bluuebunny/crossref_metadata_2025 huggingface dataset:
Analysis of inhibitor binding to the mitochondrial cytochrome c reductase by fluorescence quench titration
Authors: BRANDT Ulrich , von JAGOW Gebhard
Published: March 2005

The binding characteristics of inhibitors of the mitochondrial cytochrome c reductase were studied by fluorescence quench titration. Based on the standard binding equation, the applied numerical method allowed the online recorded titration curves to be interpreted by fitting the Kd, the number of binding sites, and the specific fluorescence of the free and the bound inhibitor. For the Qi, center, 2‐n‐nonyl‐4‐hydroxyquinoline N‐oxide and for the Qo center (E)‐β‐methoxyacrylate‐stilbene (MOA‐stilbene) were used as fluorescing inhibitors. The experiments could be extended to other, non‐fluorescing inhibitors by competition analysis. Using this method we were able to compare the binding behaviour of Qi and Qo center inhibitors under different redox states of the enzyme using the same experimental set up.

We studied the competition between inhibitors of the cytochrome c reductase representative for all subgroups and demonstrated that at least three inhibitor binding sites exist, two located in the Qo center, one located in the Qi center.

Determination of the dissociation constants of the oxidized, the partially reduced and the fully reduced enzyme showed that inhibitor binding at the Qi center is not redox‐dependent. In contrast, the binding of MOA‐stilbene to the Qo center is decreased after reduction of the iron‐sulfur center and cytochrome c1 whereas this redox change increases the affinity for a Qo center inhibitor of the hydroxynaphthoquinone type, 3‐n‐undecyl‐2‐hydroxy‐naphthoquinone.

From these results, aware of the fact that the inhibitory mechanism at the Qo center is a non‐competitive one, we made the hypothesis of a ‘catalytic switch’ to explain both the bifurcation of electron flow and the inhibition at the Qo center. A steric blockage of one of two conformational states could serve as a cogent explanation for the great structural variability of the inhibitors and differential effects on the redox centers exerted by the inhibitors. Moreover, the proposed ‘switch’ gives some insight into other experimental results which are difficult to explain with the ubiquinone cycle as currently formulated.
Construction and characterization of a functional chimeric murine – human antibody directed against human fibrin fragment‐D dimer
Authors: BULENS Frank , VANDAMME Anne‐Mieke , BERNAR Hilde , NELLES Luc , LIJNEN Roger H. , COLLEN Désiré
Published: March 2005

Fibrin‐directed monoclonal antibodies may be clinically useful for in vivo thrombus imaging and for the targeting of fibrinolytic agents to blood clots. One such murine monoclonal antibody, (mAb‐15C5), raised against the fragment‐D dimer epitope of cross‐linked human fibrin, was previously characterized [Holvoet, P., Stassen, J. M., Hashimoto, Y., Spriggs, D., Devos, P. & Collen, D. (1989) Thromb. Haemostasis 61, 307–313] has recently been cloned and expressed [Vandamme, A.‐M., Bulens, F., Bernar, H., Nelles, L., Lijnen, H. R. & Collen, D. (1990) Eur. J. Biochem. 192, 767–775]. In order to reduce the immunogenicity of the murine mAb‐15C5 in man, we have now constructed a murine – human chimera of mAb‐15C5, by substituting the cDNA sequences encoding the constant regions of the murine K light chain and γ1 heavy chain by the corresponding human genomic sequences. Both chimeric murine – human Ig chains were cloned into two separately selectable expression vectors, which were cotransfected into Chinese hamster ovary (CHO) cells. Murine – human chimeric mAb‐15C5 (mAb‐15C5Hu) was purified from the conditioned medium of selected cell lines by chromatography on Zn‐chelating Sepharose, protein‐A – Sepharose and on insolubilized antigen (fragment‐D dimer), with a final yield of 29 μg/l and a recovery of 33%. SDS/PAGE without reduction revealed a homogeneous band with a mobility similar to that of natural mAb‐15C5, whereas after reduction, both the heavy and the light chains had slightly slower mobilities than their natural counterparts. Expression in the presence of tunicamycin suggested that the differences in 71‐chain mobility were due to different N‐glycosylation patterns. Immunoblotting of proteins from SDS gels showed immunological reactivity of recombinant mAb‐15C5Hu with goat anti‐(human IgG) IgG and of recombinant and natural murine mAb‐15C5 with goat anti‐(mouse IgG) IgG. Competitive binding revealed a comparable affinity of recombinant murine mAb‐15C5, recombinant mAb‐15C5Hu and natural mAb‐15C5, for fragment‐D dimer, indicating that recombinant mAb‐15C5Hu was obtained in a functionally intact form. Thus, mAb‐15C5Hu may constitute a useful alternative to mAb‐15C5 for in vivo use in man.
Submicromolar manganese dependence of Golgi vesicular galactosyltransferase (lactose synthetase)
Authors: KUHN Nicholas J. , WARD Simon , LEONG Weng S.
Published: March 2005

Manganese(II) buffers were set up with inorganic triphosphate, trimethylenediaminetetraacetate and tetramethylenediaminetetraacetate to study the Mn dependence of β, 4‐galactosyltransferase (lactose synthetase) in preparations of rat mammary gland.

In intact particulate preparations, treated with the calcium ionophore A23187, lactose synthesis was abolished by chelators and restored by bivalent transition metal ions in a manner characteristic of activation site I of the pure enzyme. Ni(II) also activated, as did Mg at high concentration.

Only Mn(II) could restore endogenous rates, giving an apparent Km of 0.1–0.2 μM, and eliciting about 70% full activity without addition of a site II activator.

In purified Golgi membrane vesicles, Mn gave an apparent Km of 0.4 μM. This increased sharply to about 10 μM on permeabilization with filipin, lysis with detergents, solubilization with Triton X‐100, or in the pure enzyme. Preparations of chemically undamaged Golgi vesicles, known to include a proportion of the enzyme on exposed membranes, exhibited both low‐Km and high‐Km components.

The response of particulate galactosyltransferase to apparently physiological concentrations of free Mn(II) ion is interpreted as either due to a sensitizing factor within the Golgi lumen, or to the accumulation of Mn at elevated concentrations. Alternatively, the high Km of the soluble enzyme may reflect proteolytic damage.
Multiple α‐tubulin isoforms in cilia and cytoskeleton of Tetrahymena pyriformis generated by post‐translational modifications
Authors: PENQUE Deborah , GALEGO Lisete , RODRIGUES‐POUSADA Claudina
Published: March 2005

α‐Tubulin microheterogeneity was studied in Tetrahymena pyriformis. Using two‐dimensional electrophoretic analysis, we found between five and seven α‐tubulin and four β‐tubulin isoforms in cilia and four or five α‐tubulins and two β‐tubulins in cytoskeleton. Immunoblotting assay with anti‐(acetyl α‐tubulin) monoclonal antibody 6‐11B‐1 and [3H]acetate labelling revealed that the α‐tubulin isoforms are post‐translationally modified by acetylation. Our results also show that tubulins in the soluble cytoplasmic fraction are not acetylated. Nevertheless, a slight reaction with the antibody 6‐11B‐1 can be observed in the taxol and vinblastine‐treated cytoplasmic pool. Pulse/chase experiments using [35S]methionine during cell reciliation have demonstrated that the basic α‐tubulin isoforms are converted into acidic isoforms in the absence of protein synthesis, suggesting that the basic α‐tubulin is the precursor of the acidic forms which are found in cilia and cytoskeleton. In‐vivo‐translation selection demonstrated the existence of a single precursor molecule which corresponded to the most basic α‐tubulin. Taken together, our results provide evidence for the existence of post‐translational modifications, namely acetylation. Nevertheless, other post‐translational mechanisms involved in the biosynthesis of microtubules of cilia and cytoskeleton are required to explain the whole α‐tubulin heterogeneity.
The Bacillus subtilis cytochrome‐c oxidase
Authors: SARASTE Matti , METSO Tuula , NAKARI Tiina , JALLI Tuulikki , LAURAEUS Marko , VAN DER OOST John
Published: March 2005

The structural genes of cytochrome‐c oxidase in Bacillus subtilis have been isolated and sequenced. Five genes, ctaB–F, are closely spaced. ctaC, ctaD, ctaE and ctaF are the genes for subunits II, I, III and IVB, respectively. ctaB, which may encode an assembly factor, is separated and upstream from the others. In comparison to its mitochondrial counterparts, subunit I has an extended C‐terminus with two additional transmembrane segments, whereas subunit III has lost two such segments from its N‐terminus. The C‐terminal extension in subunit II is a covalent cytochrome‐c domain, previously characterized only in the thermophilic oxidases. Subunit IVB, a small hydrophobic protein, is a novel subunit. These predictions suggest that the B. subtilis cytochrome‐c oxidase is structurally more related to the four‐subunit Escherichia coli cytochrome‐bo complex than, for instance, to the Paracoccus denitrificans enzyme. Cytochrome aa3, which was previously isolated from B. subtilis [de Vrij, W., Azzi, A. & Konings, W. N. (1983) Eur. J. Biochem. 131, 97–103] is not encoded by the ctaC – F genes; thus, there seems to be two different cytochrome‐aa3‐type oxidases in this Gram‐positive bacterium.
Structural requirements for the binding of the pituitary adenylate‐cyclase‐activating peptide to receptors and adenylate‐cyclase activation in pancreatic and neuronal membranes
Authors: GOURLET Philippe , WOUSSEN‐COLLE Marie‐Claire , ROBBERECHT Patrick , DE NEEF Philippe , CAUVIN Annick , VANDERMEERS‐PIRET Marie‐Claire , VANDERMEERS André , CHRISTOPHE Jean
Published: March 2005

PACAP (pituitary adenylate‐cyclase‐activating peptide)‐binding receptors were investigated in membranes from the rat pancreatic acinar cell line, AR 4‐2J, the rat hippocampus and the human neuroblastoma cell line NB‐OK, by 125I‐PACAP(1–27) (amino acid residues 1–27 of N‐terminal amidated PACAP) binding and adenylate cyclase activation. The relative binding of 125I‐PACAP(1–27) to the receptor, and ability to activate adenylate cyclase were PACAP ≥ PACAP(1–27) > PACAP(2–38) > PACAP(1–9)‐VIP(10–28)(PACAP‐VIP) > PACAP(2–27) > [Ser9, Tyr13]VIP > [Tyr13]VIP ≥ [Ser9]VIP ≥ VIP(1–23)‐PACAP(24–27)(VIP‐PACAP) > VIP (vasoactive intestinal peptide). The N‐terminal moiety of PACAP(1–27) was more important than the three amino acids at the C‐terminus for 125I‐PACAP(1–27)‐binding site recognition. For rat pancreatic 125I‐VIP‐binding sites tested with 125I‐VIP, the order of binding affinity was PACAP = PACAP(1–27) ≥ VIP = [Ser9]VIP = [Tyr13]VIP = [Ser9, Tyr13]VIP ≥ PACAP‐VIP ≥ VIP‐PACAP > PACAP(2–38) = PACAP(2–27). Pancreatic 125I‐VIP‐binding sites, when compared to 125I‐PACAP(1–27)‐binding sites, showed little specificity and only weak coupling, so that PACAP and VIP‐PACAP acted only as partial VIP agonists on adenylate cyclase.
Caldesmon‐induced polymerization of actin from profilactin
Authors: GAŁA̧ZKIEWICZ Barbara , BUSS Folma , JOCKUSCH Brigitte M. , DA̧BROWSKA Renata
Published: March 2005

We have investigated the effect of caldesmon, a Ca2+/calmodulin‐regulated actin‐binding protein, on the complex between profilin and G‐actin (profilactin). We found that smooth muscle caldesmon dissociates this complex rapidly and induces the polymerization of the released actin. Native profilactin (e. g. the complex isolated from calf thymus) proved more resistant to the attack of caldesmon than a heterologous complex reconstituted from calf thymus profilin and skeletal muscle actin. The mode of caldesmon‐induced profilactin dissociation was similar to that described for Mg2+, and 2 mM MgCl2 potentiated the caldesmon effect. Since both caldesmon and profilin have been found enriched in ruffling membranes of animal cells, our in vitro findings may be relevant to the regulation of actin filaments in living cells.
Genomic organization of the human procollagen α1 (II) collagen gene
Authors: HUANG Min‐Chi , SEYER Jerome M. , THOMPSON James P. , SPINELLA Dominic G. , CHEAH Kathy S. E. , KANG Andrew H.
Published: March 2005

The nucleotide sequence of the human procollagen α1 (II) collagen gene extending from within the first intron through exon 15, and part of the 15th intron has been determined. This sequence analysis (7056 bases) identifies the intron/exon organization of the region of this gene encoding the N‐propeptide and part of the triple‐helical domain. Structural comparison of this with the genes of other human fibrillar collagens shows considerable diversity in terms of size and number of introns and exons that encodes the N‐propeptide domain. Although the genomic structure of the human procollagen α1 (II) gene is quite different from the rat procollagen α1 (II) gene, the nucleotide coding sequences are 89% identical.
Kinetics of the conformational transition of the spinach chloroplast fructose‐1,6‐bisphosphatase induced by fructose 2,6‐bisphosphate
Authors: SOULIÉ Jean‐Michel , RIVIÈRE Mireille , BALDET Pierre , RICARD Jacques
Published: March 2005

The activation of oxidized chloroplast fructose‐1,6‐bisphosphatase by fructose 2,6‐bisphosphate and magnesium previously described at pH 7.5 [Soulié et al. (1988) Eur. J. Biochem. 176, 111 – 117] has now been studied at pH 8, the pH which prevails under light conditions in the chloroplast stroma. The process obeys a hysteretic mechanism but the rate of activation is considerably increased with half‐times down to 50 s and the apparent dissociation constant of fructose 2,6‐bisphosphate from the enzyme is lowered from 1 mM at pH 7.5 to 3.3 μM at pH 8. The process is strictly metal‐dependent with a half‐saturation concentration of 2.54 mM for magnesium.

The conformational transition postulated in our hysteretic model has been investigated through both the spectrophometric and chemical modification approaches. The activation of the enzyme by fructose 2,6‐bisphosphate in the presence of magnesium results in a slow modification of the ultraviolet absorption spectrum of the enzyme with an overall increase of 3% at 290 nm. The same treatment leads to the protection of two free sulfhydryls and an increased reactivity of one sulfhydryl group/enzyme monomer to modification by 5,5′‐dithiobis(2‐nitrobenzoic acid). The titration of the exposed cysteinyl residue prevents the relaxation of enzyme species induced by fructose 2,6‐bisphosphate to the native form.

The activation of chloroplast fructose‐1,6‐bisphosphatase by fructose 2,6‐bisphosphate is discussed both with respect to the understanding of the overall regulation properties of the enzyme and to a possible physiological significance of this process.
An additional promoter functions in the human aldolase A gene, but not in rat
Authors: MUKAI Tsunehiro , ARAI Yuji , YATSUKI Hitomi , JOH Keiichiro , HORI Katsuji
Published: March 2005

The aldolase A gene was isolated from a human DNA library, mapped and sequenced. This gene comprises 12 exons and spans 6.5 kb. From the genomic DNA sequence and from the previous sequence analysis of the cDNA, it was revealed that the first exon L1 and the second exon encode the 5′ non‐coding sequence of mRNA L1, while the third and forth exons (corresponding to exons M and L2) encode different mRNA, mRNA M and L2, respectively; the following eight exons (exons 5–12) are shared commonly by all the mRNA species. These results indicate that the mRNA species are generated from a single aldolase A gene from one of exons L1, M or L2, in addition to exons 5–12, and also that the usage of a leader exon is similar but clearly distinct from that of rat aldolase A gene which we analyzed [Joh, K., Arai, Y., Mukai, T. & Hori, K. (1986) J. Mol. Biol. 190, 401–410]. By comparing the promoter regions in the human and rat aldolase A genes, we found similar sequences in the rat genome corresponding to those of the human L1, M and L2 promoter. We could not, however, detect any transcripts starting from sequences corresponding to the human L1 promoter in the rat genome, although the products corresponding to human M and L2 were detected. Thus, we concluded that the L1 promoter was either acquired by the human genome or deleted from the rat genome after human and rat diverged during evolution.
Transcription of synthetic DNA containing sequences with dyad symmetry by wheat‐germ RNA polymerase II
Authors: JOB Dominique , JOB Claudette , MERCOYROL Laure de , SHIRE David
Published: March 2005

Interaction of purified eukaryotic RNA polymerase II with various synthetic palindromic DNA sequences is associated with the formation of transcriptional complexes of different stabilities, i.e. having different propensities for releasing the nascent transcript. This phenomenon was observed by using wheat‐germ RNA polymerase II and a series of double‐stranded template polymers containing palindromic repeating motifs of 6–16 bp, with regulatory alternating purine and pyrimidine bases such as d[ATA(CG)nC] · d[TAT(GC)nG], with n= 1, 3 or 6 referred to as d(GC), d(GC)3 or d(GC)6, respectively. We also synthesized two double‐stranded methylated polymers, containing the repeating units d(ATAm5CGm5C) · d(TATGm5CG) and d[ATA(m5CG)6m5C] · d[TAT(Gm5C)6G] [designated d(GmC) and d(GmC)6, respectively]. All of these polymers served as templates for the reaction of single‐step addition of CTP to a CpG primer catalysed by wheat‐germ RNA polymerase II, to an extent that seems well correlated with the number of potential initiation sites within the DNA molecules. Furthermore, in these reactions, the enzyme appears to form relatively stable transcriptional complexes, as trinucleotide product was released only very slowly. In marked contrast to the results with the CpG primer, the single‐step addition reaction primed by UpA, i.e. the synthesis of UpApU proceeded at a much higher velocity and was strongly enhanced by increasing the d(G‐C) content of the repeating units of the DNA polymers. Thus, taking into account the number of potential sites at which UpApU synthesis could occur, the extent of UpApU synthesis was increased about 12‐fold with d(GC)6 compared to that with the d(GC) template. The catalytic nature of the reaction necessarily implies that the stability of the transcription complexes with the plant RNA polymerase II decreased as the d(G‐C) content of the repeating motif increased. Furthermore, although the synthesis of CpGpC could be demonstrated with d(GmC)6 as template, the UpA‐primed synthesis of UpApU could not be detected with this polymer. The results obtained in transcription of these polymers are discussed in relation to the potential involvement of palindromic DNA in transcription termination and attenuation in the presence of RNA polymerase II.
Investigations on microbial sulfur respiration
Authors: ZÖPHEL Andreas , KENNEDY M. Claire , BEINERT Helmut , KRONECK Peter M. H.
Published: March 2005

The sulfur‐reducing bacterium Spirillum 5175 was investigated with regard to membrane constituents that might be part of the sulfur oxidoreductase which converts elemental sulfur to hydrogen sulfide. Regardless of the electron acceptor used for cultivation of the bacteria, i.e. elemental sulfur, fumarate, or nitrate (Sp. 5175S,F,N), the qualitative pattern of cytochromes and Fe‐S proteins did not change significantly, as documented by ultraviolet/visible and electron paramagnetic resonance spectroscopy of oxidized (as isolated) and reduced (dithionite) samples. With elemental sulfur the prominent cytochrome exhibited absorption maxima at 553, 522.5 and 426 nm in the reduced state. In fumarate‐grown cells two prominent cytochromes were found with maxima at 561, 551, 530, 521 and 430 nm. Two b‐type cytochromes with Em at −198 mV and −20 mV vs the standard hydrogen electrode were identified in the membrane fraction of Sp. 5175F.

A yellow pigment was extracted and identified as a flexirubin‐type pigment. Although present in large quantities, it seemed not to be involved in the reduction of elemental sulfur.

Menaquinone, MK 6 (Mr 580) was the prominent quinone identified in Sp. 5175. Characterization of a second quinone was not attempted because of its much lower concentration.

The membrane constituents of Sp. 5175 were solubilized by a variety of detergents and detergent mixtures. A colorimetric procedure with photochemically reduced phenosafranin as the electron donor and cysteamine trisulfide (RS‐S‐SR, R =−CH2CH2NH2) as the electron acceptor was used to detect sulfur oxidoreductase activity.

Three membrane proteins of Sp. 5175 were purified: (1) an [NiFe] hydrogenase, homogeneous by SDS/polyacrylamide gel electrophoresis, with electron paramagnetic resonance signals as isolated at gx,y,z= 2.01, 2.16, 2.33 (100 K), and a strong signal at g= 2.02 below 20 K; (2) a cytochrome b, Fe‐S‐dependent fumarate reductase, and (3) a protein apparently linked to the sulfur oxidoreductase activity. In contrast to fumarate reductase, no b‐type cytochrome was present in the fractions exhibiting sulfur oxidoreductase activity. The presence of Fe‐S centers was demonstrated by electron paramagnetic resonance spectroscopy at 10 K. It is not clear whether the c‐type cytochrome in the same fractions is part of the sulfur‐reducing apparatus of Sp. 5175.
Phosphatidylinositol‐specific phospholipase C induces biosynthesis of acetylcholinesterase via diacylglycerol in Schistosoma mansoni
Authors: ESPINOZA Bertha , SILMAN Israel , ARNON Ruth , TARRAB‐HAZDAI Rebeca
Published: March 2005

We have previously shown that two ectoenzymes, acetylcholinesterase (AChE) and alkaline phosphatase, are released from the surface and from particulate fractions of the parasite Schistosoma mansoni, by a phosphatidylinositol‐specific phospholipase C (PtdIns‐PLC) of bacterial origin. Exposure to PtdIns‐PLC not only removes large amounts of AChE from the surface of intact, viable Schistosoma in culture, but is accompanied by a concomitant increase in overall levels of AChE in the parasite. The same phenomenon is observed with PtdIns‐PLC from two different bacterial sources; Staphylococcus aureus and Bacillus thuringiensis. The increase in AChE levels may be ascribed to de novo synthesis since exposure to PtdIns‐PLC, in the presence of the protein‐synthesis inhibitor cycloheximide, totally blocked the increase in AChE activity. Furthermore, PtdIns‐PLC induced an increased incorporation of [35S]methionine into the AChE immunoprecipitated by a specific anti‐AChE serum. This increase is selective for AChE, since total protein synthesis remained almost unchanged after PtdIns‐PLC addition, and little or no effect was observed on the enzymatic activity of alkaline phosphatase, which is also glycophosphatidylinositol anchored. Since cleavage of the phosphatidylinositol anchor by PtdIns‐PLC should liberate diacylglycerol, which may act as second messenger, we investigated the effect of exogenous diacylglycerols on the synthesis of AChE in S. mansoni. Three different diacylglycerols were tested as possible inducers of AChE activity in the parasite. Both 1‐oleoyl‐2‐acetyl‐sn‐glycerol and 1,2‐dimyristoyl‐sn‐glycerol were able to increase AChE activity by 35–40% at concentrations of 25 μg/ml. A higher concentration of 1,2‐dioctanoyl‐sn‐glycerol (70 μg/ml) was needed to produce an equivalent effect. Moreover, addition of phorbol‐12‐myristate‐13‐acetate, together with the calcium ionophore A23187, produced a similar increase in AChE activity. Finally, polymixin B, a specific inhibitor of protein kinase C, partially blocked the increase in AChE activity induced by PtdIns‐PLC. Our results suggest the involvement of glycophosphatidyl membrane‐anchor breakdown products as putative second messengers in the parasite S. mansoni.
A characterization of copper/zinc superoxide dismutase mutants at position 124 Zinc‐deficient proteins
Authors: BANCI Lucia , BERTINI Ivano , CABELLI Diane E. , HALLEWELL Robert A. , TUNG James W. , VIEZZOLI Maria Silvia
Published: March 2005

Substitution of the completely conserved aspartic acid residue at position 124 of Cu,Zn superoxide dismutase with asparagine and glycine has been performed through site‐directed mutagenesis on the human enzyme. Asp124 is H‐bonded to the NH of two histidines, one of which is bound to copper and the other to zinc. The mutant proteins, as expressed in Escherichia coli, result in an essential zinc‐free enzyme which is similar to that obtained from the wild‐type derivative through chemical manipulation. Only by extensive dialysis against 0.5 M ZnCl2 or CoCl2 at pH 5.4 was it possible to reconstitute approximetely 50% of the molecules in the Cu2Zn2 or Cu2Co2 form. The new derivatives have been characterized through EPR, CD and nuclear magnetic relaxation dispersion techniques. The Cu2Cox derivatives (x ∼ 1) were used to monitor, through electronic and 1H‐NMR spectroscopies, the metal sites which are found to be similar to those of the wild type. In addition, a double substitution with asparagine has been made, replacing the invariant aspartate at position 124 and the highly conserved aspartate at position 125. The behavior is similar to that of the other mutants in most respects. The Cu2E2 (E = empty) derivatives of the mutants are stable, even in the pH range 8–10, whereas in the case of the Cu2E2 derivative of the wild type, copper migration occurs at high pH, producing both Cu2Cu2 and apo derivatives. The activity measurements indicate that the various Cu2E2 derivatives have the same activity at low pH and similar to that of the holoenzyme. A full profile up to pH 10.5 was obtained for the mutants.
Protein O‐glycosylation in Saccharomyces cerevisiae Purification and characterization of the dolichyl‐phosphate‐D‐mannose‐protein O‐D‐mannosyltransferase
Authors: STRAHL‐BOLSINGER Sabine , TANNER Widmar
Published: March 2005

The enzyme dolichyl‐phosphate‐D‐mannose: protein O‐D‐mannosyltransferase has been solubilized from Saccharomyces cerevisiae membranes and its mannosyltransferase activity demonstrated using short peptides. The specific activity of the protein was enriched 130‐fold before it was further purified by native and SDS gel chromatography. A 92‐kDa band correlated well with the enzyme activity; an antibody raised against this protein precipitated the mannosyltransferase. The 92‐kDa band was hydrolysed to 84 kDa after treatment with endoglycosidase F, indicating that the protein is a glycoprotein which may contain four carbohydrate chains. The purified mannosyltrasferase is distinctly influenced in transfer specificity by amino acids next to serine and threonine within the acceptor peptides. Thus acidic amino acids strongly inhibit acceptor activity as do glycine and proline residues as amino‐terminal and carboxy‐terminal neighbours, respectively.
Vitamin A intake and in vivo expression of the genes involved in retinol transport
Authors: PEROZZI Giuditta , MENGHERI Elena , COLANTUONI Vittorio , GAETANI Sancia
Published: March 2005

Two different metabolic alterations in vitamin A status are known to cause changes in the amount of circulating retinol‐binding protein (RBP) and cellular retinol‐binding protein (CRBP) in experimental animals; namely vitamin A deficiency, characterized by depleted retinol‐liver stores and hypervitaminosis A, characterized by hepatic accumulation of retinyl esters. We have induced vitamin A deficiency and hypervitaminosis A in two groups of rats with the aim of determining whether the expression of the genes coding for these two proteins might be directly regulated by retinol. Using human RBP and CRBP cDNAs as probes, we measured the rate of transcription of the two genes in liver nuclei from control and treated rats by run‐on transcription assays, and the steady‐state level of the mRNAs by Northern blot analysis of total liver RNA. The distribution profile of RBP and CRBP mRNAs on fractionated liver polysomes was also examined. We have found a threefold decrease in the hepatic level of CRBP mRNA in vitamin‐A‐deficient animals, while the RBP mRNA is not affected by this nutritional deprivation. The decrease does not correspond to a lower transcription rate of the gene and therefore it is likely to result from lower stability of the CRBP mRNA. In hypervitaminosis A, we do not observe any differences in both the steady‐state level of the mRNAs and in the rate of transcription of the two genes. The results are discussed in terms of retinol‐dependent stabilization of the mRNA coding for CRBP.
ATP synthesis in chromatophores driven by artificially induced ion gradients
Authors: TURINA Paola , MELANDRI B. Andrea , GRÄBER Peter
Published: March 2005

An electrochemical potential difference for protons (μH+) across the membrane of bacterial chromatophores was induced by an artificially generated pH difference (pH) and a K+/valinomycin diffusion potential, φ. The initial rate of ATP synthesis was measured with a rapid‐mixing quenched‐flow apparatus in the time range between 70 ms and 30 s after the acid–base transition. The rate of ATP synthesis depends exponentially on pH;. Increasing diffusion potentials shift the pH dependency to lower pH values. Diffusion potentials were calculated from the Goldman equation. Using estimated permeability coefficients, the rate of ATP synthesis depends only on the electrochemical potential difference of protons irrespective of the relative contribution of pH and φ.
Modification of histidine residues on proteins from the 50S subunit of the Escherichia coli ribosome
Authors: SUMPTER Vicki G. , TATE Warren P. , NOWOTNY Petra , NIERHAUS Knud H.
Published: March 2005

L2, L3, L4, L16 and L20 are proteins of the 50S ribosomal subunit of Escherichia coli which are essential for the assembly and activity of the peptidyl transferase centre. These proteins have been modified with the histidine‐specific reagent, diethylpyrocarbonate, while L17 and L18 were treated as controls. Each modified protein tested was able to participate in the reconstitution of a 50S particle when replacing its normal counterpart, although the particles assembled with modified L2 were heterogeneous. However, although they could support assembly, modified L16 and L20 were not themselves reconstituted stably, and modified L2 and L3 were found in less than stoichiometric amounts. Particles assembled in the presence of modified L16 retained significant peptidyl transferase activity (60–70% at 10 mM diethylpyrocarbonate) whereas those reconstituted with modified L2, L3, L4 or L20 had low activity (10–30% at 10 mM diethylpyrocarbonate). The particles assembled with the modified control portein, L17, retained 80% of their peptidyl transferase activity under the same conditions. The histidine residues within the essential proteins therefore contribute to ribosome structure and function in three significant ways; in the correct assembly of the ribosomal subunit (L2), for the stable assembly of the proteins within the ribosomal particle (L20 and L16 in particular), and directly or indirectly for the subsequent activity of the peptidyl transferase centre (L2, L3, L4 and L20). The essential nature of the unmodified histidines for assembly events precludes the use of the chemical‐modification strategy to test the proposal that a histidine on one of the proteins might participate in the catalytic activity of the centre.
N‐Butyl‐N‐methyl‐4‐nitrophenyl carbamate as a specific active site titrator of bile‐salt‐dependent lipases
Authors: FOURNERON Jean‐Dominique , ABOUAKIL Nezha , CHAILLAN Catherine , LOMBARDO Dominique
Published: March 2005

The effect of a series of synthetic carbamates on the human (milk or pancreatic) bile‐salt‐dependent lipase (cholesterol esterase) was examined. N‐isopropyl‐O‐phenyl, N‐methyl‐O‐phenyl, N‐butyl‐(4‐nitrophenyl), N‐phenyl‐(4‐nitrophenyl), N‐butyl‐N‐methyl and N‐pentyl‐O‐phenyl carbamates were inhibitors of the enzyme activity, while O‐isopropyl‐N‐phenyl, O‐methyl‐N‐phenyl, O‐benzyl‐N‐isopropyl and O‐cyclohexyl‐N‐phenyl carbamates were not even recognized by the enzyme. The N‐alkyl chain length is essential for the enzyme inhibition and N‐butyl‐(4‐nitrophenyl) or N‐pentyl‐O‐phenyl carbamates are more potent inhibitors than N‐methyl‐O‐phenyl or N‐isopropyl carbamates. The inhibition by reactive carbamates fits the criteria for mechanism‐based inhibition: the inhibition is first‐order with time, shows saturation kinetics with increasing carbamate concentration and leads to an inactive stoichiometric enzyme‐inhibitor complex; the enzyme activity can be protected by a competitive inhibitor. Evidence is shown that the enzymatic nucleophilic attack of carbamates is directed at the carbonyl carbon atom and not the nitrogen atom. The inhibition of bile‐salt‐dependent lipase does not occur consecutive to the formation of a reactive isocyanate derivative of carbamate but via a tetrahedral intermediate involving essential residues implicated in the enzyme catalytic site. This intermediate evolves by liberation of alcohol (or phenol) and formation of an inactive carbamyl enzyme. Among the carbamates tested, N‐butyl‐N‐methyl‐(4‐nitrophenyl) carbamate specifically inhibits the bile‐salt‐dependent lipase; the release of 4‐nitrophenol from this carbamate is directly proportional to the enzyme inhibition and it may be defined as a specific active‐site titrator for bile‐salt‐dependent lipases.
Preparation and kinetic properties of 5‐ethylphenazine–poly(ethylene‐glycol)–glutamate‐dehydrogenase conjugate
Authors: YOMO Tetsuya , URABE Itaru , OKADA Hirosuke
Published: March 2005

5‐Ethylphenazine – poly(ethylene glycol) – glutamate dehydrogenase conjugate (EP+‐PEG‐GluDH) was prepared by linking poly(ethylene glycol)‐bound 5‐ethylphenazine to glutamate dehydrogenase. The average number of the ethylphenazine moieties bound/enzyme subunit was 0.7. This conjugate is a semisynthetic enzyme having NADH oxidase activity; the ethylphenazine moiety works as a catalytic group, and the coenzyme‐binding site of glutamate dehydrogenase works as a substrate‐binding site. The effects of the presence of the substrate‐binding site near the catalytic group were studied by using EP+‐PEG‐GluDH. Before the preparation of the conjugate, the reactivity of NADH bound in the coenzyme‐binding site toward the ethylphenazine moiety was estimated for glutamate and lactate dehydrogenases. The results show that the NADH molecule bound in the site of glutamate dehydrogenase reacts with EP+‐PEG at a rate of 43% of that of free NADH, but the NADH molecule bound in lactate dehydrogenase does not react with 1‐(3‐carboxypropyloxy)‐5‐ethylphenazine. Therefore, glutamate dehydrogenase was used as the substrate‐binding site of the semisynthetic NADH oxidase. The results of the kinetic analysis of the activity of EP+‐PEG‐GluDH show that the apparent turnover number of the active site is 0.38 s−1, which corresponds to the apparent intramolecular rate constant of the oxidation of NADH bound in the active site. The apparent effective concentration of bound NADH for the catalytic group of the ethylphenazine moiety is 0.33 mM. This means that the presence of the substrate‐binding site near the catalytic group increases the local NADH concentration by at most 0.33 mM, and this is the rate‐accelerating effect of the binding site.
Development of monoclonal antibodies against different protein and carbohydrate epitopes of dipeptidyl peptidase IV from rat liver plasma membranes
Authors: HARTEL‐SCHENK Sabine , LOCH Nikolaus , ZIMMERMANN Martin , REUTTER Werner
Published: March 2005

Dipeptidyl peptidase IV (DPP IV) is a serine exopeptidase expressed at high levels in rat kidney, liver and lung. We established eight monoclonal antibodies against partially purified DPP IV from rat liver plasma membranes. By means of a competitive dot blot assay with purified DPP IV, these antibodies were shown to recognize four different epitopes of the glycoprotein, designated A–D. The epitopes are located on the extracellular domain of DPP IV, as shown by papain digestion of liver plasma membranes. Treatment of DPP IV with neuraminidase and glycopeptide N‐glycosidase F, as well as incubation of heptocytes with the α‐mannosidase I inhibitor deoxymannojirimycin, revealed that epitope A may be formed by a mannose‐rich sugar chain and epitope D might represent a complex carbohydrate structure in the mature glycoprotein, while the epitopes B and C are formed by the protein moiety. Concanavalin A reduced the binding of monoclonal antibody to epitope A by 78%. Binding to epitope D was blocked by 73% with wheat germ lectin, and by more than 99% with sialic acid; epitopes B and C were unaffected by any of the lectins or sugars tested. The immunological cross‐reactivity with DPP IV from Morris hepatoma 7777 was demonstrated with monoclonal antibodies against epitopes A–C. Epitope D was not recognized on hepatoma DPP IV. However, in addition to DPP IV, four hepatoma plasma membrane glycoproteins were precipitated by the monoclonal antibody against the epitope D, indicating that this epitope is not uniquely restricted to DPP IV.
Effects of monocyte‐lymphocyte interaction on the synthesis of leukotriene B4
Authors: JAKOBSSON Per‐Johan , ODLANDER Björn , CLAESSON Hans‐Erik
Published: March 2005

Human monocytes in monolayers were challenged with the calcium ionophore A23187. Methanol trapping of the products in the cell‐free supernatants, followed by analysis on HPLC and by ultraviolet spectroscopy, revealed the presence of two compounds, which exhibited a conjugated‐triene spectrum and chromatographed with the compounds formed when synthetic leukotriene (LT) A4 was added to warm acidified methanol. Furthermore, addition of purified LTA4 hydrolase to the cell‐free supernatant of monocytes, stimulated with the ionophore A23187, resulted in increased levels of LTB4. These results indicate that monocytes release LTA4 extracellularly after activation with the calcium ionophore.

Incubation of monocytes together with monoclonal lymphocytic cells, of both B and T cell lineage, yielded increased levels of LTB4 whereas the non‐enzymatic isomers of this compound, i.e. Δ6‐trans‐LTB4 and 12‐epi‐Δ6‐trans‐LTB4, declined. In addition, the sum of LTB4 and its non‐enzymatically formed isomers increased in mixed cultures of monocytes and monoclonal lymphocytic cells as compared to monocytes alone.

The present study indicates that activated monocytes release LTA4, which is converted into LTB4 by monoclonal lymphocytic cells. Furthermore, the increase of the total amounts of leukotrienes on incubation of monocytes with lymphocytic cells, suggests the presence of an additional mechanism leading to activation of the 5‐lipoxygenase pathway in monocytes.
Characterization of the yeast acidic ribosomal phosphoproteins using monoclonal antibodies
Authors: VILELLA Maria Dolores , REMACHA Miguel , ORTIZ Blanca L. , MENDEZ Enrique , BALLESTA Juan Pedro García
Published: March 2005

In order to characterize the acidic ribosomal proteins immunologically and functionally, a battery of monoclonal antibodies specific for L44, L44′ and L45, the three acidic proteins detected in Saccharomyces cerevisiae, were obtained. Eight monoclonal antibodies were obtained specific for L45, three for L44′ and one for L44. In addition, two mAbs recognizing only the phosphorylated forms of the three proteins were obtained. The specific immunogenic determinants are located in the middle region of the protein structure and are differently exposed in the ribosomal surface. The common determinants are present in the carboxyl end of the three proteins. An estimation of the acidic proteins by ELISA indicated that, in contrast to L44 and L45, L44′ is practically absent from the cell supernatant; this suggests that protein L44′ does not intervene in the exchange that has been shown to take place between the acidic proteins in the ribosome and in the cytoplasmic pool. It has also been found that, while IgGs specific for L44 and L45 do not inhibit the ribosome activity, the anti‐L44′ effectively blocks the polymerizing activity of the particles.

These results show for the first time that the different eukaryotic acidic ribosomal proteins play a different functional role.
The ATPase of Bacillus alcalophilus
Authors: HOFFMANN Artur , DIMROTH Peter
Published: March 2005

The purified ATPase of Bacillus alcalophilus (F1F0) was reconstituted into proteoliposomes by gradual removal of the detergent Triton X‐100 with Amberlite XAD‐2. The reconstitution was apparently highly asymmetric with nearly 100% of the F1 portion of the ATPase becoming oriented to the outside. Similar to results obtained with the soluble enzyme, the membrane‐bound ATPase required Mg2+ and methanol for maximum activity. With Ca2+ or Mg2+ without methanol, 25% and 1%, respectively, of the maximum activity were observed. The ATPase was unable to pump Na+ ions but catalyzed the translocation of protons into the reconstituted proteoliposomes. Optimum proton translocation required the presence of Mg2+, not Ca2+, as divalent metal ion. The proton pump was inhibited by dicyclohexylcarbodiimide, venturicidin and NaN3. On incubation of the reconstituted ATPase with [14C]dicyclohexylcarbodiimide, subunit c of the enzyme complex became specifically labeled. The proteoliposomes catalyzed the Mg2+‐dependent incorporation of [32P]phosphate into ATP by ATP/[32P]phosphate exchange. This exchange was little affected by monensin, but was completely abolished by the uncoupler carbonyl cyanide m‐chlorophenylhydrazone. Protons and not Na+ are thus the coupling ions of the ATPase of B. alcalophilus.
Different isoforms of an apoprotein (apolipophorin III) associate with lipoproteins in Locusta migratoria
Authors: VAN DER HORST Dick J. , VAN DOORN Jan M. , VOSHOL Hans , KANOST Michael R. , ZIEGLER Rolf , BEENAKKERS Ad M. Th.
Published: March 2005

Insects transport lipid for flight in the form of diacylglycerol‐rich low‐density lipoproteins (low‐density lipophorin, LDLp), which in the hemolymph are produced from high‐density lipophorin (HDLp) by reversible association with several molecules of an apolipoprotein, apolipophorin III (apoLp‐III, Mr∼ 18000–20000) during lipid loading.

Two isoforms of apoLp‐III (a and b) were purified both from adult Locusta migratoria migratorioides hemolymph and LDLp, which have identical apparent Mr but differ in amino acid composition, NH2‐terminal amino acid sequence, and isoelectric points (5.35 ± 0.01 for apoLp‐IIIa, 5.10 ± 0.01 for apoLp‐IIIb). The NH2‐terminal sequence of apoLp‐IIIb is identical to the primary structure of apoLp‐III deduced from cloned cDNA [Kanost et al. (1988) J. Biol. Chem. 263, 10568–10573], whereas the NH2‐terminal sequence of apoLp‐IIIa is identical to that of apoLp‐IIIb but preceded by Arg‐Pro‐, which is the C‐terminal of the putative signal peptide coded by cDNA upstream from that coding for apoLp‐IIIb. The ratio apoLp‐IIIa apoLp‐IIIb free in hemolymph is identical to that in LDLp (5:9); since 14 molecules of apoLp‐III appear to be bound in one molecule of LDLp, an average of 5 molecules of apoLp‐IIIa and 9 of apoLp‐IIIb are involved in formation of each LDLp particle. In vivo studies using 35S‐labeled apoLp‐IIIa and b demonstrate that each of the isoforms can associate with HDLp to produce LDLp reversibly; in an in vitro system, production of LDLp containing exclusively apoLp‐IIIa or apoLp‐IIIb demonstrates independent participation of each isoform in LDLp formation.
Observation of a stable intermediate form in the reaction of human hemoglobin with carbon monoxide
Authors: COLETTA Massimo , GERACI Giuseppe
Published: March 2005

The reaction of human hemoglobin with carbon monoxide has been investigated near the equilibrium isosbestic wavelength (i.e.426 nm). As previously reported by others [Gray, R. D. & Gibson, Q. H. (1971) J. Biol. Chem. 246, 5176–5178], in the presence of 0.1 M phosphate pH 7.0 a rise‐and‐fall kinetic pattern can be observed at this wavelength, which indicates the presence of at least one spectroscopically detectable intermediate species. In this paper we demonstrate that (a) the intermediate species is thermodynamically stable; (b) both phases refer to bimolecular processes; (c) only the initial fast phase is observed when deoxyhemoglobin is reacted with substoichiometric amounts of CO (i.e. final [CO]/[heme] ≤ 0.5); (d) only the second slow phase is observed when hemoglobin that is partially saturated with CO (Ȳ≤ 0.5) is reacted with saturating CO concentrations; (e) the CO dissociation rate constant measured on the intermediate formed after a partial CO saturation at a final Ȳ∼ 0.4 has a value similar to that observed starting from the fully liganded form.

These results can be accounted for by a two‐state allosteric model [Monod, J., Wyman, J. & Changeux, J.‐P. (1965) J. Mol. Biol. 12, 88–118] under the assumption that either (a) 426 nm is an isosbestic wavelength for the To–R spectral changes but not for the To–T liganded reaction; or (b) a functional heterogeneity of the two types of subunits is present in the T state and at this wavelength this feature is spectroscopically detectable.
Membrane insertion of the pore‐forming domain of colicin A
Authors: LAKEY Jeremy H. , MASSOTTE Dominique , HEITZ Frederic , DASSEUX Jean‐Louis , FAUCON Jean‐François , PARKER Michael W. , PATTUS Franc
Published: March 2005

In order to gain some insight into the mechanism of insertion into membranes of the pore‐forming domain of colicin A and the structure of its membrane‐bound form, circular dichroism (in the near and far ultraviolet), fluorescence and ultraviolet spectroscopy experiments were carried out. Because the structure of the water‐soluble form of this fragment has been determined by X‐ray crystallography, these spectroscopic methods provided valuable information on the secondary structure and the environment of aromatic residues within the two forms of the peptide. These results strongly suggest that the pore‐forming domain of colicin A does not undergo drastic unfolding upon insertion into membrane. The conformational change associated with this process is triggered by the negatively charged lipids and probably consists of a reorientation of helix pairs with respect to each other. Exposure of the aromatic residues to the aqueous phase decreases on binding to lipids whilst the exposure of the tryptophans to the membrane phase increases. This cannot occur without a reorientation of helices 3–10. All data from this study support the model presented previously in which the known crystal structure opens like an ‘umbrella’ inserting the hydrophobic hairpin (helix 8–9) perpendicular to the membrane plane and the helical pair 1–2 and the domain containing the three tryptophans (helices 3–7) lying more or less parallel to the membrane plane. Lipids are bound more tightly to the protein at acidic pH than at neutral pH although a similar lipid protein complex is formed with 1,2‐dimyristoyl‐sn‐glycero(3)‐phospho(1)‐sn‐glycerol at both pH values.
Dictyostelium cells produce platelet‐activating factor in response to cAMP
Authors: BUSSOLINO Federico , SORDANO Concetta , BENFENATI Emilio , BOZZARO Salvatore
Published: March 2005

Evidence is provided that Dictyostelium discoideum cells produce 1‐O‐alkyl‐2‐δ‐acetyl‐O‐sn‐glycero‐3‐phosphocholine (platelet‐activating factor, PAF). D. discoideum PAF has been characterized as being identical with mammalian platelet‐activating factor, based on its stimulation of rabbit platelet aggregation, its physico‐chemical properties and mass spectrum. The basal activity of PAF increases after starvation and during aggregation and declines at the slug stage. PAF is not detected in the extracellular space. Cell treatment with cAMP pulses stimulates a transient accumulation of PAF, probably via activation of a cAMP‐dependent acetyltransferase, suggesting a possible involvement of PAF in cAMP‐regulated processes in Dectyostelium.