bluuebunny/crossref_metadata_2025 huggingface dataset:
In vivo31P‐and 13C‐NMR studies of ATP synthesis and methane formation by Methanosarcina barkeri
Authors: SANTOS Helena , FARELEIRA Paula , TOCI René , LeGALL Jean , PECK Harry D. , XAVIER António V.
Published: March 2005

Carbon and phosphorus metabolism of cell suspensions of Methanosarcina barkeri strain MS (DSM 800), grown on methanol, were probed in vivo by NMR. The experimental conditions, which involved thick cell suspensions, did not significantly affect the efficiency of the rate of methanol uptake by cells. Following exposure to methanol an acidification of both the intracellular and the extracellular spaces was observed and a gradient of 0.5 pH units across the cytoplasmic membrane was determined from the 31P‐NMR data. High levels of intracellular ATP up to 4 mM were detected. The ADP concentration determined in a suspension of starved cells was only 2 mM, suggesting that a significant amount of ADP may be immobilized and is thus not detectable by NMR. In the presence of the protonophore, 3.3′,4′,5‐tetrachlorosalicylanilide, the proton gradient was dissipated and the synthesis of ATP stopped. The inhibitor of the ATP synthase, N,N′‐dicyclohexylcarbodiimide, was rather inefficient in inhibiting ATP synthesis. High concentrations of N,N′‐dicyclohexylcarbodiimide (corresponding to 300 nmol/mg protein–1) were required to decrease the ATP content by approximately 60%, and, under these conditions, formation of acetyl phosphate was detected. However, the methanol consumption rate was not affected.
Studies of the functional topography of Escherichia coli RNA polymerase
Authors: GRACHEV Mikhail A. , LUKHTANOV Evgeny A. , MUSTAEV Arkady A. , ZAYCHIKOV Evgeny F. , ABDUKAYUMOV Melis N. , RABINOV Igor V. , RICHTER Vladimir I. , SKOBLOV Yury S. , CHISTYAKOV Pavel G.
Published: March 2005

A method is proposed for localization of the sites of affinity labelling of the β subunit of Escherichia coli RNA polymerase. The principle of this method is similar to that of the methods of rapid sequencing of nucleic acids.

The polypeptide bearing a radioactive affinity label at one of the amino acid residues is subjected to short‐term treatment with cyanogen bromide. The conditions of this reaction are selected in such a way that less than one cleavage occurs on average per polypeptide chain. Two series of radioactive peptides are formed, one involving all the possible N‐terminal peptides and the other the C‐terminal peptides. The distribution of the lengths of these peptides is studied by means of gel electrophoresis and compared with the theoretical ones based on the known amino acid sequence of the β subunit. Obviously, the affinity label resides between the C‐terminus of the shortest N‐terminal radioactive peptide and the N‐terminus of the shortest C‐terminal radioactive peptide. In order to increase reliability and resolution of the method, partial trypsinolysis may be employed.

The evidence obtained suggests that lysine residues over the regions 1036–1066, 1234–1242, and histidine‐1237 are situated in the nearest neighbourhood to, or directly involved in the formation of the active center of initiating substrate binding of the β subunit of E. coli RNA polymerase.
Metabolism of aspartate in Mycobacterium smegmatis
Authors: SRITHARAN Venkataraman , WHEELER Paul R. , RATLEDGE Colin
Published: March 2005

Mycobacterium smegmatis grows best on l‐asparagine as a sole nitrogen source; this was confirmed. [14C]Aspartate was taken up rapidly (46 nmol · mg dry cells−1· h−1 from 1 mM l‐asparagine) and metabolised to CO2 as well as to amino acids synthesised through the aspartate pathway. Proportionately more radioactivity appeared in the amino acids in bacteria grown in medium containing low nitrogen.

Activities of aspartokinase and homoserine dehydrogenase, the initial enzymes of the aspartate pathway, were carried by separate proteins. Aspartokinase was purified as three isoenzymes and represented up to 8% of the soluble protein of M. smegmatis. All three isoenzymes contained molecular mass subunits of 50 kDa and 11 kDa which showed no activity individually; full enzyme activity was recovered on pooling the subunits. Km values for aspartate were: aspartokinases I and III, 2.4 mM; aspartokinase II, 6.4 mM. Aspartokinase I was inhibited by threonine and homoserine and aspartokinase III by lysine, but aspartokinase II was not inhibited by any amino acids. Aspartokinase activity was repressed by methionine and lysine with a small residue of activity attributable to unrepressed aspartokinase I. Homoserine dehydrogenase activity was 96% inhibited by 2 mM threonine; isoleucine, cysteine and valine had lesser effects and in combination gave additive inhibition. Homoserine dehydrogenase was repressed by threonine and leucine. Only amino acids synthesised through the aspartate pathway were tested for inhibition and repression. Of these, only one, meso‐diaminopimilate, had no discernable effect on either enzyme activity.
Comparison of the two classes of binding sites (A and B) of type I and type II cyclic‐AMP‐dependent protein kinases by using cyclic nucleotide analogs
Authors: ØGRIED Dagfinn , EKANGER Roald , SUVA Robert H. , MILLER Jon P. , DØSKELAND Stein Ove
Published: March 2005

cAMP analogs, all 96 of which were modified in the adenine moiety, were examined quantitatively for their ability to inhibit the binding of [3H]cAMP to each of the two classes (A and B) of cAMP‐binding sites of type I (rabbit skeletal muscle) and type II (bovine heart) cAMP‐dependent protein kinase. The study showed that analogs can be constructed that have a higher affinity than cAMP for a binding site. N6‐phenyl‐cAMP had 18‐fold increased affinity for site A of RI (AI) and 40‐fold increased affinity for site AII. 2‐chloro‐8‐methylamino‐cAMP had a 7‐fold increased affinity for BI, and 8‐(4‐chlorophenylthio)‐cAMP had 17‐fold increased affinity for BII.

Analogs could discriminate between the two classes of binding sites by more than two orders of magnitude in binding affinity: 2‐chloro‐8‐methylamino‐cAMP had 170‐fold higher affinity for BI than for AI, and 2‐n‐butyl‐8‐thiobenzyl‐cAMP had 700‐fold higher affinity for BII than for AII. Analogs could also discriminate between the homologous binding sites of the isozymes: 2‐n‐butyl‐8‐bromo‐cAMP had 260‐fold higher affinity for AI than for AII (22‐fold higher for BII than BI), and 8‐piperidino‐cAMP had 50‐fold higher affinity for BII than for BI (and 50‐fold higher for AI than for AII).

The data suggest the following conclusions. (a) Stacking interactions are important for the binding of cAMP to all the binding sites. (b) Subtle differences exist between the sites as to the optimal electron distribution in the adenine ring since modifications that withdraw electrons at C2 and donate at C8 favour binding to BI, and disfavour binding to AI and AII. (c) There are no hydrogen bonds between the adenine ring of cAMP and any of the binding sites. (d) All sites bind cAMP in the syn conformation. (e) The subsites adjacent to the N6 and C8 positions may have nonpolar neighbouring regions since hydrophobic substituents at N6 could increase the affinity for AI and AII and similar substituents at C8 could increase the affinity for BII. Finally, (f) the sites differed in their ability to accomodate bulky substituents at C2 and C8.

For all compounds tested, their potency as activators of protein kinases I and II was found to correlate, in a predictable fashion, to their mean affinity for the two classes of binding sites, rather than to the affinity for only one of the sites.
Isolation and molecular and functional properties of the amino‐terminal domain of colicin A
Authors: KNIBIEHLER Martine , HOWARD S. Peter , BATY Daniel , GELI Vincent , LLOUBÈS Roland , SAUVE Paul , LAZDUNSKI Claude
Published: March 2005

A plasmid was constructed which allowed easy and efficient production and purification of the NH2‐terminal domain of colicin A. In only three steps, an homogenous 18‐kDa polypeptide was obtained. The NH2‐ and COOH‐terminal sequences of the protein were determined and showed that it corresponded to the NH2‐terminal 171 amino acid residues of the 63‐kDa colicin A. Although colicin A is a highly asymmetric protein, hydrodynamic studies indicated that the NH2‐terminal domain (designated AT) has a globular structure. This fragment is not the receptor‐binding domain of colicin A but is required for the transfer of colicin A across the outer membrane of sensitive cells. However, it has a low affinity for phospholipid films and this affinity is not pH‐dependent, in contrast to that of colicin A.
Myosin isoenzymes and their subunits in urodelan amphibian fast skeletal muscle
Authors: CHANOINE Christophe , GALLIEN Claude‐Louis
Published: March 2005

The distributions of native myosin isoforms were examined by electrophoresis under non‐dissociating conditions, in the fast twitch dorsal skeletal muscle of young larvae, neotenic adults and metamorphosed adults of urodelan amphibians. Both heavy and light chains of myosin isoenzymes were analysed. In pyrophosphate acrylamide gel electrophoresis three isoenzymes were demonstrated in larval myosin; other isoforms of lower electrophoretic mobility were observed in metamorphosed adults myosin. Larval and adult isoenzymes were shown to coexist in myosin from neotenic adults. Analysis of heavy chains in denaturing conditions and proteolytic digestion revealed the sequential occurrence during development of two types of heavy chains, one larval and one adult, that coexist in the myosin of neotenic adults only. Analysis of light chain patterns under denaturing conditions revealed the existence of three fast light chains which displayed no modification during the course of development. The neotenic urodelan amphibian species model represents actually the only model in which the coexistence of larval (or neonatal) and adult heavy chains is maintained throughout life in adults.
Comparison of the NH2‐terminal amino acid sequences of the four non‐identical subunits of the NAD‐linked hydrogenases from Nocardia opaca 1b and Alcaligenes eutrophus H16
Authors: ZABOROSCH Christiane , SCHNEIDER Klaus , SCHLEGEL Hans G. , KRATZIN Hartmut
Published: March 2005

The cytoplasmic, NAD‐linked hydrogenase of the Gram‐positive hydrogen‐oxidizing bacterium Nocardia opaca 1b was compared with the analogous enzyme isolated from the Gram‐negative bacterium Alcaligenes eutrophus H16. The hydrogenase of N. opaca 1b was purified by a new procedure applying chromatography on phenyl‐Sepharose and DEAE‐Sephacel with two columns in series. A homogeneous enzyme preparation with a specific activity of 74 μmol H2 oxidized · min−1· mg protein−1 and a yield of 32% was isolated. The A. eutrophus enzyme was purified as previously published. Both enzymes are tetrameric proteins composed of four non‐identical subunits (α, β, γ, δ). The four subunits of both of these enzymes were separated and isolated as single polypeptides by preparative polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate.

Immunological comparison of the four subunits of the Nocardia hydrogenase with those of the Alcaligenes enzyme showed that the α, β, γ, and δ subunits of one organism were serologically related to the analogous subunits of the other organism. Among themselves, the four subunits do not have any serological relationship.

The eight individual polypeptides were also compared with respect to the NH2‐terminal amino acid sequences determined by automated Edman degradation and to the amino acid compositions. Strong sequence similarities exist between the analogous subunits isolated from the two bacteria. Within the established N‐terminal sequences the similarities between both (α, β, γ, and δ) subunits amount to 63%, 79%, 80% and 65%, respectively. No similarities exist between the different, non‐analogous subunits α, β, γ, and δ.
Apolipoprotein‐E‐gene expression in rat liver during development in relation to insulin and glucagon
Authors: MANGENEY Marise , CARDOT Philippe , LYONNET Stanislas , COUPE Christine , BENAROUS Richard , MUNNICH Arnold , GIRARD Jean , CHAMBAZ Jean , BEREZIAT Gilbert
Published: March 2005

An apolipoprotein‐E (apo‐E) cDNA probe, cloned by immunoscreening of a λGT11 rat liver cDNA library, was used to further characterize the expression of the apo‐E gene in rat liver during development, in relation to plasma insulin and glucagon levels.

The apo‐E mRNA level was low in fetus liver, then abruptly increased at birth and rose further during the suckling period. It returned to the level at birth in 10‐week‐old adults. These variations were paralleled with dramatic changes in plasma glucagon, which rose at birth and remained high during suckling. At the same time, the insulin/glucagon molar ratio fell.

Administration of N6, O2‐dibutyryl cAMP to 5‐day‐old rats resulted in a significant induction of liver apo‐E mRNA. Moreover, liver apo‐E mRNA rose in 10‐h‐fasted suckling rats as compared to controls, while plasma glucagon increased and the insulin/glucagon ratio decreased. Conversely, glucose feeding of suckling rats did not induce any increase in liver apo‐E mRNA, the insulin/glucagon ratio was 10‐fold higher than in fasted animals.

Our results are consistent with liver apo‐E gene expression being under the control of plasma glucagon and of the glucagon/insulin balance.
Interrelated regulation of sulphur‐containing amino‐acid biosynthetic enzymes and folate‐metabolizing enzymes in Aspergillus nidulans
Authors: NADOLSKA‐LUTYK Janina , BALINSKA Malgorzata , PASZEWSKI Andrzej
Published: March 2005

In Aspergillus nidulans homocysteine can be metabolized both to cysteine and methionine. Mutants impaired in the main pathway of cysteine synthesis or in the sulphate assimilation pathway show a low pool of glutathione and elevated levels of homocysteine synthase and of the homocysteine‐to‐cysteine pathway enzymes. On the other hand, the level of methionine synthase and other enzymes of folate metabolism is depressed in these mutants. This anticoordinated regulation provides a mechanism controlling the partition of homocysteine between the two diverging pathways. Homocysteine synthase was found derepressed, along with folate enzymes, in a strain carrying a mutation which suppresses mutations in met A, met B and metG genes. These results indicate that homocysteine synthase can be regarded as the enzyme of an alternative pathway of methionine synthesis and strongly suggest that the regulatory mechanisms governing sulphur‐containing amino acid and folate metabolisms are interrelated.
N‐linked oligosaccharide changes with oncogenic transformation require sialylation of multiantennae
Authors: SANTER Ursula V. , DeSANTIS Rosaria , HÅRD Karl Johan , van KUIK J. Albert , VLIEGENTHART Johannes F. G. , WON Bokran , GLICK Mary Catherine
Published: March 2005

Glycopeptides derived from NIH 3T3 fibroblasts and these cells transformed by transfection with human DNA containing oncogene H‐ras were analyzed by 500‐MHz 1H‐NMR spectroscopy and binding to immobilized lectins. The cells were metabolically labeled with D‐[3H]glucosamine or L‐[3H]fucose and the glycopeptides included in Bio‐Gel P‐10 (Mr 5000–3500) were separated into neutral and charged fractions on DEAE‐cellulose. The major portion (80%) of these [3H]fucose glycopeptides from the non‐transformed NIH 3T3 fibroblasts were neutral or contained one or two charged residues, whereas 90% of the glycopeptides from the transformed cells contained two or more charged residues.
Evidence for cytochrome oxidase subunit I and a cytochrome c– subunit II fused protein in the cytochrome ‘c1aa3’ of Thermus thermophilus
Authors: BUSE Gerhard , HENSEL Sieghard , FEE James A.
Published: March 2005

The terminal cytochrome c1aa3 of the respiratory chain of Thermus thermophilus has been isolated and purified to homogeneity by a novel procedure. The two subunit proteins (55 and 33 kDa) have been characterized chemically. Computer searches with partial amino acid sequences obtained from both subunits show that the larger subunit belongs to the cytochrome oxidase subunit I protein family while the smaller covalently heme‐binding subunit is not a cytochrome c1 but appears to be a fused protein between cytochrome c and cytochrome oxidase subunit II.

With respect to the 16‐S rRNA‐derived phylogeny of procaryotes, the results show that the genetic information for an O2‐reacting cytochrome oxidase (EC 1.9.3.1) existed already in early eubacteria.
Molecular structure of an A‐DNA decamer d(ACCGGCCGGT)
Authors: FREDERICK Christin A. , QUIGLEY Gary J. , TENG Mai‐Kun , COLL Miquel , van der MAREL Gijs A. , van BOOM Jacques H. , RICH Alexander , WANG Andrew H.‐J.
Published: March 2005

The molecular structure of the DNA decamer d(ACCGGCCGGT) has been solved and refined by single‐crystal X‐ray‐diffraction analysis at 0.20 nm to a final R‐factor of 18.0°. The decamer crystallizes as an A‐DNA double helical fragment with unit‐cell dimensions of a=b= 3.923 nm and c= 7.80 nm in the space group P6122. The overall conformation of this A‐DNA decamer is very similar to that of the fiber model for A‐DNA which has a large average base‐pair tilt and hence a wide and shallow minor groove. This structure is in contrast to that of several A‐DNA octamers in which the molecules all have low base‐pair‐tilt angles (8–12°) resulting in an appearance intermediate between B‐DNA and A‐DNA. The average helical parameters of this decamer are typical of A‐DNA with 10.9 base pairs/turn of helix, an average helical twist angle of 33.1°, and a base‐pair‐tilt angle of 18.2°. However, the CpG step in this molecule has a low local‐twist angle of 24.5°, similar to that seen in other A‐DNA oligomers, and therefore appears to be an intrinsic stacking pattern for this step. The molecules pack in the crystal using a recurring binding motif, namely, the terminal base pair of one helix abuts the surface of the shallow minor groove of another helix. In addition, the GC base pairs have large propeller‐twist angles, unlike those found most other A‐DNA structures.
Purification and characterization of ribonuclease M and mRNA degradation in Escherichia coli
Authors: CANNISTRARO Vincent J. , KENNELL David
Published: March 2005

A previously unreported endoribonuclease has been identified in Escherichia coli, which has a preference for hydrolysis of pyrimidine‐adenosine (Pyd‐Ado) bonds in RNA. It was purified about 7000‐fold to give a single band after SDS/polyacrylamide gel electrophoresis; the eluted protein gave the same RNase specificity. The sizes of the native and denatured enzymes agreed suggesting that the enzyme exists as a monomer of approximately 26 kDa. It is called RNase M.

The only other reported broadly specific endoribonuclease in E. coli is RNase I, a periplasmic enzyme. Based on differences in charge, heat stability and substrate specificity, it was clear that RNase M is not RNase I. The specificity of RNase M was remarkably similar to that of pancreatic RNase A even though the two enzymes differ in charge characteristics and size.

Earlier studies had shown that mRNA from the lactose operon of E. coli is hydrolyzed in vivo primarily between Pyd‐Ado bonds [Cannistraro et al. (1986) J. Mol. Biol. 192, 257–274]. We propose that this major RNase activity accounts for these cleavages observed in vivo and that it is the endonuclease for mRNA degradation in E. coli.
Basic proline‐rich proteins of murine parotid glands
Authors: BANNISTER Andrew J. , DIVECHA Nullin , ASHMORE Michael , McDONALD Charles J.
Published: March 2005

Five major basic polypeptides with characteristics typical of proline‐rich proteins, accumulated in parotid glands after long term isoprenaline treatment of Balb C mice. They were studied by two‐dimensional gel electrophoresis and designated B1°, B2′°, B2°, B3° and B4° on the basis of pI‐dependent mobility. They were not observed in the glands of normal mice and were precipitated when glands were homogenized in 10% trichloroacetic acid unlike the three isoprenaline‐induced proline‐rich proteins of murine parotid glands reported previously.

Isoprenaline induced six proline‐rich in vitro translation products which were absent normally. Four of these species had pI‐dependent mobilities almost identical to B1°, B2°, B3° and B4°, indicating not only precursor/product relationships, but also that isoprenaline induced the accumulation of the proteins by regulating the mRNA.

Identical salivary counterparts of the basic glandular proline‐rich proteins were not detected whereas a series of smaller and more basic isoprenaline‐induced polypeptides were observed in saliva (major speices B1s–B4s). The glandular proline‐rich proteins were secreted from parotid tissue in vitro and the data indicate that proline‐rich proteins are synthesised as precursors and processed into salivary form in the parotid glands after secretion. The relationships between the B‐type in vitro translation products, parotid gland precursors and salivary proteins were also confirmed immunologically.
Effect of the microenvironment on the tertiary structure of cytochrome P‐450 LM2
Authors: UVAROV Valentin Yu. , TRETIAKOV Vadim E. , LESHCHENKO Albina V. , RUKAVISHNIKOV Igor G. , DZHUZENOVA Cholpon S. , TRETIAKOVA Larisa Z. , ARCHAKOV Alexander I.
Published: March 2005

The relation between microenvironment and the tertiary structure of cytochrome P‐450 LM2 has been investigated. No complete relaxation to the most active state of the native enzyme took place in the case of membrane‐incorporated hemoprotein with three or four intramolecular cross‐links.

The spatial organization of the enzyme was predicted to determine the cross‐link location on the hemoprotein surface and membrane‐incorporated parts of the polypeptide chain. It was concluded on the basis of the predicted structure that hemoprotein has an amphipathic structure and, thus, the greater part of molecule is exposed to the water phase. Not more than one NH2‐terminal α helix is able to incorporate into the membrane. The location of this region is believed to control the formation of the catalytically‐active‐conformational state of cytochrome P‐450 LM2.
Sequence of the cysteinyl‐containing peptides of 4‐aminobutyrate aminotransferase
Authors: KIM Young Tae , CHURCHICH Jorge E.
Published: March 2005

Three cysteine‐containing tryptic peptides were isolated and sequenced from mitochondrial 4‐aminobutyrate aminotransferase using DABIA (4‐dimethylaminoazobenzene‐4‐iodoacetamide) as specific labeling reagent for sulfhydryl groups. The enzyme is a dimer made up of two identical subunits, but four out of the six cysteinyl residues/dimer form disulfide bonds when treated with iodosobenzoate to yield inactive enzyme species.

To identify the cysteinyl residues undergoing reversible oxidation/reduction, the S‐DABIA‐labeling patterns of the fully reduced (active) and fully oxidized (inactive) forms of the enzyme were compared. Tryptic digests of the reduced enzyme contained three labeled peptides. If the enzyme was treated with iodosobenzoate prior to reaction with DABIA and tryptic digestion, only one labeled peptide was detected and identified (peptide I), indicating that the two missing cysteinyl‐containing peptides (peptides II, III) have been oxidized.

The sulfhydryl groups undergoing oxidation/reduction were found to be intersubunit, based on SDS/polyacrylamide gel electrophoresis results. The loss of catalytic activity of 4‐aminobutyrate aminotransferase by oxidation of sulfhydryl residues is related to constraints imposed at the subunit interface by the insertion of disulfide bonds.
Purification and properties of 5,6‐dihydropyrimidine amidohydrolase from calf liver
Authors: KAUTZ Jürgen , SCHNACKERZ Klaus D.
Published: March 2005

5,6‐Dihydropyrimidine amidohydrolase was isolated from an acetone powderof calf liver and purified to homogeneity. Purification made use of heat treatment, ammonium sulfate fractionation and chromatography on Chelating Sepharose and DEAE‐Sepharose with 44% recovery of total activity. The native enzyme has a molecular mass of 217 kDa consisting of four subunits with a molecular mass of 54 kDa each. The amidohydrolase is a metalloenzyme containing one zinc atom/subunit. The enzyme can slowly be inactivated by chelating agents. The kinetic parameters for substrates, 5,6‐dihydrouracil, 5,6‐dihydrothymine and glutarimide were determined. From log Vmax/KM data, a pKa of 7.6 could be calculated suggesting the formation of a zinc‐bound hydroxyl ion which carries out the nucleophilic attack on the C‐4 of dihydrouracil.
Cross‐reconstitution of the FOF1‐ATP synthases of chloroplasts and Escherichia coli with special emphasis on subunit δ
Authors: ENGELBRECHT Siegfried , DECKERS‐HEBESTREIT Gabriele , ALTENDORF Karlheinz , JUNGE Wolfgang
Published: March 2005

FOF1‐ATP synthases catalyse ATP formation from ADP and Pi by using the free energy supplied by the transmembrane electrochemical potential of the proton. The δ subunit of F1 plays an important role at the interface between the channel portion Fo and the catalytic portion F1. In chloroplasts it can plug the protonic conductance of CFo and in Escherichia coli it is required for binding of EF1 to EFo.

We wanted to know whether or not δ of one species was effective between Fo and F1 of the other species and vice versa. To this end the respective coupling membrane (thylakoids, everted vesicles from E. coli) was (partially) depleted of F1 and purified F1, F1(‐δ), and δ were added in various combinations to the F1‐depleted membranes. The efficiency or reconstitution was measured in thylakoids via the rate of phenazinemethosulfate‐mediated cyclic photophosphorylation and in E. coli everted vesicles via the degree of 9‐amino‐6‐chloro‐2‐methoxyacridine fluorescence quenching.

Addition of CF1 to partially CF1‐depleted thylakoid vesicles restored photophosphorylation to the highest extent. CF1(‐δ)+chloroplast δ, EF1, EF1(‐δ)+E. coliδ were also effective but to lesser extent. CF1(‐δ)+E. coliδ and EF1(‐δ)+chloroplast δ restored photophosphorylation to a small but still significant extent. With F1‐depleted everted vesicles prepared by repeated EDTA treatment of E. coli membranes, addition of CF1, CF1(‐δ)+chloroplast δ and CF1(‐δ)+E. coliδ gave approximately half the extent of 9‐amino‐6‐chloro‐2‐methoxyacridine fluorescence quenching as compared to EF1 or EF1(‐δ)+E. coliδ by energization of the vesicles with NADH, while EF1(‐δ)+chloroplast δ was ineffective. All ‘mixed’ combinations were probably reconstitutively active only by plugging the protonic leak through the exposed Fo (structural reconstitution) rather than by catalytic activity. Nevertheless, the cross‐reconstitution is stunning in view of the weak sequence similarity between chloroplast δ and E. coliδ. It favors a role of δ as a conformational transducer rather than as a proton conductor between F0 and F1.
Structural requirements for protein N‐glycosylation
Authors: ROITSCH Thomas , LEHLE Ludwig
Published: March 2005

To understand better the structural requirements of the protein moiety important for N‐glycosylation, we have examined the influence of proline residues with respect to their position around the consensus sequence (or sequon) Asn‐Xaa‐Ser/Thr. In the first part of the paper, experiments are described using a cell‐free translation/glycosylation system from reticulocytes supplemented with dog pancreas microsomes to test the ability of potential acceptor peptides to interfere with glycosylation of nascent yeast invertase chains. It was found that peptides, being acceptors for oligosaccharide transferase in vitro, inhibit cotranslational glycosylation, whereas nonacceptors have no effect. Acceptor peptides do not abolish translocation of nascent chains into the endoplasmic reticulum. Results obtained with proline‐containing peptides are compatible with the notion that a proline residue in an N‐terminal position of a potential glycosylation site does not interfere with glycosylation, whereas in the position Xaa or at the C‐terminal of the sequon, proline prevents and does not favour oligosaccharide transfer, respectively.

This statement was further substantiated by in vivo studies using site‐directed mutagenesis to introduce a proline residue at the C‐terminal of a selected glycosylation site of invertase. Expression of this mutation in three different systems, in yeast cells, frog oocytes and by cell‐free translation/glycosylation in reticulocytes supplemented with dog pancreas microsomes. leads to an inhibition of glycosylation with both qualitative and quantitative differences. This may indicate that host specific factors also contribute to glycosylation.
Evidence that the upstream stimulatory factor and the Sp1 transcription factor bind in vitro to the promoter of the human‐growth‐hormone gene
Authors: LEMAIGRE Frédéric P. , COURTOIS Stéphane J. , LAFONTAINE Dominique A. , ROUSSEAU Guy G.
Published: March 2005

Expression of the human‐growth‐hormone gene is restricted to pituitary somatotrophs. Two protein‐DNA complexes that are specific to the pituitary, and two that are not, had been demonstrated in vitro on the promoter of this gene. The two pituitary‐specific footprints had been ascribed to a single protein called growth hormone factor 1. We have now characterized the factors responsible for the two other footprints by means of deoxyribonuclease‐I protection and gel‐retardation experiments.

The first footprint, located between –257 and –290 relative to the transcription initiation site, involves at least two factors present in pituitary cells. One of these factors binds between nucleotides –257 and –267, and is indistinguishable from the upstream stimulatory factor, also called major late transcription factor or upstream element factor, initially described in HeLa cells. Earlier work by others had shown that the activator protein 2 purified from HeLa cells can bind to nucleotides –263 and –290. Our experiments suggest that a factor different from activator protein 2 is involved in the protection of this region against deoxyribonuclease I.

The second footprint, located between nucleotides –116 and –140, involves only one factor. This factor, present in pituitary cells, recognizes a GC box and is indistinguishable from transcription factor Sp1, previously described in HeLa cells. The human‐growth‐hormone gene is therefore a candidate for regulation by these factors in vivo.
Ecdysteroid receptors of the blowfly Calliphora vicina
Authors: LEHMANN Michael , KOOLMAN Jan
Published: March 2005

Ecdysteroids, the molting hormones of arthropods, act like vertebrate steroid hormones by binding to an intracellular receptor protein. We have recently isolated a protein from nuclei of blowfly larvae which has satisfied the requirements of an ecdysteroid receptor. The receptor was partially purified and its ecdysteroid‐binding properties were characterized. The availability of receptor preparations which have been stabilized by partial purification now enables us to study the general DNA‐binding properties of ecdysteroid receptors.

DNA‐binding characterstics of ecdysteroid receptors were studied with calf thymus DNA. Affinity for DNA was observed both in the presence and in the absence of steroid ligand but the ligand clearly enhanced binding of receptors to DNA. Receptor preparations contained a heterogeneous mixture of receptors; up to 25% of DNA‐binding receptors, and nonbinding forms of ecdysteroid receptors. The ability to bind to DNA was subject to inactivation which was not affected by partial purification, but which could be decelerated by dilution of the receptor preparation. Thus, dilution resulted in a spurious activation of DNA binding. A genuine activation, which would have led to an increase in the percentage of the DNA‐binding form of the ecdysteroid receptor complex, was not observed.
Mouse U3‐RNA‐processed pseudogenes are nonrandomly integrated into genomic DNA
Authors: MAZAN Sylvie , MICHOT Bernard , BACHELLERIE Jean‐Pierre
Published: March 2005

We have characterized three mouse U3‐RNA‐processed pseudogenes. Together with previous analyses of mouse functional genes and of rat genes and pseudogenes, these data provide some insights into the processes of U3 retrogene formation during the evolution of rodents. All the mouse retrogenes correspond to a full‐length U3B‐coding sequence with a 3′‐poly(A) tail and are precisely flanked by a pair of direct repeats, in agreement with formation through an RNA intermediate followed by insertion at staggered nicks in the genome. All the rodents U3 retrogenes identified so far are produced from a U3B‐RNA form, with two of them (one for each rodent) formed from a 3′‐elongated U3‐RNA precursor, pointing to the particular susceptibility of RNA precursors forms to serve as templates for retrogene formation. Rodent full‐length U3 retrogenes are not inserted randomly in the genome but are systematically found in a context of simple sequences, prone to slipped‐strand mispairings and likely to favour the appearance of single‐stranded DNA. Moreover their flanking repeats share not only the same size (15 bp) but also common sequence features (which extend to vicinal upstream nucleotides) suggesting that common mechanisms, specific to this class of retrogenes, have been involved in their formation. For the first time, a model accounting for the generation of full‐length cDNA copies from nonpolyadenylated RNA templates is proposed. Rodent U3 retrogenes appear to be of rather recent origin (posterior to the mouse/rat divergence) and some of them could have undergone subsequent genetic exchanges with the functional genes.
Cycloeucalenol — obtusifoliol isomerase
Authors: RAHIER Alain , TATON Maryse , BENVENISTE Pierre
Published: March 2005

The molecular features of 19 synthetic substrates and ground‐state analogues of cycloeucalenol, the natural substrate of cycloeucalenol — obtusifoliol isomerase, a membrane‐bound enzyme specific to higher plants, and of 9 synthetic carbocationic analogues of the high‐energy intermediate occurring during the reaction catalyzed by the isomerase, were related to their ability to be transformed by this enzyme (catalytical competence) and their potency as inhibitors of this enzyme. With substrates and ground‐state analogues it has been possible to determine at‐least two critical domains: significant binding requires the presence of the 3β‐hydroxyl group on the ring A with the correct stereochemistry together with absence of a 4β‐methyl group. Moreover initial enzyme‐substrate interaction appears to be dependent upon the assessibility of the 3β‐oxygen. Substitutions on the ring B do not preclude binding whereas they are of great influence on substrate transformation. Modifications of the ring A domain affects binding of high‐energy intermediate analogues to a much lesser extent. The effects of these and other modifications suggest that ground‐state and high‐energy intermediate analogues bind two different conformations of the isomerase active site.
N‐Acetylglucosaminyltransferase III, IV and V activities in Novikoff ascites tumour cells, mouse lymphoma cells and hen oviduct
Authors: KOENDERMAN Anky H. L. , KOPPEN Piet L. , KOELEMAN Carolien A. M. , van den EIJNDEN Dirk. H.
Published: March 2005

A specific and fast method for the determination of N‐acetylglucosaminyltransferase III, IV and V activity in one assay is described. The method is based on the separation by HPLC of the three tranferase products formed from the common acceptor oligosaccharide substrate GlcNAcβ1→2Manα1→3(GlcNAcβ1→2Manαl→6)Manβ1→4GlcNAc. Assays are not interfered with by substances that result from enzymatic or chemical breadkdown of the donor substrate UDP‐[14C]GlcNAc. Using this assay system N‐acetylglucosaminyltransferase III, IV and V activities were estimated in Novikoff ascites tumour cells, mouse lymphoma BW 5147 cells and hen oviduct.
Spectroscopic investigations of Panulirus interruptus hemocyanin in the crystalline state
Authors: VOLBEDA Anne , FEITERS Martin C. , VINCENT Michael G. , BOUWMAN Elisabeth , DOBSON Barry , KALK Kor H. , REEDIJK Jan , HÓL Wim G. J.
Published: March 2005

Single‐crystal ultraviolet spectroscopy, X‐ray absorption spectroscopy and EPR measurements have been used to examine the oxidation and oxygenation state of the dinuclear copper site of several types of hemocyanin crystals. The crystals contain Panulirus interruptus hemocyanin which forms hexameric molecules with a molecular mass of approximately 470 kDa.

Three types of crystals have been investigated. Type‐I monoclinic crystals, which have been used for the X‐ray structure determination, contain virtually only deoxyhemocyanin. Type‐II monoclinic crystals, which are less well ordered than the type‐I crystals, contain a mixture of deoxy, oxy and met forms. Older crystals contain relatively more methemocyanin. A third, hexagonal, crystal form is also partially oxygenated, and, like the type‐II monoclinic form, subject to gradual conversion to methemocyanin.
Thrombospondin is synthesized and secreted by human osteoblasts and osteosarcoma cells
Authors: CLEZARDIN Philippe , JOUISHOMME Hervé , CHAVASSIEUX Pascale , MARIE Pierre J.
Published: March 2005

In this study we have shown by both immunofluorescence and immunoprecipitation techniques that human osteoblasts and osteosarcoma cells synthesize and secrete thrombospondin, a 450‐kDa glycoprotein initially found in platelets. Immunofluorescence with a mouse monoclonal antibody to human platelet thrombospondin yielded specific granular staining within the cytoplasm of human osteoblasts. SDS/polyacrylamide gel electrophoresis analysis of immunoprecipitates obtained with polyclonal and monoclonal anti‐thrombospondin antibodies allows the identification of thrombospondin in the cellular lysates and the culture media of biosynthetically labelled osteoblasts and osteosarcoma cells. Kinetic and dose/response studies of osteoblasts and of two osteosarcoma cell lines (MG‐63, SaOs‐2) were performed to assess the ability of these cells to adhere to thrombospondin and type‐I collagen. Thrombospondin promoted the attachment of human osteoblasts whereas it inhibited the adhesion of MG‐63 and SaOs‐2 cells, both when it was directly adsorbed to plastic and when it was bound to type‐I collagen. Therefore osteoblasts and osteosarcoma cells may be valuable tools to study the role of thrombospondin in cell adhesion.
Cloning and sequencing of the genes of 2‐hydoxyglutaryl‐CoA dehydratase from Acidaminococcus fermentans
Authors: DUTSCHO Roland , WOHLFARTH Gert , BUCKEL Peter , BUCKEL Wolfgang
Published: March 2005

Two genomic libraries from Acidaminococcus fermentans DNA constructed with the λ vectors gt11 and EMBL 3 were screened with antisera raised against 2‐hydroxyglutaryl‐CoA dehydratase. Two clones giving the strongest reaction in the immunoassay were analyzed further, one was a λgt11 clone with an insert of 2050 bp and one was a λEMBL‐3 clone with an insert of approximately 11000 bp. Escherichia coli cells infected with the λgt11 clone expressed the α subunit of the dehydratase (Mr, 53870), whereas with the λEMBL‐3 clone, the α and β subunits (Mr, 41857) were detected on Western blots. Restriction fragments of both clones were subcloned in pUC 8 and sequenced by the chain termination method. Thus the complete sequence of the genes of both subunits, hgdA (α) and hgdB (β) were obtained. The genes have the following order: A‐B, with an intergenic region of only 2 bp. The deduced amino acid sequences for the α and β subunits were confirmed by four peptides sequenced by protein chemical methods. Both chains are extremely rich in cysteine (13 in α, including a CNC and two CC clusters, and nine in (β) but no similarities to other known protein sequences were found.
A common liver‐specific factor binds to the rat albumin and α‐foetoprotein promoters in vitro and acts as a positive trans‐acting factor in vivo
Authors: JOSE‐ESTANYOL Matilde , POLIARD Anne , FOIRET Danièle , DANAN Jean‐Louis
Published: March 2005

Rat liver nuclear extracts were tested for the presence of factors which might be common to the transcriptional regulation of both the albumin and α‐foetoprotein genes. Gel shift assay showed the formation of three complexes (I, II and III) with the albumin probe. Two of them (I and III) could be displaced by the α‐foetoprotein promoter. Analysis of nuclear extracts from liver, kidney, spleen and brain and competition experiments using several oligonucleotides covering regions from the albumin and α‐foetoprotein promoters showed that complex III results from the binding of the ubiquitous nuclear factor 1, while complex II involves a CCAAT‐box‐binding protein also detected in brain and spleen extracts. Complex I is formed upon binding of a liver‐specific factor to a proximal element of the rat albumin promoter. This factor also binds to a similar sequence in the α‐foetoprotein promoter and is closely related to the hepatocyte nuclear factor 1, as shown by competition experiments using an oligonucleotide covering its target sequence on the β‐fibrinogen promoter.

Transfection competition experiments indicated that, in vivo, this factor acts as a positive trans‐acting element in the expression of both the rat albumin and α‐foetoprotein genes.
Isolation and characterization of hagfish thyroid iodoprotein by its non‐thyroglobulin nature, very high iodine and carbohydrate contents and low hormone/iodine ratio
Authors: OHMIYA Yoshihiro , SUZUKI Shintaro , KONDO Yoichi
Published: March 2005

We have characterized the thyroid iodoprotein of a hagfish, Eptatretus burgeri, one of the lowest marine vertebrates. The iodoprotein was not very homogeneous in its apparent molecular mass which decreased with the increase in hormone/iodotyrosine ratio. Four subfractions with an apparent molecular mass of about 400 kDa were purified from one major fraction by size‐exclusion and Mono Q ion‐exchage HPLC. The subfractions appeared to have the same peptide backbone, since they showed a single band with the same mobility as a 160‐kDa protein in SDS/PAGE and the same amino acid composition. However they differed from each other in having increasing iodine contents (1.9% to 5.9% by mass of total amino acids) associated with the increase in hormonal iodine proportion (8.4% to 16.7% of total iodine) and carbohydrate content (35.6% to 53.5% by mass). These values are strikingly different from those of thyroglobulin with an iodine content of less than 1%, hormonal iodine of 20–40% and carbohydrate content of less than 10%. The amino acid composition of the hagfish iodoprotein, especially the cysteine content of less than 1%, was also entirely different from that of thyroglobulin. These results suggest that most, if not all, tyrosine residues of the hagfish thyroid glycoprotein with a less rigid structure are susceptible to an iodinating system, but hormone residues are formed by a much less efficient mechanism than those in thyroglobulin, when poorly iodinated.
Analysis of genomic clones of the murine U1RNA‐associated 70‐kDa protein reveals a high evolutionary conservation of the protein between human and mouse
Authors: HORNIG Horst , FISCHER Utz , COSTAS Maria , RAUH Andreas , LÜHRMANN Reinhard
Published: March 2005

We have isolated and characterised two overlapping λEMBL3 clones carrying sequences of the gene for the murine U1RNA‐associated 70‐kDa protein. The two clones cover around 23 kb of the 70‐kDa protein gene including its 3′ end. Southern blot hybridisation revealed the existence of a single copy of the 70‐kDa protein gene in the mouse genome. The 23‐kb‐long portion of the 70‐kDa protein gene is divided into eight exons. While most of the exons are quite small and are widely scattered throughout the DNA sequence, the last one consists of about 830 bp and encodes 226 amino acids of the 70‐kDa protein, including the C‐terminus. The predicted amino acid sequence of the region of the 70‐kDa protein encoded by the genomic clones reveals high conservation of structure when it is compared with the sequence of the human 70‐kDa protein. Interestingly, all deletions, additions and substitutions are localised exclusively within the C‐terminus of the protein, accounting for a 5′‐3′ polarity with respect to protein conservation. Moreover, the analysis of the genomic sequences predicts the existence of multiple subclasses of mRNAs that may arise by alternative pre‐mRNA splicing. A 72‐bp alternative exon harboring an in‐frame termination codon was also found in the mouse 70‐kDa gene and shows, surprisingly, 100% nucleotide identity to its human counterpart.
EPR study of the redox interactions in cytochrome c3 from Desulfovibrio vulgaris Miyazaki
Authors: BENOSMAN Haféda , ASSO Marcel , BERTRAND Patrick , YAGI Tatsuhiko , GAYDA Jean‐Pierre
Published: March 2005

We present a new examination of the EPR redox titration data for the tetraheme cytochrome c3 from Desulfovibrio vulgaris Miyazaki. Our analysis includes the contribution of the interaction potentials between the four redox sites and is based on the model previously developed for the study of cytochrome c3 from Desulfovibrio desulfuricans Norway. We observed, as for D. desulfuricans Norway cytochrome c3, that the conformation of the heme with the lowest redox potential, heme 4, is sensitive to the redox state of the heme with the highest potential, heme 1. However in D. vulgaris Miyazaki cytochrome c3 spectral simulations show that heme 4 is present in two conformational states which interconvert partially when heme 1 is reduced. The sets of redox parameters which satisfy the fitting procedure of the titration curves are in the following domain: –250 mV ≤e14≤–220 mV, –325 mV ≤e2≤–320 mV, –335 mV ≤e3≤–330 mV, –360 mV ≤e4≤–355 mV, –5 mV ≤I12≤ 20 mV, –10 mV ≤I13≤ 5 mV, –15 mV ≤I23≤–5 mV, –15 mV ≤I24≤–10 mV, –25 mV ≤I34≤–15 mV. As in D. desulfuricans Norway cytochrome c3 the interactions are moderate. Simple electrostatic considerations suggest that these moderate values could be related to the large accessibility of the hemes to the solvent. Our work does not confirm the existence of a cooperative interaction between heme 2 and heme 3 which has been proposed on the basis of electrochemical measurements.
The influence of tryptophan on mobility of residues in the trp repressor of Escherichia coli
Authors: LANE Andrew N.
Published: March 2005

The relative mobility of residues in the trp repressor of Escherichia coli has been examined in the absence and presence of the corepressor l‐tryptophan by one‐ and two‐dimensional 1H NMR. A comparison of relative intensities of cross peaks in NOESY and COSY spectra allowed a rigid Tyr and a mobile Tyr residue, three mobile Ser residues and three mobile Lys residues to be detected. The two Tyr residues were assigned by selective nitration with tetranitormethane. The singly nitrated molecule (on Tyr7) binds the trp operator with an affinity close to that of the unmodified repressor. Measurements of the intraring cross‐relaxation rate constant as a function of temperature for Tyr7 shows the presence of considerable internal motion on the subnanosecond time scale in the flexible N‐terminal arm. The order parameter, S2 characterising the motion is 0.35, which increases to about 0.5 in the presence of Trp. Trp decreases both the amplitude of the motion and the rate of the motion. At least three of the six Ser residues of the trp repressor have greater mobility than expected for a rigid body, and two of the Ser residues are sensitive to the presence of Trp. The more mobile Ser residues are probably those on the N‐terminal arm and the C‐terminal sequence. These results complement the single‐crystal X‐ray diffraction studies for which the electron density of the first ten and last three amino acid residues is weak. The solution data are consistent with proposals that the flexible N‐terminal arm of the trp repressor makes important contacts with the DNA.