bluuebunny/crossref_metadata_2025 huggingface dataset:
Two Subsites on the Active Center of Pig Kidney Trehalase
Authors: Asano Naoki , Kato Atsushi , Matsui Katsuhiko
Published: July 2004

A kinetic analysis of the active site of pig kidney trehalase was made by examining two types of inhibitors that are monosaccharide analogs and cause a Competitive inhibition of the trehalase. Trehalase hydrolyzes trehalose (α‐d‐glycopyranosyl α‐d‐glucopyranoside) to give an equimolar mixture of α‐d‐glucose and, by inversion of configuration, β‐d‐glucose. 1,4‐Dideoxy‐1,4‐imino‐d‐arabinitol is considered to be a transition state (glucosyl cation) analog, while methyl β‐d‐glucoside, 1,5‐dideoxy‐1,5‐imino‐d‐glucitol (1‐deoxynojirimycin), fagomine, and 1‐epivalidamine are considered to be analogs of the β‐d‐glucose that is derived by hydrolysis of trehalose. These glucosyl cation inhibitor and β‐d‐glucose analog inhibitors competed with each other at the same site on the active center of pig kidney trehalase and were therefore put together in one group (group A). Methyl α‐d‐mannoside and 1‐deoxymannojirimycin were also competitive inhibitors of trehalase and competed with each other for the same site. However, an inhibitor in group A did not compete with the methyl α‐d‐mannoside or 1,5–dideoxy‐1,5‐imino‐d‐mannitol (1‐deoxymannojirimycin). Thus these latter two inhibitors were placed in group B. These results support the hypothesis that the active center of trehalase may comprise two subsites, one for catalysis and one for recognition, that act separately on each of the glucoses of the trehalose. The catalysis site requires the correct d‐glucose configuration at carbons 2, 3, 4, and 5 or a good superimposition onto the glucosyl cation intermediate. The C2 equatorial OH group of a glucopyranosyl residue appears to be important for binding at the catalytic site since 1‐deoxynojirimycin is more tightly bound by two orders of magnitude over its 2‐deoxy derivative, fagomine. The β‐d‐glucose and glucosyl cation analogs best fit this site. The recognition site is compatible with d‐glucose and its analogs bearing the α configuration at the anomeric position. α‐d‐Mannose analogs are much more tightly bound than the corresponding d‐gluco compound at this site. The extremely high affinity (Ki= 0.52 nM) of validoxylamine A, a mimic of the substrate in the transition state, derives from the synergistic interactions of two cyclitol units with two subsites. The value obtained by multiplying the Ki (1.2 μM) for 1‐epivalidamine times that for 1‐deoxymannojirimycin (K i= 0.39 mM) is very close to that for validoxylamine A. The results described here may be applicable to other trehalase molecules.
Cloning, Sequencing, Mapping and Hyperexpression of the ribC Gene Coding for Riboflavin Synthase of Escherichia coli
Authors: Eberhardt Sabine , Richter Gerald , Gimbel Wolfgang , Werner Thomas , Bacher Adelbert
Published: July 2004

The gene coding for riboflavin synthase of Escherichia coli has been cloned by marker rescue on a 6‐kb fragment that has been sequenced. The riboflavin synthase gene is identical to the ribC locus and codes for a protein of 213 amino acids with a miss of 23.4 kDa. It was mapped to a position at 37.5 min on the physical map of the E. coli chromosome. The 3′ end of the ribC gene is directly adjacent to the cfa gene, which codes for cyclopropane‐fatty‐acid synthase. This gene is followed by two open reading frames designated ydhC and ydhB, which are predicted to code for putative proteins with 403 amino acids and 310 amino acids, respectively. The gene ydhC is similar to genes coding for resistance against various antibiotics (cmlA, bcr) and probably codes for a transmembrane protein. The protein specified by ydhB shows sequence similarity to a large family of DNA‐binding proteins and probably represents a helix‐turn‐helix protein. The ydhB gene is directly adjacent to the regulatory gene purR. A 288‐bp segment of the cfa gene has earlier been mapped incorrectly to a position adjacent to greA at 67 min. The ribC gene was hyperexpressed in recombinant E. coli strains to a level of about 30 % of cellular protein. The protein was purified to homogeneity by chromatography. The specific activity was 26000 nmol · mg−1· h−1. The protein sediments at a velocity of s20= 3.8 S. Sedimentation‐equilibrium centrifugation indicated a molecular mass of 70 kDa, consistent with a trimer structure. The primary structure of riboflavin synthase is characterized by internal sequence similarity (25 identical amino acids in the C‐terminal and N‐terminal parts) suggesting two structurally similar folding domains.
Microsomal Triacylglycerol Transfer Protein Prevents Presecretory Degradation of Apolipoprotein B‐100
Authors: Benoist Fabienne , Nicodeme Edwige , Grand‐Perret Thierry
Published: July 2004

The role of microsomal triacylglycerol transfer protein (MTP) in the secretion of apolipoprotein B‐100 (apoB‐100) has been studied using an inhibitor of MTP:4′‐bromo‐3′‐methylmetaqualone. In vitro, this compound inhibits trioleoylglycerol transfer between lipid vesicles mediated by MTP with an IC50 of 0.9 μM whereas it does not inhibit the lipid transfer mediated by the cholesteryl ester transfer protein. In HepG2 cells, 4′‐bromo‐3′‐methylmetaqualone inhibits the secretion of apoB‐100 with an IC50, of 0.3 μM, without affecting the secretion of several other proteins like apoA‐I or albumin. Moreover, there is no accumulation of apoB‐100 in treated cells. Oleic acid, which increases apoB‐100 secretion, only slightly modifies the IC50, of 4′‐bromo‐3′‐methylmetaqualone (0.5 μM). The latter has no effect on the synthesis of major lipids within the cell, but decreases the secretion of triacylglycerol into apoB‐100‐containing lipoproteins. Pulse/chase experiments reveal that 4′‐bromo‐3′‐methylmetaqualone acts on apoB‐100 production either at the co‐translational or post‐translational level. The cysteine protease inhibitor N‐acetyl‐leucyl‐leucyl‐norleucinal does not protect apoB‐100 from the 4′‐bromo‐3′‐methyl‐metaqualone effect but seems to be involved in a later step of apoB‐100 intracellular degradation. By contrast, dithiothreitol can totally reverse the effect of the MTP inhibitor on apoB‐100 production. The mechanism of MTP‐mediated lipid assembly with apoB‐100 is discussed.
Separate Compartments for the Production of Sterols, Carotenoids and Gibberellins in Gibberella fujikuroi
Authors: Domenech Carlos Eduardo , Giordano Walter , Ávalos Javier , Cerdá‐olmedo Enrique
Published: July 2004

Substrate flows in the sterol, carotenoid and gibberellin pathways of Gibberella fujikuroi were examined by isotope‐dilution experiments. The wild type and two carotenoid mutants of this fungus were grown in minimal medium with abundant glucose, limiting ammonium nitrate and a radioactively labelled precursor (either acetate, mevalonate or leucine). The precursors did not affect growth or terpenoid production, with two exceptions; leucine allowed additional growth, as expected from the nitrogen limitation in the medium, and mevalonate inhibited the accumulation of gibberellins, but only if added before the onset of gibberellin production.

The relative contributions of glucose, mevalonate, leucine and acetate as terpenoid precursors, calculated from the specific radioactivities of ergosterol, neurosporaxanthin and phytoene, were different for different products and different precursors. We conclude that the biosyntheses of sterols, gibberellins and carotenoids in Gibberella are physically separated in different subcellular compartments with independent substrate pools. The same results were obtained with the three strains, except for carotenoid production, indicating that this pathway is regulated independently from other terpenoid pathways.
Experimental Verification of the Secondary Structures of Guide RNA–Pre‐mRNA Chimaeric Molecules in Trypanosoma Brucei
Authors: Schmid Beate , Read Laurie K. , Stuart Kenneth , Göringer H. Ulrich
Published: July 2004

RNA editing in kinetoplastid organisms is an RNA‐processing reaction that adds and deletes U nucleotides at specific sites in mitochondrial pre‐mRNAs. The edited sequence is specified by guide RNAs and the processing presumably occurs within a high‐molecular‐mass ribonucleoprotein complex containing several enzymatic activities. Although the mechanism is not currently known, potential intermediates or by‐products of the editing process are chimaeric RNAs where guide (g) RNAs are covalently attached, via their non‐encoded U‐tail, to their cognate pre‐mRNAs. We determined the secondary structures of three different ATPase 6 chimaeras of Trypanosoma brucei using a set of structure‐sensitive chemical and enzymatic probes. The experiments revealed a bipartite domain structure consisting of a gRNA/pre‐mRNA interaction hairpin and an independently folding mRNA stem/loop in all three RNAs. The connecting U‐tail was a determinant for the length of the interaction stems with the oligo(U) nucleotides base pairing to internal gRNA sequences. The probed structures have calculated ΔG270 values of –92 kJ/mol to –134 kJ/mol, somewhat less stable than the predicted minimal free energy structures and support previously proposed models for the interaction between gRNAs and pre‐mRNAs. Optical melting studies indicated additional, higher order structural features for all three molecules with four defined melting transitions between 10°C and 90°C. A comparison of CD spectra in the absence and presence of mitochondrial protein extracts demonstrated no gross structural changes of the RNA structures induced by the association with polypeptides.
Purification and Partial Sequencing of High‐Affinity Progesterone‐Binding Site(s) from Porcine Liver Membranes
Authors: Meyer Christiane , Schmid Roland , Scriba Peter C. , Wehling Martin
Published: July 2004

High‐affinity progesterone‐binding sites have been identified, characterized in and purified from porcine liver membranes. They were functionally solubilized by the non‐denaturing zwitterionic detergent 3‐[(3‐cholamidopropyl)dimethylammonio]‐1‐propanesulfonic acid (Chaps, 20 mM, detergent/protein mass ratio 4:1) at a yield of 75–80%. Using [3H]progesterone as radioligand, binding studies showed high‐affinity and low‐affinity binding sites in microsomal preparations with an apparent Kd1 of 11 nM and an apparent Kd2 of 286 nM. In solubilized fractions the high‐affinity binding sites were present at an apparent Kd of 69 nM. In both preparations, progesterone binding was time‐dependent, saturable, reversible, and showed a similar hierachy of affinities for related steroids. A purification scheme was developed based on anion‐exchanger procedures. The purified fraction as identified by maximum specific progesterone‐binding activity contained two major polypeptides of apparent molecular masses (SDS/PAGE) of 28 kDa and 56 kDa, respectively. Sequencing of both polypeptides showed an identical amino terminus without significant identity in the amino acid sequence to any known protein primary structure.
The Binding of Nucleotides to Domain I Proteins of the Proton‐Translocating Transhydrogenases from Rhodospirillum rubrum and Escherichia coli as Measured by Equilibrium Dialysis
Authors: Bizouarn Tania , Diggle Christine , Jackson J. Baz
Published: July 2004

Transhydrogenase catalyses the transfer of reducing equivalents between NAD(H) and NADP(H) coupled to the translocation of protons across a membrane. The NAD(H)‐binding domain of transhydrogenase (domain I protein) from Rhodospirillum rubrum and from Escherichia coli were overexpressed and purified. Nucleotide binding to the domain I proteins was determined by equilibrium dialysis. NADH and its analogue, acetylpyridine adenine dinucleotide (reduced form), bound with relatively high affinity (Kd= 32 μM and 120 μM, respectively, for the R. rubrum protein). The binding affinity was similar at pH 8.0 and pH 9.0 in zwitterionic buffers, and at pH 7.5 in sodium phosphate buffer. NAD+ bound with lower affinity (Kd= 300 μM). NADPH bound only very weakly (Kd>1 mM).

Using a centrifugation procedure, Yamaguchi and Hatefi [Yamaguchi, M. & Hatefi, Y. (1993) J. Biol. Chem. 268, 17871–17877] found that mitochondria1 transhydrogenase, and a proteolytically derived domain I fragment from that enzyme, bound one NADH per dimer. They suggested that this result implied half‐of‐the‐sites reactivity for the interaction between the nucleotide ligand and the protein. However, our studies on both the E. coli and the R. rubrum recombinant transhydrogenase domain I proteins using equilibrium dialysis show that the binding stoichiometry for both NADH and the reduced form of acetylpyridine adenine dinucleotide (AcPdADH) is two nucleotides per dimer: no interaction between the monomeric units is evident. Reasons for the discrepancies between the work on bacterial and mitochondrial transhydrogenases are discussed.
Evidence for Glycosylation of the Juvenile‐Hormone‐Binding Protein from Galleria mellonella Hemolymph
Authors: Duk Maria , Krotkiewski Hubert , Forest Eric , Rodriguez Parkitna Jan M. , Kochman Marian , Lisowska Elwira
Published: July 2004

The juvenile‐hormone‐binding protein (JHBP) from Galleria mellonella hemolymph, which is a member of the high‐affinity/low‐molecular‐mass group of JHBP proteins, was found to be glycosylated. Glycosylation was confirmed by the following evidence. Carbohydrate gas‐liquid chromatography analysis of the purified JHBP preparations showed the presence of a low amount of sugars (Man and GlcNAc were the major components). The JHBP electrophoretic band blotted onto nitrocellulose was stained with GlycoTrack (a reagent kit used for the detection of protein glycosylation) and showed strong binding of concanavalin A (ConA). JHBP was fractionated on a ConA‐Sepharose 4B column into ConA‐bound (strongly stained with ConA) and ConA‐unbound (hardly stained with ConA) portions. Both fractions showed juvenile‐hormone‐binding activity and were glycosylated, as revealed by staining both of them with GlycoTrack. Electrospray‐ionization mass spectrometry of JHBP suggested the presence of a small amount of presumably nonglycosylated protein (24988 Da) and five glycoforms, two of which (containing Man2 GlcNAc2 or Man2Fuc1GlcNAc2 chain) were not bound or were weakly bound to ConA, and three (with Man3GlcNAc2 Man3Fuc1GlcNAc2 or Man5GlcNAc2 chain) were present in the fraction strongly bound to ConA. In conclusion, the monosugar composition, GlycoTrack staining, ConA‐binding properties and molecular mass analyses of JHBP supplied convincing evidence for its glycosylation and some information on the character of the oligosaccharide chains.
Pre‐tRNA 3′‐Processing in Saccharomyces cerevisiae
Authors: Papadimitriou Apollon , Gross Hans J.
Published: July 2004

We investigated ribonucleases from Saccharomyces cerevisiae which are active in pre‐tRNA 3′‐processing in vitro. Two pre‐tRNA 3′‐exonucleases with molecular masses of 33 and 60 kDa, two pre‐tRNA 3′‐endonucleases with molecular masses of 45 kDa60 kDa and 55 kDa and a 70‐kDa 3′‐pre‐tRNase were purified from yeast whole cell extracts by several successive chromatographic purification steps. The purified exonucleases are non‐processive 3′‐exonucleases that catalyze the exonucleolytic processing of 3′‐trailer sequences of pre‐tRNAs to produce mature tRNAs. The 45‐kDa60‐kDa 3′‐endonuclease is tRNA‐specific and catalyzes the processing of pre‐tRNAs in a single endonucleolytic step. Two isoenzymes of this activity (p45 and p60) were identified by chromatography. The second endonuclease, p55, is dependent on monovalent ions and cleaves about three nucleotides downstream the mature 3′‐end. All of the purified 3′‐pre‐tRNases accept homologous as well as heterologous pre‐tRNA substrates. Pre‐tRNAs carrying a 5′‐leader are processed with almost the same efficiency as those lacking this 5′‐leader. Mature tRNAs carrying the CCA 3′‐sequence and tRNA pseudogene products carrying mutations in the mature domain are processed by the 3′‐exonucleases, not by the 3′‐endonucleases. The specific endonuclease p45/p60 discriminates between UUUOH as a 3′‐flank, which is cleaved, and the CCA 3′‐end of mature tRNAs, which is not cleaved. This study suggests that several 3′‐pre‐tRNases are active on tRNA precursors in vitro and might therefore participate in pre‐tRNA 3′‐processing in yeast, partly in a cooperative manner.
Molecular‐Dynamics Simulation of Domain Movements in Aspartate Aminotransferase
Authors: Kasper Patrik , Sterk Michael , Christen Philipp , Gehring Heinz
Published: July 2004

Mitochondrial aspartate aminotransferase is a homodimeric protein with 2×402 amino acid residues. The enzyme in solution undergoes ligand‐induced and syncatalytic conformational changes which appear to correspond to shifts in the equilibrium between the crystallographically defined open and closed conformation. In the closed conformation, the small domain of each subunit has rotated as a rigid body by 13° and 14° towards the large coenzyme‐binding domain and has closed the active‐site pocket.

Molecular dynamics simulations at 300 K of 120‐ps duration were started from the crystal structures of the unliganded pyridoxal form (open form) of the dimeric enzyme and the 2‐methylaspartate‐liganded closed form in which the 2‐methyl group had been removed. Both structures contained the crystal water molecules and were placed in a 5‐Å shell of water. The rms fluctuations of the individual Cα atoms during the simulations agreed well with the corresponding B factors of the crystal structures. Superposition of the initial structures and the average structures of the last 20 ps showed in both simulations extensive Cα deviations in the case of the whole subunit but much smaller changes in the individual large and small domains, indicating a movement of the two domains relative to each other. In the simulation of the open form, superposition of the large domains made evident a displacement of the small domain towards its position in the closed crystal structure, which can be described by a rotation of the small domain by about 13° around the twofold symmetry (z) axis. A significantly less extensive rearrangement of parts of the small domain, i.e. a rotation of about 5° around the z axis, was observed in the simulation of the substrate‐liganded enzyme (closed form) which, in contrast to the open form, showed only small conformational changes around the active site. In both simulations an additional rotation of the small domain by 9° around the x axis occurred. The actual domain movement is estimated to occur in a time range at least two orders of magnitude larger than the simulation time of 120 ps. Apparently, the surface tension of the unrestrained nonspherical shell of water accelerates the simulated conformational change which, however, quite closely imitates the geometric features of the extensive movement of the small domains (each ≈130 residues).
The Purification and Characterization of the Catalytic Domain of Src Expressed in Schizosaccharomyces Pombe
Authors: Weijland Albert , Neubauer Gitte , Courtneidge Sara A. , Mann Matthias , Wierenga Rik K. , Superti‐Furga Giulio
Published: July 2004

The catalytic domain of chicken Src including the C‐terminal tail (Src‐CD), has been expressed in Schizosaccharomyces pombe and purified to homogeneity. The expressed protein is a mixture of unphosphorylated (80%) and mono‐phosphorylated (20%) species, that can be separated from each other by Mono Q chromatography. By a novel mass spectrometric method that utilizes parent ion scans of unseparated peptide mixtures, we found that the mono‐phosphorylated form is phosphorylated either at Tyr416 or at Tyr436. The stability of Src‐CD is comparable to the wild‐type protein. Src‐CD auto‐phosphorylates and efficiently phosphorylates substrate peptides and proteins. Auto‐phosphorylation occurs by an intermolecular mechanism and is completely inhibited by an excess of substrate peptide. Kinetic measurements for two exogenous substrates, the Src substrate peptide (AEEEIYGEFEAKKKK) and denatured enolase, showed that the overall activity (kcat) of the Src‐CD molecule is about 10 times higher than that of wild‐type Src. The kcat, values for phosphorylation of the Src substrate peptide are similar for the unphosphorylated and monophosphorylated Src‐CD (50 min−1), but the apparent Km values differ significantly (approximately 3 μM and 10 μM, respectively). Therefore, at low substrate concentrations in vitro the mono‐phosphorylated form is more active, in agreement with the importance of Tyr416 for in vivo activity. The apparent Km values of the mono‐phosphorylated Src‐CD and wild‐type Src for the Src substrate peptide and enolase are similar, indicating that, under these conditions, the kinase domain is mainly responsible for substrate binding.
Transcription of the AAC1 Gene Encoding an Isoform of Mitochondrial ADP/ATP Carrier in Saccharomyces cerevisiae is Regulated by Oxygen in a Heme‐Independent Manner
Authors: Gavurníková Gabriela , Šabova Ludmila , KiŠŠová Ingrid , Haviernik Peter , Kolarov Jordan
Published: July 2004

The mitochondrial ADP/ATP carrier in Saccharomyces cerevisiae is encoded by three independent genes, AAC1, AAC2, and AAC3. In this work, we analysed the 5′ upstream region of AAC1 by sequencing and by mapping the transcription initiation site of the gene. By monitoring the level of AAC1 mRNA and the β‐galactosidase activity of AAC1‐lacZ fusion constructs, we showed that expression of AAC1 is subjected to regulation by oxygen. In contrast to the other two AAC genes, the effect of oxygen on AAC1 is not mediated by heme and heme‐dependent transcription factors. The AAC1 expression was reduced eightfold in anaerobically grown cells compared to expression in cells grown aerobically, but it was not affected by the nature of carbon source used for growth. The data presented show that AAC1 expression, while constitutive under all aerobic conditions tested, is repressed during anaerobiosis in a heme‐independent manner.
Probing the Active Site of Tritrichomonas foetus Hypoxanthine‐Guanine‐Xanthine Phosphoribosyltransferase Using Covalent Modification of Cysteine Residues
Authors: Kanaani Jamil , Somoza John R. , Maltby David , Wang Ching C.
Published: July 2004

The hypoxanthine‐guanine‐xanthine phosphoribosyltransferase (HGXPRTase) of Tritrichomonas foetus was inactivated by the thiol reagents iodoacetate and 5,5′‐dithiobis(2‐nitrobenzoic acid) (Nbs2). Iodoacetate inactivates the enzyme in a time‐dependent and concentration‐dependent manner that follows pseudo‐first‐order kinetics. However, the observation that total inactivation with iodoacetate was not achieved suggests that none of the reactive cysteine residues is directly involved in the catalytic activity of the enzyme. Nbs2 caused 50% inactivation rapidly, which was followed by gradual modifications of an additional three cysteine residues leading to complete enzyme inactivation. Analysis of the inactivation using the method developed by Tsou (1962) revealed that modification of two cysteine residues by Nbs2 is sufficient to impair the HGXPRTase activity. Tryptic digestion of HGXPRTase labeled with iodo[2‐14C]acetic acid, followed by fractionation of the digest by HPLC and sequence analysis of the labeled peptides allowed the identification of Cys71, Cysl29, Cys132, and Cys148 as the reactive cysteine residues. GMP and 5‐phosphoribosyl‐1‐diphosphate provided complete protection against HGXPRTase inactivation by iodoacetate and against carboxymethylation of Cys129, Cys132, and Cys148. Cys71 was not protected by either substrate against iodoacetate, but its carboxymethylation caused no loss in enzyme activity either. There was also no substrate protection of Cys71 against Nbs2, which, however, caused 50% inactivation of the enzyme. Replacing the thionitrobenzoate (Nbs) moiety from Cys71 with cyanide resulted in a gradual recovery of the enzyme activity, which indicates that a steric hindrance at the active site was introduced by Nbs but removed by cyanide. Thus, our results demonstrate that although the reactive cysteine residues in HGXPRTase are not directly involved in the catalytic activity, modification of cysteine residues 129, 132, and 148 by iodoacetate or Nbs2 hinders substrate binding which can, in turn, protect the cysteine residues from modifications. The substrate protection of Cys129 and Cys148 is probably also indicative of a conformational change in the protein structure brought about by substrate binding.
Mathematical Models for Determining Metabolic Fluxes through the Citric Acid and the Glyoxylate Cycles in Saccharomyces cerevisiae by 13C‐NMR Spectroscopy
Authors: Tran‐Dinh Son , Bouet Françoise , Huynh Quang‐Tru , Herve Martine
Published: July 2004

We propose, first, a practical method for studying the isotopic transformation of glutamate or any other metabolite isotopomers in the citric acid and the glyoxylate cycles; second, two mathematical models, one for evaluating the flux through the citric acid cycle and the other for evaluating the flux through the latter coupled to the glyoxylate cycle in yeast. These models are based on the analysis of 13C‐NMR spectra of glutamate obtained from Saccharomyces cerevisiae, NCYC strain, fed with 100% enriched [2‐13C]acetate. The population of each glutamate isotopomer, the change in intensity of each multiplet component or the enrichment of any glutamate carbon is expressed by a specific analytical equation from which the flux in the citric acid and the glyoxylate cycles can be deduced.

The aerobic metabolism of 100% [2‐13C]acetate in acetate‐grown S. cerevisiae cells was studied as a function of time using 13C‐NMR, 1H‐NMR and biochemical techniques. The C1 and C6 doublet and singlet of labeled trehalose increase continuously with time indicating that there is no isotopic transformation between trehalose isotopomers even though the corresponding formation rates are different. By contrast, the glutamate C4 singlet increases then decreases with time. The C4 doublet, which is lower than the singlet for t < 60 min, increases continuously and becomes higher than the singlet for t 90 min. A similar observation was made for the C2 resonance singlet and doublet. In addition, the glutamate C2 multiplet consists of only seven instead of nine peaks as in random labeling. These results agree well with our models and demonstrate that, in the presence of acetate, anaplerotic carbon sources involved in the synthesis of acetyl‐CoA are negligible in yeast.

The flux in the citric acid cycle was deduced from a plot of the C4 area versus incubation time, while the flux within the glyoxylate cycle was determined from the relative intensity of the glutamate C4 doublet and singlet. The fluxes in the citric acid and the glyoxylate cycles were found to be comparable. The proportion of glutamate in isotopic exchange with the citric acid cycle is about 2.5% min ‐1 in yeast.
Analysis of Global Responses by Protein and Peptide Fingerprinting of Proteins Isolated by Two‐Dimensional Gel Electrophoresis
Authors: Quadroni Manfredo , Staudenmann Werner , Kertesz Michael , James Peter
Published: July 2004

A set of 8 proteins (SSI, sulfate‐starvation‐induced proteins) was observed by comparative two‐dimensional electrophoresis to be induced when Escherichia coli were grown using compounds other than sulfate or cysteine as the sole sulfur source. These proteins were isolated after two‐dimensional gel electrophoresis, digested with trypsin and the masses of the resulting peptides determined by mass spectrometry. The list of peptide masses served as a protein fingerprint which was used to search the databases, allowing four of the SSI proteins (SSI 2, 5, 7, 8) to be identified with a high degree of confidence.

To identify the other SSI proteins, and to obtain sequence information for as many of the proteins as possible, automated on‐line HPLC MS/MS (fragmentation analysis using coupled mass scanning devices) data collection was performed. The uninterpreted MS/MS spectra were used as peptide fingerprints to search the databases. Genes encoding two further proteins (SSI 1 and 3) were identified in the 8.5′ region of the Escherichia coli genome. N‐terminal sequencing of all of the proteins confirmed the results of protein and peptide fingerprinting and in addition showed that SSI 6 shows 50% similarity to the Bacillus subtilis orfM gene product. SSI 4 was not found in the databases by any of these methods.

The methods described are of general use for the rapid analysis of complex cell responses. MS data accumulation takes about 5 min/protein for protein fingerprinting and 30 min for peptide fingerprinting and requires approximately 100 fmol of material. N‐terminal sequencing however, takes about 5 h/protein and approximately 1 pmol to obtain a 10 amino acid sequence for a search.
Interaction of Human Immunodeficiency Virus Type 1 Reverse Transcriptase with Primer tRNALys3 and Affinity Modification of The Enzyme by tRNALys3 Derivatives
Authors: Nevinsky Georgy A. , Zakharova Ol'ga D. , Fournier Michel , Andreola Marie Line , Litvak Simon , Tarrago‐Litvak Laura
Published: July 2004

The recognition of primer tRNA by retroviral reverse transcriptase is a crucial step in the replication of retroviruses. In the complex formed by HIV‐1 reverse transcriptase and its natural primer tRNALys3, the heterodimeric enzyme, p66/p51, binds two molecules of tRNALys3 with different affinities. The same complex but in the presence of a non‐complementary template, poly(A), gave higher Kd values. Preincubation of the reverse transcriptase with tRNA at concentrations comparable to the Kd2 value results in different levels of stimulation of the DNA polymerase activity: 300% in the absence and 70–80% in the presence of poly(A). The activation of the catalytically active p66 subunit is most probably mediated through tRNA interaction with the site of reverse transcriptase presenting the lower affinity.

In this article, we describe the results obtained with new chemically reactive derivatives of tRNA bearing three or seven hydrophobic residues. Incubation of reverse transcriptase with tRNA derivatives, in the presence or absence of poly(A), leads to covalent binding of the reagents and inactivation of the enzymatic activity. However, during the initial step of the modification reaction, in the absence of poly(A), a slight stimulation of reverse transcriptase by tRNA derivatives took place, followed by a decrease in the enzymatic activity due to the covalent binding of tRNA derivatives to reverse transcriptase. In the presence of poly(A), enzyme inactivation occurs according to pseudo‐first‐order reaction kinetics. The affinities of tRNA derivatives for the p66/p51 heterodimer estimated from affinity modification data (Ki values) and from the inhibition of polymerization reaction (Ki values) were determined. Each analog of tRNA presented two Kd and two Ki values.
Kinetic Evidence for Protein Complexes Between Thioredoxin and NADP‐Malate Dehydrogenase and Presence of a Thioredoxin Binding Site at the N‐Terminus of the Enzyme
Authors: Braun Harald , Lichter Alexander , Häberlein Ingo
Published: July 2004

The kinetics of activation of NADP–malate dehydrogenase (MDH; EC 1.1.1.82) from soybean and spinach leaves by the chloroplast thioredoxins isolated from the same plants, by the corresponding storage forms of the soybean chloroplast thioredoxins from soybean seeds, and by the bacterial Escherichia coli thioredoxin have been studied. The Hill equation has been applied to evaluate the saturation kinetics. The observed variable thioredoxin saturation characteristics (Vmax 0.37–14.5 μmol NADPH min−1mg enzyme−1; K0.5 0.15–1.33 μM; Hill coefficient h 0.90–3.04) indicate that the activation of NADP–MDH depends strongly on the individual thioredoxin used. Thus, thioredoxin action is not solely due to simple reductive activation of the NADP–MDH. Specific thioredoxin complex formation between thioredoxin and NADP–MDH must be included into the mechanism of the activation process. To study the regulatory consequences of the specific thioredoxin/NADP‐MDH complexes we investigated the saturation kinetics of the substrates NADPH and oxaloacetate in presence of different concentrations of each individual thioredoxin species. The kinetic characteristics of the substrates (S0.5, Vmax, and Hill coefficients h) varied individually in response to the different thioredoxin species substantiating our model of thioredoxin/NADP‐MDH complex formation. Aminopeptidase–K‐truncated pea NADP‐MDH has been used to demonstrate that the N‐terminal 37 amino residues are involved in providing a specific thioredoxin binding site. The fact that the versatile light‐dependent regulation of numerous enzyme activities by only two thioredoxin species in chloroplasts cannot be accomplished without the formation of thioredoxin/target enzyme complexes is discussed in detail.
Polydispersity in Sulfation Profile of Oligosaccharide Alditols Isolated from the Protein‐Linkage Region and the Repeating Disaccharide Region of Chondroitin 4‐Sulfate of Bovine Nasal Septal Cartilage
Authors: De Beer Tonny , Inui Atsuko , Tsuda Hiromi , Sugahara Kazuyuki , Vliegenthart Johannes F. G.
Published: July 2004

Proteoglycans of bovine nasal septal cartilage bear predominantly chondroitin 4‐sulfate. After exhaustive chondroitinase ABC digestion of a chondromucoprotein preparation rich in proteoglycans and subsequent reductive β‐elimination, five hexasaccharide alditols were isolated from the glycosaminoglycan‐protein linkage region. They were analyzed by enzymatic digestion in conjunction with HPLC and by one‐dimensional and two‐dimensional 1H‐NMR spectroscopy. They share the conventional core saccharide structure Δ4,5HexAα1–3GalNAcβ1–4GlcAβ1–3Ga1β1–3Ga1β1‐4Xyl‐ol (where Δ4,5HexA is 4,5‐unsaturated hexuronic acid), but have different sulfation profiles. One compound (I) does not contain sulfate. Two of the three monosulfated compounds (II and III) have an O‐sulfate group at either C6 or at C4 of the GalNAc residue. The other monosulfated compound (IV) is hitherto unreported and has a O‐sulfate at C4 of the Gal residue preceding the GlcA residue, whereas the GalNAc is not sulfated. The disulfated compound (V) has sulfate groups at C4 of both the Gal residue preceding GlcA and the GalNAc residue. The molar ratio of compounds I–V is 38.3:5.9:43.0:1.6:11.2. The structural heterogeneity of these hexasaccharide alditols reflects the polydispersity in the linkage region of the chondroitin sulfate chains. In addition, two trisaccharide and two tetrasaccharide alditols derived from the repeating disaccharide region of the chondroitin sulfate chains were also isolated. Their structures were unambiguously determined by enzymatic analysis and by 1H‐NMR spectroscopy as Δ4,5HexAα1– 3GalNAc(4‐O‐ or 6‐O‐sulfate)β1–4GlcA‐ol and Δ4,5HexAα1–3GalNAc(4‐O‐ or 6‐O‐sulfate)β1–4GlcAβ1‐3GalNAc(4‐O‐sulfate)‐ol, respectively.
Expression, Subcellular Distribution and Response to Phorbol Esters of Protein Kinase C (PKC) Isozymes in Drug‐Sensitive and Multidrug‐Resistant KB Cells Evidence for Altered Regulation of PKC‐α
Authors: Cloud‐Heflin Billie A. , Mcmasters Robyn A. , Osborn Maudie T. , Chambers Timothy C.
Published: July 2004

Protein kinase C (PKC) comprises a family of related phospholipid‐dependent serine/threonine protein kinases. PKC has been implicated in the induction and maintenance of the multidrug‐resistance (MDR) phenotype but the role of different isozymes is not well understood. We compared the expression and subcellular distribution, and membrane association and down‐regulation induced by phorbol esters, of individual PKC isozymes in drug‐sensitive KB‐3 and multidrug‐resistant KB‐V1 human carcinoma cell lines. Immunoblotting with isozyme‐specific antibodies indicated the presence of PKCα (cytosol only), PKCβ (membrane only), PKCɛ (mainly membrane associated) and PKCζ (both fractions). PKCδ and PKCγ were not detected. The expression levels of PKCβ, PKCɛ and PKCζ were unchanged in KB‐V1 cells; PKCα was modestly increased (≈ 65%) in the resistant cells as further determined by enzyme assay. The cytosolic nature and increased expression of PKCα were confirmed by immunofluorescent localization studies. Revertant cells, obtained by culturing KB‐V1 cells in a drug‐free medium, regained drug sensitivity with a loss of P‐glycoprotein and a concomitant decrease in expression of PKCα. KB‐V1 cells were found to differ markedly from KB‐3 cells with respect to the translocation and down‐regulation specifically of PKCα upon exposure to 12‐O ‐tetradecanoyl‐1‐phorbol‐13‐acetate (TPA). Treatment with 30 nM TPA for 24 h completely depleted KB‐3 cells of PKCα whereas 1 μM TPA was required to deplete KB‐V1 cells of PKCα. Similar results were obtained when phorbol‐12,13‐dibutyrate was used instead of TPA. Defective TPA‐mediated down‐regulation of PKCα was also observed in another PKCα‐overexpressing MDR cell line, KB‐A1. Importantly, cellular uptake of radiolabeled phorbol ester was similar for both drug‐sensitive and MDR cells. Sensitive and resistant cells exhibited similar expression levels of RACK1, a PKC‐binding protein important in activation‐induced translocation. These findings further highlight the importance of PKCα in the MDR phenotype, and suggest that this isozyme may be expressed in a modified form or be subject to an altered regulation in MDR cells.
Effects of Ultraviolet‐B Radiation on Photosystem II of the Cyanobacterium Synechocystis sp. PCC 6083
Authors: Giacometti Giorgio Mario , Barbato Roberto , Chiaramonte Simona , Friso Giulia , Rigoni Fernanda
Published: July 2004

The effects of ultraviolet‐B radiation (280–320 nm) on photosystem II of Synechocystis sp. PCC 6803 were investigated at the functional and structural levels. Loss of oxygen‐evolving and electron‐transport activity, measured by various techniques including Clark electrode polarography, fluorescence induction and fluorescence relaxation after a single turnover flash, are discussed in terms of two types of damage caused by ultraviolet‐B radiation: (a) depletion of the plastoquinone pool; (b) perturbation and degradation of the D1 protein, with cleavage in the second transmembrane segment. These findings are in full agreement with those obtained, both in vivo and in vitro for higher plants for which a donor‐side mechanism involving the water‐splitting Mn cluster has been proposed for the main cleavage of the D1 protein. At the structural level, complete disruption of the photosystem II core is documented as a consequence of (or in parallel with) degradation of the D1 protein. From this point of view, ultraviolet‐B‐induced photoinhibition is unlike the visible‐induced type and less susceptible to repair by synthesis and reinsertion of new D1 protein
The Association of Different Detergents with the Photosynthetic Reaction Center Protein of Rhodobacter sphaeroides R26 and the Effects on its Photochemistry
Authors: Gast Peter , Hemelrijk Petra W. , Van Gorkom Hans J. , Hoff Arnold J.
Published: July 2004

Detergent‐free reaction centers from Rhodobacter sphaeroides R26 were used to study the solubilization of reaction centers in various detergents and their effects on reaction center photochemistry. 500 ± 100 n ‐octyl‐β‐d‐glucopyranoside or 51 ± 5 Triton X‐100 molecules were associated with one reaction center. For N,N‐alkylamine N‐oxide detergents with chain lengths in the range from 8–13 carbon atoms, the number of detergent molecules associated with the reaction centers increased with decreasing chain length. The amount of detergent molecules associated with the reaction centers decreased almost tenfold if the pH was increased from pH 6 to pH 10. The addition of 5% 1,2,3‐heptanetriol to various detergents lowered the detergent/reaction center ratio by a factor of two compared to that for the pure detergent. The detergent concentration at which solubilization of the reaction center occurs was close to the critical micelle concentration for all detergents studied. The absorption band at 865 nm of the primary donor in the reaction center shifts to 846 nm when detergent was removed from the reaction center; upon re‐solubilization with various detergents, this band shifts back to 865 nm. In 80–90% of the detergent‐free reaction centers, the secondary electron transfer from QA to QB was inhibited; this electron transfer was restored after re‐addition of detergent.
Solubilization and Characterization of a Thyroid Ca2+‐Dependent and NADPH‐Dependent K3Fe(CN)6 Reductase
Authors: Gorin Yves , Ohayon Renée , Carvalho Denise P. , Dème Danielle , Leseney Anne‐Marie , Haye Bernard , Kaniewski Jacques , Pommier Jacques , Virion Alain , Dupuy Corinne
Published: July 2004

The thyroid plasma membrane contains a Ca2+‐regulated NADPH‐dependent H2O2‐generating system which provides H2O2 for the thyroid‐peroxidase‐catalyzed biosynthesis of thyroid hormones. The molecular nature of the membrane‐associated electron transport chain that generates H2O2 in the thyroid is unknown, but recent observations indicate that a flavoprotein containing a FAD prosthetic group is involved. Solubilization was reinvestigated using 3‐[(3‐cholamidopropyl)dimethylammonio]‐1‐propanesulfonate (Chaps), Triton X‐100, and high salt concentrations. Chaps eliminated about 30% of the proteins, which included a ferricyanide reductase, without affecting the H2O2‐generating system. Similarly, Triton X‐100 alone did not extract the NADPH oxidase. An NADPH‐oxidase activity, which was measured in the presence of the artificial electron acceptor potassium ferricyanide, was solubilized by increasing the ionic strength to 2 M KCl. This NADPH‐ferricyanide reductase activity was shown to belong to the H2O2‐generating system, although it did not produce H2O2. It was still Ca2+ dependent and H2O2 production was restored by decreasing the ionic strength by overnight dialysis. No H2O2 production activity was detected after sucrose density gradient centrifugation of the dialyzed solubilized enzyme, but a well‐defined peak of NADPH oxidation activity with a sedimentation coefficient of 3.71 S was found in the presence of K3Fe(CN)6. These results suggest that some unknown component(s) (phospholipid or protein) is removed during sucrose density gradient centrifugation. Finally, thyrotropin, which induces NADPH oxidase and regulates H2O2 production in porcine thyrocytes in primary culture, also induced the NADPH‐K3Fe(CN)6 reductase activity associated with the H2O2‐generating system. Thus, this enzyme seems to be another marker of thyroid differentiation.
cDNA Cloning, Expression and Characterization of Nitric‐oxide Synthase from the Salivary Glands of the Blood‐Sucking Insect Rhodnius prolixus
Authors: Yuda Masao , Hirai Makoto , Miura Ken , Matsumura Haruo , Ando Katsuhiko , Chinzei Yasuo
Published: July 2004

Rhodnius prolixus, a blood‐sucking bug, is a unique insect that is known to produce nitric oxide (NO) in the salivary glands to use as a vasodilator for blood sucking. We report here the cloning of the NO synthase (NOS) cDNA from these salivary glands and its expression in a baculovirus system. This cDNA encodes a protein of 1174 amino acids with a calculated molecular mass of 132331 Da. The primary structures of mammalian NOS, including the putative cofactor‐recognition sites for heme, tetrahydrobiopterin (BH4), calmodulin, FMN, FAD and NADPH are all conserved in salivary‐gland NOS. Recombinant salivary‐gland NOS differed from nerve NOS and endothelial NOS in that it lacked a large N‐terminal domain and an N‐terminal myristylation sequence, respectively. Salivary‐gland NOS produced in a baculovirus system showed NOS activity and demonstrated that salivary‐gland NOS was soluble and was Ca2+ and calmodulin dependent, similarly to mammalian constitutive NOS isoforms. Recombinant salivary‐gland NOS was purified to near homogeneity and migrated at 130 kDa on SDS/PAGE.
Membrane Topology of the Mannose Transporter of Escherichia coli K12
Authors: Huber François , Erni Bernhard
Published: July 2004

The mannose transporter of the bacterial phosphotransferase system mediates carbohydrate transport across the cytoplasmic membrane concomitant with carbohydrate phosphorylation. It also functions as a receptor for bacterial chemotaxis [Adler, J. & Epstein, W. (1974) Proc. Natl Acad. Sci. USA 71, 2895–2899] and is required for infection of the cell by bacteriophage λ where it most likely functions as a pore for penetration of phage DNA [Elliott, J. & Arber, W. (1978) Mol. & Gen. Genet. 161, 1–8]. The transporter consists of two transmembrane subunits (27‐kDa IICMan and 31‐kDa IIDMan) and a hydrophilic subunit (35‐kDa IIABMan). Protein fusions of IICMan and IIDMan with β‐galactosidase (LacZ) and with alkaline phosphatase (PhoA) were analyzed to determine the membrane topology of the two proteins. Protein fusions were obtained by progressively deleting the manY and manZ genes from their 3′ ends and ligating them to lacZ and ṕhoA that lack promotor and leader sequences. Based on the analysis of 30 IICMan‐PhoA, 10 IICMan‐LacZ, 12 IIDMan‐PhoA, and 30 IIDMan‐LacZ fusions, it is predicted that IICMan has six membrane‐spanning segments with the N‐ and C‐termini on the cytoplasmic face of the membrane. IIDMan is anchored in the membrane by a single membrane‐spanning segment at the end of the C‐terminus, while most of the protein (250 residues) protrudes into the cytoplasm.
Reversible, Non‐Denaturing Metal Substitution in Bovine Adrenodoxin and Spinach Ferredoxin and the Different Reactivities of [2Fe‐2S]‐Cluster‐Containing Proteins
Authors: Iametti Stefania , Uhlmann Heike , Sala Nicla , Bernhardt Rita , Ragg Enzio , Bonomi Francesco
Published: July 2004

The non‐denaturing substitution of cluster iron by other metals was studied in spinach ferredoxin and in bovine adrenodoxin. Only some of several metal species tested (Cd2+, Zn2+, VO2+, Mn2+, Co2+, Ni2+) caused bleaching of the residual visible absorbance and of the EPR signals of the reduced ferredoxins. No formation of mixed‐metal cluster was observed. The most reactive metal species were Cd2+ and Zn2+, and Cd2+ was found to react also with oxidized adrenodoxin. Metal‐treated proteins were resolved into a mixture of apoprotein, metal‐substituted protein and unreacted holoprotein. Their biological activity was proportional to the residual holoprotein concentration. Spinach ferredoxin and adrenodoxin were found to differ substantially with regard to their metal‐substitution reactivity under oxidizing and reducing conditions, reaction time, and formation of apoprotein, which was more pronounced for spinach ferredoxin. Exchange of cluster iron with Cd2+ in adrenodoxin generated stable species containing 2 mol sulfide/mol protein, and 2 or 5 mol cadmium/mol protein, respectively. The relative amount of the two substitution products depended on the experimental conditions. CD and NMR data on all the cadmium‐substituted proteins suggest that iron replacement led to a significant structural rearrangement. Nevertheless, all the metal‐substituted proteins could be re‐converted into the native iron‐containing form upon incubation with iron in the absence of reductants, of denaturing agents, and of an external source of sulfide. The different reactivity of the two proteins is discussed in terms of the cluster environment, along with the possible physiological relevance of these findings.