bluuebunny/crossref_metadata_2025 huggingface dataset:
Influence of polyols on the structural properties of Kluyveromyces lactisβ‐galactosidase under high hydrostatic pressure
Authors: Athès Violaine , Lange Reinhard , Combes Didier
Published: March 2003

The conformational changes in dimeric Kluyveromyces lactisβ‐galactosidase induced by hydrostatic pressure were investigated by means of its intrinsic tryptophan fluorescence. At high pressure, the fluorescence emission spectrum was shifted to the red, indicating the exposure of buried Trp residues to the aqueous solvent. This spectral change was paralleled by a loss of enzyme activity. The shift of the emission spectrum was quantified by evaluating the centre of spectral mass (〈νg〉), which is an intensity‐weighted mean wavenumber. The experimental data could be fitted to a two‐state transition (native  denatured), corrected for a linear pressure dependence of 〈νg〉, and allowed the determination of thermodynamic parameters Δ.cf2.G.es0.rb.eiapp.rb, Vapp and P1/2. The results were consistent with a partial unfolding of the protein and not simply with dissociation of this dimeric enzyme. In the presence of polyols, the native conformation of β‐galactosidase was considerably more resistant to pressure. This protective effect of polyols is probably due to a reduced accessibility of water inside the protein structure, through the direct or indirect action of these additives on the enzyme.
Interactions between serine acetyltransferase and O‐acetylserine (thiol) lyase in higher plants
Authors: Droux Michel , Ruffet Marie‐Line , Douce Roland , Job Dominique
Published: March 2003

The last steps of cysteine synthesis in plants involve two consecutive enzymes. The first enzyme, serine acetyltransferase, catalyses the acetylation of L‐serine in the presence of acetyl‐CoA to form O‐acetylserine. The second enzyme, O‐acetylserine (thiol) lyase, converts O‐acetylserine to
L‐cysteine in the presence of sulfide. We have, in the present work, over‐produced in Escherichia coli harboring various type of plasmids, either a plant serine acetyltransferase or this enzyme with a plant O‐acetylserine (thiol) lyase. The free recombinant serine acetyltransferase (subunit mass of 34 kDa) exhibited a high propensity to form high‐molecular‐mass aggregates and was found to be highly unstable in solution. However, these aggregates were prevented in the presence of O‐acetylserine (thiol) lyase (subunit mass of 36 kDa). Under these conditions homotetrameric serine acetyltransferase associated with two molecules of homodimeric O‐acetylserine (thiol) lyase to form a bienzyme complex (molecular mass ≈300 kDa) called cysteine synthase containing 4 mol pyridoxal 5′‐phosphate/mol complex. O‐Acetylserine triggered the dissociation of the bienzyme complex, whereas sulfide counteracted the action of O‐acetylserine. Protein−protein interactions within the bienzyme complex strongly modified the kinetic properties of plant serine acetyltransferase : there was a transition from a typical Michaelis‐Menten model to a model displaying positive kinetic co‐operativity with respect to serine and acetyl‐CoA. On the other hand, the formation of the bienzyme complex resulted in a very dramatic decrease in the catalytic efficiency of bound O‐acetylserine (thiol) lyase. The latter enzyme behaved as if it were a structural and/or regulatory subunit of serine acetyltransferase. Our results also indicated that bound serine acetyltransferase produces a build‐up of O‐acetylserine along the reaction path and that the full capacity for cysteine synthesis can only be achieved in the presence of a large excess of free O‐acetylserine (thiol) lyase. These findings contradict the widely held belief that such a bienzyme complex is required to channel the metabolite intermediate O‐acetylserine.
Dissection of drug‐binding‐induced conformational changes in P‐glycoprotein
Authors: Wang Guichun , Pincheira Roxana , Zhang Jian‐Ting
Published: March 2003

P‐glycoprotein (Pgp) is a membrane‐transport ATPase. It uses energy from ATP hydrolysis to transport pleiotropic cytotoxic drugs from inside to outside of cells. Thus, elevated expression of Pgp in cancer cells causes multidrug resistance. It is now known that the conformational state of Pgp changes during its catalytic cycle. However, how ATP hydrolysis relates to drug binding by Pgp is yet to be determined. In this study, we used limited trypsin digestion of Pgp in isolated inside‐out membrane vesicles to investigate the effects of drugs on Pgp conformation and to determine the drug‐bound conformational states of Pgp in the catalytic cycle. We found that (a) binding of vinblastine or verapamil alone can cause a conformational change in Pgp, but the change induced by the drug binding is different from that induced by nucleotide binding, (b) there may be at least two binding sites for Pgp substrates, one for drugs such as vinblastine and verapamil and the other for drugs such as colchicine and adriamycin, (c) the conformation of Pgp bound by ATP and vinblastine is different from the conformation bound by either one alone, and (d) the ADP‐bound Pgp does not bind vinblastine. Based on these observations and our previous studies, we propose a model for drug binding and transport in the catalytic cycle of Pgp.
Modulation of calmodulin function by ubiquitin−calmodulin ligase and identification of the responsible ubiquitylation site in vertebrate calmodulin
Authors: Laub Markus , Steppuhn Johannes A. , Blüggel Martin , Immler Dorian , Meyer Helmut E. , Jennissen Herbert P.
Published: March 2003

Calmodulin is the universal calcium modulator in eukaryotic cells. Its biological activity is closely regulated by the second messenger Ca2+. Previous studies in cell‐free extracts [Laub, M. & Jennissen, H. P. (1997) Biochim. Biophys. Acta 1357, 173−191] have shown that calmodulin is reversibly ubiquitylated by ubiquityl−calmodulin synthetase (ubiquitin−calmodulin ligase,
EC6.3.2.21) in the presence of Ca2+ without being channeled to degradation by the 26S proteasome. As shown here monoubiquitylation strongly decreases the biological activity of calmodulin towards phosphorylase kinase by reducing its affinity approximately threefold and the maximal degree of activation approximately twofold. Thus, a structural clarification of the ubiquitylation site on calmodulin has become crucial for advancing our knowledge in this field on a molecular level. As demonstrated by sequence analysis and mass spectrometry of conjugates, the ubiquitylation site is located in the first Ca2+‐binding loop of calmodulin and has the octapeptide structure ‐L‐F‐D‐K21‐D‐G‐D‐G‐ with Lys21 being the ubiquitylated residue in vertebrate and other calmodulins. This catalytic recognition sequence is, however, not the only structural requirement for calmodulin ubiquitylation by ubiquityl−calmodulin synthetase. Removal of the 41 C‐terminal amino acids (fourth Ca2+‐binding loop) separated by several nanometers from Lys21 drastically decreases the affinity and reactivity of the synthetase for calmodulin, indicating a more extensive structural requirement for the substrate binding site i.e. binding recognition. This allows the enzyme to discriminate in a site‐specific manner between two nearly identical catalytic recognition sites in vertebrate calmodulin of which the second site ‐V‐F‐D‐K94‐D‐G‐N‐G‐ in the third Ca2+‐binding loop is apparently not ubiquitylated by the synthetase.
Cleavage of Escherichia coli tryptophan indole‐lyase by trypsin at Lys406 affects the transmission of conformational changes associated with monovalent cation activation
Authors: Phillips Robert S. , Doshi Kokila J.
Published: March 2003

Escherichia coli tryptophan indole‐lyase (Trpase) is a pyridoxal 5′‐phosphate(pyridoxal‐P)‐dependent enzyme which catalyzes the hydrolytic cleavage of
L‐tryptophan to indole and ammonium pyruvate. This enzyme is strongly activated by K+ and similar monovalent cations, and the spectrum of the pyridoxal‐P cofactor is also affected by pH and cations. Treatment of Trpase with trypsin results in a 20−100‐fold decrease in elimination activity, depending on the substrate, concomitant with a change in the relative amounts of the 337 nm and 420 nm forms of the bound pyridoxal‐P, and a shift in the λmax from 420 nm to 423 nm. In addition, the pH sensitivity of the pyridoxal‐P cofactor is eliminated after trypsin treatment. Nicked Trpase exhibits only fourfold activation by K+, compared with about 50‐fold for native enzyme, but the KA for K+ is unaffected. Both the native and trypsin‐nicked Trpase react with amino acids to form equilibrating mixtures of external aldimine and quinonoid intermediates in rapid‐scanning stopped‐flow experiments. However, the rate constant for quinonoid intermediate formation from
L‐tryptophan is reduced by at least 400‐fold by treatment with trypsin. In contrast, the rate constant for formation of quinonoid intermediates of
L‐alanine and S‐ethyl‐
L‐cysteine is affected only twofold or less by trypsin treatment. The site of trypsin cleavage was identified by electrospray‐ionization mass spectrometry as Lys406, which is predicted to lie on a flexible surface loop. Some active‐site residues, particularly Arg419, which is predicted by sequence similarity to be the substrate α‐carboxylate‐binding site, and His463, are located in the sequence between Lys406 and the C‐terminus. Hence, cleavage of the peptide bond of E. coli Trpase at Lys406 probably affects the change from active to inactive conformations that normally takes place in the presence of activating monovalent cations.
The binding sites for Xenopus laevis FIII/YY1 in the first exon of L1 and L14 ribosomal protein genes are dispensable for promoter expression
Authors: De Rinaldis Emanuele , Pisaneschi Giuseppe , Camacho‐Vanegas Olga , Beccari Elena
Published: March 2003

The genes coding for the ribosomal proteins (rp genes) L14 and L1 in the toad Xenopus laevis are contacted in the first exon by the frog protein, FIII/YY1, homolog of the human zinc‐finger protein YY1, acting as repressor, activator and initiator of transcription. To investigate the functional significance of FIII/YY1 in the context of the two rp genes, the L14 region at nucleotide positions −105 to +44, including all of the first exon was linked to the chloramphenicol acetyltransferase (CAT) reporter gene; constructs with wild‐type and mutated sites for FIII/YY1 were injected into nuclei of stage V−VI oocytes and analyzed for CAT activity. The same procedure was followed for constructs made with L1 sequences at nucleotide positions −17 to +1567. Mutations in the sites for FIII/YY1 did not change reporter activity, nor did overexpression of FIII/YY1 in the oocytes prior to injection with L1 and L14 constructs. Since oocytes are non‐dividing cells, transfections were made of Xenopus kidney cells in culture with the same constructs and the results obtained in oocytes confirmed.
Effect of molybdate and tungstate on the biosynthesis of CO dehydrogenase and the molybdopterin cytosine‐dinucleotide‐type of molybdenum cofactor in Hydrogenophaga pseudoflava
Authors: Hänzelmann Petra , Meyer Ortwin
Published: March 2003

The molybdenum‐containing iron‐sulfur flavoprotein CO dehydrogenase is expressed in a catalytically fully competent form during heterotrophic growth of the aerobic bacterium Hydrogenophaga pseudoflava with pyruvate plus CO. We have adopted these conditions for studying the effect of molybdate (Mo) and tungstate (W) on the biosynthesis of CO dehydrogenase and its molybdopterin (MPT) cytosine‐dinucleotide‐(MCD)‐type molybdenum cofactor. W was taken up by the Mo transport system and, therefore, interfered with Mo transport in an antagonistic way. Depletion of Mo from the growth medium as well as inclusion of excess W both resulted in the absence of intracellular Mo and led to the biosynthesis of CO dehydrogenase species of proper L2M2S2 subunit structure that carried the two 2Fe : 2S type‐I and type‐II centers and two FAD molecules. EPR, ultraviolet/visible and CD spectroscopies established the full functionality of the cofactors. Due to the absence of the Mo‐MCD cofactor, the enzyme species were catalytically inactive. Unexpectedly, the following cytidine nucleotides were present in inactive CO dehydrogenase : CDP, dCDP, CMP, dCMP, CTP or dCTP. The sum of cytidine nucleotides was two/mol enzyme. The binding specificities of inactive CO dehydrogenase for cytidine nucleotides (oxy > deoxy; diphosphate > monophosphate > triphosphate), and the absence of MPT suggest that, in active CO dehydrogenase, the cytidine diphosphate moiety of Mo‐MCD provides the strongest interactions with the protein and determines the specificity for the type of nucleotide. In H. pseudoflava, the biosynthesis of MPT (identified as form A) was independent of Mo. Mo was, however, strictly required for the conversion of MPT to MCD (identified as form‐A−CMP) as well as the insertion of Mo‐MCD into CO dehydrogenase. These data support a model for the involvement of Mo in the biosynthesis of the Mo‐MCD cofactor and of fully functional CO dehydrogenase in which the synthesis and insertion of Mo‐MCD require Mo, and protein synthesis including integration of the FeS‐centers and FAD are independent of Mo.
Modular evolution of the Glx‐tRNA synthetase family
Authors: Siatecka Miroslawa , Rozek Marek , Barciszewski Jan , Mirande Marc
Published: March 2003

The accuracy of protein biosynthesis generally rests on a family of 20 aminoacyl‐tRNA synthetases, one for each amino acid. In bacteria, archaea and eukaryotic organelles, the formation of Gln‐tRNAGln is prevalently accomplished by a transamidation pathway, aminoacylation of tRNAGln with Glu by glutamyl‐tRNA synthetase (GluRS) followed by a tRNA‐dependent transamidation of Glu from Glu‐tRNAGln. A few bacterial species, such as Escherichia coli, possess a glutaminyl‐tRNA synthetase (GlnRS), responsible for Gln‐tRNAGln formation. Phylogenetic analysis of the GluRS or GlnRS families (GlxRS) suggested that GlnRS has a eukaryotic origin and was horizontally transferred to a restricted set of bacteria. We have now isolated an additional GlnRS gene from the plant Lupinus luteus and analyzed in more details the modular architecture of the paralogous enzymes GluRS and GlnRS, starting from a large data set of 33 GlxRS sequences. Our analysis suggests that the ancestral GluRS‐like enzyme was solely composed of the catalytic domain bearing the class‐defining motifs of aminoacyl‐tRNA synthetases, and that the anticodon‐binding domain of GlxRSs was independently acquired in the bacteria and archaea branches of the universal tree of life, the eukarya sub‐branch arising as a sister group of archaea. The transient capture of UAA and UAG codons could have favored the emergence of a GlnRS in early eukaryotes.
Interactions of non‐detergent sulfobetaines with early folding intermediates facilitate in vitro protein renaturation
Authors: Vuillard Laurent , Rabilloud Thierry , Goldberg Michel E.
Published: March 2003

Non‐detergent sulfobetaines (NDSB) are a family of solubilizing and stabilizing agents for proteins. In a previous study [Goldberg, M. E., Expert‐Bezancon, N., Vuillard, L. & Rabilloud, T. (1996) Folding & Design 1, 21−27] we showed that the amount of active protein recovered in in vitro folding experiments could be significantly increased by some NDSBS. In this work we investigated the mechanisms by which these molecules facilitate protein renaturation. Stopped‐flow and manual‐mixing fluorescence and enzyme activity measurements were used to compare the kinetics of protein folding in the presence and absence of N‐phenyl‐methyl‐N,N‐dimethylammonium‐propane‐sulfonate (NDSB 256). Hen lysozyme and the β2 subunit of Escherichia coli tryptophan synthase were chosen as model systems since their folding pathways had been previously investigated in detail. It is shown that, massive aggregation of tryptophan synthase occurs within less than 2.5 s after dilution in the renaturation buffer, but can be prevented by NDSB 256; only very early folding phases (such as the formation of a loosely packed hydrophobic core able to bind 8‐anilino‐1‐naphthalenesulphonic acid, and the initial burying of tryptophan 177) are significantly altered by NDSB256; none of the later phases is affected. Furthermore, NDSB 256 did not significantly affect any of the kinetic phases observed during the refolding of denatured lysozyme retaining intact disulphide bonds. This shows that NDSB 256 only interferes with very early steps in the folding process and acts by limiting the abortive interactions that could lead to the formation of inactive aggregates.
A regulatory factor, Fil1p, involved in derepression of the isocitrate lyase gene in Saccharomyces cerevisiae
Authors: Kanai Tamotsu , Takeshita Shigeru , Atomi Haruyuki , Umemura Ken , Ueda Mitsuyoshi , Tanaka Atsuo
Published: March 2003

A mutant was isolated that failed to derepress the 5′ upstream region of Candida tropicalis isocitrate lyase gene (UPR‐ICL)‐mediated gene expression in acetate medium, and the gene (FIL1) that complemented this mutation was isolated. The fil1 null mutant in which FIL1 is disrupted (Δfil1 strain) could not grow on acetate or ethanol, and the derepression of the isocitrate lyase encoded by ICL1 in Saccharomyces cerevisiae was also defected. The amino acid sequence of Fil1p (230 amino acids) showed similarity to ribosome recycling factors (RRFs) of prokaryotes. Compared to prokaryotic RRFs, Fil1p had an N‐terminal 46‐amino‐acid extension which was shown to be able to function as a mitochondrial‐targeting sequence. The subcellular fractionation of the Δfil1 strain showed that protein constituents of the mitochondrial fraction of the Δfil1 strain differed from those of the wild‐type strain, but resembled those of chloramphenicol‐treated cells or ρ+ cells. The specific activity of cytochrome c oxidase, was severely decreased in Δfil1, ρ+ and chloramphenicol‐treated cells compared with wild‐type cells, while enzymatic levels of mitochondrial NAD+‐linked isocitrate dehydrogenase, which is encoded by nuclear DNA, were not affected. These results suggest that Fil1p is necessary for protein synthesis in mitochondria of S. cerevisiae. Furthermore, cells treated with antimycin A, along with chloramphenicol‐treated, ρ+, and Δfil1 cells, showed deficiency in derepression of isocitrate lyase. Northern‐blot analysis showed that this can be ascribed to no increase in transcription of ICL1 and FBP1 encoding fructose 1,6‐bisphosphatase. The results indicate the presence of a communication pathway between mitochondria and the nucleus which represses expression of genes encoding the key enzymes of the glyoxylate cycle and gluconeogenic pathway when there is a deficiency in the mitochondrial respiratory chain.
Changing transcription start sites in H‐type α(1,2)fucosyltransferase Gene (FUT1) during differentiation of the human erythroid lineage
Authors: Koda Yoshiro , Soejima Mikiko , Kimura Hiroshi
Published: March 2003

Recent studies have suggested that at least three transcription‐initiation sites were present in the human H‐type α(1,2)fucosyltransferase gene (FUT1). In the present study, we have investigated these transcription start sites of FUT1 in undifferentiated leukemic cells (K562) that have erythroid characteristics, in erythroleukemia cells (HEL), and in bone marrow cells. K562 cells used exclusively exon 1 as the start site. While HEL cells used mainly exon 2 as the start site, the major start site for bone marrow cells was within exon 7. In addition, we investigated the transcription start site(s) in vascular endothelial cells (ECV304) as an example of mature cells and found that the start site was predominantly within exon 7. The promoter activities were found in the 5′ flanking regions of these three start sites after transfection of constructs with luciferase reporter gene into K562 and HEL cells. These findings suggested that the transcription start sites of FUT1 changed during differentiation of the erythroid lineage and that the tissue‐specific and stage‐specific expressions of the FUT1 were regulated by three distinct promoters. We also found that the 5′ flanking region of exon 2 (intron 1) consisted of repetitive sequences (chromosome 19‐specific 37‐bp minisatellite repeats, Alu sequence and long terminal repeat) and that the start site of exon 2 was within the long terminal repeat. Thus, these repetitive sequences may play a role in the expression of the FUT1.
Biochemical characterization and crystal structure of a recombinant hen avidin and its acidic mutant expressed in Escherichia coli
Authors: Nardone Errica , Rosano Camillo , Santambrogio Paolo , Curnis Flavio , Corti Angelo , Magni Fulvio , Siccardi Antonio G. , Paganelli Giovanni , Losso Romeo , Apreda Biancamaria , Bolognesi Martino , Sidoli Alessandro , Arosio Paolo
Published: March 2003

The mature hen avidin encoded by a synthetic cDNA was expressed in Escherichia coli in an insoluble form. After resolubilization, renaturation and purification, a recovery of about 20 mg/l cell culture was obtained. ELISA assays indicated no apparent differences in biotin binding between the natural and recombinant avidins. In addition, an acidic avidin mutant, bearing the substitutions Lys3←Glu, Lys9←Glu, Arg26←Asp and Arg124←Leu of four exposed basic residues, was produced. The protein, expressed and renatured as wild‐type avidin, showed unaltered biotin‐binding activity. The acidic pI (≈5.5) and lack of aggregation of the mutant allowed easy electrophoretic analysis under non‐denaturing conditions of the protein alone and of its complexes with biotin, biotinylated transferrin or peroxidase. Analysis of the sera from sensitized subjects revealed that the avidin mutant has altered antigenicity. Both recombinant avidins were crystallized and the three‐dimensional structures solved by molecular replacement and refined to 0.22 nm resolution. The three‐dimensional structures of the two recombinant molecules, in the absence of biotin and of glycosylation, are fully comparable with those of the natural hen avidin previously reported.
Phosphorylation‐specific antibodies for human cardiac troponin‐I
Authors: Al‐Hillawi Eman , Chilton Derrick , Trayer Ian P. , Cummins Peter
Published: March 2003

A panel of seven monoclonal antibodies (mAbs) raised against cardiac troponin‐I (CdTnI) isolated from canine and human hearts, which have been shown to be cardiac‐specific but cross‐species reactive [Cummins, B., Aukland, M. L. & Cummins, P. (1987) Amer. Heart J. 113, 1333−1344], were used in this study. These mAbs were tested against recombinant wild‐type and mutant human CdTnI proteins to assess their value as probes for the phosphorylation status of CdTnI. Four mAbs were found to react positively with the recombinant wild‐type protein and their epitopes were contained in residues 31−210 of the human cardiac protein. Two of these mAbs appeared to be directed against the same epitope site within this region. The remaining three mAbs only reacted against the recombinant wild‐type protein when it was phosphorylated, showing that these three antibodies were directed against the phosphate group(s) on Ser23 and/or Ser24. In order to investigate this further, a series of single and double mutants of CdTnI were used in which either Ala (to direct the enzymatic phosphorylation) or Asp (to mimic the phosphate group) replaced the Ser23 and/or Ser24. It was found the all three mAbs were able to react with the mono‐phosphorylated form of the [Ala23]CdTnI single mutant but not the mono‐phosphorylated form of the [Ala24]CdTnI single mutant, showing that they specifically required phosphorylation at Ser24. Experiments with a synthetic peptide composed of residues 1−29 of human CdTnI confirmed these data. Two of the three phosphorylation‐specific mAbs were able to react with mutants containing either two Asp residues replacing Ser23 and Ser24 or one Asp residue instead of Ser24, indicating that a negative charge at position Ser24 is sufficient to invoke a reaction. The other mAb was more specific in that it would only react with CdTnI species with a phosphate group on Ser24.
Uracil DNA glycosylase from Mycobacterium smegmatis and its distinct biochemical properties
Authors: Purnapatre Kedar , Varshney Umesh
Published: March 2003

Deamination of cytosine residues contributes to the appearance of uracil in DNA. Uracil DNA glycosylase (UDG) initiates uracil excision repair to safeguard the genomic integrity. To study the mechanism of uracil excision in mycobacteria (organisms with G+C rich genomes), we have purified UDG from Mycobacterium smegmatis by more than 3000‐fold. The molecular mass of M. smegmatis UDG, as determined by SDS/PAGE, is ≈25 kDa and it shows maximum activity at pH 8.0. The N‐terminal sequence analysis shows that the initiating amino acid, formyl‐methionine is cleaved from the mature protein. More interestingly, unlike Escherichia coli UDG, which forms a physiologically irreversible complex with the inhibitor protein Ugi, M. smegmatis UDG forms a dissociable complex with it. M. smegmatis UDG excises uracil from the 5′‐terminal position of the 5′‐phosphorylated substrates. However, its excision from the 3′‐penultimate position is extremely poor. Similar to E. coli UDG, M. smegmatis UDG also uses pd(UN)p as its minimal substrate. However, in contrast to E. coli UDG, which excises uracil from different loop positions of tetraloop hairpin substrates with highly variable efficiencies, M. smegmatis UDG excises the same uracil residues with comparable efficiencies. Kinetic parameters (Km and Vmax) for uracil release from synthetic substrates suggest that M. smegmatis UDG is an efficient enzyme and better suited for molecular biology applications. We discuss the usefulness of the distinct biochemical properties of M. smegmatis UDG in the possible design of selective inhibitors against it.
The propeptide of prohormone convertase PC2 acts as a transferable aggregation and membrane‐association signal
Authors: Jan Gwénaël , Taylor Neil A. , Scougall Kathleen T. , Docherty Kevin , Shennan Kathleen I. J.
Published: March 2003

Prohormone convertase 2 (PC2) is a subtilisin‐like protease involved in the intracellular processing of prohormones and proneuropeptides. Like its substrates, it is synthesised as a prepropeptide which undergoes proteolysis during transit through the regulated secretory pathway. Previous studies have shown that aggregation and membrane association of proPC2 occurs in a calcium‐dependent and pH‐dependent manner and that the pro‐region of PC2 may be involved in this process. These events may be involved in the sorting of proteins to the regulated secretory pathway. To investigate this further, we made a chimeric protein containing both the signal peptide and pro‐region of PC2 and the N‐terminal part of α1‐antitrypsin, called pro2α1. PC2, α1‐antitrypsin and pro2α1 were compared with regard to their membrane association and aggregation properties using, respectively, sucrose gradient centrifugation after expression in Xenopus oocytes, and an in vitro aggregation assay. The chimeric protein, pro2α1, underwent low‐pH‐dependent aggregation and membrane association similar to wild‐type PC2. Membrane association occurred at pH 5.5 in the absence of calcium and at pH 6.0 in the presence of 10 mM calcium but not at pH 6.5 or 7.0. α1‐antitrypsin, as expected of a constitutively secreted protein, did not aggregate at low pH, nor associate with membranes. Pro2α1 thus exhibits the membrane association and aggregation properties of PC2, confirming the role of the pro‐region in these processes.

A series of deletions were performed within the 84‐residue propeptide in order to define the sequences involved. Deletion of amino acids 52−77 reduced aggregation but large deletions in the pro‐region had only a minimal effect on membrane association. These data suggest that several regions within the pro‐peptide are important in these events.
Structure and alternative splicing of the ketohexokinase gene
Authors: Hayward Bruce E. , Bonthron David T.
Published: March 2003

Ketohexokinase (fructokinase, KHK) catalyses the phosphorylation of fructose to fructose‐1‐phosphate. It thereby initiates the intracellular catabolism of a large proportion of dietary carbohydrate. Although found at high level in liver, renal cortex and small intestine, fructokinase activity has also been known for many years to be present at lower levels in most other tissues. We previously found that there appeared to be two isoforms of human KHK, and have now investigated the molecular basis for this in human, rat and mouse. Cloning of the human KHK gene, on chromosome 2p23.2‐2p23.3, shows that it has nine exons, spanning 14 kb. An intragenic duplication has resulted in two similar 135‐bp exons (designated 3a and 3c), separated by a short intron. Exon 3a and exon 3c are mutually exclusively spliced into KHK mRNA. This exon−intron structure and the pattern of alternative splicing are conserved in both the rat and mouse, suggesting distinct conserved functions for the two KHK isoforms. The alternative splicing is also tissue specific, since in both rat and human, tissues expressing high levels of KHK (liver, kidney and duodenum) utilise exclusively the 3c exon, while other tissues use only 3a. Furthermore, comparison of human foetal and adult tissues indicates a developmental splicing shift from use of exon 3a to exon 3c.
Characterization of brain PCTAIRE‐1 kinase immunoreactivity and its interactions with p11 and 14‐3‐3 proteins
Authors: Le Bouffant Françoise , Capdevielle Joël , Guillemot Jean‐Claude , Sladeczek Fritz
Published: March 2003

An antibody directed against the C‐terminal part of PCTAIRE‐1 recognized three proteins in rodent brain. The high‐molecular‐mass band is most abundant in the cerebellum, hippocampus and cortex. It migrated at the same apparent molecular mass as recombinant PCTAIRE‐1 and interacted, like recombinant PCTAIRE‐1, with p11 and 14‐3‐3 proteins. Combination of p11 or 14‐3‐3 affinity resins with immunoprecipitation and peptide elution allowed us to obtain a purified full‐length PCTAIRE‐1 preparation having significant kinase activity. These results suggest that PCTAIRE‐1 is an active kinase in brain. The catalytic core region of PCTAIRE‐1 which is common for all cyclin‐dependent kinases, does not interact with p11 and 14‐3‐3 proteins in the two‐hybrid assay. Full interaction with p11 and 14‐3‐3 proteins requires both, the N‐terminal and C‐terminal ends of PCTAIRE‐1, suggesting that complex three‐dimensional arrangements are responsible for these interactions. A low‐molecular‐mass protein (migrating at about 30 kDa) that was also recognized by the antibody directed against the carboxy‐terminal part of PCTAIRE‐1, is abundant and almost homogeneously distributed in all brain areas investigated. Database searches starting with the amino acid sequences of two peptides obtained by tryptic digestion of this protein yielded cDNA and genomic (a gene of about 10 kb on human chromosome 1q24‐1q25 and clone 262D12) sequences, allowing us to compose a DNA sequence coding for a putative 26 kDa protein containing both peptides. This protein has no important sequence similarity with any other known protein. But many DNA sequences are found in databases with an almost 100 % identity with parts of the 26 kDa protein coding sequence. Our results allow us to attribute these widely distributed cDNA sequences to an existing 26‐kDa protein and to localize a gene within two recently published genomic sequences.
Purification and characterization of a flavoprotein involved in the degradation of epoxyalkanes by Xanthobacter Py2
Authors: Westphal Adrie H. , Swaving Jelto , Jacobs Linda , de Kok Aart
Published: March 2003

Recently a newly discovered pyridine nucleotide‐disulfide oxidoreductase was reported to be essential for the degradation of epoxyalkanes by the Xanthobacter Py2 [Swaving, J., De Bont, J. A. M., Westphal, A. & De Kok, A. (1996) J. Bacteriol. 178, 6644−6646]. The disulfide oxidoreductase has now been purified from propene‐grown Xanthobacter Py2. This enzyme (component II) is a NADPH‐dependent FAD‐containing homodimeric protein. The physiological substrate for this enzyme is unknown. The enzyme was active with the following dithiol substrates in decreasing order : 1,3‐propanedithiol, reduced lipoamide and dithiothreitol, and inactive with glutathione and monothiols. In the reversed direction, only activity with 5,5′‐dithiobis(2‐nitrobenzoate) could be measured. Compared with other disulfide reductases it has a high activity with 5,5′‐dithiobis(2‐nitrobenzoate) and a low diaphorase and oxidase activity. Steady‐state kinetic studies at pH 8.5 with 1,3‐propanedithiol show that the enzyme operates by a ternary complex mechanism in the direction of NADP+ reduction. Anaerobic incubation of the enzyme with 1,3‐propanedithiol resulted in slow reduction of the enzyme to yield the thiolate−FAD charge‐transfer complex, the rate depending on the pH. At pH 7, where reduction was not detectable within 2 h, rapid mixing of NADP+ with the enzyme‐propanedithiol mixture resulted in the formation of a complex between the reduced enzyme and NADP+ within the dead time of the instrument (5.6 ms). This is followed by slow formation of NADPH, concomitant with the appearance of the flavin C(4a)‐thiol adduct, as judged from the spectral changes. This suggests that the rate‐limiting step is the transfer of a hydride ion from the half‐reduced enzyme to NADP+. Stopped‐flow experiments involving reduction by NADPH show a biphasic behavior. The rapid formation (kobs = 40 s−1) of a transient intermediate with little absorption decrease at 460 nm and long wavelength absorption was followed by the slow formation (kobs = 4 s−1) of a species characterized as the thiolate−FAD charge‐transfer complex with bound NADP+. Some formation of the FAD C(4a)‐thiol adduct was also observed. Photoreduction in the presence of deazaflavin results in rapid bleaching at 450 nm, followed by the slow formation of a stable semiquinone. Full reduction could not be achieved, either by photoreduction or with NADPH, and was incomplete even with dithionite or NADPH in the presence of arsenite. The results indicate a low redox potential of the FAD and a slow rate of electron transfer from the pyridine nucleotide to the redox active disulfide and vice versa. From a sequence alignment with other disulfide reductases, it appears that the active site His−Glu diad is absent in this enzyme. The kinetic and spectral features described above will be discussed in this context.
Localization of phospholipase C δ3 in the cell and regulation of its activity by phospholipids and calcium
Authors: Pawelczyk Tadeusz , Matecki Andrzej
Published: March 2003

The localization of phospholipase C δ3 (PLC δ3) in the cell and its regulatory properties has been investigated. Western blotting showed that human platelet PLC δ3 is located in the membrane and cytosolic fraction. The enzyme amount in the cytosolic fraction was significantly lower than that in the membrane fraction. In rat liver, PLC δ3 was present in both the membrane and cytosolic fraction and was absent in nuclei. Examination of the effects of phospholipids on PLC δ3 revealed that this enzyme is inhibited by phosphatidylethanolamine (PtdEtn) and phosphatidylcholine (PtdCho). Similar inhibition was observed in the presence of sphingomyelin and phosphatidylserine (PtdSer). This is in contrast to PLC δ1, which is activated by PtdCho and PtdEtn. In a detergent assay, PLC δ1 is activated by spermine and sphingosine, whereas PLC δ3 was inhibited by both these compounds at concentrations that maximally stimulated PLC δ1. A deletion mutant of PLC δ3, lacking the entire pleckstrin homology (PH) domain (residues 1−137), was fully active in the detergent assay, and it was inhibited by spermine, sphingosine and phospholipids to the same extent as the native enzyme. PLC δ3 activation required calcium ions. The relationship between the Ca2+ concentration and enzymatic activity was almost identical for the deletion mutant and the native enzyme. However, in the liposome assay, PLC δ3 was less sensitive to Ca2+ stimulation. This is in contrast to PLC δ1, which is equally sensitive to Ca2+ stimulation in both the detergent and liposome assays. We conclude that Ca2+ is necessary to induce specific conformational changes of PLC δ3, which leads to a productive orientation of the catalytic domain relative to the membrane. The regulatory properties of PLC δ3 described in this report suggest that PLC δ3 has a relatively low activity in cellular conditions that fully activate PLC δ1.
Conformation analysis of eel calcitonin
Authors: Ogawa Kiyoshi , Nishimura Shigenori , Uchiyama Susumu , Kobayashi Kaoru , Kyogoku Yoshimasa , Hayashi Mitsuo , Kobayashi Yuji
Published: March 2003

The solution structure of eel calcitonin in a mixture of 60 % water and 40 % trifluoroethanol has been determined in this study by the combined use of 1H‐NMR spectroscopy and distance geometry calculations. 1H‐NMR spectroscopy provided 181 distance constraints, 5 dihedral angle constraints and 14 hydrogen bond constraints. The observed NOEs demonstrated the presence of an amphiphilic α‐helix in position Leu4−Gln20. The seven best converged structures exhibit backbone atomic rmsd of 0.027 nm for the backbone atoms from the averaged coordinate position in the region of Cys1−Leu19. In the previous study [Ogawa, K., Nishimura, S., Doi, M., Kyogoku, Y., Hayashi, M. & Kobayashi, Y. (1994) Eur. J. Biochem. 222, 659−666], the conformation of elcatonin, an analogue of eel calcitonin, was characterized by an amphiphilic α‐helix between Thr6 and Thr21 and a turn structure in the first five residues of the N‐terminus. The major difference of structure between eel calcitonin and elcatonin exists within the cyclic moiety at the helical N‐terminus. Some of the turn structure detected at the N‐terminus in elcatonin is not found in eel calcitonin. This is attributed to the difference in the ring formation caused by the disulfide bridge and ethylene bridge. A medium‐range NOE, dαN(i,i+2), is detected between the CαH of Arg24 and the NH of Asp26, so a turn structure occurs in this segment. This NOE connectivity profile in the C‐terminal region is also the same in elcatonin, suggesting that this is important for receptor binding and immunological properties.
Structure‐specific binding recognition of a methanogen chromosomal protein
Authors: Paradinas Carine , Gervais Alain , Maurizot Jean‐Claude , Culard Françoise
Published: March 2003

The archaeon Methanosarcina thermophila expresses large amounts of a small basic protein, called MC1 (methanogen chromosomal protein), which was previously identified as a DNA‐binding protein possibly involved in DNA compaction in some methanogenic species. We have investigated the binding of MC1 to various kinds of branched DNA molecules whose double helix axis is severely kinked. We show that MC1 is able to distinguish and to bind preferentially to four‐way junctions. This preferential binding is observed in the absence and presence of divalent cations. However, we find that MC1 has a low affinity for bulged DNA structures. These results show how MC1 is able to discriminate between different deformations of the DNA double helix.
Solution conformation of the Pseudomonas syringae MSU 16H phytotoxic lipodepsipeptide Pseudomycin A determined by computer simulations using distance geometry and molecular dynamics from NMR data
Authors: Coiro Vincenza Maria , Segre Anna Laura , Di Nola Alfredo , Paci Maurizio , Grottesi Alessandro , Veglia Gianluigi , Ballio Alessandro
Published: March 2003

Pseudomycin A is a cyclic lipodepsinonapeptide phytotoxin produced by a strain of the plant pathogenic bacterium Pseudomonas syringae. Like other members of this family of bacterial metabolites, it is characterised by a fatty acylated cyclic peptide with mixed chirality and lactonic closure. Several biological activities of Pseudomycin A are lower than those found for some of its congeners, a difference which might depend on the diverse number and distribution of charged residues in the peptide moiety. Hence, it was of interest to investigate its conformation in solution. After the complete interpretation of the two‐dimensional NMR spectra, NOE data were obtained and the structure was determined by computer simulations, applying distance geometry and molecular dynamics procedures. The conformation of the large ring of Pseudomycin A in solution includes three rigid structural regions interrupted by three short flexible regions that act as hinges. The overall three‐dimensional structure of the cyclic moiety is similar to that of previously studied bioactive lipodepsinonapeptides produced by other pseudomonads.
EPR and CD spectroscopy of fast myosin light chain conformation during binding of trifluoperazine
Authors: Huang William , Wilson Gregory J. , Brown Louise J. , Lam Harold , Hambly Brett D.
Published: March 2003

The conformations of isolated rabbit fast myosin light chains (LCs) were modified using trifluoperazine (TFP), the hydrophobic calmodulin inhibitor. CD spectroscopy showed that TFP altered secondary structural content of the LCs, with half‐maximal effects at TFP concentrations of ≈14−50 μM, which is within the range required to alter muscle fiber contraction in both agonistic and antagonistic ways [Kurebayashi, N. & Ogawa, Y. (1988) J. Physiol. 403, 407−424]. EPR spectroscopy provided structural information from paramagnetic probes on C‐terminal domain surfaces. In the absence of TFP, τR (rotational correlation time) was 1.6 ns for both alkali light chains (ALCs) and 1.8 ns for light chain 2 (LC2). This was faster than expected for proteins of this size (≈10 ns). TFP progressively recruited the probes into populations with τR sevenfold to 12‐fold slower, with half‐maximal effects at a TFP concentration of ≈370−800 μM. The differences probably indicate that CD spectroscopy detects changes in protein conformation due to ’specific' TFP binding at the LC hydrophobic core, while less specific binding at higher TFP concentrations is required to effect conformational changes on the protein surfaces near the paramagnetic probes. TFP binding was generally not cooperative. Comparative sequence analysis between calmodulin, troponin C, and myosin LCs indicated considerable conservation between residues expected to bind TFP.
Mutational analysis of the conserved cationic residues of Bacillus stearothermophilus 6‐phosphoglucose isomerase
Authors: Meng Menghsiao , Chen Yen‐Ting , Hsiao Yi‐Yuong , Itoh Yoshifumi , Bagdasarian Michael
Published: March 2003

The importance in catalysis of the conserved arginine (R207) and lysine residues (K144, K294, K356, and K425) of 6‐phosphoglucose isomerase from Bacillus stearothermophilus was assessed by site‐directed mutagenesis and kinetic analysis. In general mutations had minor effects on the Km for fructose 6‐phosphate. More dramatic effects were seen on kcat. The R207A mutant had a five orders of magnitude decrease in kcat relative to the wild‐type enzyme. There was a significant recovery, by three orders of magnitude, in the kcat for the R207K mutant. The results suggest that the positive charge provided by R207 plays a critical role in the isomerization reaction. K425 was substituted with alanine, valine, phenylalanine, tryptophan and aspartate. All mutant enzymes at position 425 had kcat decreased in the range of several‐hundred‐fold. For the other mutants, K294A and K144A, the kcat values were 3.5 % and 27 % of the wild‐type enzyme, respectively. No effects on catalysis were observed for the K356A mutant. The results suggest that R207, K144, K294, and K425 are located in the active site of the enzyme. The active‐site location and the catalytic roles of K425 and K294 are supported further by the inhibitory effects of pyridoxal 5′‐phosphate on enzymatic activities. The data also confirm the importance of K425 and K144 anticipated by the affinity labeling studies of the corresponding residues by pyridoxal 5′‐phosphate in pig muscle phosphoglucose isomerase.
Antisense oligonucleotide targeting the transforming growth factor β1 increases expression of specific genes and functions of Leydig cells
Authors: Le Roy Christine , Leduque Patrick , Yuan Li Jacques , Saez José Maria , Langlois Dominique
Published: March 2003

Transforming growth factor β1 (TGFβ1) has been reported to be a potent inhibitor of differentiated functions of many steroidogenic cells. Porcine Leydig cells (LC), as well as Sertoli cells (SC), express TGFβ1 mRNA and secrete this peptide, suggesting that it might play an autocrine role. Moreover, many studies have suggested a possible paracrine regulation of LC by SC‐secreted factors. To assess whether TGFβ1 plays an autocrine/paracrine role on these steroidogenic cells, we attempted to inhibit TGFβ1 protein synthesis by transfecting LC, SC and LC+SC for 24 h with 10 μM of an unmodified antisense oligonucleotide (AON) complementary to the translation‐initiation region of the TGFβ1 mRNA and, as controls, with the corresponding sense (SON) or scrambled (SCRON) oligonucleotides. First, we determined at which level, transcriptional or translational, the TGFβ1 AON acts. Neither TGFβ1 AON, SON nor SCRON modified TGFβ1 mRNA levels in LC, SC or LC+SC. However, TGFβ1 AON caused the disappearance of TGFβ1 immunoreactivity in both cell types. In addition, TGFβ1 AON reduced the attachment of TGFβ1 mRNA in ribosomal and polyribosomal fractions. Then, we showed that the decrease of the TGFβ1 protein induced by the AON results in an increase of the expression of LC specific genes and of LC steroidogenic capacity. In LC and LC+SC, TGFβ1 AON increased the mRNA levels of both LH/hCG receptor (1.9‐fold and 3.5‐fold, respectively) and P450 c17 (5‐fold and 8‐fold, respectively). This was associated with an enhancement of hCG‐induced testosterone production by both LC and LC+SC (1.6‐fold and 2.2‐fold, respectively) when compared with untransfected cells. The TGFβ1 AON effects were always more pronounced on LC+SC than on LC. The present findings show that TGFβ1 has an autocrine/paracrine inhibitory effect on cultured porcine Leydig cells, an effect that can be overcome by TGFβ1 AON.
Priority Paper The metabolic pathway of visual pigment chromophore formation in Drosophila melanogaster
Authors: Seki Takaharu , Isono Kunio , Ozaki Kaoru , Tsukahara Yasuo , Shibata‐Katsuta Yuko , Ito Masayoshi , Irie Toshiaki , Katagiri Masanao
Published: March 2003

Carotenoid‐depleted fruit flies, Drosophila melanogaster, were reared on yeast/glucose medium containing lipid‐depleted white corn grits and cholesterol. After rearing for more than a year, the yield of flies remained constant and the content of 3‐hydroxyretinal in a head was three logarithmic units less than that of normal flies reared on medium containing yellow corn grits. When all‐trans retinal was supplied as the sole source of retinoids, the flies formed and accumulated all‐trans 3‐hydroxyretinal in the dark. To examine the metabolic pathway to produce (3S)‐3‐hydroxyretinal in Drosophila, all‐trans retinal was supplemented for two hours to carotenoid‐depleted flies in the dark, and the subsequent changes in the composition of 3‐hydroxyretinal enantiomers were analyzed using a chiral column on HPLC. The results indicated initial formation of (3R)‐3‐hydroxyretinal followed by isomerization into the 3S enantiomer. In another set of experiments, the membrane fraction was obtained from the head homogenate of retinoid‐depleted flies and an in vitro assay of 3‐hydroxyretinal formation from retinal was performed. The 3‐hydroxyretinal produced was the 3R enantiomer, supporting the result obtained from the in vivo experiment whereby (3S)‐3‐hydroxyretinal is produced from retinal via (3R)‐3‐hydroxyretinal. Addition of NADPH enhanced 3‐hydroxyretinal formation and the presence of carbon monoxide inhibited it, suggesting that hydroxylation at the C3 position of retinal occurred via the monooxygenase activity of cytochrome P‐450.