bluuebunny/crossref_metadata_2025 huggingface dataset:
Sequence of a Gene Cluster from Malonomonas Rubra Encoding Components of the Malonate Decarboxylase Na+ Pump and Evidence for Their Function
Authors: Berg Michael , Hilbi Hubert , Dimroth Peter
Published: July 2004

Malonate decarboxylation in Malonomonas rubra involves the formation of malonyl‐S‐[acyl‐carrier protein] from acetyl‐S‐[acyl‐carrier protein] and malonate, carboxyltransfer to a biotin protein and its decarboxylation that is coupled to δNa+ generation. The genes encoding components of the malonate decarboxylase enzyme system have been cloned and sequenced. These are located within a gene cluster of ≈11 kb comprizing 14 genes that have been termed madYZGBAECDHKFLMN in the given order. Upstream of madY an open reading frame pointing into the opposite direction of the mad genes was found with structural similarities to insertion‐sequence elements. The upstream region also contains DNA regions which are typical for an Escherichia coliσ70 promoter. Within 950 bp downstream of madN no other open reading frame was found. This region contains a putative terminator sequence. The intergenic regions within the mad gene cluster are short (usually <70 bp, maximum 302 bp) and ribosome binding sites were defined before all 14 genes. Thus, this DNA region could form a transcriptional unit and all 14 genes could be translated into proteins. The genes madABCDEF encode the structural proteins of the malonate decarboxylase as yet identified. By comparing protein and DNA sequences and by data bank searches for related proteins with known function the following assignments could be made: MadA represents the acyl‐carrier‐protein–transferase component, MadB is the integral membrane‐bound carboxybiotin protein decarboxylase, MadC and MadD are the two subunits of the carboxyltransferase, MadE is the acyl carrier protein and MadF is the biotin protein. Sequence comparison further indicates that MadH could be involved in the acetylation of the phosphoribosyl‐dephospho‐CoA prosthetic group and MadG could be involved in its biosynthesis. MadL and MadM are membrane proteins that could function as malonate carrier. The function of the madY,Z,K and N gene products is as yet unknown.
Efficiency of P‐glycoprotein–Mediated Exclusion of Rhodamine Dyes from Multidrug‐Resistant Cells is Determined by their Passive Transmembrane Movement Rate
Authors: Eytan Gera D. , Regev Ronit , Oren Galit , Hurwitz Charles D. , Assaraf Yehuda G.
Published: July 2004

The aim of the present study was to examine the relationship between the rate of the passive transmembrane movement of multidrug resistance (MDR)‐type substrates and the ability of P‐glycoprotein to extrude them from MDR cells. For this purpose, seven rhodamine dyes were examined for their P‐glycoprotein–mediated exclusion from MDR cells, their localization in wild‐type drug‐sensitive cells, their capacity to stimulate the ATPase activity of P‐glycoprotein reconstituted in proteoliposomes, and their transmembrane movement rate in artificial liposomes. All these rhodamine dyes were accumulated in wild‐type drug‐sensitive cells and were localized mainly in the mitochondria. All the dyes tested were substrates of reconstituted P‐glycoprotein and cellular P‐glycoprotein and were excluded to a variable degree from MDR cells. The transmembrane movement rate proved the major factor determining the efficacy of the P‐glycoprotein–mediated exclusion of rhodamine dyes from MDR cells. Thus, rhodamine B, the poorest cellular P‐glycoprotein substrate, exhibited a high affinity toward reconstituted P‐glycoprotein, but was the fastest membrane‐traversing dye. In contrast, tetramethylrosamine, the best cellular MDR probe, exhibited high affinity toward reconstituted P‐glycoprotein and slow transmembrane movement rate. Therefore, an anticancer drug with a fast transmembrane movement rate is expected to over‐come the MDR phenomenon. Furthermore, the widely used MDR marker, rhodamine 123, was a poor cellular MDR substrate compared with other rhodamine dyes, especially tetramethylrosamine, which was a superior cellular MDR substrate for functional dye‐exclusion studies.
Expression of the Zymomonas Mobilis Gfo Gene for NADP‐Containing Glucose: Fructose Oxidoreductase (GFOR) in Escherichia Coli— Formation of Enzymatically Active Pregfor but Lack of Processing Into a Stable Periplasmic Protein
Authors: Wiegert Thomas , Sahm Hermann , Sprenger Georg A.
Published: July 2004

Glucose:fructose oxidoreductase (GFOR) of the gram‐negative bacterium Zymomonas mobilis is a periplasmic enzyme with tightly bound cofactor NADP. The preprotein carries an unusually long N‐terminal signal peptide of 52 amino acid residues. Expression of the gfo gene in cells of Escherichia coli K12, under the control of a tac promoter, led to immunologically detectable proteins in western blots, and to the formation of an enzymatically active precursor form (preGFOR), located in the cytosol. Processing of preGFOR to the mature form was not observed in E. coli. Replacement of the authentic GFOR signal peptide by the shorter signal peptides of PhoA or OmpA from E. coli led to processing of the respective GFOR precursor proteins. However, the processed proteins were unstable and rapidly degraded in the periplasm unless an E. coli mutant was used that carried a triple lesion for periplasmic and outer‐membrane proteases. When fusion‐protein export was inhibited by sodium azide or carboxylcyanide m‐chlorophenylhydrazone, the cytoplasmic precursor forms of the respective preGFOR were not degraded. A major protease‐resistant GFOR peptide from the OmpA‐GFOR fusion was found within spheroplasts of E. coli to which NADP had been added externally. The formation of this peptide did not occur in the presence of NAD. It is concluded that NADP is required for GFOR to fold into its native conformation and that its absence from the E. coli periplasm is responsible for failure to form a stable periplasmic protein. The results strongly suggest that, in Z. mobilis, additional protein factors are required for the transport of NADP across the plasma membrane and/or incorporation of NADP into the GFOR apoenzyme.
Purified Methyl‐Coenzyme‐M Reductase is Activated when the Enzyme‐Bound Coenzyme F430 is Reduced to the Nickel(I) Oxidation State by Titanium(III) Citrate
Authors: Goubeaud Marcel , Schreiner Guido , Thauer Rudolf K.
Published: July 2004

The nickel porphinoid, coenzyme F430, is the prosthetic group of methyl‐coenzyme M reductase. The active form of the enzyme exhibits Ni‐EPR signals designated as MCR‐red1 and MCR‐red2. The inactive form of the enzyme is either EPR silent or it exhibits a distinct Ni‐EPR signal designated MCR‐0x1. Evidence is presented here that the MCR‐ox1 form of the enzyme can be converted in vitro to the MCR‐red1 form by reduction with titanium(III) citrate at pH 9. During conversion, the specific activity increases with increasing MCR‐red1 spin concentration from 2 U/mg to approximately 100 U/mg at spin concentrations higher than 80%. The reduced methyl‐coenzyme‐M reductase shows an ultraviolet/visible spectrum characteristic for coenzyme F430 in the Ni(I) oxidation state, with maxima at 386 nm and at 750 nm. The results indicate that methyl‐coenzyme‐M reductase is activated when the enzyme‐bound coenzyme F430 is reduced to the Ni(I) oxidation state. The experiments were performed with purified methyl‐coenzyme‐M reductase isoenzyme I of Methanobacterium thermoautotrophicum (strain Marburg).
Dexamethasone Increases Expression of 5‐Lipoxygenase and its Activating Protein in Human Monocytes and THP‐1 Cells
Authors: Riddick Carl A. , Ring William L. , Baker Joseph R. , Hodulik Craig R. , Bigby Timothy D.
Published: July 2004

The aim of this study was to assess the effect of dexamethasone on 5‐lipoxygenase pathway expression in human peripheral blood monocytes and the acute monocytic leukemia cell line, THP‐1. Cells were conditioned over a period of days with dexamethasone, at concentrations relevant in vivo, to study the effect of the glucocorticoid on calcium‐ionophore‐stimulated 5‐lipoxygenase product and arachidonic acid release. The effect of dexamethasone on levels of immunoreactive protein and steady‐state messenger RNA encoding for 5‐lipoxygenase and its activating protein (5‐LAP) was also assessed. Dexamethasone increased the stimulated release of 5‐lipoxygenase products from both monocytes and THP‐1 cells in a dose‐dependent fashion. The increase in product generation was not due to changes in the availability of arachidonic acid. However, immunoreactive protein and steady‐state messenger RNA encoding for 5‐lipoxygenase and 5‐LAP were increased by conditioning with dexamethasone. There was no apparent effect of the glucocorticoid on LTA4‐hydrolase‐immunoreactive protein levels or specific activity. We conclude that dexamethasone increases 5‐lipoxygenase pathway expression in both monocytes and in THP‐1 cells. This effect is due, at least in part, to increases in immunoreactive protein and steady‐state messenger RNA encoding for 5‐lipoxygenase and 5‐LAP. These results suggest a role for glucocorticoids in the regulation of 5‐lipoxygenase pathway expression in mononuclear phagocytes.
Phospholipase‐C‐Independent Inositol 1,4,5‐Trisphosphate Formation in Dictyostelium Cells — Activation of a Plasma‐Membrane‐Bound Phosphatase by Receptor‐Stimulated Ca2+ Influx
Authors: Van Dijken Peter , Bergsma Jan C. T. , Van Haastert Peter J. M.
Published: July 2004

Dictyostelium cells have enzyme activities that generate the inositol polyphosphate Ins(1,4,5)P3 from Ins(1,3,4,5,6)P5 via the intermediates Ins(1,3,4,5)P4 and Ins(1,4,5,6)P4. These enzyme activities could explain why cells with a deletion of the single phospholipase C gene (plc− cells) possess nearly normal Ins(1,4,5)P3 levels. In this study the regulation and the subcellular localization of the enzyme activities was investigated. The enzyme activities performing the different reaction steps from Ins(1,3,4,5,6)P3 to Ins(l,4,5)P3 are probably due to a single enzyme. Indications for this are the previously shown similar Ca2+ dependencies of the various reaction steps. Furthermore, the activities mediating the complete conversion of Ins(1,3,4,5,6)P5 to Ins(1,4,5)P3 co‐purify after subcellular fractionation, solubilization, and chromatography of the proteins. Subcellular fractionation studies demonstrate that the enzyme is localized mainly at the inner face of the plasma membrane. The enzyme activity could not be stimulated in vitro by guanosine 5′‐(3‐thio)triphosphate, a procedure known to activate G‐protein‐coupled enzymes in Dictyostelium. Still, in plc− cells the level of Ins(1,4,5)P3 was increased significantly after stimulation with high concentrations of the extracellular ligand cAMP. This stimulation is most likely due to the influx of Ca2+ because no increase of Ins(1,4,5)P3 could be detected in the absence of extracellular Ca2+. The results demonstrate the existence of a new receptor‐controlled route for the formation of Ins(1,4,5)P3 that is independent of phospholipase C.
Influence of Charge Differences in the C‐Terminal Part of Nisin on Antimicrobial Activity and Signaling Capacity
Authors: Van Kraaij Cindy , Breukink Eefjan , Rollema Harry S. , Siezen Roland J. , Demel Rudy A. , De Kruijff Ben , Kuipers Oscar P.
Published: July 2004

Three mutants of the lantibiotic nisin Z, in which the Val32 residue was replaced by a Glu, Lys or Trp residue, were produced and characterized for the purpose of establishing the role of charge differences in the C‐terminal part of nisin on antimicrobial activity and signaling properties. 1H‐NMR analyses showed that all three mutants harbor an unmodified serine residue at position 33, instead of the usual dehydroalanine. Apparently, the nature of the residue preceding the serine to be dehydrated, strongly affects the efficiency of modification. Cleavage of [Glu32,Ser33]nisin Z by endoproteinase Glu‐C yielded [Glu32]nisin Z(1–32)‐peptide, which has a net charge difference of ‐2 relative to wild‐type nisin Z. The activity of [Lys32, Ser33]nisin Z against Micrococcus flavus was similar to that of wild‐type nisin, while [Trp32,Ser33]nisin Z, [Glu32, Ser33]nisin Z and [Glu32]nisin Z(1–32)‐peptide exhibited 3–5‐fold reduced activity, indicating that negative charges in the C‐terminal part of nisin Z are detrimental for activity. All variants showed significant loss of activity against Streptococcus thermophilus. The potency of the nisin variants to act as signaling molecules for auto‐induction of biosynthesis was significantly reduced. To obtain mutant production, extracellular addition of (mutant) nisin Z to the lactococcal expression strains was essential.
Characterisation of the Molybdenum‐Responsive ModE Regulatory Protein and its Binding to the Promoter Region of the modABCD (Molybdenum Transport) Operon of Escherichia Coli
Authors: Anderson Lisa A. , Palmer Tracy , Price Nicholas C. , Bornemann Stephen , Boxer David H. , Pau Richard N.
Published: July 2004

Molybdenum‐dependent repression of transcription of the Escherichia coli modABCD operon, which encodes the high‐affinity molybdate transporter, is mediated by the ModE protein. This regulatory protein was purified as an N‐terminal His6‐tagged derivative and characterised both with and without the N‐terminal oligohistidine extension. Equilibrium centrifugation showed that ModE is at least a 57‐kDa homodimer. Circular dichroism spectroscopy indicated that when molybdate or tungstate bind to ModE there is little change in its α‐helical content, but a major change in the environment of tryptophan and tyrosine residues occurs. Addition of molybdate or tungstate to the protein results in almost 50% quenching of the fluorescence attributed to tryptophan. Titration of fluorescence quenching showed that two molecules of molybdenum bind to each dimer of ModE with a Kd of 0.8 μM. DNA mobility‐shift assays showed that ModE requires molybdenum, or tungstate, to bind with high affinity (approximate Kd of 30 μM ModE) to the modABCD promoter region. In accord with ModE's role as a molybdenum‐dependent transcriptional represser, DNase I footprinting experiments showed that the ModE‐molybdenum complex binds to a single 31‐bp region around the transcription start of the modABCD promoter. This region contains a 6‐base palindromic sequence CGTTAT‐N12‐ATAACG.
Cerastotin, a Serine Protease from Cerastes Cerastes Venom, with Platelet‐Aggregating and Agglutinating Properties
Authors: Marrakchi Naziha , Barbouche Rym , Guermazi Sami , Karoui Habib , Bon Cassian , El Ayeb Mohamed
Published: July 2004

Cerastotin, a thrombin‐like enzyme from the venom of the desert viper Cerastes cerastes, has been purified by gel filtration on Sephadex G‐75 and two ion‐exchange chromatographies on Mono S columns. It is a neutral glycoprotein (pI = 6.6), present as a single polypeptide chain of 40 kDa. Its N‐terminal sequence shows strong similarity with those of other thrombin‐like enzymes from snake venoms. Cerastotin possesses esterase and amidolytic activities measured with Nα‐tosyl‐l‐arginine methyl ester and the thrombin chromogenic substrate d‐phenylalanyl‐l‐pipecolyl‐l‐arginine p‐nitroanilide, respectively. The amidolytic activity is inhibited by phenylmethylsulfonyl fluoride, Nα‐tosyl‐l‐lysine chloromethane, Nα‐tosyl‐l‐phenylalanyl chloromethane, d‐phenylalanyl‐l‐prolyl‐l‐arginyl chloromethane and benzamidine, suggesting that cerastotin is a serine protease. Cerastotin efficiently clots human plasma and cleaves preferentially the a chain of fibrinogen. Cerastotin did not induce aggregation of washed normal platelets, but did aggregate platelets in the presence of exogenous fibrinogen. A monoclonal antibody directed against glycoprotein (GPIb), which specifically inhibits induced agglutination by ristocetin also completely blocks platelet aggregation induced by cerastotin. However, another anti‐GPIb monoclonal antibody, which specifically inhibits α‐thrombin binding to GPIb, did not prevent this aggregation. Furthermore, platelets which were desensitised by α‐thrombin still aggregate in the presence of cerastotin, but not α‐thrombin. Similarly a monoclonal antibody, anti‐GPIIb‐IIIa, which blocks fibrinogen binding, did not inhibit cerastotin‐induced platelet aggregation. This activity is abolished in the presence of 1 mM phenylmethylsulfonyl fluoride and/or 10 mM EDTA. Cerastotin also agglutinates formalin‐fixed and washed platelets, only in the simultaneous presence of fibrinogen and of Von Willebrand factor.
Binding of Thyroxine Analogs to Human Liver Aldehyde Dehydrogenases
Authors: Zhou Jianzhong , Weiner Henry
Published: July 2004

A fragment of a cytosolic thyroid‐hormone‐binding protein from Xenopus liver was reported to be 92–100% identical to residues 236–258 in several cytosolic aldehyde dehydrogenases [Yamauchi, K. & Tata, J. R. (1994)Eur. J. Biochem. 225, 1105–1112], which have been proposed to form part of the hinge region necessary to bind the adenosine moiety of NAD. Here we investigated the effects of two thyroxine analogs, 3,3′,5–triiodo‐l‐thyronine and 3,3′,5‐triiodothyroacetic acid, on purified human liver mitochondrial and cytosolic aldehyde dehydrogenases. The compounds were found to be competitive inhibitors against NAD and uncompetitive inhibitors with respect to aldehyde. At pH 7.4, the apparent Ki values were in the micromolar range when the concentration of NAD was varied. The inhibition against recombinantly expressed mutant forms of aldehyde dehydrogenase, which possessed diminished NAD binding, was determined. Essentially no differences were found between the native enzyme and the mutants, showing that the analog binding was not affected by altering the NAD‐binding site. Furthermore, the analogs could displace NAD but not NADH from the enzyme. These findings indicated that the binding of NAD differed from that of NADH, and that aldehyde dehydrogenases, like other dehydrogenases, can be inhibited by thyroxine analogs.
Characterization of The Active Site of Schwanniomyces Occidentalis Glucoamylase by In Vitro Mutagenesis
Authors: Hülseweh Birgit , Dahlems Ulrike M. , Dohmen Jürgen , Strasser Alexander W. M. , Hollenberg Cornelis P.
Published: July 2004

Site‐directed mutagenesis was performed to define the active site of the Schwanniomyces occidentalis glucoamylase. The mutated GAM1 genes were expressed in Saccharomyces cerevisiae, and enzymatic and growth properties of the transformants were determined. Mutants were transcribed and translated similar to the wild‐type glucoamylase. Therefore, all effects on enzymatic activity could be referred to single amino acid substitutions.

Asp470 was shown to be essential for the enzyme activity. Replacement of Asp470 by glycine led to a complete loss of activity. We suppose that Asp470 serves as a general acid‐base and stabilizes the formation of the intermediate carbenium ion. Substitution of Trp468 by alanine affected predominantly the α‐1,6 activity and not the α‐1,4 activity of the enzyme. The exchange impaired substrate binding as well as enzymatic catalysis. An influence of amino acid 474 on the substrate specificity could not be demonstrated. Exchanges at position 474 exhibited Kn, and Vmax values similar to wild‐type glucoamylase.
7α‐Hydroxylation and 3‐Dehydrogenation Abolish the Ability of 25‐Hydroxycholesterol and 27‐Hydroxycholesterol to Induce Apoptosis in Thymocytes
Authors: Zhang Jie , Xue Yintong , Jondal Mikael , Sjövall Jan
Published: July 2004

Oxygenated derivatives of sterols (oxysterols), including 25‐hydroxy cholesterol and 27‐hydroxycholesterol, have immunosuppressive effects. Oxysterols can directly induce apoptosis in immature thymocytes, cells which are inherently sensitive to induction of programmed cell death. For that reason, the metabolism of 25‐hydroxycholesterol and 27‐hydroxycholesterol in mouse thymus has been studied. When incubated with thymic tissue, both oxysterols were found to be 7α‐hydroxylated with subsequent oxidation to 7α‐hydroxy‐3‐oxo‐4 steroids. A minor fraction of 27‐hydroxycholesterol was also metabolised to 3β‐hydroxy‐5‐cholestenoic, 3β,7α‐dihydroxy‐5‐cholestenoic and 7α‐hydroxy‐3‐oxo‐4‐choleste‐noic acids. The 7α‐hydroxylase was found to be localised to the thymic epithelial cells and the reaction was stimulated by interleukin‐1β and inhibited by metyrapone and RU486. In contrast to 25‐hydroxycholesterol and 27‐hydroxycholesterol, the 7α‐hydroxylated metabolites, 7α,25‐dihydroxycholesterol, 7α,25‐dihydroxy‐4‐cholesten‐3‐one and 7α,27‐dihydroxy‐4‐cholesten‐3‐one did not induce thymocyte apoptosis. The results suggest that 7α‐hydroxylation may be of regulatory importance, possibly by protecting the developing thymocytes against toxic effects by oxysterols.
An Aspartic Endopeptidase is Involved in the Breakdown of Propeptides of Storage Proteins in Protein‐Storage Vacuoles of Plants
Authors: Hiraiwa Nagako , Kondo Maki , Nishimura Mikio , Hara‐Nishimura Ikuko
Published: July 2004

To understand the mechanism of the maturation of various proteins in protein‐storage vacuoles, we purified a 48‐kDa aspartic endopeptidase composed of 32‐kDa and 16‐kDa subunits from castor bean. Immunocytochemical and cell fractionation analyses of the endosperm of maturing castor bean seed showed that the aspartic endopeptidase was localized in the matrix of the protein‐storage vacuoles, where a variety of seed storage proteins were also present. The amount of the aspartic endopeptidase increased at the mid‐maturation stage of the seeds before accumulation of the storage proteins. To determine how the aspartic endopeptidase is responsible for maturation of seed proteins in concert with the vacuolar processing enzyme, we prepared 35S‐labeled proproteins of seed proteins from the endoplasmic reticulum fraction of pulse‐labeled maturing endosperm and used the authentic proproteins as substrates for in vitro processing experiments. The purified aspartic endopeptidase was unable to convert any of three endosperm proproteins, pro2S albumin, proglobulin, and proricin, into their mature sizes, while the purified vacuolar processing enzyme could convert all three proproteins. We further examined the activity of aspartic endopeptidase on the cleavage of an internal propeptide of Arabidopsis pro2S albumin, which is known to be removed post‐translationally. The aspartic endopeptidase cleaved the propeptide at three sites under acidic conditions. These results suggest that aspartic endopeptidase cannot directly convert pro2S albumin into the mature form, but it may play a role in trimming the C‐terminal propeptides from the subunits that are produced by the action of the vacuolar processing enzyme.
Influence of Divalent Cations on Inner‐Arm Mutants of Restriction Endonuclease EcoRI
Authors: Windolph Stephanie , Alves Juergen
Published: July 2004

To understand the functional role of the inner arm region of EcoRI for the interaction with its DNA substrate, we mutated each of the positively charged amino acid residues Lys130 and Arg131 to Ala or Glu. The resulting cleavage activities are only about tenfold reduced but DNA binding in the absence of divalent cations is totally disrupted for three of the mutants ([Ala130]EcoRI, [Glu130]EcoRI and [Glu131]EcoRI). The binding can be rescued by adding 1 mM Ca2+. This binding behaviour resembles that of the blunt end cutter EcoRV. Furthermore, the cleavage activity of the [Glu130]EcoRI is stimulated by Ca2+. Therefore, a second metal binding site is involved in [Glu130]EcoRI catalyzed DNA cleavage. Its location is postulated to be in the inner arm region at positions 133 and 135 presenting an Asp‐Xaa‐Asp motif typical for Ca2+ binding. A net positive charge at the tip of the inner arm due to the basic amino acids Lys130 and Arg131 in the wild‐type enzyme or due to binding of a divalent cation in the mutants is important for DNA recognition by EcoRI. However, a direct phosphate contact providing an indirect readout is not observed. Implications of the binding and cleavage behaviour with regard to mechanistic differences between blunt end and sticky end cutters and a general catalytic mechanism of restriction enzymes are discussed.
On the Mechanism of Biosynthesis of Divinyl Ether Oxylipins by Enzyme from Garlic Bulbs
Authors: Grechkin Alexander N. , Ilyasov Akhat V. , Hamberg Mats
Published: July 2004

The microsomal fraction of homogenate of garlic (Allium sativum L.) bulbs contains a divinyl ether synthase which catalyzes conversion of (9Z,11E,13S)‐13‐hydroperoxy‐9,11‐octadecadienoic acid and (9Z,11E,13S,15Z)‐13‐hydroperoxy‐9,11,15‐octadecatrienoic acid into (9Z,11E,1′E)‐12–(1′‐hexenyloxy)‐9,11‐dodecadienoic acid (etheroleic acid) and (9Z,11E,1′E,3′Z)‐12–(1′,3′‐hexadienyloxy)‐9,11‐dodecadienoic acid (etherolenic acid), respectively. Two isomers of etheroleic acid were isolated. As shown by NMR spectrometry, the double bond configurations of these compounds were (9E,11E,1′E) and (9Z,11Z,1′E). Experiments with linoleic acid (13R,S)‐hydroperoxide demonstrated that the S enantiomer was a much better substrate for the divinyl ether synthase compared to the R enantiomer. Incubation of (9Z,11E,13S)‐[18O2]hydroperoxy‐9,11‐octadecadienoic acid led to the formation of etheroleic acid which retained 18O in the ether oxygen. An intermediary role of an epoxyallylic cation in etheroleic acid biosynthesis is postulated.
Interactions between Ovine Cathepsin L, Cystatin C and α2‐Macroglobulin — Potential Role in The Genital Tract
Authors: Peloille Sylvie , Esnard Annick , Dacheux Jean‐Louis , Guillou Florian , Gauthier Francis , Esnard Frédéric
Published: July 2004

The specific inhibitor of cysteine proteinases, cystatin C, was purified from ram rete testis fluid and the conditioned medium of Sertoli cells. This molecule associated with sheep liver cathepsin L at one of the fastest rates ever described for a proteinase/inhibitor interaction (1.75±0.20×108M−1ċ s−1). But the association rate constant for the interaction of cathepsin L with α2‐macroglobulin, a non‐specific inhibitor of proteinases, was also extremely high (8.8±0.75×106M−1ċ s−1). Cathepsin L complexed with α2‐macroglobulin was protected from inhibition by type 2 and type 3 cystatins. The data indicate that cystatin C is the most potent inhibitor of cathepsin L in mammalian male genital tract fluids, whereas α2‐macroglobulin may act as a terminal acceptor of this enzyme. These inhibitors could therefore inhibit the activated form of procathepsin L which may appear during the complex process of spermatozoa production and maturation in the testis and epididymis.
Periplasmic Barl Protease of Saccharomyces Cerevisiae is Active Before Reaching its Extracellular Destination
Authors: Ballensiefen Wolfgang , Schmitt Hans Dieter
Published: July 2004

Saccharomyces cerevisiae MATa and MATα cells secrete α‐factor and α‐factor pheromones. These peptides act on cells of the opposite mating type. They induce physiological changes which allow the formation of diploid cells. MATa strains produce an extracellular protease which cleaves, and thus inactivates the MATα cell‐specific α‐factor pheromone. This pepsin‐like enzyme is encoded by the BAR1(SST1) gene and is secreted into the periplasmic space of MATa cells. We found that the Bar1p protease is already active in early compartments of the secretory pathway. Our results indicate that Bar1 protease tolerates large N‐terminal extensions of its substrate and does not require Golgi‐specific modifications such as outer‐chain glycosylation for activity.
Presence of UDP‐d‐Xylose:β‐D‐Glucoside α‐1,3‐D‐Xylosyltransferase Involved in the Biosynthesis of the Xylαl‐3Glcβ‐Ser Structure of Glycoproteins in the Human Hepatoma Cell Line HepG2
Authors: Omichi Kaoru , Aoki Kyoko , Minamida Satsuki , Hase Sumihiro
Published: July 2004

HepG2 cells were examined for the presence of UDP‐d‐xylose:β‐d‐glucoside α‐1,3‐xylosyltransferase activity by using 2‐[(2‐pyridyl)amino]ethyl β‐d‐glucopyranoside (Glcβ‐R) as a fluorogenic acceptor. UDP‐d‐xylose and the acceptor were incubated with the HepG2 microsomal fraction, and the enzymatic reaction mixture was analyzed by HPLC. Structural analysis of the fluorogenic product by α‐d‐xylosidase digestion, FAB‐MS, and Smith degradation revealed that it was Xylα1‐3Glcβ‐R, thus demonstrating the existence of β‐d‐glucoside α‐1,3‐xylosyltransferase. A solubilization study of the enzyme from the microsomal fraction indicated that it differed from UDP‐d‐xylose:α‐d‐xyloside α‐1,3‐xylosyltransferase of the HepG2 microsomal fraction producing Xylα1‐3Xylα1‐3Glc‐R′ [R′, (2‐pyridyl)amino‐] from UDP‐d‐xylose and Xylα1‐3Glc‐R′ [Minamida, S., Aoki, K., Natsuka, S., Omichi, K., Fukase, K., Kusumoto, S. & Hase, S. (1996)J. Biochem. (Tokyo) 120, 1002–1006]. The newly detected enzyme appears to be involved in the biosynthesis of the Xylα1‐3Glcβ‐Ser structure of glycoproteins such as human blood coagulation factors VII and IX.
Involvement of Phosphatidylinositol 3′‐Kinase in Stem‐Cell‐Factor‐Induced Phospholipase D Activation and Arachidonic Acid Release
Authors: Kozawa Osamu , Blume‐Jensen Peter , Heldin Carl‐Henrik , Rönnstrand Lars
Published: July 2004

We have shown previously that the stem cell factor (SCF) receptor undergoes phosphorylation on serine residues following ligand stimulation, and that this phopshorylation is dependent mainly on the activity of protein kinase C (PKC). In the present study, we have further investigated the molecular mechanisms behind SCF‐stimulated activation of PKC, and found that SCF does not activate phosphati‐dylinositol‐specific phospholipase C. In contrast, phospholipase D (PLD) is activated in response to SCF in a dose‐dependent manner. Activation of PLD was not inhibited by calphostin C, an inhibitor of PKC. On the other hand, inhibitors of phosphatidylinositol Ptdlns 3'‐kinase (Ptdlns 3'‐kinase), i.e. wortmannin and LY294002, inhibited SCF‐induced PLD activation. Moreover, a mutant SCF receptor in which Tyr721, which is responsible for activation of Ptdlns 3'‐kinase, is mutated to a phenylalanine residue was unable to mediate activation of PLD. Thus, PtdIns 3'‐kinase appears to be essential for SCF‐induced PLD activation. Furthermore, we demonstrate that phosphatidic acid (PtdH), generated through the action of PLD in response to SCF, is metabolized to diacylglycerol by dephosphorylation. Diacylglycerol can then activate PKC, and, moreover, after deacylation by a diacylglycerol lipase, yield arachidonic acid, an important second messenger in cell signaling.
Requirement for the Proton‐Pumping NADH Dehydrogenase I of Escherichia Coli in Respiration of NADH to Fumarate and Its Bioenergetic Implications
Authors: Tran Quang Hon , Bongaerts Johannes , Vlad Dorina , Unden Gottfried
Published: July 2004

In Escherichia coli the expression of the nuo genes encoding the proton pumping NADH dehydrogenase I is stimulated by the presence of fumarate during anaerobic respiration. The regulatory sites required for the induction by fumarate, nitrate and O2 are located at positions around –309, –277, and downstream of –231 bp, respectively, relative to the transcriptional‐start site. The fumarate regulator has to be different from the O2 and nitrate regulators ArcA and NarL. For growth by fumarate respiration, the presence of NADH dehydrogenase I was essential, in contrast to aerobic or nitrate respiration which used preferentially NADH dehydrogenase II. The electron transport from NADH to fumarate strongly decreased in a mutant lacking NADH dehydrogenase I. The mutant used acetyl‐CoA instead of fumarate to an increased extent as an electron acceptor for NADH, and excreted ethanol. Therefore, NADH dehydrogenase I is essential for NADH → fumarate respiration, and is able to use menaquinone as an electron acceptor. NADH → dimethylsulfoxide respiration is also dependent on NADH dehydrogenase I. The consequences for energy conservation by anaerobic respiration with NADH as a donor are discussed.
Mapping of Nuclease‐Sensitive Sites in Native Reticulocyte Ribosomes — An Analysis of the Accessibility of Ribosomal RNA to Enzymatic Cleavage
Authors: Holmberg Lovisa , Nygårrd Odd
Published: July 2004

Treatment of ribosomes in reticulocyte lysates with low concentrations of the calcium‐dependent nuclease from Staphylococcus aureus resulted in cleavage of rRNA. The positions of the cleaved phosphodiester bonds were localised by primer extension and polyacrylamide gel electrophoresis. S. aureus nuclease‐induced strand scissions were found in the 5′‐domain of 18S rRNA and in domains II, IV and VI of 28S rRNA. The majority of the cleavage sites were located in eukaryote‐specific expansion segments and only one cleavage site was found in a region suggested to be directly involved in ribosomal function. Treatment of the reticulocyte lysate with increasing amounts of S. aureus nuclease resulted in the introduction of new cleavage sites. However, even at the highest nuclease concentration used, large parts of the rRNAs were protected from nuclease digestion. Removal of translational components, by salt wash of isolated reticulocyte polysomes, exposed additional rRNA sequences to S. aureus nuclease cleavage. These sequences were found in the 3′‐major domain of 18S rRNA and in domains II, IV, and V of 28S rRNA. These sites are located at the putative translational surface of the ribosome. The translational activity of the S. aureus nuclease‐treated ribosomes, determined after addition of exogenous mRNA, was directly correlated to the extent of nuclease digestion of the ribosomes. However, the decrease in translational activity observed in lysates treated with low amounts of S. aureus nuclease was not due to a preferential exclusion of damaged ribosomes from polysome formation. This suggests that the induced cleavages were not detrimental to ribosomal function but could influence the rate of ribosomal movement along the mRNA.
The N‐terminal Region of α‐Dystroglycan is an Autonomous Globular Domain
Authors: Brancaccio Andrea , Schulthess Therese , Gesemann Matthias , Engel Jürgen
Published: July 2004

The structure of the N‐terminal region of mouse α‐dystroglycan (DGN) was investigated by expression of two protein fragments (residues 30–180 and 30–438) in Escherichia coli cells. Trypsin susceptibility experiments show the presence of a stable α‐dystroglycan N‐terminal region (approximately from residue 30 to 315). In addition, guanidinium hydrochloride (Gdn/HCl) denaturation of DGN‐(30–438)‐peptide, monitored by means of tryptophan fluorescence, produces a cooperative transition typical of folded protein structures. These results strongly suggest that the α‐dystroglycan N‐terminal is an autonomous folding unit preluding a flexible mucin‐like region and that its folding is not influenced by the absence of glycosylation. In order to obtain more information on the structural features of the N‐terminal domain we have also used circular dichroism, analytical sedimentation and electron microscopy analysis. Circular dichroic spectra show the absence of typical secondary structure (e.g. α‐helix or β‐sheet) and closely resemble those recorded for loop‐containing proteins. This is consistent with a sequence similarity of the α‐dystroglycan domain with the loop‐containing protein elastase. Analytical ultracentrifugation and electron microscopy analysis reveal that the N‐terminal domain has a globular structure. DGN‐(30–438)‐peptide does not bind in the nanomolar range to an iodinated agrin fragment which binds with high affinity to tissue purified α‐dystroglycan. No binding was detected also to laminin. This result suggests that the α‐dystroglycan N‐terminal domain does not contain the binding site to its extracellular matrix binding partners. It is less likely than the lack of glycosylation reduces its binding affinity, because the N‐terminal globular domain only contains two glycosylation sites.
Enhanced Stability of Urokinase‐Type Plasminogen Activator mRNA in Metastatic Breast Cancer MDA‐MB‐231 Cells and LLC‐PK1 Cells Down‐Regulated for Protein Kinase C — Correlation with Cytoplasmic Heterogeneous Nuclear Ribonucleoprotein C
Authors: Nanbu Rika , Montero Lilian , D'orazio Daniel , Nagamine Yoshikuni
Published: July 2004

In LLC‐PK1 cells, urokinase‐type plasminogen activator (uPA) mRNA has a short half‐life of 70 min. We have previously demonstrated that most of the regulatory regions responsible for the rapid turnover of uPA mRNA in LLC‐PK1 cells reside in its 3′ untranslated region (3′ UTR), where there are at least three regulatory sites, one of which is A+U‐rich. This A+U‐rich sequence mediates uPA mRNA stabilization induced by protein kinase C (PKC) down‐regulation. In this work, we found that uPA mRNA is rather stable in MDA‐MB‐231 cells with a half‐life of 17 h. We compared the stability of hybrid globin mRNA containing different parts of uPA mRNA in its 3′ UTR and found that the A+U‐rich sequence of uPA mRNA renders otherwise stable globin mRNA unstable in LLC‐PK1 cells but not in MDA‐MB‐231 cells. We identified a cytoplasmic protein of 40 kDa (p40) which specifically interacts with the A+U‐rich sequence. Levels of p40 activity as detected by ultraviolet cross‐linking were higher in MDA‐MB‐231 and PKC‐down‐regulated LLC‐PK1 cells than in untreated LLC‐PK, cells. Prior treatment of the cytoplasm with a specific antibody against heterogeneous nuclear ribonucleoprotein C (hnRNP C) significantly reduced p40 activity. These results suggest a correlation between the A+U‐rich sequence‐dependent uPA mRNA stabilization in vivo and the binding of hnRNP C to the A+U‐rich sequence in vitro.
Mutational Analysis of Extracellular Cysteine Residues of Rat Secretin Receptor Shows that Disulfide Bridges are Essential for Receptor Function
Authors: Vilardaga Jean‐Pierre , Di Paolo Emmanuel , Bialek Christopher , De Neef Philippe , Waelbroeck Magali , Bollen Alex , Robberecht Patrick
Published: July 2004

We attempted to express point‐mutant secretin receptors where each of the 10 extracellular Cys residues was replaced by a Ser residue, in Chinese hamster ovary (CHO) cells. Six of the point‐mutant receptors (C24→S, C44→S, C53→S, C67→S, C85→S and C101→S) could not be detected by binding or functional studies: the mutations resulted in functional inactivation of the receptor. In contrast, the four other point‐mutant receptors (C11→S, C186→S, C193→S and C263→S) were able to bind poorly 125I‐secretin, and to activate adenylate cyclase with high secretin EC50 values. These results suggest that cysteine residues 24, 44, 53, 67, 85 and 101 are necessary for receptor function, and that the two putative disulfide bridges formed by cysteine residues 11, 186, 193 and 263 are functionally relevant, but not essential for receptor expression. Secretin activated the adenylate cyclase through the quadruple mutant (C11,186,193,263→S), the four triple mutants, and through double mutants C186,193→S and Cl86,263→S with a very high (μM) EC30 value, suggesting that, in the wild‐type receptor, disulfide bridges are formed between C11–C186, and between C193–C263. Prior treatment with dithiothreitol resulted in a marked EC50 increase of the wild‐type receptor and of those receptors with at least the two cysteine residues in positions 11 and 186, suggesting that the C11–C186 (but not the C193–C263) disulfide bridge was accessible to this reducing agent. Several results nevertheless indicated that, in mutant receptors, alternative disulfide bridges can be formed between cysteine 186 and cysteine 193 or 263, suggesting that these three residues are in close spatial proximity in the wild‐type receptor.
Role of the Stimulatory Proteins Sp1 and Sp3 in the Regulation of Transcription of the Rat Pyruvate Kinase M Gene
Authors: Netzker Roland , Weigert Cora , Brand Karl
Published: July 2004

Site‐directed mutagenesis of cis‐regulatory elements in the 5′ flanking region of the rat pyruvate kinase M gene revealed that two out of the three GC boxes (–1337–124 and–48/–39) are involved in the stimulation of a core promoter (–35/+46). These two regions were also protected in DNasel foot‐printing assays. Sp1 and Sp3 were identified as binding proteins to all three GC boxes by supershift experiments.

Cotransfections in Drosophila SL2 cells revealed a strong stimulatory function of Sp1 and a synergistic effect of Sp3 to Sp1 in the activation of the pyruvate kinase M promoter. No inhibitory effect of Sp3 was detected.

These data indicate that binding of Sp1 at two GC boxes is required for full promoter activity of the pyruvate kinase M gene and thus contributes to the observed cell‐cycle‐dependent expression of this enzyme in proliferating rat thymocytes.
Evolution of The Aminoacyl‐tRNa Synthetase Family and The Organization of the Drosophila Glutamyl‐Prolyl‐tRNA Synthetase Gene — Intron/Exon Structure of the Gene, Control of Expression of the Two mRNAs, Selective Advantage of the Multienzyme Complex
Authors: Cerini Claire , Semeriva Michel , Gratecos Danielle
Published: July 2004

In Drosophila, glutamyl‐prolyl‐tRNA synthetase is a multifunctional synthetase encoded by a unique gene and composed of three domains: the amino‐ and carboxy‐terminal domains catalyze the aminoacylation of glutamic acid and proline tRNA species, respectively, and the central domain is made of 75 amino acids repeated six times amongst which 46 are highly conserved and constitute the repeated motifs [Cerini, C., Kerjan, P., Astier, M., Gratecos, D., Mirande, M. & Sémériva, M. (1991) EMBO J. 10, 4267–4277].

The intron/exon organization of the Drosophila gene reveals the presence of six exons among which four are in the 5′‐end encoding glutamic acid activity. Only one exon encodes the repeated motifs. A comparison of introns positions, intron classes and intron/exon boundaries in the Drosophila gene and in its human counterpart is compatible with the intron‐early hypothesis presiding, at least in part, to the evolution of the synthetases.

The full‐length fly protein is encoded by a 6.1‐kb mRNA which is expressed throughout development. In addition, a shorter transcript encompasses the 3′‐end of the cDNA and it is especially abundant in 5–10‐h embryos until the first larval stage. Expression of these two mRNAs seems to be controlled by two independent promoters. The 6.1‐kb mRNA promoter is probably localized in the 5′‐end of the gene. The small mRNA promoter resides in the 4th intron and evidence is provided that the mRNA encodes only the domain corresponding to prolyl‐tRNA synthetase and is functional in vivo.

Finally, transgenic flies have been established by using constructs corresponding to the three domains of the protein. Overexpression of the repeated motifs leads to a sterility of the flies that suggests a role of these motifs in linking the multienzyme complex to an, as yet, unknown structure of the protein synthesis apparatus.
The Complete cDNA Sequence and Expression of the First Major Allergenic Protein of Malassezia Furfur, Mal f 1
Authors: Schmidt Margit , Zargari Arezou , Holt Palle , Lindbom Lars , Hellman Ulf , Whitley Paul , Van Der Ploeg Ingeborg , Härfast Bengt , Scheynius Annika
Published: July 2004

For the first time the complete cDNA encoding a major allergen and novel protein of the yeast Malassezia furfur, Mai f 1, has been sequenced and expressed. The amino acid sequences of nine tryptic peptides of the protein were determined. Oligonucleotides were designed from these amino acid sequences. The cDNA sequence was obtained by hybridizing these primers to mRNA and enhancement by reverse‐transcriptase PCR techniques. The cDNA is 1176 bp in length. It shows an open reading frame of 1050 bp coding for a protein of 38178 Da and a deduced amino acid sequence containing 350 residues. The hydropathy plot and the tryptic digest indicate that the first 22 amino acids represent a leader sequence determining a mature protein of 35988 Da. The complete encoding cDNA was expressed as a maltose‐binding protein fusion protein in Escherichia coli. The recombinant fusion protein reacted with our specific monoclonal antibody and with IgE from patients with atopic dermatitis.
Molecular Cloning of the Hamster CMP‐sialic Acid Transporter
Authors: Eckhardt Matthias , Gerardy‐Schahn Rita
Published: July 2004

Chinese hamster ovary (CHO) glycosylation mutants of the Lec2 complementation group are unable to express sialylated glycoproteins and glycolipids due to a defect in the Golgi CMP‐sialic acid transporter (CMP‐Sia‐Tr). Using an expression cloning strategy, we isolated a cDNA encoding the hamster CMPSia‐Tr which complements the Lec2 phenotype. The deduced amino acid sequence of the cloned cDNA shows 95% identity to the recently cloned murine CMP‐Sia‐Tr. The expression of a hamster CMP‐Sia‐Tr fusion protein with an N‐terminal MDYKDDDDK (FLAG) sequence revealed Golgi localisation of the transporter. Amino acid sequence comparison revealed strong similarity (44.6% identity and 19.3% similarity) of CMP‐Sia‐Tr to the recently cloned human UDP‐galactose transporter (UDP‐Gal‐Tr). In contrast, sequence similarities to the yeast UDP‐N‐acetylglucosamine transporter (UDP‐GlcNAc‐Tr) and the GDP‐mannose transporter (GDP‐Man‐Tr) of Leishmania donovani are restricted to a region encoding the two most C‐terminally located transmembrane helices. A computer‐based structural analysis of CMPSia‐Tr proposes an eight transmembrane helix model with the N‐ and C‐termini located on the cytosolic side of the Golgi membrane.
Genetic and Biochemical Characterization of the Broad Spectrum Chlorobenzene Dioxygenase from Burkholderia Sp. Strain PS12 — Dechlorination of 1,2,4,5‐Tetrachlorobenzene
Authors: Beil Stefan , Happe Birgitta , Timmis Kenneth Nigel , Pieper Dietmar Helmut
Published: July 2004

The bacterium, Burkholderia (previously Pseudomonas) sp. strain PS12, reported earlier to degrade 1,2,4‐trichlorobenzene is shown here to utilize also 1,2,4,5‐tetrachlorobenzene (Cl4‐benzene) as a growth substrate. To investigate the possibility that this organism attacks Cl4‐benzene with a chlorobenzene dioxygenase which concomitantly causes dehalogenation, and to analyze the substrate range of the initial enzyme, a 5503‐bp DNA fragment from PS12, exhibiting high similarity to genes coding for class IIB dioxygenases, was cloned and expressed in Escherichia coli. The sequence includes the tec genes coding for the α‐subunit and β‐subunit of a terminal dioxygenase, a ferredoxin and a reductase. E. coli cells producing these proteins were able to dioxygenolytically attack a range of aromatic compounds including chlorinated benzenes and toluene, and also dinuclear aromatics such as biphenyl and dibenzo‐p‐dioxin. The enzyme was shown by 18O2 incorporation experiments to dioxygenolytically attack a chlorosubstituted carbon atom of Cl4‐benzene, thereby forming an unstable diol intermediate which spontaneously rearomatizes with concomitant chloride elimination to the corresponding 3,4,6‐trichlorocatechol (Cl3‐catechol).
Sequence, Structure and Evolution of the Ecdysone‐InducibleLsp‐2Gene ofDrosophila Melanogaster
Authors: Mousseron‐Grall Sylvie , Kejzlarová‐Lepesant Jana , Burmester Thorsten , Chihara Carol , Barray Martine , Delain Etienne , Pictet Raymond , Lepesant Jean‐Antoine
Published: July 2004

TheLsp‐2gene encodes a major larval serum protein (hexamerin) ofDrosophila melanogaster. Transcription ofLsp‐2is controlled by 20‐hydroxyecdysone. Here we report the analysis of the structure of theLsp‐2gene including the adjacent 5′ and 3′ sequences. In contrast to all other known hexamerin genes,Lsp‐2does not contain an intron. TheLsp‐2mRNA measures 2312 bases, as deduced from experimental determination of the transcription‐start and stop sites and conceptual translation results in a 718 amino acid hexamerin subunit, including a 21‐amino‐acid signal peptide. While the calculated molecular mass of the native 697‐amino‐acid subunit is 83.5 kDa, mass spectrometry gave a value of 74.5 kDa. We detected in theLsp‐2gene a 2052‐bp antisense ORF that probably does not code for any protein. An unusual accumulation of rarely used codon triplets was found at the 5′ and 3′ ends of theLsp‐2ORF. The calculated secondary structure matches well with that of arthropod hemocyanins. Electron micrographs show for LSP‐2 hexamers a cubic shape, which can not be easily reconciled with its hexameric structure. Phylogenetic analysis revealed that LSP‐2 diverged from the LSP‐1‐like hexamerins after separation of the Diptera from other insect orders.