bluuebunny/crossref_metadata_2025 huggingface dataset:
Do ATP4− and Mg2+ bind stepwise to the F1‐ATPase of Halobacterium saccharovorum ?
Authors: Schobert Brigitte
Published: March 2003

It is commonly believed that MgATP2− is the substrate of F1‐ATPases and ATP4− acts as a competitive inhibitor. However, the velocity equation for such competitive inhibition is equivalent to that for a rapid equilibrium ordered binding mechanism in which ATP4− adds first and the binding of Mg2+ is dependent on the formation of the E ATP4− complex. According to this ordered‐binding model, solution formed MgATP2− is not recognized by the ATPase as a direct substrate, and the high‐affinity binding of Mg2+ to the E ATP4− complex is the key reaction towards the formation of the ternary complex. These models (and others) were tested with an F1‐ ATPase, isolated from Halobacterium saccharovorum, by evaluating the rate of ATP hydrolysis as a function of free [ATP4−] or free [Mg2+]. The rates were asymmetrical with respect to increasing [ATP4−] versus increasing [Mg2+]. For the ordered‐binding alternative, a series of apparent dissociation constants were obtained for ATP4− (.cf2.K.cf2..cf1..esapp.rb.eiA.rb), which decreased as [Mg2+] increased. From this family of .cf2.K.cf2..cf1..esapp.rb.eiA.rb the true KA was retrieved by extrapolation to [Mg2+] = 0 and was found to be 0.2 mM. The dissociation constants for Mg2+, established from these experiments, were also apparent (.cf2.K.cf2..cf1..esapp.rb.eiB.rb) and dependent on [ATP4−] as well as on the pH. The actual KB was established from a series of .cf2.K.cf2..cf1..esapp.rb.eiB.rb by extrapolating to [ATP4−] = ∞ and to the absence of competing protons, and was found to be 0.0041 mM. The pKa of the protonable group for Mg2+ binding is 8.2. For the competitive inhibition alternative, rearrangement of the constants and fitting to the velocity equation gave an actual binding constant for MgATP2− (KEAB) of 0.0016 mM and for ATP4− (KEA) of 0.2 mM. Decision between the two models has far‐reaching mechanistic implications. In the competitive inhibition model MgATP2− binds with high affinity, but Mg2+ cannot bind once the E ATP4− complex is formed, while in the ordered‐binding model binding of Mg2+ requires that ATP4− adds first. The steric constraints evident in the diffraction structure of the ATP binding site in the bovine mitochondrial F‐ATPase [Abrahams, J. P., Leslie, A. G. W., Lutter, R. & Walker, J. E. (1994) Nature 370, 621−628] tend to favor the ordered‐binding model, but the final decision as to which kinetic model is valid has to be from further structural studies. If the ordered‐binding model gains more experimental support, a revision of the current concepts of unisite catalysis and negative cooperativity of nucleotide binding will be necessary.
Mutagenesis of the fructose‐6‐phosphate−binding site in the 2‐kinase domain of 6‐phosphofructo‐2‐kinase/fructose‐2,6‐bisphosphatase
Authors: Bertrand Luc , Vertommen Didier , Freeman Phillip M. , Wouters Johan , Depiereux Eric , Di Pietro Attilio , Hue Louis , Rider Mark H.
Published: March 2003

Multiple alignment of several isozyme sequences of the bifunctional enzyme 6‐phosphofructo‐2‐kinase/fructose‐2,6‐bisphosphatase revealed conserved residues in the 2‐kinase domain. Among these residues, three asparagine residues (Asn76, Asn97 and Asn133; numbering refers to the liver isozyme sequence) and three threonine residues (Thr132, Thr134 and Thr135) are located near the fructose 6‐phosphate−binding site in the crystal structure of the bifunctional enzyme. The role of these residues in substrate binding and catalysis in the 6‐phosphofructo‐2‐kinase domain has been studied by mutagenesis to alanine. Since the crystal structure of 6‐phosphofructo‐2‐kinase does not contain fructose 6‐phosphate, this substrate was docked into the putative binding site by computer modelling, and its interactions with the protein were predicted. Analysis of the mutagenesis‐induced changes in kinetic properties and of the substrate‐docking model revealed that all these residues are directly or indirectly involved in fructose‐6‐phosphate binding. All the mutants displayed an increased Km for fructose 6‐phosphate (10−200‐fold). We propose that Asn133 stabilises Arg138, which itself makes a direct electrostatic bond with the 6‐phosphate group of fructose 6‐phosphate, that Asn76 interacts with the C3 hydroxyl group of fructose 6‐phosphate, that Thr132 makes a hydrogen bond with the C6 oxygen of this substrate, and that Thr134 interacts with two residues involved in fructose‐6‐phosphate binding, Thr132 and Tyr199. On the other hand, Asn97 and Thr135 play structural roles, by maintaining the structure of the fructose‐6‐phosphate‐binding pocket.
Negative cooperativity in the steady‐state kinetics of sugar oxidation by soluble quinoprotein glucose dehydrogenase from Acinetobacter calcoaceticus
Authors: Olsthoorn Arjen J. J. , Otsuki Tetsuo , Duine Johannis A.
Published: March 2003

Steady‐state‐kinetics investigations were carried out for the oxidation of aldose sugars by soluble quinoprotein glucose dehydrogenase (GDH) from Acinetobacter calcoaceticus using N‐methylphenazonium methyl sulfate (PMS) as artificial electron acceptor. As is not uncommon for a dye‐linked dehydrogenase, the enzyme showed ping‐pong behaviour and double‐substrate inhibition. However, under conditions that avoided its masking by sugar‐substrate inhibition as much as possible, negative kinetic cooperativity with respect to sugar substrate oxidation by this enzyme was demonstrated. Arguments are presented that exclude trivial factors as a cause for the phenomenon observed. Experimental data could be fitted with an equation accounting for biphasic cooperativity containing two sets of apparent kinetic parameters, V1 and K1, and V2 and K2, representing the enzyme's Michaelis‐Menten behaviour at low and high substrate concentrations, respectively. Assuming that subunit interaction causes the cooperativity effect, the sets express the performance of soluble GDH's two subunits in two states of mutual interaction. From fitting the experimental data for several sugars with this equation, it appeared that their V1 values were similar, although their K1 values varied considerably. This showed that the cooperativity effect dramatically changes the performance of soluble GDH, as reflected by the V2 and K2 values for glucose (in phosphate buffer) being about 10‐fold and 100‐fold higher than the V1 and K1 values, respectively. Substituting the Ca2+ involved in activation of pyrroloquinoline quinone (PQQ) in soluble GDH by Sr2+ affected the cooperativity effect (an increase of the K2 value) but not the two turnover rates of the hybrid enzyme for glucose. The data suggest that the two catalytic cycles of soluble GDH have different rate‐limiting steps compared with that of PQQ‐containing methanol dehydrogenase.
Binding characteristics of Ustilago maydis topoisomerase I to DNA containing secondary structures
Authors: Thiyagarajan Manimekalai M. , Waldman Scott A. , Noè Mariadele , Kmiec Eric B.
Published: March 2003

The binding affinity of purified native Ustilago maydis topoisomerase I enzyme for radiolabeled DNA substrates with various secondary structures was determined by gel shift and equilibrium binding analysis. Topoisomerase I exhibited cooperativity in binding to DNA regardless of the substrate structure. Further analysis demonstrated that cruciform DNA has two populations of binding sites for topoisomerase I while the other substrates (single‐stranded DNA, DNA molecules containing six or one mismatched base pairs, hairpin, and fully homologous duplex DNA) have a single population of binding sites. The affinity of topoisomerase I for cruciform was found to be an order of magnitude higher affinity than for any of the other substrates. The high affinity of topoisomerase I for cruciform and specificity of topoisomerase I‐cruciform structure interaction were confirmed by competition experiments. These studies demonstrate the high affinity of topoisomerase I for cruciform structure.
Flexibility of the nascent polypeptide chain within the ribosome
Authors: Choi Kyoung Moo , Atkins John F. , Gesteland Raymond F. , Brimacombe Richard
Published: March 2003

The ribosomal environment of the N‐terminus of the nascent polypeptide chain has been investigated using peptides of different lengths, synthesized in situ on Escherichia coli ribosomes; the peptides each carry a photoreactive diazirine moiety at their N‐terminus, so as to generate cross‐links to neighbouring ribosomal components. Our previous studies [Choi, K. M. & Brimacombe, R. (1998) Nucleic Acids Res. 26, 887−895] with three independent families of peptides, derived from the E. coli ompA protein gene, the tetracycline‐resistance gene and the bacteriophage T4 gene 60, identified a series of sites within the 23S rRNA to which the peptides became cross‐linked. The distribution of these cross‐links indicated that the nascent peptide is very flexible within the 50S subunit. Here, we demonstrate that the N‐termini of the ompA and gene‐60 peptides can, in addition, even become concomitantly cross‐linked to the 30S subunit. The cross‐linking is predominantly to 30S ribosomal proteins S1, S2, S4 and (to a lesser extent) S3, which form a cluster near to the decoding region. This result is discussed in terms of the flexibility of the nascent peptide during the co‐translational folding process, and in terms of the ’ribosomal bypass' phenomenon which is known to occur during translation of the gene 60 mRNA.
Stability of neutral trehalase during heat stress in Saccharomyces cerevisiae is dependent on the activity of the catalytic subunits of cAMP‐dependent protein kinase, Tpk1 and Tpk2
Authors: Zähringer Harald , Holzer Helmut , Nwaka Solomon
Published: March 2003

In Saccharomyces cerevisiae cAMP‐dependent protein kinase (cAPK) is involved in nutrient sensing and growth regulation via the Ras/cAMP pathway. Target enzymes, e.g. neutral trehalase, are activated or inactivated rapidly by cAPK‐mediated phosphorylation. In addition, stress‐induced transcription of genes of the general stress‐response, e.g. HSP12, is negatively regulated via cAPK.

We have investigated the effect of low cAPK activity on the stress‐induced expression of neutral trehalase Nth1p. For this purpose we used mutants (tpk1tpk2TPK3, tpk1TPK2tpk3 and TPK1tpk2tpk3) with double knockouts of the three TPK genes encoding catalytic subunits of cAPK. It is shown that the tpk1tpk2TPK3 mutant, which has very low cAPK activity, exhibits a heat‐stress‐induced inactivation of neutral trehalase that is not observed in tpk1TPK2tpk3, TPK11tpk2tpk3 mutants and wild‐type cells. However, heat stress induces an increase in NTH1 mRNA in the tpk1tpk2TPK3 mutant. Introduction of a plasmid carrying the TPK1 or TPK2 gene into tpk1tpk2TPK3 cells restores the heat‐induced increase of neutral trehalase activity. In vitro and in vivo results suggest that the heat induced inactivation of neutral trehalase is due to a reversible inactivation of Nth1p. Our data indicate that a certain level of phosphorylation is essential for maintenance of neutral trehalase activity during heat shock in S. cerevisiae. Two identical putative cAPK phosphorylation sites have been found in the sequence predicted for the Nth1p. Stabilization and activation of neutral trehalase may be regulated by these sites. Furthermore, our data suggest that the heat‐stress‐induced transcription of the NTH1 gene is not negatively regulated by cAPK, that the TPK genes have no effect on the glucose repression of the NTH1 gene, and that non‐detectable neutral trehalase activity in derepressed tpk1tpk2TPK3 cells is correlated with the reduced thermotolerance observed in this strain, similar to the heat‐shock‐recovery defect reported for the nth1Δ mutant.
Interaction of vanadate oligomers and permolybdate with the 90‐kDa heat‐shock protein, Hsp90
Authors: Sőti Csaba , Radics Lajos , Yahara Ichiro , Csermely Péter
Published: March 2003

The 90‐kDa heat‐shock protein (Hsp90) is a molecular chaperone that aids the folding of nuclear hormone receptors and protein kinases. Hsp90 protein complexes can be stabilized by molybdate and by other transition metal oxyanions such as vanadate. Our earlier findings [Csermely, P., Kajtár, J., Hollósi, M., Jalsovszky, G., Holly, S., Kahn, C. R., Gergely, P. Jr, So†ti, C., Mihály, K. & Somogyi, J. (1993) J. Biol. Chem. 268, 1901−1907] showed that vanadate and molybdate can induce a large conformational change of Hsp90. Here we provide direct evidence for the binding of vanadate and molybdate to Hsp90 by demonstrating that surface‐plasmon‐resonance measurements indicate binding of various vanadate oligomers to Hsp90, 51V‐NMR measurements show an extensive interaction of decavanadate with the chaperone, and permolybdate treatment of Hsp90 induces a marked mobility shift of the protein and its tryptic fragments. Our results indicate the flexibility of molybdate/vanadate‐binding sites of Hsp90, which are able to accomodate various species of these transition metal anions.
Isolation, cDNA cloning and gene expression of an antibacterial protein from larvae of the coconut rhinoceros beetle, Oryctes rhinoceros
Authors: Yang Jun , Yamamoto Masanori , Ishibashi Jun , Taniai Kiyoko , Yamakawa Minoru
Published: March 2003

An antibacterial protein, designated rhinocerosin, was purified to homogeneity from larvae of the coconut rhinoceros beetle, Oryctes rhinoceros immunized with Escherichia coli. Based on the amino acid sequence of the N‐terminal region, a degenerate primer was synthesized and reverse‐transcriptase PCR was performed to clone rhinocerosin cDNA. As a result, a 279‐bp fragment was obtained. The complete nucleotide sequence was determined by sequencing the extended rhinocerosin cDNA clone by 5′ rapid amplification of cDNA ends. The deduced amino acid sequence of the mature portion of rhinocerosin was composed of 72 amino acids without cystein residues and was shown to be rich in glycine (11.1 %) and proline (11.1 %) residues. Comparison of the deduced amino acid sequence of rhinocerosin with those of other antibacterial proteins indicated that it has 77.8 % and 44.6 % identity with holotricin 2 and coleoptrecin, respectively. Rhinocerosin had strong antibacterial activity against E. coli, Streptococcus pyogenes, Staphylococcus aureus but not against Pseudomonas aeruginosa. Results of reverse‐transcriptase PCR analysis of gene expression in different tissues indicated that the rhinocerosin gene is strongly expressed in the fat body and the Malpighian tubule, and weakly expressed in hemocytes and midgut. In addition, gene expression was inducible by bacteria in the fat body, the Malpighian tubule and hemocyte but constitutive expression was observed in the midgut.
Purification, characterization and complete amino acid sequence of nuclease Cl from Cunninghamella echinulata var. echinulata
Authors: Ho Heng‐Chien , Shiau Pyng‐Fang , Liu Fon‐Chang , Chung Jing‐Gung , Chen Ling‐Yun
Published: March 2003

It is known, from the zymogram method of nuclease activity assay, that the crude extracts of Cunninghamella echinulata var. echinulata contained at least three distinct extracellular nucleases. Among them, the major form was 30 kDa in molecular mass and termed nuclease C1. In this report, nuclease C1 was purified to apparent homogeneity by chromatography on Cibacron blue‐3GA, phenyl‐Sepharose 4B and HiTrap Heparin. Nuclease C1 acquired enzymatic activity in the presence of Mn2+ or Mg2+ and was inhibited by EDTA. The activity was maximal at pH 7−8.5. The primary structure of nuclease C1 was completely determined using enzymatic digestion and gene cloning. The N‐terminal 49 residues of nuclease C1 were first elucidated from a tryptic digest. Two degenerate upstream primers were subsequently designed to amplify the cDNA encoding nuclease C1. The resulting protein sequence of nuclease C1 was shown to be composed of 252 residues. It was intriguing to find that the protein sequence of nuclease C1 showed significant similarities with the sequences of the mitochondrial nucleases of Saccharomyces cerevisiae (44 % identity) and Schizosaccharomyces pombe (42 % identity). Residue His87 of nuclease C1 was postulated to be located at the active site from sequence similarity with secreted nuclease from Serratia marcescens.
Use of a photoactivatable lipid to probe the topology of PA63 of Bacillus anthracis in lipid membranes
Authors: Wang Xiao‐Ming , Wattiez Ruddy , Brossier Fabien , Mock Michèle , Falmagne Paul , Ruysschaert Jean‐Marie , Cabiaux Véronique
Published: March 2003

The protective antigen of Bacillus anthracis is a key protein that promotes the translocation of the enzymatic moieties of the two toxins of B. anthracis into the cell cytoplasm. The membrane topology of the active form of the protective antigen (PA63) was investigated by proteolysis of PA63 inserted into liposomes containing a photoactivatable, radioactive lipid, and characterization of the N‐terminal moiety of the deeply‐inserted (and therefore radiolabeled) peptides. A single sequence starting at residue Ala258 was identified. Fourier‐transform infrared spectroscopy showed that the protected peptide was mainly adopting a β‐sheet structure whose orientation was compatible with a transmembrane organization.
Adrenocorticotropic hormone stimulates CYP11B1 gene transcription through a mechanism involving AP‐1 factors
Authors: Mukai Kuniaki , Mitani Fumiko , Agake Reiko , Ishimura Yuzuru
Published: March 2003

To elucidate the mechanism(s) by which adrenocorticotropic hormone (corticotropin) stimulates transcription of the steroid 11β‐monooxygenase gene (CYP11B1) in adrenocortical cells, the 5′‐flanking region of rat CYP11B1 was analyzed using transient transfection and protein‐binding assays with mouse adrenocortical Y1 cells. The results indicated that both basal and corticotropin‐induced transcriptional activation of CYP11B1 required a common regulatory element containing a binding site for activator protein‐1 (AP‐1) transcription factors (dimers of the Jun and Fos family proteins) in the 5′‐flanking region. Other DNA‐binding protein(s) such as transcription factor Ad4BP was not required for either basal or corticotropin‐induced transcriptional activation. Corticotropin stimuli were found to induce expression of a subset of the jun and fos family gene products in Y1 cells significantly, while total amounts of AP‐1 factors capable of binding to its site in the CYP11B1 promoter did not change greatly. Treatment of rats with corticotropin had similar effects on mRNA levels of the jun and fos family genes in the adrenocortical zona fasciculata cells together with an enhancing effect on the level of CYP11B1 mRNA in the tissue. The effects of corticotropin on mRNA levels of the jun and fos family genes as well as transcription of CYP11B1 in Y1 cells were mimicked by treatment of the cells with dibutyryl cAMP. Furthermore, when components of AP‐1 factors were overexpressed by transfecting Y1 cells with their expression vectors, a paired expression of AP‐1 components such as c‐Jun and c‐Fos, which were inducible by corticotropin, transactivated the CYP11B1 promoter more strongly in the absence of corticotropin than other combinations such as JunD and Fra‐2 expressed constitutively. These results suggest that corticotropin regulates transcription of the CYP11B1 gene by causing compositional changes in AP‐1 transcription factors in the adrenocortical cells via a cAMP‐dependent pathway.
Solution structures of human immunodeficiency virus type 1 (HIV‐1) and moloney murine leukemia virus (MoMLV) capsid protein major‐homology‐region peptide analogs by NMR spectroscopy
Authors: Clish Clary B. , Peyton David H. , Barklis Eric
Published: March 2003

The capsid domain of retroviral Gag proteins possesses a single highly conserved subdomain termed the major homology region (MHR). While the mutagenesis of residues in the MHR will impair virus infectivity, the precise solution structure and function of the MHR is not known. To aid the structure/function characterization of the MHR, the structures of synthetic peptides encompassing the MHR of the human immunodeficiency virus type I (HIV‐1) and Moloney murine leukemia virus (MoMLV) capsid proteins were investigated by several techniques. Homology‐based secondary‐structure prediction suggested that the HIV‐1 and MoMLV peptides could form 50 % and 38 %α‐helix, respectively. CD studies indicated that, in the presence of 50 % trifluoroethanol, the HIV‐1 peptide adopts an α‐helical structure over half of its length, while the MoMLV peptide is over one third α‐helix. Further analysis by 1H‐NMR suggested that the C‐terminal portion of the MHR of each virus forms a helix in aqueous solution. Distance‐geometry structures of each peptide were calculated from NOE distance restraints and were refined by restrained molecular dynamics. The C‐terminal halves of both peptides were observed to be in an α‐helical conformation, while the N‐terminal halves were disordered. Furthermore, both helices were amphipathic with high conservation of amino acid side‐chain character, suggesting that a conserved helical MHR C‐terminus is essential to retroviral capsid protein function.
Quantitative structure/activity relationship for the rate of conversion of C4−substituted catechols by catechol‐1,2‐dioxygenase from Pseudomonas putida (arvilla) C1
Authors: Ridder Lars , Briganti Fabrizio , Boersma Marelle G. , Boeren Sjef , Vis Eric H. , Scozzafava Andrea , Veeger Cees , Rietjens Ivonne M. C. M.
Published: March 2003

The influence of various C4/C5 substituents in catechol (1,2‐dihydroxybenzene) derivatives on the overall rate of conversion by catechol‐1,2‐dioxygenase from Pseudomonas putida (arvilla) C1 was investigated. Using catechol, 4‐methylcatechol, 4‐fluorocatechol, 4‐chlorocatechol, 4‐bromocatechol, 4,5‐difluorocatechol and 4‐chloro‐5‐fluorocatechol, it could be demonstrated that substituents at the C4 and/or C5 position decrease the rate of conversion, from 62 % (4‐methylcatechol) down to 0.7 % (4‐chloro‐5‐fluorocatechol) of the activity with non‐substituted catechol. The inhibition was reversible upon addition of excess catechol for all substrates tested. This indicates that the lower activities are neither due to irreversible inactivation of the enzyme nor to product inhibition. Based on the reaction mechanism proposed in the literature [Que, L. & Ho, R. Y. N. (1996) Chem. Rev. 96, 2606−2624], the nucleophilic reactivity of the catecholate was expected to be an essential characteristic for its conversion by catechol‐1,2‐dioxygenase. Therefore, the rates of conversion were compared with calculated energies of the highest occupied molecular orbital (EHOMO) of the substrates. A clear quantitative relationship (R > 0.97) between the ln kcat and the calculated electronic parameter EHOMO was obtained. This indicates that the rate‐limiting step of the reaction cycle is dependent on the nucleophilic reactivity of the substrate and not sterically hindered by the relatively large bromine or methyl substituents used in the present study. Possible steps in the reaction mechanism determining the overall rate at 20 +C are discussed.
Purification and molecular cloning of porcine intestinal glycerol‐ester hydrolase
Authors: David Laetitia , Guo Xiao‐Jun , Villard Claude , Moulin André , Puigserver Antoine
Published: March 2003

A glycerol‐ester hydrolase was purified to homogeneity from porcine intestinal mucosa using a partial delipidation method and an eight‐step purification procedure. The isolation scheme used gave a 483‐fold purification, resulting in a pure enzyme with a specific activity on tributyrin of 290 μmol  min−1  mg−1. The molecular mass of the enzyme was estimated at 240 kDa, based on the results of size‐exclusion chromatography, and at 60 kDa, as determined by SDS/PAGE analysis. The isoelectric focusing data obtained indicated that only one isoform with a pI of 5.1 was present. Complete identity was found to exist between the N‐terminal sequence of the first 25 amino acid residues and that of a porcine liver carboxylesterase. A full‐length cDNA coding for the enzyme was isolated from pig small intestine. We observed that the corresponding protein originally named intestinal glycerol‐ester hydrolase definitely belongs to the carboxylesterase family. The deduced amino acid sequence consisted of 565 residues and showed 97 % identity with that of porcine liver carboxylesterase and more than 50 % identity with those of other carboxylesterases from different mammalian species.
Spectroscopic studies of the interaction of Ca2+‐ATPase‐peptides with dodecyl maltoside and its brominated analog
Authors: Soulié Stéphanie , de Foresta Béatrice , Møller Jesper V. , Bloomberg Graham B. , Groves Jonathan D. , le Maire Marc
Published: March 2003

The transmembrane sector of sarcoplasmic reticulum Ca2+‐ATPase comprises ten putative transmembrane spans (M1−M10) in current topology models. We report here the structure and properties of three synthetic peptides with a single Trp representing the M6 and M7 regions implicated in Ca2+ binding : peptide M6 (amino acid residues 785−810), peptide M7‐L (amino acid residues 808−847) corresponding to loop 6‐7 and the majority of span M7, and peptide M7‐S (amino acid residues 818−847) which contains a shorter version of loop 6‐7 than M7‐L. After uptake of the peptides in the hydrophobic environment of dodecyl maltoside micelles, the peptides gain a significant amount of secondary structure, as indicated by their CD spectra. However, the α‐helical content of M6 is lower than would be expected for a classical transmembrane segment. For M7‐L peptide, the L6‐7 loop is subject to specific proteolytic cleavage by proteinase K, as in intact Ca2+‐ATPase. The formation of the peptide‐detergent complexes was followed from the resulting fluorescence intensity changes, either enhancement using n‐dodecyl β‐
D‐maltoside or quenching using the recently introduced brominated analog of n‐dodecyl β‐
D‐maltoside : 7,8‐dibromododecyl β‐maltoside [de Foresta, B., Legros, N., Plusquellec, D., le Maire, M. & Champeil, P. (1996) Eur. J. Biochem. 241, 343−354]. Our results indicate that M7‐L and M7‐S are completely taken up by the detergent micelles. In contrast, the M6 peptide, which is highly water soluble, is more loosely associated with the detergent, as is also demonstrated by size‐exclusion chromatography. The location of Trp in micelles was evaluated from the quenching observed in mixed micelles of n‐dodecyl β‐
D‐maltoside/7,8‐dibromododecyl β‐maltoside, using tryptophan octyl ester and solubilized Ca2+‐ATPase as reference compounds. We conclude that W832 in M7 appears to be located near the surface of the micelle, in agreement with its membrane interfacial localization predicted in most Ca2+‐ATPase topology models. In contrast, our data suggest that W794 in M6 has a deeper insertion in the micelle although not to the extent predicted by current models of Ca2+‐ATPase and the rather short α‐helix span of M6 may lead to exposure of a significant part of the C‐terminal of this peptide to the micelle surface. The results are discussed in relation to the proposed roles of these membrane segments in active transport of Ca2+ ions, in particular, the demonstration that M6 does not behave as a classical transmembrane helix may be correlated with the evidence, from site‐directed mutagenesis, that this transmembrane segment should be essential in Ca2+ binding.
Crystal structure of subtilisin DY, a random mutant of subtilisin Carlsberg
Authors: Eschenburg Susanne , Genov Nicolay , Peters Klaus , Fittkau Siegfried , Stoeva Stanka , Wilson Keith S. , Betzel Christian
Published: March 2003

The crystal structure of subtilisin DY inhibited by N‐benzyloxycarbonyl‐Ala‐Pro‐Phe‐chloromethyl ketone has been solved by molecular replacement with subtilisin Carlsberg as the starting model. The model has been refined to a crystallographic R factor (= Σ∣∣Fo∣−∣Fc∣∣ / Σ∣Fo∣) of 15.1 % using X‐ray diffraction data to 0.175 nm resolution. Subtilisin DY is an alkaline proteinase from the X‐irradiated Japanese strain DY of Bacillus licheniformis, which normally produces subtilisin Carlsberg. It has very similar properties to subtilisin Carlsberg, with a slightly enhanced resistance to heat and guanidine hydrochloride‐induced denaturation, in spite of the fact that the sequences of the two enzymes differ in 31 positions out of 274 residues. The close similarity in overall three‐dimensional structure of subtilisins DY and Carlsberg and also their physicochemical properties, such as activity and stability, shows that nature aided by X‐irradiation for rapid ’evolution' is able to accommodate considerable changes in sequence without substantial changes in property.
Differential stimulation of proline‐rich tyrosine kinase 2 and mitogen‐activated protein kinase by sphingosine 1‐phosphate
Authors: Guo Chunmei , Zheng Chuanhai , Martin‐Padura Ines , Bian Z. Christine , Guan Jun‐Lin
Published: March 2003

Sphingosine 1‐phosphate (SphP), a metabolite of cellular sphingolipids, has been shown to induce cell proliferation by activating the mitogen‐activated protein kinase (MAPK) pathway. Proline‐rich tyrosine kinase 2 (Pyk2) is a novel cytosolic tyrosine kinase which mediates activation of the MAPK or c‐Jun N‐terminal kinase (JNK) signaling pathways in response to a variety of stimuli that elevate intracellular calcium. In this report, we show that SphP stimulates both tyrosine phosphorylation of Pyk2 and MAPK activation in a transient and dose‐dependent manner in rat aortic smooth muscle cells. Further studies indicate that Pyk2 phosphorylation, but not MAPK activation, is dependent on a pertussis toxin‐sensitive G‐protein‐coupled receptor as well as partially on actin cytoskeleton. In addition, both intracellular calcium mobilization and protein kinase C (PKC) are required for optimal Pyk2 phosphorylation while either calcium increase or PKC activation is sufficient for MAPK activation in response to SphP. Finally, we show that a tyrosine kinase(s) other than Pyk2 is necessary for MAPK activation by SphP. Together, these results suggest that SphP stimulates tyrosine phosphorylation of Pyk2 through a G‐protein coupled receptor, which is dissociated from its activation of the MAPK pathway in these cells.
Biochemical characterization of the SecA protein of Streptomyces lividans
Authors: Blanco Jorge , Driessen Arnold J. M. , Coque Juan José R. , Martin Juan F.
Published: March 2003

The SecA protein of Streptomyces lividans was purified to near electrophoretic homogeneity by means of FPLC from an overproducing strain harbouring plasmid pULA400, in which the secA gene (Blanco, J., Coque, J. J. R. & Martín, J. F. (1996) Gene (Amst.) 176, 61−65) was expressed from the strong promoter of the Streptomyces griseus saf gene. The native form of SecA was shown to be a dimer (Mr 209 kDa) by gel filtration. It crossreacted with antibodies raised against Escherichia coli or Bacillus subtilis SecA proteins. Purified S. lividans SecA showed a low endogenous ATPase activity that was stimulated by addition of a S. lividans lipid fraction. SecA contains a high‐affinity and a low‐affinity nucleotide‐binding site (NBS). [α‐32P]ATP could be crosslinked by ultraviolet radiation at the high‐affinity site. The intrinsic tryptophan fluorescence of SecA decreased on addition of increasing concentrations of ADP and reached a saturation level at about 1 μM (the range of saturation at the NBS I). The calculated Kd of the high‐affinity binding site for ADP was 150 nM. Millimolar concentrations of ATP or ADP did not render the S. lividans SecA protein resistant to V8 protease degradation, in contrast to what occurs with the E. coli and B. subtilis SecA proteins. SecA was found to bind to urea‐washed S. lividans membrane vesicles with high‐affinity, i.e. 10 nM. SecA‐dependent binding of E. coli SecB to membrane vesicles was observed when E. coli SecA was used, but not with the S. lividans SecA, suggesting that this interaction may be specific for the Gram‐negative bacteria. An in vitro translocation system has been developed using inverted membrane vesicles of S. lividans. SecA supported in vitro translocation of proAmy into S. lividans membrane vesicles in an ATP‐dependent manner.
Carbon dioxide fixation by reversible pyrrole‐2‐carboxylate decarboxylase from Bacillus megaterium PYR2910
Authors: Wieser Marco , Fujii Noriko , Yoshida Toyokazu , Nagasawa Toru
Published: March 2003

Pyrrole‐2‐carboxylate decarboxylase from Bacillusmegaterium PYR2910 attains a balanced reaction equilibrium with an equilibrium constant of 0.3−0.4 M. Therefore, the enzyme catalyzes the reverse carboxylation of pyrrole after addition of bicarbonate. For the synthesis of pyrrole‐2‐carboxylate, the reverse reaction was optimized and the equilibrium was shifted towards the carboxylate. The product yield was 230 mM (25.5 g/l) pyrrole‐2‐carboxylate from 300 mM pyrrole in a batch reaction and 325 mM (36.1 g/l) from 400 mM pyrrole in a fed‐batch reaction, using both whole cells and the purified enzyme in a pH 8.0 reaction mixture with bicarbonate saturation of 1.9 M. Kinetic studies indicated, that bicarbonate is the reactive species used by this carbon dioxide‐fixation enzyme.
Modulation of carcinoembryonic antigen release by glucosylceramide
Authors: Babia Teresa , Veldman Robert Jan , Hoekstra Dick , Kok Jan Willem
Published: March 2003

Previous work suggested that glucosylceramide (GlcCer) plays a role in the regulation of cell differentiation of HT29 human colon tumor cells [1]. In the present study, we investigated the role of GlcCer in the cellular release of carcinoembryonic antigen (CEA), a marker for cell differentiation. This was done by modulating the intracellular level of the glycolipid, according to two different approaches. The cells were treated with D,
L‐threo‐1‐phenyl‐2‐decanoylamino‐3‐morpholino‐1‐propanol (PDMP), which resulted in a specific lowering of the cellular GlcCer pool. Alternatively, by exogenous addition of a short‐chain analog of the lipid, hexanoyl(C6)‐GlcCer, the cellular pool was enhanced. The results demonstrate that PDMP causes an increase in the release of CEA, while exogenous C6‐GlcCer suppresses its release. Furthermore, the enhanced release of CEA in the presence of PDMP, could be completely reversed upon exogenous addition of C6‐GlcCer. Control experiments reveal that a potential interference of the well‐known modulator of cell physiology, ceramide (Cer), can be excluded. Long‐term depletion of GlcCer resulted in a change in a morphological feature of differentiation of the cells, i.e. an increase in apical membrane surface with microvilli brush borders, accompanied by an enhanced expression of the cytoskeletal protein villin. These results, together with the observations on modulation of the differentiation marker CEA by GlcCer, provide support for the conclusion that GlcCer interferes with the differentiation of HT29 cells.
Sialylated N‐glycans in adult rat brain tissue
Authors: Zamze Susanne , Harvey David J. , Chen Yi‐Ju , Guile Geoffrey R. , Dwek Raymond A. , Wing David R.
Published: March 2003

This paper extends our earlier work on the analysis of neutral N‐glycans from adult rat brain to glycans carrying NeuAc residues as their sole charged groups. These structures comprised at least 40 % of the total (acidic and neutral) N‐glycan pool. Compounds were identified by a combination of endoglycosidase and exoglycosidase digestions, anion‐exchange chromatography, normal and reverse‐phase high‐performance liquid chromatography, matrix‐assisted laser desorption/ionisation‐mass spectrometry and combined gas chromatography/mass spectrometry. Mono‐, di‐ and trisialylated components, together with components substituted with four (or more) NeuAc residues, showed abundances of approximately 12, 10, 7 and 7 %, respectively, relative to the total N‐glycan pool. In addition, neuraminidase digestion resulted in the neutralisation of a fraction of highly charged species, possibly indicating the presence of N‐glycans substituted with short chains of polysialic acid. Sialylated bi‐, tri‐ [mainly the (2,4)‐branched isomer], tetraantennary complex, polylactosamine and hybrid structures were detected. Typically, for ’brain‐type' N‐glycosylation, these sialylated structures were variously modified by the presence of core α1−6‐linked and outer‐arm α1−3‐linked fucose residues and by a bisecting GlcNAc. Structural groups such as sialyl Lewisx and NeuAcα2−3 substituted Galβ1−4GlcNAc antennae were common. In contrast to the neutral glycans, however, a widespread distribution of terminal β1−3‐linked galactose residues was observed. The presence of β1−3‐linked galactose allowed for a high degree of sialylation as afforded by the presence of the NeuAcα2−3Galβ1−3(NeuAcα2−6)GlcNAc structural group. This revealed a number of novel structures including the presence of tetraantennary N‐glycans with more than one β1−3galactose residue and (2,4)‐branched triantennary oligosaccharides containing three such residues. Disialylated hybrid glycans containing β1−3‐linked galactose and ’polylactosamine' N‐glycans with one to three terminal β1−3galactose residues were additional novel features. The N‐glycans modified by polysialylation lacked outer‐arm fucose and bisecting GlcNAc residues but all contained one or more terminal β1−3‐linked galactose residues. These may be representative, therefore, of the polysialylated N‐glycans expressed mainly on neural cell‐adhesion molecules and known to be present in adult rat brain. The diversity of presentation of terminal sialylated groups in rat brain implies potential specificity for possible charge or lectin‐mediated interactions. The distinguishing sets of sialylated structures described here are indicative of differences in the natural glycosylation processing pathways in different cell types within the central nervous system, a specificity that may be further magnified on the individual glycoproteins.
Inhibition of Streptomyces griseus aminopeptidase and effects of calcium ions on catalysis and binding
Authors: Papir Galia , Spungin‐Bialik Anya , Ben‐Meir Daniella , Fudim Ella , Gilboa Rotem , Greenblatt Harry M. , Shoham Gil , Lessel Uta , Schomburg Dietmar , Ashkenazi Ruth , Blumberg Shmaryahu
Published: March 2003

Streptomyces griseus aminopeptidase is a zinc metalloenzyme containing 2 mol zinc/mol protein, similar to the homologous enzyme Aeromonas proteolytica aminopeptidase. In addition, a unique Ca2+‐binding site has been identified in the Streptomyces enzyme, which is absent in the Aeromonas enzyme. Binding of Ca2+ enhances stability of the Streptomyces enzyme and modulates its activity and affinity towards substrates and inhibitors in a structure‐dependent manner. Among the three hydrophobic 4‐nitroanilides of alanine, valine and leucine, the latter displays the largest overall activation (increase in kcat/Km). Large enhancements in affinity (1/Ki) upon Ca2+ binding have been observed for inhibitors with flexible (leucine‐like) residues at their N‐termini and smaller enhancements for inhibitors with rigid (phenylalanine‐like) residues.
The amino acid sequence of the ram spermatidal protein 3
Authors: Chevaillier Philippe , Chirat Frédéric , Sautiere Pierre
Published: March 2003

As in other mammals, several nuclear basic proteins replace histones during the differentiation of germinal cells into spermatozoa in the ram. These proteins called transition proteins (TP) are later replaced by protamines. The amino acid sequence of the ram spermatidal protein 3 has been established by Edman degradation of the protein and of its fragments generated from digestion with endoproteinase Lys‐C and pepsin and from the coding sequence of the gene and of the cDNA. The ram protein 3 is a basic protein of 109 residues (calculated Mr 13 200) with arginine and lysine residues uniformly distributed along the polypeptide chain. Of the 13 serine and threonine residues, 9 are located in structural motifs where they could be phosphorylated and, thus, modulate the binding of the protein to DNA. The tyrosine residues at position 33 and position 93, located in a basic environment, and the tryptophan residue at position 29 could be involved in the interactions of the protein with DNA through the stacking of their aromatic ring between the nucleotide bases. The ram protein 3 differs completely from the two well‐defined transition protein families TP1 and TP2, which are also synthesised transiently during mammal spermiogenesis. In contrast with the rat TP3, ram protein 3 does not correspond to a precursor of a protamine. However, it shares structural similarities with both transition proteins TP3 and TP4 of the boar. The ram protein 3 and the boar transition proteins TP3 and TP4 probably belong to the same transition‐protein group and would play similar functions in the chromatin remodelling during spermiogenesis. As protamine and transition‐protein TP1 and TP2 genes from mammals, the coding sequence of the gene of ram protein 3 is interrupted by one intron but its organisation is different.
Analysis of the clumping‐mediating domain(s) of sex pheromone plasmid pAD1‐encoded aggregation substance
Authors: Muscholl‐Silberhorn Albrecht
Published: March 2003

Aggregation substance, a cell surface adhesin encoded on sex‐pheromone plasmids exclusively found in the opportunistic pathogen Enterococcus faecalis, displays a dual function in that it mediates (a) adhesion to host tissues and (b) aggregation of E. faecalis cells to result in efficient plasmid transfer. While there have been many investigations regarding the regulation of inducible plasmid transfer and involvement in pathogenicity, nearly nothing is known about the structural basis of clumping capacity intrinsic to aggregation substance. Here, data are presented regarding the respective domain(s) of Asa1 (the adhesin encoded on plasmid pAD1) by analyzing the effects of in‐frame deletions on clumping and by measuring binding of E. faecalis cells to surface‐bound subfragments of aggregation substance. These data indicate that the N‐terminal part of the adhesin is responsible for clumping, with a region between amino acid 525 and amino acid 617 playing a dominant role. Furthermore, by raising antibodies against different fragments of Asa1, it is shown that aggregation substance contains cross‐reacting epitopes in its C‐terminal and N‐terminal parts, which could not be derived from their primary sequences.
Characterization of a calcium‐dependent calmodulin‐binding domain in the 135‐kD human protein 4.1 isoform
Authors: Leclerc Estelle , Vetter Stefan
Published: March 2003

The putative calmodulin binding domain of non‐erythroid protein 4.1, previously suggested by Kelly et al. [Kelly, G. M., Zelus, B. D. & Moon, R. T. (1991) J. Biol. Chem. 266, 12 469−12 473] has been synthesized, and its binding to calmodulin has been studied by fluorescence spectroscopy. For this purpose, the peptide has been N‐terminally dansylated. The 4.1 peptide Dns‐Abu‐S76RGLSRLFSSFLKRPKS92, binds calmodulin in a calcium‐dependent way with high affinity (Kd = 23 ± 6 nM). The peptide inhibits bovine‐heart phosphodiesterase with an IC50 of 50 nM. Since the sequence of the peptide shows two putative consensus sites of phosphorylation by cAMP‐dependent protein kinase or Ca2+−calmodulin protein‐kinase II, the interaction of the two mono‐phosphorylated peptides (P4.1 Ser80‐P and P4.1 Ser84‐P) and the di‐phosphorylated peptide (P4.1 Ser80‐P/Ser84‐P) with calmodulin has been investigated. A decrease of affinity by a factor 1.5−8 has been observed for the phosphorylated peptides. CD measurements have shown an increase of the content of α helices in the peptides when bound to calmodulin.
Purification and properties of the chloroplastic form of biotin holocarboxylase synthetase from Arabidopsis thaliana overexpressed in Escherichia coli
Authors: Tissot Ghislaine , Pepin Régis , Job Dominique , Douce Roland , Alban Claude
Published: March 2003

Holocarboxylase synthetases (HCSs) are key enzymes in biotin utilisation in both prokaryotes and eukaryotes. In a previous study, we demonstrated that, in plants, HCS activity is localised in cytosol, chloroplasts and mitochondria. We also described the cloning and sequencing of a full‐length cDNA encoding an Arabidopsis thaliana HCS isoform with a putative organelle‐transit peptide. In the study reported here, this cDNA was used to construct an overproducing Escherichia coli strain. The recombinant enzyme was isolated using an efficient three‐step purification procedure. Polyclonal antibodies raised against pure HCS were produced to elucidate the subcellular localisation of this protein. Immunodetection carried out by Western blotting of isolated pea leaf subcellular compartments specifically revealed a single polypeptide that was ascribed to the chloroplast compartment. Immunocytochemistry of thin‐cut sections from tobacco leaves, transformed by the complete coding sequence of A. thaliana HCS cDNA via Agrobacterium tumefaciens, confirmed that the enzyme encoded by this cDNA is the chloroplastic isoform. Moreover, physicochemical, biochemical and kinetic properties of the pure recombinant HCS were determined. The native recombinant enzyme is a 37‐kDa monomer. In contrast to the major part of HCS activity measured in leaf extracts, the recombinant chloroplastic enzyme did not require addition of Mg2+ to be fully active, but was substantially inhibited by EDTA. This suggested that the chloroplastic HCS may contain a tightly‐bound divalent cation required for enzyme activity. The recombinant enzyme was able to biotinylate efficiently apo‐biotin carboxyl carrier protein (BCCP) from E. coli and apo‐methylcrotonoyl‐CoA carboxylase (MCCase) from A. thaliana. Apparent Km values for the enzyme substrates
D‐biotin, ATP and apo‐MCCase were found to be 130 nM, 4.4 μM and 32 μM, respectively. Steady‐state kinetic analyses of the HCS‐catalysed reaction were investigated with respect to reaction mechanism and inhibition by AMP, one of the end‐products of the enzyme‐catalysed reaction. Substrate interaction and product inhibition patterns indicated that ATP and
D‐biotin bind sequentially, in an ordered manner, to the enzyme and that ATP or
D‐biotin and apo‐BCCP bind in ping‐pong fashion.
Kinetic properties of a single nucleotide binding site on chloroplast coupling factor 1 (CF1)
Authors: Günther Stephan , Huchzermeyer Bernhard
Published: March 2003

The kinetics of nucleotide binding to spinach chloroplast coupling factor CF1 in a fully inhibited state were investigated by stopped‐flow experiments using the fluorescent trinitrophenyl analogue (NO2)3Ph‐ADP. The CF1 was in a state in which two of the three binding sites on the β subunits were irreversibly blocked with ADP, Mg2+ and fluoroaluminate, while the three binding sites on the α subunits were occupied by nucleotides [Garin, J., Vincon, M., Gagnon, J. & Vignais, P. V. (1994) Biochemistry 33, 3772−3777)]. Thus, it was possible to characterise a single nucleotide‐binding site without superimposed nucleotide exchange or binding to an additional site. (NO2)3Ph‐ADP binding to the remaining site on the third β subunit was characterised by a high dissociation rate of 15 s−1, leading to a very low affinity (dissociation constant higher than 150 μM). Subsequent to isolation, CF1 preparations contained two endogenously bound nucleotides. Pre‐loading with ATP yielded CF1 with five tightly bound nucleotides and one free nucleotide‐binding site on a β subunit. Pre‐loading with ADP, however, resulted in a CF1 preparation containing four tightly bound nucleotides and two free nucleotide binding sites. One of the two free binding sites was located on a β subunit, while the other was probably located on an α subunit.