1 and phorbol ester. In partially purified receptor preparations from eight different tissues insulin best stimulated (highest V) phosphorylation of a random copolymer composed of glutamic and tyrosine residues at a 4:1 ratio (Glu/Tyr, 4:1). The insulin‐stimulated phosphorylation of this polymer was highly significant also in receptor preparations from fresh human monocytes, where insulin binding and autophosphorylation were difficult to detect. Other tyrosine‐containing polymers Ala/Glu/Lys/Tyr (6:2:5:1) and Glu/Ala/Tyr (6:3:1) were also phosphorylated by the insulin‐stimulated kinase but to a lower extent. A tyrosine kinase stimulated by insulin‐like growth factor‐1, and one stimulated by phorbol ester also best phosphorylated the polymer Glu/Tyr (4:1). The three kinases differed only in their capability to phosphorylate Glu/Ala/Tyr (6:3:1) or Ala/Glu/Lys/Tyr (6:2:5:1). Glu/Tyr (4:1) was a poor substrate for the epidermal growth factor receptor kinase which best phosphorylated the polymer Glu/Ala/Tyr (6:3:1). Three additional polymers: Glu/Tyr (1:1), Glu/Ala/Tyr (1:1:1), and Lys/Tyr (1:1) failed to serve as substrates for all four tyrosine kinases tested. Taken together these findings suggest that. (a) Hormone‐sensitive tyrosine kinases have similar yet distinct substrate specificity and are likely to phosphorylate their native substrates on tyrosines adjacent to acidic (glutamic) residues. (b) Tyrosine‐containing polymer substrates are highly sensitive and convenient tools to study (hormone‐sensitive) tyrosine kinases whose native substrates are unknown or present at low concentrations.
Nucleotide sequence of cloned cDNA coding for preproricin
Authors: LAMB F. Ian , ROBERTS Lynne M. , LORD J. Michael
Published: March 2005

The primary structure of a precursor protein that contains the toxic (A) and galactose‐binding (B) chains of the castor bean lectin, ricin, has been deduced from the nucleotide sequence of cloned DNA complementary to preproricin mRNA. A cDNA library was constructed using maturing castor bean endosperm poly(A)‐rich RNA enriched for lectin precursor mRNA by size fractionation. Clones containing lectin mRNA sequences were isolated by hybridization using as a probe a mixture of synthetic oligonucleotides representing all possible sequences for a peptide of the ricin B chain. The entire coding sequence of preproricin was deduced from two overlapping cDNA clones having inserts of 1614 and 1049 base pairs. The coding region (1695 base pairs) consists of a 24‐amino‐acid N‐terminal signal sequence (molecular mass 2836 Da) preceding the A chain (267 amino acids, molecular mass 29 399 Da), which is joined to the B chain (262 amino acids, molecular mass 28 517) by a 12‐amino‐acid linking region (molecular mass 1385 Da).
In vitro transcription initiation from three different Escherichia coli promoters
Authors: EHRLICH Ricardo , LAROUSSE Annick , JACQUET Marie‐Ange , MARIN Monica , REISS Claude
Published: March 2005

Transcription initiation from β‐lactamase, tetracycline resistance and RNA1 promoters, present in plasmid pAT 153, were studied employing the abortive initiation technique. Assays appear to be promoter‐specific with supercoiled and linear templates. Supercoiling enhances the isomerization rate constant of the open RNA‐polymerase–promoter complex formation. Results agree with the in vivo behaviour of the corresponding promoters, and allow us to propose a hypothesis about the effect of supercoiling on transcription initiation.
Localization of the sites of ADP‐ribosylation and GTP binding in the eukaryotic elongation factor EF‐2
Authors: NILSSON Lars , NYGÅRD Odd
Published: March 2005

Tryptic cleavage of EF‐2, molecular mass 93 kDa, produced an 82‐kDa polypeptide and a 10‐kDa fragment, which was further degraded. By a slower reaction the 82‐kDa polypeptide was gradually split into a 48‐kDa and a 34‐kDa fragment. Similarly, treatment with chymotrypsin resulted in the formation of an 82‐kDa polypeptide and a small fragment. In contrast to the tryptic 82‐kDa polypeptide the corresponding chymotryptic cleavage product was relatively resistant to further attack. The degradation of the 82‐kDa polypeptide with either trypsin or chymotrypsin was facilitated by the presence of guanosine nucleotides, indicating a conformational shift in native EF‐2 upon nucleotide binding. No effect was observed in the presence of ATP, indicating that the effect was specific for guanosine nucleotides. After affinity labelling of native EF‐2 with oxidized [3H]GTP and subsequent trypsin treatment the radioactivity was recovered in the 48‐kDa polypeptide showing that the GTP‐binding site was located within this part of the factor. Correspondingly, tryptic degradation of EF‐2 labelled with [14C]NAD+ in the presence of diphtheria toxin showed that the site of ADP‐ribosylation was within the 34‐kDa polypeptide. By cleavage with the tryptophan‐specific reagent N‐chlorosuccinimide the site of ADP‐ribosylation could be located at a distance of 40–60 kDa from the GTP‐binding site and about 4–11 kDa from the nearest terminus.
Steady‐state kinetic analysis of transcription of cloned tRNASer genes from Drosophila melanogaster
Authors: ST. LOUIS Daniel , SPIEGELMAN George B.
Published: March 2005

Drosophila melanogaster Schneider II cells contain a factor which inhibits transcription in vitro of cloned tRNA genes in crude extracts made from these cells. The inhibitor could, however, be effectively neutralized by addition of certain non‐template DNAs.

In the absence of the transcription inhibitor activity, the steady‐state kinetics of tRNA production from cloned genes followed one‐substrate enzyme kinetics to a high degree of accuracy. Maximal rates of transcription and apparent affinity constants were analyzed for a collection of cloned D. melanogaster tRNASer genes. The stability of the complex formed by the transcription proteins and the template DNA was found to be nearly constant for the genes examined. The transcription rates, however, were greatly influenced by the DNA sequences flanking the tRNA genes.

Analysis of transcription competition between DNA templates showed pure competitive behavior. Inhibition constants derived from these experiments indicated that the formation of the transcription complex was affected by sequences flanking the tRNA genes. Furthermore, the rate‐limiting step in complex formation was independent of the stability of the final form of the complex.
Studies of the cellulolytic system of Trichoderma reesei QM 9414
Authors: van TILBEURGH Herman , PETTERSSON Göran , BHIKABHAI Ramagauri , DE BOECK Hilde , CLAEYSSENS Marc
Published: March 2005

1. The 1,4‐β‐glucan cellobiohydrolase II (CBH II) from Trichoderma reesei QM 9414 catalyses the hydrolysis of the 4‐methylumbelliferyl β‐d‐glycosides derived from cellotriose, cellotetraose and cellopentaose [MeUmb(Glc)n; n= 3–5]. The reaction has been followed by quantitative high‐performance liquid chromatography. Specific activity for cellobiose removal at apparent substrate saturation were determined as (0.8 ± 0.2) min−1 for MeUmb(Glc)3 and (9 ± 2) min−1 for MeUmb(Glc)4. The enzyme showed a deviant specificity with MeUmb(Glc)5 as substrate. Two chromophoric products were formed simultaneously [MeUmb(Glc)3 and MeUmb(Glc)2] with turn‐over numbers (17 ± 4) min−1 and (21 ± 6) min−1, respectively.

2. Methylumbelliferyl β‐glucoside (MeUmbGlc) and the corresponding cellobioside [MeUmb(Glc)2] were used in equilibrium binding experiments. Both ligands yielded one binding site per molecule of Mr= 54000 upon forced flow dialysis (diafiltration). The association constants found were in fair agreement with those determined from MeUmb fluorescence quenching titrations. Quenching was total at all temperatures investigated for MeUmb(Glc)2, whereas for MeUmbGlc it increased from 80% to 100% between 2°C and 20°C. The association constants fitted linear van't Hoff plots in both cases.

3. MeUmb(Glc)2 and MeUmbGlc were also used as indicator ligands to determine the association constants and thermodynamic parameters of several non‐chromophoric ligands of CBH II. The binding of glucose increased the affinity for MeUmb(Glc)2 whereas it displaced MeUmbGlc from its complex.

4. A putative binding site of the CBH II containing four subsites can be proposed. The thermodynamic data for methyl β‐d‐glucopyranoside and cellobiose as ligands also point at an extended binding site.
Structural analysis of the O‐glycosidically linked core‐region oligosaccharides of human meconium glycoproteins which express oncofoetal antigens
Authors: HOUNSELL Elizabeth F. , LAWSON Alexander M. , FEENEY James , GOOI Hock C. , PICKERING Nicola J. , STOLL Mark S. , LUI Seung C. , FEIZI Ten
Published: March 2005

Glycoproteins were extracted from meconium samples of group O neonates of secretor type by pronase digestion followed by precipitation in 67% aqueous ethanol and separated into Ii antigen enriched and depleted fractions by affinity chromatography. The latter fraction strongly expressed the oncofoetal antigens recognised by natural antibodies in mouse sera and the hybridoma antibody FC 10.2, and this activity was enhanced after mild acid hydrolysis to remove sialic acid and fucose residues. Oligosaccharides were released from the mild‐acid‐treated fraction by base‐borohydride degradation and purified by gel permeation chromatography on Bio‐Gel P4 and high performance liquid chromatography on octadecylsilyl and aminopropylsilyl columns. The major oligosaccharides were characterised by fast atom bombardment and electron impact mass spectrometry, combined gas‐liquid chromatography/mass spectrometry and 500‐MHz proton NMR spectroscopy. Their structures, in order of abundance, were:
Abnormal amino acid metabolism in mutants of Saccharomyces cerevisiae affected in the initiation of sporulation
Authors: DICKINSON J. Richard , AMBLER Richard P. , DAWES Ian W.
Published: March 2005

Amino acid metabolism was examined during sporulation in a wild‐type strain of Saccharomyces cerevisiae and in a mutant homozygous for the spdl mutation (derepression of sporulation). In the wild‐type initiation of sporulation involves a rise in intracellular glutamate. In the derepressed mutant there was no further increase of intracellular glutamate: glutamate was already present at a greater concentration than in the wild‐type. These elevated glutamate levels are apparently due to reduction in the activity of 2‐oxoglutarate dehydrogenase. One consequence of the elevated glutamate levels in diploids homozygous for the spdl mutation is the production of considerable amounts of glycyl‐l‐proline, glutathione and saccharopine.
Purification and characterisation of an odorant‐binding protein from cow nasal tissue
Authors: BIGNETTI Enrico , CAVAGGIONI Andrea , PELOSI Paolo , PERSAUD Krishna C. , SORBI Robert T. , TIRINDELLI Roberto
Published: March 2005

Cow nasal tissue contains a protein which shows specific binding activity for ‘green’ smelling compounds such as 2‐isobutyl‐3‐methoxypyrazine. This protein has now been purified using anion‐exchange fast protein liquid chromatography. The protein has a relative molecular mass of 400000–44000, s= 3.1 ± 0.3 S, pI = 4.7 ± 0.1 with an absorbance maximum at 278 nm, and consists of two subunits with an identical relative molecular mass of 19000. It is localised in the soluble fraction of cells from the olfactory mucosa and respiratory mucosa from the middle part of the maxillary and nasal turbinates, and is absent from all other tissues tested.
The role of carboxyl groups in the function of endo‐β‐1, 4‐glucanase from Schizophyllum commune
Authors: CLARKE Anthony J. , YAGUCHI Makoto
Published: March 2005

The endo‐β‐1, 4‐glucanase from Schizophyllum commune was purified to homogeneity by a modified procedure that employed concanavalin A – Sepharose. The catalytic site of the enzyme has previously been proposed to consist of Glu‐33 and Asp‐50 that act in a manner analogous to lysozyme [Yaguchi, M., Roy, C., Rollin, C. F., Paice, M. G. & Jurasek, L. (1983) Biochem. Biophys. Res. Commun. 116, 408–411]. The role of carboxyl groups in the mechanism of action was delineated through kinetic and chemical modification studies. The rate of endoglucanase‐catalysed hydrolysis of CM‐cellulose was determined viscometrically at 30°C, 0.06 M ionic strength and pH 2.5–9.0. The pH profile for maximum velocity gave the kinetic apparent pK values 3.8 and 6.6 and for initial velocity the pK values 3.7 and 6.1. Treatment of the endoglucanase with diethylpyrocarbonate resulted in the modification of all the four histidyl residues present in the enzyme with the retention of 95% of the original enzymatic activity. A water‐soluble carbodiimide completely inactivated the cellulase and kinetic analysis indicated that at least one molecule of carbodiimide binds to the enzyme for inactivation. The pH dependence of the inactivation is consistent with the modification of carboxyl groups. The binding of an inhibitor, cellobiose, prior to carbodiimide modification protected the enzyme from rapid inactivation. Titration of the enzyme with dithiobis(2‐nitrobenzoic acid) indicated the absence of free thiol groups. Reaction of the endoglucanase with tetranitromethane resulted in the modification of six of the fourteen tyrosyl residues of the enzyme with approximately 35% diminution in activity.
Tryptophan 5‐hydroxylase
Authors: CASH Christopher D. , VAYER Philippe , MANDEL Paul , MAITRE Michel
Published: March 2005

Tryptophan 5‐hydroxylase (EC 1.14.16.4; l‐tryptophan tetrahydropteridine: oxygen oxidoreductase) was purified to electrophoretic homogeneity from whole brain supernatant using the following steps: pteridine – argarose affinity chromatography, hydrophobic and finally hydroxyapatite chromatography. Exogenous catalase was necessary throughout most of the purification procedure in order to protect the enzyme against inactivation. The iron chelator desferrioxamine at a concentration of 10 μM or higher brought about an irreversible loss of enzyme activity of a partially purified preparation containing an excess of catalase, whereas this same chelator at a lower concentration afforded considerable protection of the enzyme's activity during the final purification stage despite the quasi‐total absence of catalase and the presence of an excess of ferrous iron. Antiserum raised in the rabbit to purified tryptophan 5‐hydroxylase appears to be monospecific for the enzyme after immunoadsorption of anti‐catalase antibodies which were present due to the trace of catalase which remained in the final enzyme preparation.
The protein phosphatases involved in cellular regulation
Authors: STRÅLFORS Peter , HIRAGA Akira , COHEN Philip
Published: March 2005

A type‐1 protein phosphatase (protein phosphatase‐1G) was purified to homogeneity from the glycogenprotein particle of rabbit skeletal muscle. Approximately 3 mg of enzyme were isolated within 4 days from 5000 g of muscle. Protein phosphatase‐1G had a molecular mass of 137 kDa and was composed of two subunits G (103 kDa) and C (37 kDa) in a 1:1 molar ratio. The subunits could be dissociated by incubation in the presence of 2 M NaCl, separated by gel‐filtration on Sephadex G‐100, and recombined at low ionic strength. The C component was the catalytic subunit, and was identical to the 37‐kDa type‐1 protein phosphatase catalytic subunit (protein phosphatase‐1C) isolated from ethanol‐treated muscle extracts, as judged by peptide mapping. The G component was the glycogen‐binding subunit. It was very asymmetric, extremely sensitive to proteolytic degradation, and failed to silver stain on SDS/polyacrylamide gels.

Protein phosphatase‐1G was inhibited by inhibitor‐1 and inhibitor‐2, but unlike protein phosphatase‐1c, the rate of inactivation was critically dependent on the ionic strength, temperature and time of preincubation with the inhibitor protein. At near physiological temperature and ionic strength, protein phosphatase‐1G was inactivated very rapidly by inhibitor‐1. Protein phosphatase‐1G interacted with inhibitor‐2 (1–2) to form an inactive species, with the structure GCI‐2. This form could be activated by preincubation with Mg‐ATP and glycogen synthase kinase‐3.

The G subunit could be phosphorylated on a serine residue(s) by cyclic‐AMP‐dependent protein kinase, but not by phosphorylase kinase or glycogen synthase kinase‐3. Phosphorylation was rapid and stoichiometric, and increased the rate of inactivation of protein phosphatase‐1G by inhibitor‐1. The relationship of the G subunit to the ‘deinhibitor protein’ is discussed.
Structural studies on the linkage unit between poly(N‐acetylglucosamine 1‐phosphate) and peptidoglycan in cell walls of Bacillus pumilus AHU 1650
Authors: KOJIMA Naoya , IIDA Johji , ARAKI Yoshio , ITO Eiji
Published: March 2005

Structural studies were carried out on the polymer chains and their linkage regions in two kinds of teichoic acids, poly(N‐acetylglucosamine 1‐phosphate) [poly(GlcNAc‐1‐P)] and glycerol teichoic acid, bound to peptidoglycan in the cell walls of Bacillus pumilus AHU 1650. The poly(GlcNAc‐1‐P)‐glycan complex isolated from lysozyme digests of the cell walls contained mannosamine and glycerol as minor components. On the basis of proton NMR spectroscopic data and isolation of N‐acetylglucosamine 4‐phosphate from acid hydrolysates, the poly(GlcNAc‐1‐P) was shown to be a polymer in which N‐acetylglucosamine 1‐phosphate units are joined at C‐4 of the glucosamine residues. Mild alkaline hydrolysis of the poly(GlcNAc‐1‐P)‐glycan complex gave a mannosamine‐linked glycan fragment and the acidic polymer fraction that contained glycerol residues. Mild acid treatment of the mannosamine‐linked glycan fragment gave the linkage disaccharide, ManNAc(β1 → 4)GlcNAc, whereas the acidic polymer fraction was degraded by this treatment into N‐acetylglucosamine 4‐phosphate and a glycerol‐containing fragment characterized as P‐(Gro‐P)7 (Gro = glycerol). On the other hand, direct mild acid hydrolysis of the complex gave a fragment characterized as P‐(Gro‐P)7‐ManNAc(β1 → 4)GlcNAc. These results lead to a conclusion that in the cell walls the poly(GlcNAc‐1‐P) chain is attached to peptidoglycan through a linkage unit, (Gro‐P)7‐ManNAc(β1 → 4)GlcNAc. By means of similar procedures, it was shown that the other cell wall polymer, glycerol teichoic acid, is also attached to peptidoglycan through the same disaccharide, ManNAc(β1 → 4)GlcNAc.
Transcription in methanogens
Authors: THOMM Michael , STETTER Karl O.
Published: March 2005

The purification of the DNA‐dependent RNA polymerase of Methanococcus thermolithotrophicus is described. As the first step of purification the endogenous template was removed from the enzyme by hydrophobic interaction chromatography. The purified enzyme consists of seven components with different molecular masses.

Transcription studies on T7 DNA and the recombinant plasmid pMV15, containing rRNA genes of Methanococcus vannielii, revealed that only the methanogen DNA is transcribed specifically, indicating a principal structural difference between archaebacterial and eubacterial promoters. This could be shown both by analysis of ternary transcription complexes and Southern hybridization. The site of initiation was found within a restriction fragment harbouring the first 390 nucleotides of the sequence coding for mature 16S rRNA and 1100 base pairs of upstream sequences. The specific initiation on this fragment strongly suggests that the enzyme can start in vitro transcription from the promoter(s) of rRNA synthesis.
Do yeast aminoacyl‐tRNA synthetases exist as ‘soluble’ enzymes within the cytoplasm?
Authors: ÇCIRAKOḠLU Beyazit , WALLER Jean‐Pierre
Published: March 2005

The aminoacyl‐tRNA synthetases from a crude extract of yeast were shown to bind to heparin‐Ultrogel through ionic interactions, in conditions where the corresponding enzymes from Escherichia coli did not. The behaviour of purified lysyl‐tRNA synthetases from yeast and E. coli was examined in detail. The native dimeric enzyme from yeast (Mr 2 × 73000) strongly interacted with immobilized heparin or tRNA, as well as with negatively charged liposomes, in conditions where the corresponding native enzyme from E. coli (Mr 2 × 65000) displayed no affinity for these supports. Moreover, the aptitude of the native enzyme from yeast to interact with polyanionic carriers was lost on proteolytic conversion to a fully active modified dimer of Mr 2 × 65500. A structural model is proposed, according to which each subunit of yeast lysyl‐tRNA synthetase is composed of a functional domain similar in size to that of the prokaryotic enzyme, contiguous to a ‘binding’ domain responsible for association to negatively charged carriers. The evolutionary acquisition of this property by lower eukaryotic aminoacyl‐tRNA synthetases suggests that it fulfils an important function in vivo, unrelated to catalysis. We propose that it promotes the compartmentalization of these enzymes within the cytoplasm, through associations with as yet unidentified, negatively charged components, by electrostatic interactions too fragile to withstand the usual extraction conditions.
Nucleotide sequence and high‐level expression of the major Escherichia coli phosphofructokinase
Authors: HELLINGA Homme W. , EVANS Philip R.
Published: March 2005

The gene for the major phosphofructokinase enzyme in Escherichia coli, pfk A, has been sequenced. Comparison of the amino acid sequence with other phosphofructokinases showed that this enzyme is related to the Bacillus stearothermophilus and rabbit muscle enzymes, but is different from the second, minor phosphofructokinase found in E. coli.

The region which has been sequenced comprises the complete pfkA – tpi interval on the E. coli genetic map. Two other genes have been identified from the nucleotide sequence: a gene for a periplasmic sulphate‐binding protein, sbp, and for a membrane‐bound enzyme, CDP‐diglyceride hydrolase, cdh. This establishes the complete gene arrangement in this region as pfkA‐sbp‐cdh‐tpi.

The pfkA gene has been subcloned into a high‐copy‐number plasmid under the control of a strong, chimaeric promoter which arose as an artefact in the construction of the plasmid gene bank from which the original pfkA recombinant was isolated. A specialised recombinant has been constructed which carries a 1.4 × 103‐nucleotide insert containing just the pfkA gene flanked by two HindIII recognition sites providing a simple system for the recloning of this gene into different vectors. This recombinant expresses the enzyme at high levels (40–50% of total cell protein is active, soluble phosphofructokinase). This expression system is now being used to study the enzyme using ‘reverse genetics’.
Mutations affecting the enzymes involved in the utilization of 4‐aminobutyric acid as nitrogen source by the yeast Saccharomyces cerevisiae
Authors: RAMOS Fernando , El GUEZZAR Mounir , GRENSON Marcelle , WIAME Jean‐Marie
Published: March 2005

We present genetic evidence for the enzymes 4‐aminobutyrate: 2‐oxoglutarate aminotransferase (EC 2.6.1.19) and succinate‐semialdehyde dehydrogenase [NAD(P)+] (EC 1.2.1.16) constituting the functional pathway for the utilization of 4‐aminobutyric acid as a nitrogen source by Saccharomyces cerevisiae. We show that the pathway is induced by 4‐aminobutyric acid and that the presence of the pathway enzymes probably requires the integrity of a positive control element.
l‐Phenylalanine ammonia‐lyase from Phaseolus vulgaris
Authors: BOLWELL G. Paul , BELL John N. , CRAMER Carole L. , SCHUCH Wolfgang , LAMB Chris J. , DIXON Richard A.
Published: March 2005

l‐Phenylalanine ammonia‐lyase (EC 4.3.1.5) has been purified over 200‐fold from cell cultures of bean (Phaseolus vulgaris L.) exposed to elicitor heat‐released from the cell walls of the phytopathogenic fungus Colletotrichum lindemuthianum. Four forms of the enzyme, with identical Mr but differing apparent pI values of 5.4, 5.2, 5.05 and 4.85, were observed following the final chromatofocussing stage of the purification.

A preparation (purified 43‐fold by ammonium sulphate precipitation, gel‐filtration and ion‐exchange chromatography) containing all four forms exhibited apparent negative rate cooperativity with respect to substrates. However, the individual forms displayed normal Michaelis‐Menten kinetics, with Km values of 0.077 mM, 0.122 mM, 0.256 mM and 0.302 mM in order of decreasing apparent pI value.

A preparation purified 200‐fold and containing all four forms was used to immunise rabbits for the production of anti‐(phenylalanine ammonia‐lyase) serum. The antiserum was characterised by: (a) immunotitration experiments; (b) solid phase enzyme‐linked immunosorbent assays; (c) comparison of immunoprecipitates of 35S‐labelled phenylalanine ammonia‐lyase subunits (synthesized both in vivo and in vitro) on both one‐dimensional and two‐dimensional polyacrylamide gels after immunoprecipitation with the bean antiserum or antisera raised against pea and parsley phenylalanine ammonia‐lyase preparations and (d) immune blotting.

Experiments involving (a) immunoprecipitation followed by analysis on SDS/polyacrylamide gels and (b) SDS/polyacrylamide gel electrophoresis followed by immune blotting, indicated that the Mr of newly synthesized (in vivo and in vitro) bean phenylalanine ammonia‐lyase subunits is 77000; a 70000‐Mr form is readily generated as a partial degradation product during purification.

Immunoprecipitates of bean phenylalanine ammonia‐lyase synthesized both in vivo and in vitro showed the presence of multiple subunit types of identical Mr but differing in pI. Furthermore, treatment of bean cultures with Colletotrichum elicitor resulted in a 10‐fold increase in phenylalanine ammonia‐lyase extractable activity within 8 h, and chromatofocussing analysis indicated that this was associated with differential increased appearance of the high‐pI, low‐Km forms as compared to the two higher Km forms. This differential induction was further confirmed by immune blotting of crude extracts subjected to isoelectric focussing.
The mitochondrial location of protoporphyrinogen oxidase
Authors: DEYBACH Jean‐Charles , DA SILVA Vasco , GRANDCHAMP Bernard , NORDMANN Yves
Published: March 2005

Using the digitonin method and subsequent fractionation of rat liver mitochondria, protoporphyrinogen oxidase (penultimate enzyme in the heme biosynthesis pathway) was found to be closely associated with the mitochondrial inner membrane fraction.

Chemical treatment with non‐specific probes (trypsin and diazobenzene sulfonate) of either intact or inverted mitoplasts, indicated that protoporphyrinogen oxidase was anchored within the lipid bilayer of the inner membrane. Protoporphyrinogen had an equal access to the active site of the enzyme from both sides of the inner membrane and its transformation to protoporphyrin did not appear to be energy‐dependent.

Studies of protoporphyrinogen synthesis from exogenously added coproporphyrinogen in either intact or hypoosmotically treated mitochondria underlined the importance of the peculiar submitochondrial location of coproporphyrinogen oxidase and protoporphyrinogen oxidase for the transfer of substrates to the inner membrane.
Amino acid biosynthesis and sodium‐dependent transport in Methanococcus voltae, as revealed by 13C NMR
Authors: EKIEL Irena , JARRELL Ken F. , SPROTT G. Dennis
Published: March 2005

Of several methanogenic bacteria examined only Methanococcus voltae readily incorporated exogenous amino acids into cell protein. This was easily shown, since growth in the presence of exogenous amino acids resulted in a loss of signal intensities from those carbon atoms normally labelled by [13C]acetate during biosynthesis. From 80% to 95% of the Ser, Lys, Pro or Val incorporated into protein could be supplied directly from the growth medium. In contrast, Asp and Glu, if supplied to the medium, accounted for only a small percentage of the total acidic amino acid used in protein synthesis. Constitutive transport systems took up a wide range of amino acids at rates of 0.1–4.1 nmol min−1 mg−1. The transport systems required Na+, with the possible exception of the basic amino acid lysine, and were inhibited by N‐ethylmaleimide or 3,3′,4′,5‐tetrachlorosalicylanilide. No interconversion of Ile to other amino acids was detected when cells were given [13C]Ile during growth, whereas the expected labelling of the Asp and Glu families of amino acids resulted when [13C]Asp was provided to the culture.

Mc. voltae synthesized its amino acids from acetate via routes fully consistent with those found in Methanospirillum hungatei [Ekiel, I., Smith, I. C. P. & Sprott, G. D. (1983) J. Bacteriol. 156, 316–326]. Propionate could substitute for an auxotrophic requirement for Ile, resulting in the synthesis of Ile with the β‐carbon originating from the carboxyl of acetate and the α‐carbon from the carboxyl of propionate. No labelling of Ile from [13C]acetate could occur without the fatty acid. These results provide strong evidence for the carboxylation of propionate to form 2‐oxobutyrate as intermediate in Ile biosynthesis, and show that the metabolic defect in Ile biosynthesis occurs prior to 2‐oxobutyrate synthesis. The presence of constitutive amino acid transport systems and multiple routes for ile biosynthesis make Methanococcus voltae an attractive methanogen for genetic studies.
Modification of the N‐linked oligosaccharides in cell surface glycoproteins during chick embryo development
Authors: CODOGNO Patrice , BOTTI Joëlle , FONT Jacqueline , AUBERY Michèle
Published: March 2005

Important differences in asparagine‐linked glycopeptides were observed in in vitro cultured fibroblasts derived from chick embryo at different stages of development. Cells from 8‐day and 16‐day embryos were labeled metabolically with [3H]mannose. Cell surface glycopeptides obtained after mild trypsin treatment were extensively digested with pronase and then chromatographed on concanavalin‐A‐Sepharose and other immobilized lectins. The most important changes concerned the complex type chains. The ratio between triantennary plus tetraantennary and biantennary chains increased about 2.5‐fold from the 8th to the 16th day of development. In the same way, complex chains with bisecting N‐acetylglucosamine increased from 8‐day to 16‐day cells as shown by Phaseolus‐vulgaris‐erythroagglutinin – agarose chromatography. In 16‐day cells, the majority of triantennary chains (60%) with α‐linked mannose substituted at C2 and C6 positions and biantennary chains (50%) wer shown to contain fucosyl (α1 → 6)N‐acetylglucosaminyl structure in the core region by their ability to bind to a lentil lectin affinity column. Similarly, in 8‐day cells, triantennary chains (50%) were more fucosylated than biantennary chains (35%). Thus, complex structures exhibited an increased fucosylation of their invariable core from the 8th to the 16th day of development, except for fucosylated triantennary chains which were retained on Phaseolus vulgaris Leucoagglutin and on lentil lectin. These latter structures were present at the surface of 8‐day cells and absent at the surface of 16‐day cells.

After chromatography on Bio‐Gel P6 and treatment with endo‐β‐N‐acetylglucosaminidase H, the [3H]mannose‐labeled glycopeptides were separated by high resolution chromatography into glycopeptides with complex chains and glycopeptides with high‐mannose chains. Analysis of the high‐mannose oligosaccharides released after endo‐β‐N‐acetylglucosaminidase H treatment by chromatography on Bio‐Gel P4 indicated that the same type of high‐mannose chains were present at the surface of 8‐day and 16‐day cells.

Quantification of mannose, galactose and sialic acid residues using gas liquid chromatography was consistent with a decrease of the relative amount of oligomannose chains and an increase of the relative amount of complex type chains in 16‐day cells compared to 8‐day cells. Thus N‐linked oligosaccharides derived from cell surface glycoproteins undergo changes during embryo development resulting in greater complexity of carbohydrate chains.
Pulse‐chase studies of the synthesis of apolipoprotein B in a human hepatoma cell line, Hep G2
Authors: WETTESTEN Margit , BOSTRÖM Kristina , BONDJERS Göran , JARFELDT Marianne , NORFELDT Per‐Ingvar , CARRELLA Massimo , WIKLUND Olov , BORÉN Jan , OLOFSSON Sven‐Olof
Published: March 2005

We have used pulse‐chase methodology to study the synthesis of apolipoprotein B in a human hepatoma‐derived cell line, the Hep G2 cells. A 2‐min pulse with [35S]methionine was followed by a chase period varying from 5–90 min. A protein of large molecular mass (estimated molecular mass: 312 ± 41 kDa, mean ± SD, n= 8) could be immunoprecipitated from the cells at all chase periods between 5 min and 60 min with both monoclonal antibodies to a narrow density cut of the low density lipoprotein LDL‐2 (density: 1.030–1.055 g/ml) and polyclonal antibodies to the apolipoprotein B apoB 100 or to a narrow density cut of LDL‐2 (density: 1.030–1.055 g/ml). In addition to this large molecular mass protein, nascent polypeptides could be precipitated after 5, 10 and 15 min of chase.

The apolipoprotein B molecules that had been labelled during the pulse disappeared from the cells after 60–90 min of chase, while they started to appear in the medium after 30–35 min of chase.

The results obtained indicate (a) that apolipoprotein B is synthesized as one polypeptide with a large molecular mass, (b) that newly synthesized apolipoprotein B molecules are secreted after a delay of 30–35 min, (c) that no intracellular accumulation of apolipoprotein B occurs, and (d) that apolipoprotein B is recovered in the density fraction < 1.21 g/ml of the medium suggesting that it is secreted in lipoprotein form.