bluuebunny/crossref_metadata_2025 huggingface dataset:
DNA‐dependent mono(ADP‐ribosyl)ation of p33, an acceptor protein in hen liver nuclei
Authors: MISHIMA Koichi , TSUCHIYA Mikako , TANIGAWA Yoshinori , YOSHIMURA Yasuro , SHIMOYAMA Makoto
Published: March 2005

A non‐histone acceptor protein for hen liver nuclear ADP‐ribosyltransferase was purified to an apparently homogeneous state through salt extraction and chromatography on hydroxyapatite, phenyl‐Sepharose, carboxymethyl‐cellulose, Sephadex G‐75, phenyl 5‐PW, mono S and Radial PAK C18. This protein was termed p33. The ADP‐ribosylation of p33 was enhanced more than 60‐fold by double‐stranded DNA. Single‐stranded DNA, RNA and poly(L‐glutamate), but not deoxyribonucleotide, were partially effective. DNA‐dependent ADP‐ribosylation was also observed when whole histones were used as acceptor. DNA required for the maximal ADP‐ribosylation depended on the dose of the acceptor protein; the optimal mass ratio of DNA to the acceptor protein was 1:1 with both p33 and whole histones. DNA decreased the Km for NAD and concomitantly increased the Vmax value, but did not alter the Km for p33. These results are consistent with the idea that p33 may participate in chromatin processes such as replication or transcription, through modification by nuclear ADP‐ribosyltransferase.
Chronic stimulation of rat skeletal muscle induces coordinate increases in mitochondrial and nuclear mRNAs of cytochrome‐c‐oxidase subunits
Authors: HOOD David A. , ZAK Radovan , PETTE Dirk
Published: March 2005

Fast‐twitch tibialis anterior muscle of the rat was subjected to chronic low‐frequency (10 Hz, 10 h daily) nerve stimulation in order to investigate the time course of changes in cytochrome‐c‐oxidase activity, as well as in tissue levels of specific mitochondrially and nuclear‐encoded, cytochrome‐c‐oxidase‐subunit mRNAs. Chronic stimulation induced a progressive increase in cytochrome‐c‐oxidase activity which was threefold elevated after 35 days. A similar increase was recorded for citrate‐synthase activity. Glyceraldehyde‐3‐phosphate dehydrogenase, which was studied as a glycolytic reference enzyme, moderately decreased, as did the tissue level of its corresponding mRNA. There was a parallel increase in the tissue levels of the two cytochrome‐c‐oxidase‐subunit mRNAs over the entire stimulation time course. The extent of increase (stimulated/control) was 2.4 ± 0.3 and 1.8 ± 0.2 (means ± SEM) for the mitochondrial and nuclear subunit mRNAs, respectively. This parallel increase suggested a coordinate regulation of the two subunits. The increase in cytochrome‐c‐oxidase activity initially corresponded to the changes at the mRNA level. However, with longer stimulation times (beyond 14 days), the increase in cytochrome‐c‐oxidase activity clearly exceeded that of the two mRNAs. This divergence was progressive and was interpreted to indicate that the increase in cytochrome‐c‐oxidase content was brought about not only by changes in the levels of the specific mRNAs, but also by alterations at the level of translation.
The temperature and pH dependence of some properties of p‐hydroxybenzoate hydroxylase from Pseudomonas fluorescens
Authors: VAN BERKEL Willem J. H. , MÜLLER Franz
Published: March 2005

The free and complexed flavoprotein, p‐hydroxybenzoate hydroxylase, was studied by light‐absorption, circular‐dichroism and fluorescence techniques as a function of the pH. The following compounds served as ligands for the enzyme: p‐hydroxybenzoate, p‐fluorobenzoate, benzoate, p‐aminobenzoate and tetrafluoro‐p‐hydroxybenzoate. Depending on the technique used, the various ligands exhibit pH‐dependent physical properties and dissociation constants. The data can be fitted with pKa values in the range 7.7 – 7.9. It is suggested that this pKa value belongs to a tyrosine residue in the active center of the enzyme. This assignment is supported by published data and additional experiments.
Preparation and use of a universal primed Sepharose for the purification of DNA‐binding proteins
Authors: ARCANGIOLI Benoit , POCHET Sylvie , SOUSA Rosemary , HUYNH‐DINH Tam
Published: March 2005

We have devised a novel method for the construction of a DNA affinity matrix and tested its use in the purification of a sequence‐specific DNA‐binding protein from the yeast Saccharomyces cerevisiae. The matrix was prepared in two steps: first, a palindromic oligonucleotide containing an XhoI cohesive end was covalently linked via its loop to a Sepharose matrix; second, directly to this ‘universal’ primed Sepharose was ligated a 37‐bp oligonucleotide, with XhoI cohesive ends, containing the sequence of the upstream activation sequence 1 (UAS1) site of the yeast iso‐1‐cytochrome c (CYC1) gene. After fractionating a yeast crude extract through DEAE‐cellulose, heparin ultrogel and Mono Q columns, a single pass through the affinity matrix allowed the purification to apparent homogeneity of the 120‐kDa protein factor P, which is responsible for the binding to the UAS1 site.
Vanadate‐mediated photocleavage of rabbit skeletal myosin
Authors: MOCZ Gabor
Published: March 2005

The heavy chain of myosin from rabbit skeletal muscle can be cleaved at three sites by irradiation with nearultraviolet light in the presence of 0.1–1.0 mM vanadate. The sigmoidal dependence upon vanadate concentration, with half‐mximal rate occurring at about 0.5 mM vanadate and a sigmoidicity of 2.7, is consistent with the chromophore responsible for cleavage being oligomeric vanadate. Cleavage occurs at two sites located within the head region of the molecule, 23 kDa and 75 kDa from the NH2‐terminus; these sites are cleaved equally well in heavy meromyosin and subfragment 1. In the presence of 1 mM vanadate, the half‐times for cleavage of the 23‐kDa and 75kDa sites are about 15 and 10 min, respectively. The rate of cleavage at both these sites is retarded 2–3‐fold by the presence of > 10 μM MgATP. The third photocleavage site is located about 5–10 kDa from the COOH terminus of the intact heavy chain, and cleaves equally well in the isolated rod and in light meromyosin. Cleavage at this site occurs with a half‐time of 138 min, and its rate is unaffected by the presence of MgATP. The vanadate‐mediated cleavage of the heavy chains is accompanied by characteristic changes in the myosin ATPase properties, with the Ca2+, Mg2+ and actin‐activated Mg2+ ATPases becoming elevated, whereas the K+/EDTA ATPase becomes inactivated. The sites of photocleavage in the myosin heavy chain might be associated with sites of phosphate binding.
Phosphorylation of avian retrovirus matrix protein by Ca2+/phospholipid‐dependent protein kinase
Authors: LEIS Jonathan , PHILLIPS Nelson , FU Xiangdong , TUAZON Polygena T. , TRAUGH Jolinda A.
Published: March 2005

The matrix protein from avian myeloblastosis virus and the Rous sarcoma virus, Prague C strain, is a phosphoprotein. A comparison of the amino acid sequences shows these phosphoproteins are very similar. The sites of phosphorylation of the matrix protein purified from virions are identified as serine residues 68 and 106. Treatment with purified rabbit skeletal‐muscle protein phosphatase 1 or 2A, selectively releases phosphate from serine 68, while alkali treatment releases phosphate from both sites. When analyzed as a substrate for six different protein kinases, only the Ca2+/phospholipid‐dependent protein kinase modifies the matrix protein. The serine residues phosphorylated in vivo are identical to those phosphorylated in vitro by this protein kinase. The role of these phosphorylation events in viral production is discussed.
Synthesis and biological activity of N6‐{N[(4‐azido‐3,5,6‐trifluoro)‐pyridin‐2‐yl]‐2‐aminoethyl}‐adenosine 5′‐monophosphate, a new AMP photoactivatable derivative
Authors: SICSIC Sames , LEONIL Joëlle , LE GOFFIC François
Published: March 2005

N
6‐{N‐[(4‐Azido‐3,5,6‐trifluoro)pyridin‐2‐yl]‐2‐aminoethyl}‐adenosine 5′‐monophosphate has been synthesized and evidence presented for its structural assignment by ultraviolet and 19F‐NMR spectroscopies. Its photolysis was shown to occur within 5 min. This AMP derivative behaves as a competitive inhibitor of NAD+ in horse‐liver‐alcohol‐dehydrogenase‐promoted oxidation of ethanol, with a Ki (0.95 mM) comparable to the Ki of AMP (1.9 mM). Moreover it is an activator of the enzyme when nicotinamide ribose is used as the oxidation cofactor. This activation is as good as that promoted by AMP or by the well known 8‐azido‐AMP. Upon photolysis of this new derivative in the presence of horse liver alcohol dehydrogenase, a covalent enzyme–analogue complex was isolated and assayed as a catalyst in the oxidation of ethanol using nicotinamide ribose as the cofactor. The reaction took place without complementation of AMP, indicating clearly that the AMP analogue is mainly covalently bound in the AMP‐binding site, and that the linkage formed between the enzyme and the azido derivative has not dramatically altered the active site of the enzyme. A similar experiment with 8‐azido‐AMP produced a completely inactive complex.
Carbohydrate structure of glycoprotein 65 encoded by the polycythemia‐inducing strain of Friend spleen focus‐forming virus
Authors: STRUBE Karl‐Hermann , GEYER Rudolf
Published: March 2005

The secondary envelope‐gene product, glycoprotein 65 (gp65), of the polycythemia‐inducing variant of Friend spleen focus‐forming virus (F‐SFFVp) was isolated from F‐SFFVp‐infected normal rat kidney cells cultivated in the presence or absence (–Glc) of glucose. Oligosaccharide side chains present were sequentially liberated by treatment of tryptic glycopeptides with endo‐β‐N‐acetylglucosaminidase H and peptide N‐glycosidase F and fractionated by high‐performance liquid chromatography. The glycans were characterized by digestion with exoglycosidases, by chromatographic comparison with oligosaccharide standards and by methylation analysis.

The results demonstrate that gp65 contains oligomannosidic, hybrid and N‐acetyllactosaminic glycans. The oligomannosidic glycans represent the same partially glucosylated species with six to nine mannose residues present in F‐SFFVp gp52, the biosynthetic precursor of gp65 [Strube, K.‐H. Schott, H.‐H. and Geyer, R. (1988) J. Biol. Chem. 263, 3762–3771]. Oligosaccharides of the hybrid type were found to comprise one sialylated lactosamine unit and three or four α‐linked mannose residues. Analysis of the N‐acetyllactosaminic glycans revealed that gp65 carries fucosylated, partially sialylated bi‐antennary, tri‐antennary and tetra‐antennary oligosaccharides, in addition to incomplete species. The glycosylation of gp65(–Glc) is characterized by the presence of oligomannosidic glycans with five to nine mannose residues, similar hybrid‐type species and by increased amounts of incomplete N‐acetyllactosaminic oligosaccharides, a decrease in sialylation and the lack of tetra‐antennary species.
Effect of papain digestion on polypeptide subunits and electron‐transfer pathways in mitochondrial b‐c1 complex
Authors: LORUSSO Michele , COCCO Tiziana , BOFFOLI Domenico , GATTI Domenico , MEINHARDT Steven , OHNISHI Tomoko , PAPA Sergio
Published: March 2005

Papain digestion of subunits of mitochondrial b‐c1 complex (ubiquinol–cytochrome‐c reductase) isolated from bovine heart and its impact on redox and proton‐motive activity of the whole complex were investigated. A 5‐min incubation of the oxidized enzyme with papain resulted in digestion of core protein II and the 14‐kDa subunit, and limited digestion of the iron‐sulfur protein. This was accompanied by a small inhibition of the rate of electron flow and a marked inhibition of proton translocation with decrease of the H+/e− ratio for proton pumping. When papain treatment was performed on the b‐c1 complex pre‐reduced with ascorbate, partial proteolysis of the iron‐sulfur protein and the 14‐kDa subunit was greatly accelerated and the electron transfer activity was more markedly inhibited. In all the conditions tested, digestion of the Rieske iron‐sulfur protein paralleled the inhibition of reductase activity. Under ascorbate‐reduced conditions, papain digestion of the complex gave rise to an alteration of the EPR line shape of the iron‐sulfur cluster, namely a broadening and shift of the gx negative peak and destabilization of the protein‐bound antimycin‐sensitive semiquinone. The latter paralleled the decrease in electron transfer activity and inhibition of antimycin‐sensitive cytochrome‐b reduction. The results obtained indicate the following.

1. Core protein II and the 14‐kDa protein may contribute to the proton‐conducting pathway(s) from the matrix aqueous phase to the primary protolytic redox center (protein‐bound semiquinone/quinone couple).

2. The iron‐sulfur protein contributes, together with other protein(s) (the 14‐kDa subunit), to the stabilization of the protein bound antimyein‐sensitive semiquinone species in a protein pocket in the complex.

3. Reduction of the high‐potential redox centers induces a change in the quaternary structure of the complex which results in an enhanced surface exposure of segments of the 14‐kDa protein and the iron‐sulfur protein.
Identification of a yeast ribonuclease H as an Sm antigen
Authors: KARWAN Robert , KINDÅS‐MÜGGE Ingela
Published: March 2005

We have isolated a 55‐kDa enzyme from Saccharomyces cerevisiae on the basis of its ability to hydrolyze specifically the RNA moiety of RNA/DNA hybrids [RNase H(55)]. Remarkably, monospecific anti‐[R Nase H(55)] antibodies revealed that the protein associates with several small RNAs, including some of the essential yeast spliceosomal snRNAs. Moreover, immunoprecipitation as well as immunoblotting experiments demonstrated that the yeast enzyme reacts (a) with human anti‐Sm autoantisera, (b) with a monoclonal antibody specific for the human snRNP proteins B/B′, but (c) not with U1‐ribonucleoprotein‐specific autoantibodies. These results disclosed a hitherto unexpected degree of evolutionary conservation in snRNP protein structure between yeast and man. Additionally, our findings suggested a re‐evaluation of the enzymatic mechanism of R Nases H which recognize both RNA and RNA/DNA hybrids.
Characterization of the ribosomal properties required for formation of a GTPase active complex with the eukaryotic elongation factor 2
Authors: NYGÅRD Odd , NILSSON Lars
Published: March 2005

The binding stability of the different nucleotide‐dependent and ‐independent interactions between elongation factor 2 (EF‐2) and 80S ribosomes, as well as 60S subunits, was studied and correlated to the kinetics of the EF‐2‐ and ribosome‐dependent hydrolysis of GTP. Empty reconstituted 80S ribosomes were found to contain two subpopulations of ribosomes, with approximately 80% capable of binding EF‐2 · Guo PP[CH2]P with high affinity (Kd < 10−9 M) and the rest only capable of binding the factor‐nucleotide complex with low affinity (Kd= 3.7 × 10−7 M). The activity of the EF‐2‐ and 80S‐ribosome dependent GTPase did not respond linearly to increasing factor concentrations. At low EF‐2/ribosome ratios the number of GTP molecules hydrolyzed/factor molecule was considerably lower than at higher ratios. The low response coincided with the formation of the high‐affinity complex. At increasing EF‐2/ribosome ratios, the ribosomes capable of forming the high‐affinity complex was saturated with EF‐2, thus allowing formation of the low‐affinity ribosome · EF‐2 complex. Simultaneously, the GTPase activity/factor molecule increased, indicating that the low‐affinity complex was responsible for activating the GTP hydrolysis. The large ribosomal subunits constituted a homogeneous population that interacted with EF‐2 in a low‐affinity (Kd= 1.3 × 10−6 M) GTPase active complex, suggesting that the ribosomal domain responsible for activating the GTPase was located on the 60S subunit. Ricin treatment converted the 80S particles to the type of conformation only capable of interacting with EF‐2 in a low‐affinity complex. The structural alteration was accompanied by a dramatic increase in the EF‐2‐dependent GTPase activity. Surprisingly, ricin had no effect on the factor‐catalyzed GTP hydrolysis in the presence of 60S subunits alone.
Isolation of a galactose‐free 20‐kDa fragment exhibiting butyrylcholine esterase and aryl acylamidase activity from human serum butyrylcholine esterase by limited α‐chymotrypsin digestion
Authors: RAO Rammohan V. , BALASUBRAMANIAN Aiylam S.
Published: March 2005

Purified human serum butyrylcholine esterase (∼ 90‐kDa subunit), which also exhibits aryl acylamidase activity, was subjected to limited α‐chymotrypsin digestion. Three major protein fragments of ∼ 50 kDa, ∼ 21 kDa and ∼ 20 kDa were found to be produced, as observed by SDS‐gel electrophoresis of the chymotryptic digest. The purified butyrylcholine esterase could fully bind to a Ricinus‐communis‐agglutinin‐Sepharose column but after chymotryptic digestion about 15–20% of the enzyme activity remained unbound and was recovered in the run‐through fractions. Sephadex G‐75 chromatography of the chymotryptic digest showed an enzymatically active fragment eluted at an approximate molecular mass of 20 kDa, apart from the undigested butyrylcholine esterase eluted at the void volume. The butyrylcholine esterase fragment that did not bind to Ricinus communis agglutinin also was eluted at an approximate molecular mass of 20 kDa from a Sephadex G‐75 column. This enzymatically active low‐molecular‐mass fragment from Sephadex G‐75 chromatography showed a single protein band of ∼ 20 kDa on SDS‐gel electrophoresis. Neutral sugar analysis of the ∼ 20 kDa fragment showed the presence of mannose only, whereas the undigested butyrylcholine esterase showed the presence of both mannose and galactose. Amino‐terminal‐sequence analysis of the ∼ 20 kDa fragment showed the sequence Arg‐Val‐Gly‐Ala‐Leu, which agrees with amino acid residues 147–151 reported for human serum butyrylcholine esterase [Lockridge et al. (1987) J. Biol. Chem. 262, 549–557]. Both cholinesterase and aryl acylamidase activities were co‐eluted in all chromatographic procedures. The results suggested that limited α‐chymotrypsin digestion of human serum butyrylcholine esterase resulted in the formation of a ∼ 20‐kDa enzymatically active fragment with Arg147 as its N‐terminal residue and which was devoid of galactose.
Synthesis of glycoconjugates derived from various lipopolysaccharides of the Vibrionaceae family
Authors: BANOUB Joseph H. , SHAW Derek H. , NAKHLA N. Anthony , HODDER Howard J.
Published: March 2005

Conjugation of simple ketoses (such as 3‐deoxy‐D‐manno‐2‐octulosonic acid and N‐acetylneuraminic acid) and of various O‐specific polysaccharides (from Aeromonas hydrophila and Aeromonas salmonicida) to the bifunctional spacer 1,6‐hexanediamine, was achieved by reductive amination. The saccharide–1‐(6‐amino)‐hexane alkyamines obtained were converted into the corresponding isothiocyanate derivatives and coupled to the free ɛ‐amino group of lysine residues of the protein carrier bovine serum albumin. In similar manner, the aldehyde group introduced by selective periodate oxidation into the partially O‐deacylated lipopolysaccharide of Vibrio anguillarum was conjugated to 1,6‐hexanediamine, converted into the corresponding isothiocyanate and covalently attached to bovine serum albumin.
Hemoglobin from the Antarctic fish Notothenia coriiceps neglecta
Authors: D'AVINO Rossana , CARUSO Carla , ROMANO Mario , CAMARDELLA Laura , RUTIGLIANO Bruno , di PRISCO Guido
Published: March 2005

The complete amino acid sequence of the α chain of the main hemoglobin of the Antarctic fish Notothenia coriiceps neglecta (family Nototheniidae) has been determined. It consists of 142 residues; an acetylated seryl residue is at the amino terminal. The molecular mass is 15 519 Da. In comparison with α‐chain sequences of non‐Antarctic poikilothermic fish hemoglobins, the homology appears to be significantly lower than that existing among the latter species. A higher homology has been found with the α‐chain sequence of the non‐poikilothermic bluefin tuna.
Purification and characterization of a methionine‐specific aminopeptidase from Salmonella typhimurium
Authors: WINGFIELD Paul , GRABER Pierre , TURCATTI Gerardo , MOVVA N. Rao , PELLETIER Michel , CRAIG Stewart , ROSE Keith , MILLER Charles G.
Published: March 2005

An aminopeptidase specific for methionine (peptidase M) has been purified from wild‐type and mutant Salmonella typhimurium strains. Recombinant peptidase M was also purified from Escherichia coli. These preparations were characterized with respect to their physicochemical properties using analytical ultracentrifugation. SDS/PAGE, isoelectric focusing, titration curve analysis, amino acid analysis, N‐ and C‐terminal sequencing and various spectroscopic methods.

Peptidase M activity is stimulated by Co2+, in agreement with previous studies using crude extracts of Salmonella. The purified preparations did not contain significant amounts of any metal. Enzymically important metal is loosely associated and lost during enzyme purification.

Peptidase M was shown to contain seven free sulphydryl residues none of which are involved in either intraor inter‐molecular disulphide bonds. Most appear solvent‐accessible as evidenced by their reactivity under native conditions. Limited modification of the sulphydryl residues with either iodoacetamide or 5.5′‐dithiobis(2‐nitrobenzoic acid) led to inactivation. Several cysteines were shown to be labelled to various degrees by peptide mapping of inactivated S‐[14C]carboxymethylated protein. Whether cysteine modification affects enzymic activity directly (blocking an active site) or indirectly (by causing conformational change) remains to be established.
Purification and characterization of yeast anthranilate phosphoribosyltransferase
Authors: HOMMEL Ulrich , LUSTIG Ariel , KIRSCHNER Kasper
Published: March 2005

Anthranilate phosphoribosyltransferase from Saccharomyces cerevisiae has been purified to homogeneity from an overproducing strain. Analytical ultracentrifugation demonstrated that the enzyme is a dimer of Mr= 83000 ± 4000 (s20,w= 4.7 S). Moreover, as shown by active enzyme sedimentation, the enzyme remains dimeric even at low concentrations. The presence of yeast phosphoribosylanthranilate isomerase in the gradient does not lead to complex fromation between the two enzymes as might be expected if phosphoribosyl anthranilate, the very labile product of the anthranilate phosphoribosyltransferase, were channelled to phosphoribosylanthranilate isomerase in vivo. The steady‐state‐kinetic behaviour of the enzyme suggests that catalysis involves a ternary enzyme‐substrate complex, with KANTm= 1.6 μM, and KPRib‐PPm= 22.4 μM. The enzyme has been used to generate phosphoribosylanthranilate in situ for kinetic studies of phosphoribosylantharnilate isomerase from Escherichia coli: KPRAm=5μM, Kcat= 40 s−1.
Gene expression in regenerating liver in relation to cell proliferation and stress
Authors: SOBCZAK Joëlle , TOURNIER Marie‐Francoise , LOTTI Anne‐Marie , DUGUET Michel
Published: March 2005

When hepatocyte proliferation is stimulated in the liver by partial hepatectomy, messenger RNAs coding for fibrinogen, actin, c‐myc and topoisomerase I are rapidly accumulated. We distinguish an early phase of accumulation (0–3 h after partial hepatectomy) which is also observed after a sham operation for the four genes, and during inflammation produced by Freund's adjuvant in the case of fibrinogen and c‐myc genes. The hepatic response to inflammation appears therefore to mimic events characteristic of the G0/G1 transition, such as the accumulation of the c‐myc mRNA. The late phase of mRNA accumulation (beyond 3 h after partial hepatectomy) is typical of liver regeneration. The level of c‐myc mRNA is transiently increased (20‐fold over normal) 20 h after partial hepatectomy, that is, at the time of DNA synthesis. Topoisomerase‐I mRNA level increases between 3 and 24 h after partial hepatectomy (5‐10‐fold over normal). These results suggest that accumulation of c‐myc and topoisomerase‐I mRNAs is associated with DNA replication in regenerating liver.
Primary structures of rat and bovine liver mitochondrial aldehyde dehydrogenases deduced from cDNA sequences
Authors: FARRÉS Jaume , GUAN Kun‐Liang , WEINER Henry
Published: March 2005

The cDNA coding for rat liver mitochondrial aldehyde dehydrogenase was cloned and sequenced. It contained an open reading frame of 1557 bp. Of the deduced 519 amino acid residues, 19 were proposed to correspond to the signal peptide necessary to allow the protein to enter the mitochondria [Farrés, J., Guan, K.‐L. and Weiner, H. (1987) Biochem. Biophys. Res. Commun. 150, 1083–1087]. The sequence of the 500 amino acid residues comprising the mature subunit was 96% identical to that of the corresponding human liver mitochondrial enzyme.

The longest cDNA isolated coding for the bovine liver enzyme contained sequence information corresponding to residues 72‐500 of the rat or human enzyme. The deduced protein sequence of these residues was 94% identical to those of the human enzyme.

Over 60% of the bases in the coding region of the rat cDNA were G and C. These residues were clustered non‐randomly and two potentially stable stem‐loop structures could be calculated as existing in the mRNA. One would be found in the region coding for amino acid residues 64‐79; a similar secondary structure could be responsible for the existing truncated cDNAs from the bovine‐cDNA libraries. Comparisons with the amino acid sequences of other aldehyde dehydrogenases support the suggestion that the NAD‐binding domain may be located in the middle portion of the mature enzyme.
Identification and localization of the membrane‐associated, ATP‐binding subunit of the oligopeptide permease of Salmonella typhimurium
Authors: GALLAGHER Maurice P. , PEARCE Stephen R. , HIGGINS Christopher F.
Published: March 2005

The OppF protein, a component of the oligopeptide permease of Salmonella typhimurium, is an ATP‐binding protein and is believed to couple ATP hydrolysis to the transport process. This protein is an example of a large family of closely related proteins which couple ATP to a variety of different biological processes. The oppF gene has been cloned and sequenced. In order to identify and characterize its protein product we overproduced the protein from the cloned gene. Anti‐OppF antibodies were raised against a synthetic peptide. Using these antibodies as a probe we identified OppF in wild‐type and overproducing strains. Protease accessibility studies showed the protein to be a peripheral membrane protein located on the cytoplasmic side of the inner membrane. These findings have general implications for the organization and function of this class of prokaryotic and eukaryotic transport system.
Inhibitors of protein phosphatase‐1
Authors: STRÅLFORS Peter , HEMMINGS Hugh C. , GREENGARD Paul
Published: March 2005

Protein phosphatase inhibitor‐1 was purified from bovine adipose tissue. The protein had an apparent molecular mass of 32 kDa by SDS/PAGE and a Stokes' radius of 3.4 nm. It was phosphorylated by cAMP‐dependent protein kinase on a threonyI residue; this phosphorylation was necessary for inhibition of protein phosphatase‐1. Bovine adipose tissue inhibitor‐1 was compared directly with rabbit skeletal muscle inhibitor‐1 and with a 32000‐Mr, dopamine‐ and cAMP‐regulated phosphoprotein from bovine brain (DARPP‐32), also an inhibitor of protein phosphatase‐1. By the following biochemical and immunochemical criteria, bovine adipose tissue inhibitor‐1 was found to be very similar and possibly identical to DARPP‐32 and was clearly distinct from skeletal muscle inhibitor‐1: molecular mass by SDS/PAGE; Stokes' radii; phosphorylation on threonine residues; Staphylococcus‐aureus‐V8‐protease‐generated peptide patterns analyzed by SDS/PAGE; tryptic phosphopeptide maps analysed by two‐dimensional thin‐layer electrophoresis/chromatography; elution on reverse‐phase HPLC; chymotryptic peptide maps as analysed by reverse‐phase HPLC; amino acid composition; antibody recognition by immunoprecipitation and immunoblotting; effect of cyanogen bromide cleavage on protein phosphatase inhibitor activity. Based on these results we conclude that bovine brain and adipose tissue contain an identical phosphoprotein inhibitor of protein phosphatase‐1 (DARPP‐32), which is distinct from that of skeletal muscle (inhibitor‐1).
Aspartate aminotransferase isoenzymes in Leotosphaeria michotii
Authors: ROMESTANT Michel , JEREBZOFF Stephan , NOAILLAC‐DEPEYRE Jacqueline , Gas Nicole , DARGENT Robert
Published: March 2005

Two of aspartate aminotransferase were obtained from the fungus Leptosphaeria michotii and purified to a state of apparent homogeneity by a five‐step purification procedure ending with blue Ultrogel chromatography. Holoenzyme specific activities were 13430 and 9110 nkat oxalacetate/mg protein−1 and isoelectric points were 7.1 and 7.0 for forms A and B, respectively. Both isoenzymes were isologous dimers ofMr 92000. They differed mainly in theirKm for 2‐oxoglutarate and aspartate, their ability to use cysteine sulfinate as a substrate and their ability in vitro to be specifically tightly associated as follows: form A with a malate dehydrogenase monomer ofMr 25000; form B with an unidentified protein ofMr 40000– 44000. Rabbit antiserum raised against the form A holoenzyme was not reactive against the form B holoenzyme and vice versa. Association of the holoenzyme with the complex essentially provoked a shift of the isoelectric point to 5.8 for form A and to 5.2 for form B, without affecting kinetic parameters. In order to localize in situ the two transaminase forms, ultrastructural detection was carried out by immunogold stainig of thin sections of Lowicryl‐K4M‐embedded colonies. Antiserum against form A essentially labelled cytoplasm and cell wall and, to some extent, mitochondria, while antiserum against form B heavily labelled mitochondria and cell wall and to a lesser extent cytoplasme. Moreover, mitochondria were isolated and purified by Percoll‐density‐density‐gradient centrifugation. Only form A was identified in this subcellular fraction using ELISA.
Neutral glycolipids of atherosclerotic plaques and unaffected human aorta tissue
Authors: PROKAZOVA Nina V. , MUKHIN Dmitry N. , OREKHOV Alexander N. , GLADKAYA Elena M. , VASILEVSKAYA Valentina V. , MIKHAILENKO Irina A. , SADOVSKAYA Valentina L. , BUSHUEV Valery N. , BERGELSON Lev D.
Published: March 2005

The composition, structure and localization of neutral glycosphingolipids of human aorta taken from subjects who had died after myocardial infarction were studied. Individual glycosphingolipids were purified by high‐performance liquid chromatography and were characterized on the basis of their chromatographic mobility, carbohydrate composition, methylation analysis and by 1H‐NMR spectroscopy. The main aortic glycosphingolipids were identified as glucosylceramide, lactosylceramide, globotriaosylceramide and globotetraosylceramide. Significant differences in the neutral glycosphingolipid composition of intima and media were detected.

The neutral glycosphingolipid profile of medial plaques resembled that of unaffected media; however, significant differences were detected between intimal plaques and unaffected intima. Whereas the latter contained trihexosylceramide and globoside as the only neutral glycolipids, the intimal plaque glycolipids consisted mainly of glucosylceramide and also contained appreciable amounts of lactosylceramide which were completely absent in the unaffected intima. In comparison to intimal plaques, unaffected intima is characterized by a much higher content of cerebrosides terminating by β‐galactosyl residues which are known to interact with growth factors and other external stimuli.

It thus seems possible that the proliferative activity of smooth muscle cells in atherosclerotic diseases is to some extent associated with their neutral glycolipid profile.
A small isoform of NADH: ubiquinone oxidoreductase (complex I) without mitochondrially encoded subunits is made in chloramphenicol‐treated Neurospora crassa
Authors: FRIEDRICH Thorsten , HOFHAUS Götz , ISE Wolfgang , NEHLS Uwe , SCHMITZ Beate , WEISS Hanns
Published: March 2005

In mitochondria of Neurospora crassa grown in the presence of chloramphenicol a small form of NADH: ubiquinone reductase in place of the normal electron‐transfer‐complex I. This smaller enzyme has a molecular mass of approximately 350 kDa and consists of (at least) 13 different subunits which are all synthesized in the sytoplasm. The complex I which is normally found in Neurospora has a molecular mass of approximately 700 kDa and consists of around 30 different subunits, of which at least six are made in the mitochondria. Immunoblotting and peptide mapping suggest that the subunits of the small enzyme are homologous to subunits of the large enzyme, one subunit might even be identical. The small and the large NADH:ubiquinone reductases have the same high‐affinity binding site for NADH but the two enzymes differ in the affinity and inhibitor sensitivity of the ubiquinone‐binding site. The possibility is discussed that the small NADH:ubiqinone reductase is primitive isoform of complex I.
Transfection of hepatic genes into adult rat hepoatocytes in primary culture and their tissue‐specific expression
Authors: GINOT Frédéric , DECAUX Jean‐François , COGNET Mireille , BERBAR Tsouria , LEVRAT François , KAHN Axel , WEBER Anne
Published: March 2005

We describe in this paper a method for studying transient gene expression in a primary culture of adult rat hepatocytes. After isolation by collagenase perfusion, hepatocytes in a monolayer were transfected with foreign DNA by the calcium phosphate precipitation technique during the first 24 hours after plating. When they were transfected with a plasmid containing the gene for chloramphenicol acetyltransferase driven by the early promoter of simian virus 40, hepatocytes reproducibly expressed high levels of chloramphenicol acetyltransferase (CAT); this transient expression was much higher than that obtained with the rat hepatoma cell line H4II. Different medium conditions have been tested: an optimal level of CAT activity can be obtained using a serum‐free, hormonally defined medium. Using these techniques, we have investigated the expression of liver‐specific genes transferred into hepatocytes. We show that the lv‐pyruvate kinase promoter is active in these hepatocytes while it is silent in fibroblasts.

Moreover, the use of serum‐free medium may allow investigation of the role of hormones and nutrients in cells which respond normally to these effectors.
Oxacillin‐hydrolysing β‐lactamases
Authors: MOSSAKOWSKA Danuta , ALI Norryai A. , DALE Jeremy W.
Published: March 2005

We have extended the sequence of the OXA‐2 β‐lactamase which together with S1 mapping has enabled us to identify the promoter site for this gene. This lies in a region that is found upstream from a variety of resistance genes on different plasmids; each gene appears to have been inserted at the same specific site and to be expressed from the same promoter. The ancestral plasmid thus appears to function as a natural expression vector. The sequence of the recombination site at the 5′ end of the OXA‐2 gene shows a marked similarity with the attP sequence of λ. DNA‐probe analysis confirmed that the OXA‐2 and OXA‐3 β‐lactamases are related, and indicated no similarity with other β‐lactamase genes. However, a comparison of amino acid sequences demonstrates that the OXA‐2, OXA‐1 and PSE‐2 β‐lactamases show some similarities to the typical class A enzymes, especially in the central helical domain of the latter, which is largely responsible for forming the active site of the enzyme. The three oxacillinases also show maked amino acid sequence similarity with the product of a regulatory gene, blaR1, required for β‐lactamase induction in Bacillus licheniformis.
A new crystal form of ricin‐OR
Authors: CHANG Wen‐Rui , CHEN Liqing , ROSE John , WEI Chin Hsuan , ABRAMS Leatrice , SAX Martin , WANG Bi‐Cheng
Published: March 2005

Ricin‐OR, an antitumor toxin, has been crystallized in space group P2 with cell parameters a= 8.77 nm, b= 4.64 nm, c= 7.64 nm and β=101°. There is one molecule in the asymmetric unit and the solvent content is estimated to be 48% by volume. The crystals diffract to 0.25 nm resolution which is higher than that of the previously reported C2 crystal form which had a solvent content of 65%.
Study of the arrangement of the functional domains along the yeast cytoplasmic aspartyl‐tRNA synthetase
Authors: PREVOST Gilles , ERIANI Gilbert , KERN Daniel , DIRHEIMER Guy , GANGLOFF Jean
Published: March 2005

Aspartyl‐tRNA synthetase from yeast (AspRS) was screened for functional domains by measuring the effect of two types of amino acid mutations on its catalytic properties: (a) insertion of a dipeptide or a tetrapeptide along the polypeptide chain, (b) deletion of various lenghts from the enzyme C‐terminal. It was shown that insertion mutations significantly affect the kinetic properties of AspRS only when occurring in the second quarter of the molecule and the two centrally located mutations even inactivate the enzyme completely. Analysis of kinetic data stronly suggests that, in fact, all the observed activity modifications result from alteration of the activation reaction rate constant, Kcat only. This led to the conclusion that the domain involved in aspartic acid activation should be located in the second quarter of the molecule.

Furthermore, a deletion mutant with a modification of the last five amino acid residues was isolated. This mutant is fully active in the activation step, but has lost 80% of the wild‐type aminoacylation activity. This involvement of the C‐terminus in acylation implies that it has to be folded towards strategic regions of the enzyme, thus favouring conformations required for catalysis or maintaining the tRNA in a functional position.
Acridine dimers: influence of the intercalating ring and of the linking‐chain nature on the equilibrium and kinetic DNA‐binding parameters
Authors: MARKOVITS Judith , GARBAY‐JAUREGUIBERRY Christiane , ROQUES Bernard P. , Le PECQ Jean‐Bernard
Published: March 2005

The rigidity of the linking chain of bifunctional intercalators in the ditercalinium series was shown to be critical for antitumor activity. In order to study the influence of the rigidity of the linking chain on the DNA‐binding properties of DNA bifunctional intercalators, fluorescent 9‐aminoacridine and 2‐methoxy‐6‐chloro‐9‐aminoacridine analogues with chains of variable rigidity were synthesized.

1H‐NMR studies show that the conformation of 9‐aminoacridine dimers is almost independent of the nature of the linking chain. A strong self‐stacking of the aromatic rings of the 2‐methoxy‐6‐chloro‐9‐aminoacridine is observed for dimers with flexible chains but not for those with rigid chains.

All the dimers having a linking chain long enough to bisintercalate in DNA according to the excluded site model are indeed bisintercalators. The kinetic association constant of all monomers and dimers for poly‐[d(A‐T)] poly[d(A‐T)] are in the same range (2–4 × 107 M−1 s−1). The large increase of DNA binding affinity observed for the dimers is always associated with the expected decrease of the dissociation rate constant.

The effect of chain rigidity and pH on the calf thymus DNA binding of 9‐aminoacridine and 2‐methoxy‐6‐chloro‐9‐aminoacridine dimers is quite different. In the series of 9‐aminoacridine the pKa of the dimers remains high and therefore no difference of DNA‐binding affinity is observed between pH 5 and 7.4. The rigidity of the linking chain does not significantly alter the DNA‐binding affinity.

In the 2‐methoxy‐6‐chloro‐9‐aminoacridine series. the pKa of all dimers became smaller than the physiological pH and a dramatic decrease of DNA‐binding affinity is observed when the pH is increased from pH 5 to 7.4. This decrease appears significantly smaller for dimers with rigid chains. A similar dramatic decrease of binding affinity at pH 7.4 is not observed for poly[d(A‐T)] · poly[d(A‐T)]. This factor makes these dimers strongly specific for the alternating polymer at pH 7.4.
A human melanoma‐derived cell line (IGR39) with a very high number of vasoactive‐intestinal‐peptide (VIP) receptors
Authors: LUIS José , MARTIN Jean‐Michel , EL BATTARI Assou , REYNIER Max , MARVALDI Jacques , PICHON Jacques
Published: March 2005

Using mono[125I]iodinated vasoactive intestinal peptide (125I‐VIP), a very high number of specific binding sites for VIP were identified at the surface of the human melanoma cell line IGR39. The Scatchard analysis of competitive displacement experiments between native VIP and 125I‐VIP was consistent with the existence of two classes of VIP‐binding sites. IGR39 cells possess 0.54 × 106 high‐affinity sites with a dissociation constant (Kd) of 0.66 nM and 1.3 × 106 sites of moderate affinity with a Kd of 4.7 nM. Pharmacological studies indicated that the order of potency in inhibiting 125I‐VIP binding of the VIP secretin family peptides was VIP Gt; peptide histidine methioninamide > human growth‐hormone‐releasing factor(1–44) > secretin. Glucagon has no effect on the binding of the labelled peptide.

By means of photoaffinity labelling a polypeptide of Mr 63000 was characterized. The labelling of this species was completely abolished by native VIP. The order of potency of VIP‐related peptides in inhibiting 125I‐VIP cross‐linking to its receptor was the same as in the competition experiments. The glycoprotein nature of the VIP‐binding sites of IGR39 cells has been investigated by affinity chromatography on wheat‐germ‐agglutinin–Sepharose.
Casein kinase II from Rana temporaria oocytes
Authors: KANDROR Konstantin V. , BENUMOV Aleksej O. , STEPANOV Aleksandr S.
Published: March 2005

A homogeneous preparation of casein kinase II has been isolated from the ribosome‐free extracts of Rana temporaria oocytes by means of chromatography on heparin‐Sepharose, phosphocellulose and mono Q. The enzyme consists of three subunits with molecular mass of 43 kDa, 41 kDa and 29 kDa. The protein kinase was labelled with radioactive iodine and injected back into oocytes. As shown by histoautoradiography the enzyme forms a diffuse ring aroung the nucleus in the oocyte cytoplasm. A part of casein kinase II is found in informosomes. During oocytes maturation casein kinase II activity increases 7 h after progesterone administration and at the final stages of maturation (20–23 h). Cycloheximide blocks the second augmentation of kinase activity and does not influence the first one.
Generation of hydrogen peroxide by cerebral‐cortex synaptosomes
Authors: ZOCCARATO Franco , DEANA Renzo , CAVALLINI Lucia , ALEXANDRE Adolfo
Published: March 2005

Guinea‐pig cerebral cortex synaptosomes steadily release H2O2 into the suspending medium, at the rate of 20–30 pmol min−1 mg protein−1. A transient increase of the H2O2 release is induced by the addition of 1 mM Ca2+, which declines within 60–90 s to a rate identical or slightly higher than that before Ca2+. The extra H2O2 following Ca2+ addition varies between 40–100 pmol/mg protein and is insensitive to verapamil. The H2O2 release increases strongly (up to 250 pmol min−1 mg−1) upon depletion of the synaptosomal glutathione by treatment with 1‐chloro‐2,4‐dinitrobenzene, a substrate for glutathione transferase. This treatment however has no effect on the Ca2+‐induced H2O2 transient. In these treated synaptosomes a further increase of the output of H2O2 is rapidly induced upon addition of the Ca2+ ionophore ionomycin. This increase (about 100 pmol min−1 mg−1) lasts several minutes and requires the presence of Ca2+. A similar, though less pronounced increased H2O2 release is obtained (also in the absence of Ca2+) upon depolarization of the synaptosomal plasma membrane with KCI or with veratridine.
Structural analysis of the nontoxic lipid A of Rhodobacter capsulatus 37b4
Authors: KRAUSS Jürgen H. , SEYDEL Ulrich , WECKESSER Jürgen , MAYER Hubert
Published: March 2005

Lipid A from Rhodobacter capsulatus 37b4 consists of a d‐glucosaminyl‐(β1–6)‐d‐glucosamine disaccharide backbone, carrying diphosphorylethanolamine at C‐1 of the reducing glucosamine and phosphorylethanolamine at C‐4′ of the nonreducing glucosamine. 1,4′‐Bisphosphorylated lipid A, lacking the polar head groups, was also encountered and contributed to the observed microheterogeneity in the phosphate substitution. The amino functions of both glucosamines are substituted almost entirely by the rare 3‐oxotetradecanoic acid, which is a characteristic constituent of lipid A in the genus Rhodobacter. 3‐Hydroxydecanoic acid is ester‐bound at C‐3 and C‐3′ of the glucosamine disaccharide and the one at the nonreducing glucosamine (C‐3′) is partially substituted by dodecenoic acid to form an ester‐bound diester. In free lipid A, hydroxy groups at C‐4 and C‐6′ of the glucosamine disaccharide are unsubstituted, C‐6′ being the putative attachment point of the lipopolysaccharide core. The nontoxic Rhodobacter capsulatus lipid A shows extensive serological cross‐reaction with the toxic Salmonella lipid A. Structural similarities in the hydrophilic part of both types of lipid A, dissimilarities in the hydrophobic part and their impacts on serologic properties are discussed.
Domain structure of mitochondrial and chloroplast targeting peptides
Authors: von HEIJNE Gunnar , STEPPUHN Johannes , HERRMANN Reinhold G.
Published: March 2005

Representative samples of mitochondrial and chloroplast targetting peptides have been analyzed in terms of amino acid composition, positional amino acid preferences and amphiphilic character. No highly conserved ‘homology blocks’ are found in either class of topogenic sequence. Mitochondrial‐matrix‐targeting peptides are composed of two domains with different amphiphilic properties. Arginine is frequently found either at position –10 or –2 relative to the cleavage site, suggesting that some targeting peptides may be cleaved twice in succession by two different matrix proteases. In stroma‐targeting chloroplast transit peptides three distinct regions are evident: an uncharged amino‐terminal domain, a central domain lacking acidic residues and a carboxy‐terminal domain with the potential to form an amphiphilic β‐strand. Targeting peptides that route proteins to the mitochondrial intermembrane space or the lumen of chloroplast thylakoids have a mosaic design with an aminoterminal matrix‐or stroma‐targeting part attached to a carboxy‐terminal extension that shares many characteristics with secretory signal peptides.
Changes in mRNA levels of rat pancreatic lipase in the early days of consumption of a high‐lipid diet
Authors: WICKER Catherine , PUIGSERVER Antoine
Published: March 2005

The time‐course response of rat pancreatic enzymes to a diet containing 25% sunflower oil was investigated. A 1.2‐fold enhancement in lipase specific activity was observed as early as the first day of diet consumption and was further increased up to 1.9‐fold on the 5th day. On the other hand, colipase activity was slightly decreased during the first two days of high‐lipid diet intake and then increased. An immediate and direct effect was also exerted by the 25% lipid diet on lipase biosynthesis. Both fractional synthetic rate and specific activity of lipase were comparably induced. Due to a 1.6‐fold increase in the overall protein synthesis following 5 days of lipid diet consumption, the absolute synthesis of lipase and amylase was increased by 3.5‐fold and 0.98‐fold, respectively as compared to control animals, the synthesis of procarboxypeptidases and serine proteases did not increase before day 5, probably as the result of a distinct adaptative mechanism. The pancreatic mRNA levels in control and adapted animals, which were determined by dot‐blot hybridization with amylase and lipase cDNAs, were consistent with a biphasic induction of lipase synthesis since a first increase in the level of the enzyme‐specific mRNA during the first two days of diet intake (4‐fold on day 1) was followed by a second increase after the fourth day (6.5‐fold on day 5). On the other hand, amylase mRNA level was unchanged during the dietary manipulation. Thus, hyperlipidic diets exerted an immediate effect on both lipase activity and synthesis but a delayed effect on procarboxypeptidase and serine protease synthesis, In a similar manner, the immediate induction of lipase mRNA level by dietary fat, followed by another increase a few days later, suggested that at least two different mechanisms are involved in lipase mRNA induction.