bluuebunny/crossref_metadata_2025 huggingface dataset:
The sex pheromone system of Enterococcus faecalis
Authors: WIRTH Reinhard
Published: March 2005

The sex pheromone system of Enterococcus faecalis was discovered by observing a clumping reaction of E. faecalis strains during conjugative transfer of plasmids. It was found that only a special type of E. faecalis plasmids, the so‐called sex pheromone plasmids, are transferred via this mechanism. Various experiments, especially by the group of D. B. Clewell, led to the formulation of a model describing how the sex pheromone system works. Small linear peptides, the so‐called sex pheromones, are excreted by strains not possessing the corresponding sex pheromone plasmid. Donor strains harboring the plasmid do not produce the corresponding sex pheromone; they react to the presence of the peptide by production of a plasmid‐encoded adhesin, the so‐called aggregation substance. This adhesin allows contact between the non‐motile mating partners; after conjugative transfer of the plasmid, the former recipient possesses and replicates the new plasmid. Thereby the population of E. faecalis strains is shifted to a high percentage of donor strains. This is especially true because a donor strain will still excrete sex pheromones corresponding to plasmids it does not harbor; therefore, such a strain can also function as recipient for other sex pheromone plasmids it does not possess.

Various aspects of this unique plasmid collection mechanism have been studied during the last few years. The data indicate that, with the exception of pAM373, all sex pheromone plasmids possess one DNA region which is highly similar to and codes for the adhesin. It is also becoming more and more clear that regulatory functions/proteins are not conserved between different sex pheromone plasmids. Induction of adhesin synthesis needs the action of a regulatory cascade composed of unique features; at the moment we are just beginning to understand this cascade. By sequencing the first structural gene for one of those adhesins, we realized that the aggregation substance might act also as an adhesin for eucaryotic cells, probably by interaction with integrins. At least in the case of the in vitro cultured pig kidney tubulus cell line LLC‐PK1 this idea could be verified. An interesting aspect of the sex pheromone system of E. faecalis is its evolution. I will discuss the idea that two different components, both of which well might contribute to virulence of the opportunistic pathogenic bacterium, were combined in the species E. faecalis to result in this unique plasmid collection system.
Homology modelling of the catalytic domain of human furin
Authors: SIEZEN Roland J. , CREEMERS John W. M. , VAN DE VEN Wim J. M.
Published: March 2005

A model is presented for the three‐dimensional structure of the catalytic domain of the human serine proteinase furin and its interaction with model substrates. This homology model is based on the crystal structures of subtilisin BPN' and thermitase in complex with the inhibitor eglin, and it also applies to other members of the eukaryotic subtilisin‐like proprotein convertases. Predictions are made of the general protein fold, inserted loops, disulfide bonds, Ca2+‐binding sites and salt bridges. A detailed prediction of the substrate‐binding region attempts to explain the basis of specificity for multiple basic residues preceding the cleavage site. Specific acidic residues in the S1, S2 and S4 subsites of the substrate‐binding region of furin are identified which appear to be of particular importance, while residues of the S2′, S3, S5 and S6 subsites may also contribute to substrate binding.

Based on this model, protein engineering can be employed not only to test the predicted enzyme‐substrate interactions, as demonstrated for human furin, but, equally importantly, to design proprotein convertases with a desired specificity, or to design novel substrates or inhibitors.
Purification and properties of an α‐methylacyl‐CoA racemase from rat liver
Authors: SCHMITZ Werner , FINGERHUT Ralph , CONZELMANN Ernst
Published: March 2005

The (R)‐ and (S)‐isomers of α‐methyl‐branched fatty acids were shown to be rapidly interconverted as coenzyme A thioesters, by an α‐methylacyl‐CoA racemase. The enzyme was purified some 5600‐fold from rat liver, to apparent homogeneity. It is a monomer of 45 kDa with an isolectric point of pH 6.1 and is optimally active between pH 6 and pH 7. It acts only on coenzyme A thioesters, not on free fatty acids, and accepts as substrates a wide range of α‐methylacyl‐CoAs, including pristanoyl‐CoA and trihydroxycoprostanoyl‐CoA (an intermediate in bile acid synthesis), but neither 3‐methyl‐branched nor linear‐chain acyl‐CoAs. The racemase catalyzes a rapid exchange of the H atom in the α‐position of the fatty acid against a proton from water, indicating that the mechanism involves abstraction of a proton. Based on this observation, a very sensitive and convenient radiometric assay, with 2‐methyl[2‐3H]acyl‐CoAs as substrates, was developed. The enzyme was inactivated by micromolar concentrations of Hg2+ and to a lesser extent by Cu2+ but not by iodoacetamide and only slightly by N‐ethylmaleimide and thimerosal.
Botulinum A and the light chain of tetanus toxins inhibit distinct stages of Mg · ATP‐dependent catecholamine exocytosis from permeabilised chromaffin cells
Authors: LAWRENCE Gary W. , WELLER Ulrich , DOLLY J. Oliver
Published: March 2005

Susceptibilities of Mg · ATP‐independent and Mg · ATP‐requiring components of catecholamine secretion from digitonin‐permeabilised chromaffin cells to inhibition by Clostridial botulinum type A and tetanus toxins were investigated. These toxins are Zn2+‐dependent proteases which specifically cleave the 25‐kDa synaptosomal‐associated protein (SNAP‐25) and vesicle‐associated membrane protein (VAMP) II, respectively. When applied to permeabilised chromaffin cells they rapidly inhibited secretion in the presence of Mg · ATP but the catecholamine released in the absence of Mg · ATP, thought to represent fusion of primed granules, was not perturbed. The toxins can exert their effects per se in the absence of the nucleotide complex; therefore, Mg · ATP‐requiring steps of secretion are implicated as roles for their targets. Primed release was lost rapidly after permeabili‐sation of the cells but could be maintained by including Mg · ATP during the incubation before stimulating release with Ca2+. This ability of Mg · ATP to maintain primed release was only partially inhibited by botulinum neurotoxin A whereas it was abolished by tetanus toxin, consistent with the distinct substrates for these toxins. This study reveals a component of release within which these proteins are either resistant to cleavage by these toxins or in such a position that degradation can no longer prevent granule fusion. Differences in the steps of release at which these toxins can affect inhibition are also revealed.
A few amino acid substitutions are responsible for the higher thermostability of a novel NAD+‐dependent bacillar alcohol dehydrogenase
Authors: CANNIO Raffaele , ROSSI Mosè , BARTOLUCCI Simonetta
Published: March 2005

The gene adh‐hT encoding a thermostable and thermophilic NAD+‐dependent alcohol dehydrogenase (ADH) from the novel and more thermophilic Bacillus stearothermophilus LLD‐R strain was cloned and its nucleotide sequence determined. The deduced protein sequence shows remarkable amino acid substitutions when compared to the sequence of the protein isolated from strain NCA1503 and significant similarity with the highly thermostable ADH from the thermoacidophilic archaebacterium Sulfolobus solfataricus. The alignment of these sequences led to the identification of three amino acid replacements probably responsible for the higher thermostability of the novel bacillar ADH.

Adh‐hT gene expression in Escherichia coli, a fast purification procedure and the characterization of the recombinant enzyme are also described.
Mass spectrometric analysis of human soluble catechol O‐methyltransferase expressed in Escherichia coli
Authors: VILBOIS Francis , CASPERS Patrick , DA PRADA Mosé , LANG Gabrielle , KARRER Catherine , LAHM Hans‐Werner , CESURA Andrea M.
Published: March 2005

Technological advances in the field of mass spectrometry (MS) are providing powerful analytical means for the investigation of proteins and peptides. In the present work we have used pneumatically assisted electrospray (ion‐spray) MS for the biochemical characterization of recombinant human catechol O‐methyltransferase (rhCOMT). hCOMT could be expressed in Escherichia coli in large quantities but in two forms of different size, both enzymically active. Electrospray MS analysis showed that the smaller rhCOMT protein had a molecular mass of 24352±2Da, corresponding to the calculated value for native hCOMT (without the initiating methionine), whereas that mass of the larger protein was of 25775±4Da.

To investigate the molecular differences between the two proteins, they were digested with trypsin and the peptides produced analysed by electrospray MS. Neither protein apparently contained disulfide bridges and the observed molecular masses of the tryptic peptides corresponded to the calculated values. It was possible to determine, however, that the larger protein contained an extended C‐terminus with the correct sequence GPGSEAGP plus an additional stretch, EDLR. This C‐terminal extension resulted from ribosomal frameshift at the codon of the last proline (CCC, rare codon in prokaryotes). In fact, rightward frameshifting would produce a hCOMT form with an additional stretch of 11 amino acid (EDLRSHHHHHH) and the calculated molecular mass of this protein (25773.5Da) is in good agreement with our experimental result.

The differential reactivity of the cysteine residues of the correct rhCOMT enzyme, in the presence and in the absence of S‐adenosyl‐L‐methionine (AdoMet) and MgCl2, was also studied. 5‐Iodoacetamido fluorescein (5‐IAF) was used as thiol‐modifying reagent. Under the conditions used, 5‐IAF rapidly inactivated rhCOMT but the presence of AdoMet and MgCl2 partially protected it from inactivation. The 5‐IAF‐labeled tryptic peptides were separated by HPLC and then submitted to electrospray MS and tandem MS. Several cysteine residues appeared to be readily available to chemical modification by 5‐IAF. Incorporation of 5‐IAF occurred to a larger extent into Cys32, Cys68, Cys94 and Cys172. AdoMet and MgCl2 markedly reduced the label incorporation into Cys68 and Cys94, therefore suggesting that these residues belong to a region at or near the binding site of AdoMet.
Enhancement of the reductive activation of chloroplast fructose‐1,6‐bisphosphatase by modulators and protein perturbants
Authors: BALLICORA Miguel A. , WOLOSIUK Ricardo A.
Published: March 2005

To characterize the mechanism of chloroplast fructose‐1,6‐bisphosphatase activation, we have examined kinetic and structural changes elicited by protein perturbants and reductants. At variance with its well‐known capacity for enzyme inactivation, 150 mM sodium trichloroacetate yielded an activatable chloroplast fructose‐1,6‐bisphosphatase in the presence of 1.0 mM fructose 1,6‐bisphosphate and 0.1 mM Ca2+. Other sugar bisphosphates did not replace fructose 1,6‐bisphosphate whereas Mg2+ and Mn2+ were functional in place of Ca2+. Variations of the emission fluorescence of intrinsic fluorophores and a noncovalently bound extrinsic probe [2‐(P‐toluidinyl)naphthalene‐6‐sulfonate] indicated the presence of conformations different from the native form. A similar conclusion was drawn from the analysis of absorption spectra by means of fourth‐derivative spectrophotometry. The effect of these conformational changes on the reductive process was studied by subsequently incubating the enzyme with dithiothreitol. The reaction of chloroplast fructose‐1,6‐bisphosphatase with dithiothreitol was accelerated 13‐fold by the chaotropic anion: second‐order rate constants were 48.1 M−1· min−1 and 3.7 M−1· min−1 in the presence and in the absence of trichloroacetate, respectively. Thus, the enhancement of the reductive activation by compounds devoid of redox activity illustrated that the modification of intramolecular noncovalent interactions of chloroplast fructose‐1,6‐bisphosphatase plays an essential role in the conversion of enzyme disulfide bonds to sulfhydryl groups. In consequence, a conformational change would operate concertedly with the reduction of disulfide bridges in the light‐dependent activation mediated by the ferredoxin–thioredoxin system.
Electrostatic effects in trypsin reactions
Authors: TÕUGU Vello , TIIVEL Toomas , TALTS Peeter , ŠIKŠNIS Virginius , POYARKOVA Svetlana , KESVATERA Tõnu , AAVIKSAAR Aavo
Published: March 2005

The influence of inorganic salts on trypsin‐catalyzed reactions has been studied. It is shown that: (a) monovalent cations are reversible competitive inhibitors of tryptic hydrolysis of cationic substrates, whereas their binding has no effect on the reaction of neutral substrates; (b) a nonelectrostatic salt effect on the binding of both cationic and non‐ionic substrates is caused by changes in the thermodynamic activity coefficient of the substrate; (c) the rate of trypsin active‐site acylation is not affected by inorganic salts with monovalent cations. The data suggest that low‐molecular‐mass substrates are extracted into the enzyme microphase during substrate binding and further chemical transformations proceed without an access from surrounding medium. It is proposed that formation of a properly oriented dipole in the trypsin binding pocket by the cationic group of the substrate and Asp189 carboxyl is responsible for the elevated acylation rate of trypsin active site by substrates containing lysine and arginine. Introduction of additional negative charges into the enzyme molecule by chemical modification of lysyl residues by pyromellitic anhydride increased the specificity of trypsin towards cationic substrates and inhibitors. Lysine residues are therefore considered as suitable targets for site‐directed mutagenesis aimed at the improvement of selectivity and catalytic properties of trypsin.
Regulation of protein kinase A subunits during germination of Mucor rouxii sporangiospores
Authors: ROSSI Silvia , MORENO Silvia
Published: March 2005

Levels of protein kinase A (PKA) subunits and of cAMP have been measured during aerobic germination of the sporangiospores of the dimorphic fungus Mucor rouxii; further, the holoenzyme and its catalytic (C) and regulatory (R) subunits have been visualized through sucrose gradient centrifugation. Sporangiospores contain around 0.06 μM of a dimeric holoenzyme species of 5.5 S and a sixfold excess of a free R subunit of 2.7 S. Both these species are proposed to be derived by proteolysis from the native forms. Enzymic activity at this stage is highly inhibited, as demonstrated with permeabilized cells. Immediately upon germination, and after a transient increase in cAMP concentration from 10 μM to 90 μM, C‐subunit levels fall to 30%. After the onset of germination, the specific activity and concentration of both the 5.5 S holoenzyme species and the 2.7 S species of free R subunit decrease in parallel to the increase in total protein and volume. Net synthesis of C and R subunits to form a native holoenzyme species of 8.8 S is apparent 4 h onwards after germination. A significant increase in cellular concentration is observed at 6 h. At 7 h of growth, when germ‐tube emission is complete, the holoenzyme concentration is around 0.23 μM; there is almost no free R subunit and the intracellular concentration of cAMP is around 3 μM. A role for PKA during germination and morphogenesis is discussed.
Regulation of the apolipoprotein AIV gene expression by estrogen differs in rat and mouse
Authors: SRIVASTAVA Rai Ajit K. , KITCHENS Robert Tom , SCHONFELD Gustav
Published: March 2005

Previously we have shown that estrogen administration to Sprague Dawley rats and to the inbred C3H/HeJ mouse strain produced different effects on plasma lipoproteins [Srivastava, R. A. K., Baumann, D. & Schonfeld, G. (1993) Eur. J. Biochem. 216, 527–538]. While low‐density lipoprotein (LDL) levels fell in rats, they rose in mice. Plasma apoprotein (apo) AI levels and high‐density lipoprotein (HDL) cholesterol fell in both species but by much less in mice than in rats. Since apolipoproteins AIV and AII are two other protein constituents of HDL, we wished to test the hypothesis that estrogen would produce different effects on these apoproteins in mice and rats. Male rats and C3H/HeJ mice were administered 17β‐estradiol at 5 μg · g body mass−1· day−1 for six consecutive days. In a separate experiment, castrated male C3H/HeJ mice were administered β‐estradiol [(0.16 μg · g body mass−1· day−1 or 5.0 μg · g body mass−1· day−1, or testosterone (1 μg/g)] for 14 days. ApoAIV mRNA levels were determined in total liver, in liver nuclei and in total intestine. Rat hepatic apoAIV mRNA decreased twofold (from 16.5±3pg/μg total RNA to 7.1±2.5 pg/μg total RNA) while mouse hepatic and nuclear apoAIV mRNA both increased 1.5–2‐fold. Intestinal apoAIV mRNA decreased in mice and increased in rats. Testosterone had no effects. Nuclear apoAIV mRNA transcription rates in rat and mouse liver changed little, if at all, indicating that estrogen‐induced changes in steady‐state levels of apoAIV mRNA were not determined by hepatic transcriptional mechanisms. Both species possessed similar apoAIV mRNA transcription start sites. To assess whether other mouse strains also differed from rats, we surveyed 13 other inbred mouse strains. Some strains increased hepatic apoAIV mRNA, some did not change but, in contrast to rat, no strain experienced a fall in mRNA levels. Estrogen‐induced changes in plasma apoAIV levels were not correlated with changes in the levels of hepatic apoAIV mRNA levels. These data indicate that (a) apoAIV mRNA levels are regulated differently by estrogen in mouse and rat livers and intestines, (b) regulation of apoAIV mRNA by estrogen is both mouse strain and tissue specific and (c) regulation of plasma apoAIV is achieved by mechanisms other than those depending on the steady‐state levels of hepatic apoAIV mRNA.

In contrast with apoAIV mRNA, estrogen decreased hepatic apoAII mRNA both in rat (threefold) and in mouse (twofold) and parallel changes were observed in transcription rates. Thus, estrogen‐mediated regulation of ApoAII mRNA levels occurred similarly in the two rodent species, mainly by transcriptional mechanisms.
The purification of early‐pregnancy factor to homogeneity from human platelets and identification as chaperonin 10
Authors: CAVANAGH Alice C. , MORTON Halle
Published: March 2005

Early‐pregnancy factor (EPF), first discovered in the early stages of gestation, is associated with and necessary for cell proliferation in a wide variety of biological situations. Like many other growth factors, EPF is present in platelets, and, by titration studies with a neutralising anti‐EPF monoclonal antibody, platelets were identified as an extremely rich source of this growth factor. EPF has been purified from clinically outdated human platelets by heat extraction, ion‐exchange and affinity chromatographies on SP‐Sephadex and heparin‐Sepharose respectively, high‐performance hydrophobic interaction chromatography and three reverse‐phase HPLC steps, with an average yield of 15 μg/100 platelet units (equivalent to ∼ 50 1 blood). Using SDS/PAGE, EPF migrated as a single band with approximate Mr 8500, coincident with biological activity. Mass spectrometry provided an accurate and precise determination of the molecular mass as Mr 10843.5±2, along with definitive evidence of the homogeneity of the preparation. Attempts at Edman degradation indicated that the molecule was blocked at the N‐terminus and sequencing of proteolytic fragments was undertaken. The amino acid sequence of approximately 70% of the molecule was determined which, with a single exception, is identical with rat chaperonin 10. This structural relationship was shown to extend to functional identity by studies using chaperonin 10 and its functional associate chaperonin 60. Investigations with the latter confirmed that chaperonin 10 is the moiety in pregnancy serum which initiates response in the EPF bioassay. Our studies identify EPF as a member of the highly conserved heat‐shock family of molecules and demonstrate a molecular chaperone performing an extracellular role.
Transport of C4‐dicarboxylates by anaerobically grown Escherichia coli
Authors: ENGEL Peter , KRÄMER Reinhard , UNDEN Gottfried
Published: March 2005

Transport activities for uptake, efflux and exchange of C4‐dicarboxylates were observed in anaerobically grown Escherichia coli. All three transport modes were only present in strains containing the transcriptional activator FNR of anaerobic respiration, and were repressed by nitrate and O2. The kinetic and energetic parameters of C4‐dicarboxylate transport and the mechanism of the uptake, efflux and exchange reactions were analyzed in whole cells and in membrane vesicles.

Fumarate/succinate exchange could be characterized as homologous or heterologous 1:1 counter‐exchange. The external substrate was determined as divalent fumarate2‐ (or succinate2‐) at pH 6–9, whereas monovalent H‐fumarate dominated as the substrate at pH 3–4. The exchange was not inhibited by dissipation of p or constituents of it (Ψ or pH). We conclude that this transport mode functions as an electroneutral exchange of C4‐dicarboxylates. The uptake of C4‐dicarboxylates did not depend on internal counter‐substrate and resulted in an accumulation of the substrate. Similar to antiport, fumarate was accepted in the divalent form at pH values greater than or equal to 6 and in the monovalent form at pH 3.5–6. The uptake was inhibited by dissipation of p or Ψ. Artificially imposed pH, Ψ or fumarate gradients were able to drive fumarate uptake. An involvement of Na+ could not be detected. Thus the uptake is likely to operate as an electrophoretic H+/fumarate symport.

Independent of the presence of an external counter‐substrate, the substrates were secreted from cells or membrane vesicles loaded with succinate or fumarate. The efflux was electrogenic. Energizing the cells or membrane vesicles inhibited efflux, maximal efflux rates were obtained only after dissipation of p or Ψ. An imposed K+‐diffusion potential (outside positive) inhibited succinate excretion. The efflux of succinate from de‐energized membrane vesicles generated a Ψ of –70 mV. It is thus suggested that succinate efflux functions as a H+/succinate symport.
The genetic interaction of kar2 and wbp1 mutations
Authors: TE HEESEN Stephan , AEBI Markus
Published: March 2005

The endoplasmic binding protein BiP and N‐linked glycosylation are proposed to be essential components in the processing pathway of secreted protein. In Saccharomyces cerevisiae, BiP is encoded by the KAR2 gene; WBP1 encodes an essential component of the N‐oligosaccharyltransferase complex. wbp1 mutations result in reduced oligosaccharyltransferase activity and a temperature‐sensitive phenotype. We show that a combination of kar2 and wbp1 mutations results in a synthetic phenotype with a strongyl reduced growth rate at the permissive temperature. To investigate the role of N‐linked glycosylation in BiP function, the processing of non‐glycosylated carboxypeptidase was followed in different kar2 strains at the permissive temperature. In all kar2 strains, the processing of non‐glycosylated carboxypeptidase Y was drastically reduced. A specific BiP/non‐glycosylated carboxypeptidase Y complex was detected in kar2‐159 and kar2‐203 cells whereas the kar2‐1 mutation did not result in such a complex. Our data show that BiP and N‐linked glycosylation are directly involved in the processing of secreted proteins. The results support the hypothesis that BiP stabilizes the folding‐competent and assembly‐competent state of a polypeptide, whereas N‐linked oligosaccharides are structural components required in the folding process after the polypeptide is released from BiP.
Three‐dimensional crystal structure of the A‐tract DNA dodecamer d(CGCAAATTTGCG) complexed with the minor‐groove‐binding drug Hoechst 33258
Authors: VEGA M. Cristina , GARCÍA SÁEZ Isabel , AYMAMÍ Joan , ERITJA Ramón , VAN DER MAREL Gijs A. , VAN BOOM Jaques H. , RICH Alexander , COLL Miquel
Published: March 2005

The molecular structure of the DNA A‐tract dodecamer d(CGCAAATTTGCG) complexed with the drug Hoechst 33258 has been determined by X‐ray diffraction analysis. The Hoechst molecule binds in the DNA minor groove covering the sequence AATTT of the central A‐tract, with the piperazine group close to one of the GC regions. The drug molecule makes two three‐centered hydrogen bonds from the nitrogen atoms of the benzimidazole rings to the N3 and O2 atoms of the DNA bases. Although a high propeller twist is observed in the A‐tract, only one unsymmetrical three‐centered hydrogen bond is present in the DNA major groove. The structure is compared with other minor‐groove‐binding drug complexes and the influence of these drugs on DNA A‐tracts is discussed.
Molecular cloning of a novel luteinizing‐hormone/ human‐chorionic‐gonadotropin‐receptor cDNA
Authors: LU Deborah L. , MENON K. M. J.
Published: March 2005

The biological action of luteinizing hormone/human chorionic gonadotropin (lutropin/choriogonadotropin) in the ovary is mediated by interaction with its specific receptor. Lutropin/choriogonadotropin‐receptor hnRNA is processed into multiple mRNAs. However, nucleotide sequences for many of the transcripts, including the major form (6.7 kb), have yet to be determined. In an attempt to identify a cDNA encoding the major transcript, we have isolated a 3.5‐kb cDNA clone from a rat ovary cDNA library. The 3.5‐kb cDNA recognized only two (6.7 kb, 4.4 kb) of the three (6.7, 4.4, 2.6 kb) ovarian lutropin/choriogonadotropin‐receptor transcripts when used as a probe. The first 732 nucleotides of the newly identified 3.5‐kb cDNA showed 98% identity to the 3′ untranslated region (3′ UTR) of the previously cloned cDNA corresponding to the 4.4‐kb transcript. Southern blot analysis indicated that the 3.5‐kb cDNA and the C‐terminal domain of the lutropin/choriogonadotro‐pin‐receptor originate from the same gene. Oligonucleotide‐directed cleavage of the 6.7‐kb lutropin/ choriogonadotropin‐receptor mRNA by RNase H revealed that the newly identified 3.5‐kb cDNA is a 3′ extension of the 4.4‐kb transcript. We propose that the nucleotide sequence of the 6.7‐kb lutropin/choriogonadotropin‐receptor transcript, the major form found in rat ovary, contains a long 3′ UTR, which has not been previously identified.
Regulatory mechanisms of cAMP‐dependent and cell‐specific expression of human steroidogenic cytochrome P450scc (CYP11A1) gene
Authors: WATANABE Nobuaki , INOUE Hideshi , FUJII‐KURIYAMA Yoshiaki
Published: March 2005

Cytochrome P450scc (CYP11A1) is the enzyme that catalyzes the side‐chain cleavage reaction of cholesterol, the first and rate‐limiting reaction in the biosynthesis of steroid hormones in the adrenal cortex. DNase‐I‐footprinting analysis using nuclear extracts from the bovine adrenal cortex and the 5′ upstream regulatory region (nucleotides –1697 to – 1523) of the CYP11A1 gene, which is mainly required for response to cAMP [Inoue, H., Watanabe, N., Higashi, Y. & Fujii‐Kuriyama, Y. (1991) Eur. J. Biochem. 195, 563–569], revealed that some protein factors bound to that region. One of the sequences protected by the binding factors is a cAMP‐responsive‐element (CRE)‐like sequence, which is known to be recognized by CRE‐binding protein (CREB) or its related proteins, and another is a sequence designated Ad4 which is bound by a tissue‐specific factor, Ad4‐binding protein (Ad4BP). The region containing the two closely arranged DNA sequences showed a high level of cAMP responsive and cell‐specific expression when it was fused to the basal promoters. Introduction of point mutations in these sequences demonstrated that the CREB/ATF factors and Ad4BP bound to the sequences showed synergistic enhancer effects on cAMP‐responsive and cell‐specific expression of the CYP11A1 gene.
Characterization of recombinant plant cinnamate 4‐hydroxylase produced in yeast
Authors: URBAN Philippe , WERCK‐REICHHART Danièle , TEUTSCH Hermann G. , DURST Francis , REGNIER Sylvie , KAZMAIER Michael , POMPON Denis
Published: March 2005

Helianthus tuberosus cinnamate 4‐hydroxylase (CYP73 or CA4H), a member of the P450 superfamily which catalyses the first oxidative step of the phenylpropanoid pathway in higher plants by transforming cinnamate into p‐coumarate, was expressed in the yeast Saccharomyces cerevisiae. The PCR‐amplified CA4H open reading frame was inserted into pYeDP60 under the transcriptional control of a galactose‐inducible artificial promoter. Engineered S. cerevisiæ strains producing human P450 reductase or normal or overproduced amounts of yeast P450 reductase were transformed to express recombinant CA4H. When grown on galactose, yeast cells produced CA4H holoprotein bound to the endoplasmic reticulum membrane as judged from the reduced iron/carbon monoxide difference spectrum centered at 452 nm and from typical cinnamate 4‐hydroxylase activity upon coupling with the different P450 reductases and NADPH. Some CA4H protein was found also addressed to the yeast mitochondria but as a low‐activity form. The spectral and kinetic characterizations of the yeast‐produced CA4H in different redox protein environments are presented using both assays on yeast microsomal fractions and bioconversions on living cells. Results indicate that the microsomal system constituted by the overexpressed yeast P450 reductase and CA4H is characterized by a 1:1 coupling between NADPH oxidation and cinnamate hydroxylation and by one of the highest turnover numbers reported for an NADPH‐dependent P450 reaction. Based on spectral perturbation and inhibition studies, coumarate appeared to have no detectable affinity for the enzyme. A possible geometry of the substrate recognition pocket is discussed in the light of these data.
β1‐integrin‐mediated adhesion of melanoma cells to the propolypeptide of von Willebrand factor
Authors: TAKAGI Junichi , SUDO Yoshimitsu , SAITO Takashi , SAITO Yuji
Published: March 2005

Cell‐adhesion activity of the bovine propolypeptide of von Willebrand factor (pp‐vWF) was assessed by means of an in vitro assay with several cell lines of both normal and tumor‐cell origin. pp‐vWF promoted adhesion and spreading of B16 mouse melanoma cells and G‐361 human melanoma cells. However, it could not induce adhesion of any other cell lines tested including endothelial cells, normal fibroblasts, and tumor cells of sarcoma, carcinoma, neuroblastoma and leukemia origin. A monospecific polyclonal antibody against pp‐vWF, but not against fibronectin, laminin, and von Willebrand factor (vWF), completely blocked the pp‐vWF‐mediated adhesion, indicating that the cell adhesion was due to the pp‐vWF molecule and not due to possible contamination of these three well‐known adhesive proteins. The cell‐adhesion activity was also observed with human pp‐vWF and, furthermore, the adhesion to both bovine and human pp‐vWF was not affected by a peptide containing the Arg‐Gly‐Asp sequence while the peptide abolished the cell adhesion to vWF. The adhesion was completely dependent on Mg2+ and inhibited by Ca2+. Inhibition by an anti‐(β1 integrin) mAb (4 B4) indicates that the receptor for this protein belongs to the β1‐integrin family. A monoclonal antibody (TC4) among several antibodies directed against bovine pp‐vWF inhibited the B16 adhesion to immobilized pp‐vWF. The epitope for this monoclonal antibody lies in a central 8‐kDa portion of pp‐vWF, suggesting that this region is important for the cell‐adhesion activity. This idea was supported by the finding that purified 8‐kDa fragment promoted adhesion of B16 cells in a concentration‐dependent manner. As pp‐vWF shows unique cell‐type specificity in its adhesion activity, which is completely different from that of fibronectin, laminin, vWF and collagen, it may be a novel type of adhesive glycoprotein that utilizes a β1‐integrin receptor.
Site‐directed mutagenesis of signal‐recognition particle RNA Identification of the nucleotides in helix 8 required for interaction with protein SRP19
Authors: ZWIEB Christian
Published: March 2005

The RNA component of signal recognition particle (SRP) consists of eight helices which form a functional unit with the proteins of the SRP. The primary binding site of the 19‐kDa protein of SRP (SRP19) is a tetranucleotide loop (tetraloop) in helix 6 of the SRP RNA, but additional determinants are located in helix 8, which might play important roles in the assembly and the function of the particle. To determine the structural features in helix 8 essential for interaction with SRP19, we altered helix 8 systematically by site‐directed mutagenesis, and determined the ability of protein SRP19 to interact with the various mutant SRP RNAs. Binding of SRP19 was affected by base changes introduced into the 5′ portion (192A, 193G, 194G in the human SRP RNA), but not into the 3′ portion (205A, 206G, 207C) of the distally located conserved internal loop of helix 8. Of the three bases at positions 192–194, only a pyrimidine at position 192 impaired the association with SPR19. An important feature of the SRP19‐RNA interaction were the three base pairs U195‐G204, C196‐G203 and G197‐C202 which shape the helix‐8 tetraloop. Some base‐specific features in the base pairs were also recognized. The tetraloop bases of helix 8 were dispensable for the interaction with SRP19.
ATPase kinetics for wild‐type Saccharomyces cerevisiae F1‐ATPase and F1‐ATPase with the β‐subunit Thr197→Ser mutation
Authors: MUELLER David M. , INDYK Virginia , MCGILL Lisa
Published: March 2005

Unisite ATPase kinetic constants were measured for wild‐type yeast Saccharomyces cerevisiae F1‐ATPase and F1‐ATPase with the Thr197→Ser mutation in the β subunit. Under unisite conditions, the concentration of ATP is greater than that of the enzyme, ATP hydrolysis is slow and the affinity of the enzyme for ATP and ADP is high. The Thr197→Ser mutation in the yeast F1‐ATPase increases the specific activity of ATP hydrolysis threefold and makes the enzyme much less sensitive to azide and oxyanions [Mueller, D. M. (1989) J. Biol. Chem. 264, 16552–16556]. A unifying hypothesis is that the affinity of F1‐ATPase for ADP is altered by azide, oxyanions and the Thr197→Ser mutation. To address this hypothesis, kinetic and thermodynamic constants were measured for the wild‐type and mutant enzymes in the absence and presence of azide and oxyanions. The results indicate that sulfite and azide do not significantly alter unisite thermodynamic binding constants of either enzyme for ADP at the catalytic site. The mutation Thr197→Ser has little effect on the binding constant for ADP, or on other unisite kinetic constants of the enzyme, in the presence or absence of azide or oxyanions. However, the binding of ADP to the enzyme was affected by oxyanions and the Thr197→Ser mutation as measured by determining the KADPi values for multisite ATPase activity (saturating ATP). The Ki for ADP on ATPase activity was measured for the wild‐type and mutant enzymes in the presence and absence of sulfite under multisite conditions. Sulfite increases the KADP values for ATP hydrolysis under multisite conditions approximately threefold for the wild‐type and mutant enzymes and the Thr197→Ser mutation increases KADPi ninefold. The effect of sulfite on KADPi is additive to the effect of the Thr197→Ser mutation, suggesting that these are distinct effects. These results indicate that the effects of azide, oxyanions, and the Thr197→Ser mutation on the biochemistry of F1‐ATPase are limited primarily to multisite conditions. Both sulfite and the Thr197→Ser mutation decrease the affinity of the enzyme for ADP, as measured by the increase in the Ki values. Furthermore, the mechanisms of activation by sulfite and the Thr197→Ser mutations are different. This difference occurs despite their common biochemical consequences on the apparent affinity for ADP.
A thermodynamic study by laser‐flash photolysis of plastocyanin and cytochrome c6 oxidation by photosystem I from the green alga Monoraphidium braunii
Authors: DÍAZ Antonio , HERVÁS Manuel , NAVARRO José A. , DE LA ROSA Miguel A. , TOLLIN Gordon
Published: March 2005

Plastocyanin and cytochrome c6 from the green alga Monoraphidium braunii reduce the photo‐oxidized algal photosystem I (PSI) reaction center chlorophyll (P700) with similar kinetics, as expected from their functional equivalence. The observed P700‐ reduction rate constants show a non‐linear dependence on metalloprotein concentration, which indicates a (minimal) two‐step kinetic mechanism involving complex formation prior to electron transfer. The dependence of the observed rate constants on NaCl concentration suggests that the electrostatic interaction forces between the negatively charged donor proteins and PSI are repulsive at neutral pH and relatively low ionic strength (I), although attractive dipole‐dipole interactions may play a role at higher ionic strengths. Activation parameters for P700+ reduction by cytochrome c6 and plastocyanin have been determined by studying the temperature dependence of the respective rate constants at varying ionic strength and pH. Changes in NaCl concentration and pH induce significant changes in the activation free energy of the overall reaction, even though the corresponding values for activation enthalpy and entropy undergo changes in opposite directions. Such a compensation effect between enthalpy and entropy is observed with both cytochrome c6 and plastocyanin. Protein concentration dependencies of the observed rate constants at different temperatures has allowed an estimate of the free energy change during complex association, as well as the activation parameters for electron transfer, according to a two‐step kinetic model.
Molecular and functional characterization of recombinant human γ‐glutamyltransferase
Authors: THIOUDELLET Christine , OSTER Thierry , WELLMAN Maria , SIEST Gérard
Published: March 2005

We previously described the establishment of a transfected cell line (V79HGGT) that stably produces the highest recombinant human γ‐glutamyltransferase (GGT) activity. We now report the utilization of V79HGGT as a model system for studying human GGT. The papain‐solubilized recombinant enzyme has been highly purified from cultured cells by a new procedure. Studies on the purified enzyme, either by N‐terminal sequencing or by characterization of its enzymic activities, confirmed that recombinant GGT shares structural and catalytic identity with native human enzymes. The circular dichroism analysis indicated an α‐helical content of 19%. Based on these data, we have undertaken a study on the functional consequences of elevated GGT activity on the reduced glutathione (GSH) content. GSH status was followed in V79 and V79HGGT cells throughout growth. A particular pattern was observed for each cell line, depending on, but differentially affected by, alteration of the culture medium. Elevated GGT activity was associated with a 2.5‐fold reduced GSH content, clearly suggesting a negative influence of the highly expressed enzyme on the GSH level under normal growth conditions. Possible mechanisms involved are proposed. Our findings pointed out that, among the GSH‐related enzymes, GGT could constitute an important factor determining the steady‐state content of GSH.
Differential polysomal localization of human insulin‐like‐growth‐factor‐2 mRNAs in cell lines and foetal liver
Authors: DE MOOR Cornelia H. , JANSEN Maarten , SUSSENBACH John S. , VAN DEN BRANDE J. Leo
Published: March 2005

Examination of the association of insulin‐like‐growth‐factor‐2 mRNAs with polyribosomes in five cell lines revealed that greater than 50% of the total mRNA population was present in the untranslated free mRNP fraction for each cell line. Of the different subtypes of insulin‐like‐growth‐factor‐2 messengers, the least abundant mRNAs, starting with exon 4 (leader 2, 5.0 kb) and exon 6 (leader 4, 4.8 kb), were found in the polysomes only, while the most abundant transcript, starting with exon 5 (leader 3, 6.0 kb and 2.1 kb) was found predominantly in the untranslated fractions. 20–30% of leader 3 mRNAs, however, were in the larger polysomes (four or more ribosomes), indicating that a subpopulation of this mRNA can be translated efficiently. The peak fraction for the leader 4 insulin‐like‐growth‐factor‐2 mRNA (4.8 kb) in the polysomes was migrating faster in the sucrose gradients than the peak fractions of leader 2 and 3 mRNAs (5.0 kb and 6.0 kb), implying that more ribosomes were associated with this type of mRNA. In foetal liver, the situation was similar, though in this case the leader 2 mRNA was most heavily loaded with polysomes. Treatment of cells with low concentrations of cycloheximide caused the polysomal RNAs to shift to even larger polysomes while the untranslated fraction of the leader 3 mRNAs stayed in the untranslated fractions. These results indicate that, both in established cell lines and in foetal liver, insulin‐like‐growth‐factor‐2 translation is influenced both by mRNP sequestration and differential translation initiation efficiency of the insulin‐like‐growth‐factor‐2 mRNAs.
The (2R)‐hydroxycarboxylate‐viologen‐oxidoreductase from Proteus vulgaris is a molybdenum‐containing iron‐sulphur protein
Authors: TRAUTWEIN Thomas , KRAUSS Friedrich , LOTTSPEICH Friedrich , SIMON Helmut
Published: March 2005

An oxidoreductase with an extremely broad substrate specificity reducing reversibly 2‐oxocar‐boxylates at the expense of reduced artificial redox mediators to (2R)‐hydroxycarboxylates has been purified to a specific activity of up to 1800 μmol · min−1· mg−1 for the reduction of phenylpyruvate. The membrane‐bound non‐pyridine nucleotide‐dependent enzyme appears in the form of various oligomers of the 80‐kDa monomer.

The isoelectric point is 5.1. Based on a molecular mass of 80 kDa the enzyme contains up to one molybdenum, four iron and four sulphur atoms. After growth on 99Mo‐labelled molybdate, enzyme and radioactivity coincided as shown by gel electrophoresis.

Permanganate oxidation delivers 0.7 mol pterin‐6‐carboxylic acid. The molybdenum cofactor is a mononucleotide. The enzyme is inhibited by cyanide. The first 20 amino acids have been determined. The enzyme belongs to the rare group of molybdoenzymes which possess no further prosthetic groups than the iron‐sulphur clusters.
Loss of phospholipid asymmetry in human platelet plasma membrane after 1–12 days of storage
Authors: GAFFET Patrick , BASSÉ François , BIENVENÜE Alain
Published: March 2005

We used paramagnetic analogs of endogenous phospholipids to study modification of phospholipid distribution in platelet plasma membranes during aging. Asymmetrical distributions and translocation kinetics were very different for spin‐labeled phosphatidylserine and spin‐labeled phosphatidylcholine in fresh platelet plasma membranes. In freshly prepared platelets and up to day 7, spin‐labeled phosphatidylserine very rapidly penetrated to the inner leaflet of the platelet plasma membrane. However, spin‐labeled phosphatidylcholine was mainly retained on the external leaflet. From day 7 to day 9, the two translocation kinetics became identical with symmetrical distribution of both spin‐labeled phospholipids at equilibrium. Inhibition of translocase activity and modification of membrane stability accounted for these transformations. The rapid re‐exposition of spin‐labeled phosphatidylserine after stimulation by the calcium ionophore A23187, measured in fresh platelet concentrates, persisted up to day 9 but disappeared between day 10 and day 12. From day 7 to day 9, a strong cytoskeleton proteolysis and marked decrease in intracellular ATP were observed. Moreover, complete suppression of β‐N‐acetyl glucosaminidase secretion and vesicle formation after A23187 stimulation of aged platelets indicated that platelets could no longer be activated beyond day 9. Taken together, these results showed that during in vitro aging there are metabolic and membrane modifications in platelet similar to those described for platelet activation. In addition, all of the observed events occurred simultaneously between day 7 and day 9. These results highlight the importance of maintaining plasma membrane asymmetry to increase the hemostatic effectiveness of transfused platelet concentrates.
Identification of a major low‐molecular‐mass thiol of the trypanosomatid Crithidia fasciculata as ovothiol A
Authors: STEENKAMP Daniel J. , SPIES Hendrik S. C.
Published: March 2005

An unidentified low‐molecular‐mass thiol, U23, previously detected as the 7‐diethylamino‐3‐(4′‐maleimidylphenyl)‐4‐methylcoumarin derivative in extracts of the trypanosome Crithidia fasciculata, was purified as the bimane derivative. Resonances attributable to U23 were discerned from those of the bimane label by comparison of the 1H‐ and 13C‐NMR spectra of monobromobimane and U23‐bimane. The complete 1H‐ and 13C‐NMR spectra of U23‐bimane were assigned by means of 1H‐1H correlation spectroscopy, 1H‐13C correlation spectroscopy and 13C multiplicity determinations. The results indicated identity of U23 with 1‐N‐methyl‐4‐mercaptohistidine (ovothiol A), previously isolated from marine sources. This assignment was confirmed by NOE difference experiments, fast‐atom‐bombardment mass spectrometry of U23‐bimane and ultraviolet/visible spectrophotometry of U23, which was isolated as the disulfide. The isolation of ovothiol A from a parasitic protozoan suggest that the 4‐mercaptohistidines may have a wider distribution and function as antioxidant thiols than was hitherto realized.
Enhanced production of pituitary adenylate‐cyclase‐activating polypeptide by 1, N6‐dibutyryladenosine 3′,5′‐monophosphate, phorbol 12‐myristate 13‐acetate and by the polypeptide itself in human neuroblastoma cells, IMR‐32
Authors: SUZUKI Nobuhiro , HARADA Masataka , HOSOYA Masaki , FUJINO Masahiko
Published: March 2005

We investigated a regulatory mechanism for the production of pituitary adenylate‐cyclase‐activating polypeptide (PACAP) using the human neuroblastoma cell line, IMR‐32. Although immunoreactive (ir–) PACAP38 was produced by these cells, it was not accumulated in the culture medium. To overcome this problem, we cultured the cells in the presence of a biotinylated monoclonal antibody to PACAP38 in order to trap ir‐PACAP38 in the medium. Then, the trapped ir‐PACAP38 was measured with a two‐site enzyme immunoassay based on the biotin‐avidin interaction. These studies showed that confluent IMR‐32 cells constitutively secreted at least 130 pg/106 cells per day of ir‐PACAP38. Treatment of the cells with 1 mM 1, N6‐dibutyryladenosine 3′,5′‐monophosphate (Bt2cAMP) or 0.1 μM phorbol 12‐myristate 13‐acetate stimulated ir‐PACAP38 production at almost twice the basal level in IMR‐32 cells, and these two reagents additively stimulated ir‐PACAP38 production. Northern blot analysis indicated that treatment of the cells with both Bt2cAMP and the phorbol ester increased the intensity of several mRNA bands that hybridized with a cDNA probe for human PACAP precursor.

We also found that IMR‐32 cells possessed high‐affinity type‐I PACAP receptors (Kd= 230 pM, binding sites: 8.6 × 104 sites/cell), which had a 1000‐fold higher affinity for PACAP38 and PACAP27 than for vasoactive intestinal polypeptide. The treatment of IMR‐32 cells with PACAP and vasoactive intestinal polypeptide increased intracellular cAMP levels, and also increased ir‐PACAP production. Northern blot analysis revealed that PACAP at 10 nM markedly stimulated the expression of a 3.7‐kb mRNA band that hybridized with PACAP precursor cDNA. These results indicate that IMR‐32 cells produce ir‐PACAP38, and that PACAP stimulates the synthesis of PACAP mRNA and peptides by increasing intracellular cAMP. Although it remains to be investigated whether PACAP secreted by IMR‐32 cells really acts in an autocrine fashion, the present study provides a useful model for further studies of the biological role of PACAP in neuronal cells that produce PACAP and also respond to PACAP.