bluuebunny/crossref_metadata_2025 huggingface dataset:
Apolipoprotein E expression by human‐monocyte‐derived macrophages
Authors: ROUIS Mustapha , NIGON Fabienne , EGGERMAN Thomas L. , BREWER H. Bryan , CHAPMAN M. John
Published: March 2005

The effects of opsonised zymosan and of acetylated low‐density lipoprotein (AcLDL) on the synthesis and secretion of apolipoprotein E (apoE), and of apoE mRNA abundance, have been studied in human‐monocyte‐derived macrophages (MDM). Stimulation by opsonised zymosan led to a concentration‐dependent increase in apoE secretion; non‐opsonised zymosan was without effect. Incubation with AcLDL led to a concentration‐dependent elevation in apoE synthesis which paralleled the increase in cellular cholesterol content. The opsonised‐zymosan‐induced stimulation of apoE production was additive to that resulting from cholesterol loading with AcLDL. Opsonised zymosan alone did not affect the cholesterol content of MDM. Cholesterol‐loaded MDM remained responsive to opsonised zymosan stimulation, displaying a 3.5‐fold elevation in apoE secretion as compared to their non‐stimulated counterparts. Cell‐associated apoE remained at trace levels under all conditions of cell treatment. Studies involving [35S]methionine incorporation showed de novo synthesis of apoE to be enhanced in both cholesterol‐loaded and opsonised‐zymosan‐stimulated macrophages. Estimation of apoE mRNA in opsonised‐zymosan‐stimulated and control MDM by dot‐blot analysis revealed similar message abundance; by contrast, elevation in cellular cholesterol content following incubation with modified LDL led to a significant increase in apoE mRNA levels. We conclude that the opsonised‐zymosan‐induced stimulation of apoE synthesis and secretion in human MDM may occur by a mechanism(s) independent of cellular cholesterol content.
Intrinsic tryptophan fluorescence measurements suggest that polylactosaminyl glycosylation affects the protein conformation of the gelatin‐binding domain from human placental fibronectin
Authors: ZHU Betty C. R. , LAINE Roger A. , BARKLEY Mary D.
Published: March 2005

Glycosylation can affect the physical and biochemical properties of the polypeptide chain in glycoproteins. Asparagine‐N‐linked polylactosaminyl glycosylation of the chymotryptic 44‐kDa gelatin‐binding domain from human placental fibronectin confers protease resistance [Zhu, B. C. R., Fisher, S. F., Panda, H., Calaycay, J., Shively, J. E. & Laine, R. A. (1984) J. Biol. Chem. 259, 3962–3970] and weakens the binding to gelatin [Zhu, B. C. R. & Laine, R. A. (1985) J. Biol. Chem. 260, 4041–4045]. Intrinsic tryptophan fluorescence of the gelatinbinding domain was used to probe glycosylation‐dependent protein conformation changes. In gelatin‐binding fragments containing incrementally smaller polylactosamine oligosaccharides, the fluorescence intensity progressively decreased and the emission spectrum shifted about 7 nm to the blue. Removal of the polylactosamine chains from a highly glycosylated fragment with endo‐β‐galactosidase from Escherichia freundii also quenched the protein fluorescence. The fluorescence lifetimes did not appear to be affected by the extent of glycosylation, suggesting static quenching of the tryptophan emission in the low glycosylated fragments. Acrylamide quenching studies showed that the accessibility of the tryptophans to small solutes was not altered by glycosylation. The steadystate emission anisotropy increased with decreasing polylactosamine chain length. The results indicate that the polylactosamine chains alter the tryptophan environments in the gelatin‐binding domain, probably by changing the polypeptide conformation. These putative protein conformation changes may be partially responsible for the altered gelatin binding, protease resistance, and cell adhesion functions of fetal tissue fibronectin.
On the domain construction of the multienzyme gramicidin S synthetase 2
Authors: SKARPEID Hans‐Jacob , ZIMMER Trine‐Lise , von DÖHREN Hans
Published: March 2005

The multienzyme gramicidin S synthetase 2, composed of one polypeptide chain, was treated with trypsin and chymotrypsin to give fragments retaining partial enzyme activities. Previously, a tryptic fragment of this multi‐enzyme has been identified as a structural and functional domain. In this study two more fragments, activating Leu and Val, respectively, are shown to represent domains. Careful inspection of the data on limited proteolysis, from this study as well as from previous work, suggests that domains are not simply connected like pearls on a string, and a model for the structure of gramicidin S synthetase, with implications for other peptide synthetase multienzymes, is presented. It is suggested that gramicidin S synthetase 2 is constructed from core catalytic domains and intervening framework. Such an interpretation is in accordance with all published data on limited proteolysis of peptide synthetases, but needs an interplay with gene structural studies in order to be validated and refined.
Secondary and tertiary structure characteristics of Megasphaera elsdenii flavodoxin in the reduced state as determined by two‐dimensional 1H NMR
Authors: van MIERLO Carlo P. M. , MÜLLER Franz , VERVOORT Jacques
Published: March 2005

The secondary structure of two‐electron‐reduced Megasphaera elsdenii flavodoxin has been determined by visual, qualitative inspection of the sequential connectivities involving CαH, CβH and NH protons observed in NOESY (two‐dimensional nuclear Overhauser enhancement spectroscopy) spectra. Results from an amide proton exchange experiment were used to confirm the secondary structure assignment and to demonstrate the compactness and stability of the protein. After the secondary structure elements were established, the global fold of the protein and the flavin binding site have been determined using nonsequential interresidual NOE connectivities as primary source of information. The secondary structure and the global fold of M. elsdenii and Clostridium MP flavodoxin appeared to be very similar, differences are observed however. M. elsdenii flavodoxin consists of a central parallel β‐sheet including five strands surrounded on both sides by a pair of α‐helices.
Rates of RNA degradation in isolated rat hepatocytes
Authors: BALAVOINE Sophie , FELDMANN Gérard , LARDEUX Bernard
Published: March 2005

RNA degradation in isolated rat hepatocytes was measured as the release of radioactive cytidine from fed rats previously labeled in vivo for 60 h with [6‐14C]orotic acid. Rates were determined from the linear accumulation of [14C]cytidine between 30 and 120 min of incubation in the presence of 0.5 mM unlabeled cytidine to suppress reutilization.

In the absence of amino acids, rates of RNA degradation in isolated hepatocytes averaged 3.97%/h. A complete mixture of amino acids added at 10–20 times normal plasma concentration inhibited RNA degradation by 65–70%. However, at physiological concentrations of amino acids, RNA degradation in isolated rat hepatocytes was less responsive as compared to perfused rat livers.

Numerous and large autophagic vacuoles at various stages of digestion were identified throughout the cytoplasm of isolated hepatocytes after 2 h of incubation in the absence of amino acids. The addition of amino acids at 20 times normal plasma concentration abolished almost completely the appearance of autophagic vacuoies. Furthermore, propylamine, which accumulates in lysosomes, suppressed RNA degradation by 65% and the inhibitor of autophagic vacuole formation, 3‐methyladenine, inhibited 70–80% of the degradation. Taken together, these results strongly suggest a contribution of the lysosomal system in the increase of RNA degradation rates in isolated rat hepatocytes.
Enhancement of tyrosine kinase activity of the Drosophila epidermal growth factor receptor homolog by alterations of the transmembrane domain
Authors: WIDES Ronald J. , ZAK Naomi B. , SHILO Ben‐Zion
Published: March 2005

The Drosophila epidermal growth factor receptor homolog (DER) displays sequence similarity to both the epidermal growth factor (EGF) receptor and the neu vertebrate proteins. We have examined the possibility of deregulating the tyrosine kinase activity of DER by introducing structural changes which mimic the oncogenic alterations in the vertebrate counterparts. Substitution of valine by glutamic acid in the transmembrane domain, in a position analogous to the oncogenic mutation in the rat neu gene, elevated the in vivo kinase activity of DER in Drosophila Schneider cells sevenfold. A chimera containing the oncogenic neu extracellular and transmembrane domains and the DER kinase region, also showed a threefold elevated activity relative to a similar chimera with normal neu sequences. Double truncation of DER in the extracellular and cytoplasmic domains, mimicking the deletions in the v‐erbB oncogene, did not however result in stimulation of in vivo kinase activity. The chimeric constructs were also expressed in monkey COS cells, and similar results were obtained. The ability to enhance the DER kinase activity by a specific structural modification of the transmembrane domain demonstrates the universality of this activation mechanism and strengthens the notion that this domain is intimately involved in signal transduction. These results also support the inclusion of DER within the tyrosine‐kinase receptor family.
Vanadate inhibits liver fructose‐2,6‐bisphosphatase
Authors: RIDER Mark H. , BARTRONS Ramon , HUE Louis
Published: March 2005

Vanadate was found to be a reversible non‐competitive inhibitor of chicken liver fructose‐2,6‐bisphosphatase. The inhibition was best observed in the presence of glycerol 2‐ or 3‐phosphate and half‐maximal effect was obtained with about 0.15 mM vanadate.

Vanadate decreased the extent of phosphorylation of the enzyme (E‐P) by fructose 2,6‐[2‐32P]bisphosphate. This did not result from an increased rate of E‐P breakdown, as is the case with phosphoglycerate mutase, an enzyme which shares structural and functional similarity to fructose‐2,6‐bisphosphatase. The data were consistent with the formation of a dead‐end transition state analogue of phosphate in the active site.

Inhibition of fructose‐2,6‐bisphosphatase by vanadate offers a likely explanation for the increase in fructose 2,6‐bisphosphate concentration brought about by vanadate in isolated rat hepatocytes.
Palmitoyl‐CoA and the acyl‐CoA thioester of the carcinogenic peroxisome‐proliferator ciprofibrate potentiate diacylglycerol‐activated protein kinase C by decreasing the phosphatidylserine requirement of the enzyme
Authors: ORELLANA Ariel , HIDALGO Perla C. , MORALES Maria N. , MEZZANO Diego , BRONFMAN Miguel
Published: March 2005

To gain insight into the mechanism by which long‐chain acyl‐CoA thioesters potentiate diacylglycerol‐activated protein kinase C, the cofactor dependence of this activating effect was studied with purified rat brain enzyme and histone H1 as substrate. Using two different assay systems, palmitoyl‐CoA was found to decrease greatly the amount of phosphatidylserine required to activate the kinase. No relative changes were observed in the dependence of the enzyme for other cofactors (diacylglycerol, ATP, and Ca2+) in the presence of palmitoyl‐CoA. The potentiating effect of palmitoyl‐CoA and the decrease in phosphatidylserine requirement of the kinase was also demonstrated using the 47‐kDa protein of human platelets as substrate and platelet protein kinase C as source of enzyme. The acyl‐CoA thioester of the carcinogenic peroxisome‐proliferator ciprofibrate was also found to decrease the phosphatidylserine requirement of protein kinase C. The data suggest that acyl‐CoAs may play a role in the regulation of protein kinase C activity.
Phalloidin‐induced accumulation of myosin in rat hepatocytes is caused by suppression of autolysosome formation
Authors: UENO Takashi , WATANABE Sumio , HIROSE Miyoko , NAMIHISA Toshihiko , KOMINAMI Eiki
Published: March 2005

Administration of phalloidin in vivo to rats causes marked changes in the distribution of actin and myosin in hepatocytes, which accompanies reduced bile flow. We have found that in hepatocytes treated with phalloidin for 3 and 7 days, cellular myosin content increased about 1.5‐fold and 4.7‐fold, respectively. In addition, total cell protein content and several marker enzyme activities were also elevated by 30–120% depending on the duration of phalloidin treatment. These observations allow us to speculate that phalloidin somehow elicits inhibition of cellular protein degradation, which results in the increase of these protein levels. To examine this possibility further, we analyzed leupeptin‐induced density shift of phagolysosomes. In normal liver, the injection of leupeptin/E64c caused an increase in the density of both heterolysosomes and autolysosomes, due to retarded digestion of sequestered proteins as a result of the inhibition of lysosomal cathepsins. Accumulation, in these denser autolysosomes, of lactic dehydrogenase, pyruvate kinase, aldolase, and myosin was demonstrated by enzyme assays and immunoblot analysis. In the phalloidin‐treated liver, the increase in the density of autolysosomes and the accumulation of above cytoplasmic enzymes were markedly inhibited. However, phalloidin did not affect the shift in the density of heterolysosomes. From these data, we concluded that autolysosome formation was specifically hindered in phalloidin‐treated rat hepatocytes, which results in the reduction of autophagic protein degradation and eventual increase in intracellular protein levels.
Studies on the structure and regulation of the human hepatic interleukin‐6 receptor
Authors: ROSE‐JOHN Stefan , SCHOOLTINK Heidi , LENZ Dorothée , HIPP Erich , DUFHUES Gabi , SCHMITZ Hildegard , SCHIEL Xaver , HIRANO Toshio , KISHIMOTO Tadamitsu , HEINRICH Peter C.
Published: March 2005

Affinity cross‐linking of 125I‐labeled recombinant human interleukin‐6 (IL‐6) to human hepatoma cells (HepG2) allowed the detection of three IL‐6‐containing complexes with molecular masses of 100 kDa, 120 kDa and 200 kDa.

Treatment of HepG2 cells with dexamethasone led to a time‐ and dose‐dependent up‐regulation of IL‐6‐receptor mRNA levels. By the use of cross‐linking this effect was also seen at the protein level, where all three IL‐6‐binding complexes increased upon incubation of HepG2 cells with dexamethasone. Under conditions of IL‐6‐receptor up‐regulation by dexamethasone, γ‐fibrinogen mRNA induction by IL‐6 is stronger and occurs earlier than without dexamethasone. We propose therefore that the expression of the IL‐6 receptor might be a rate‐limiting step in acute‐phase‐protein induction.
Isolation and characterization of UDP‐glucose dolichyl‐phosphate glucosyltransferase from human liver
Authors: MATERN Heidrun , BOLZ Rita , MATERN Siegfried
Published: March 2005

The enzyme UDP‐glucose dolichyl‐phosphate glucosyltransferase has been purified to near homogeneity from human liver microsomes. A 1100‐fold enrichment over starting microsomal membranes was achieved by selective solubilization followed by anion‐ and cation‐exchange chromatography, 5‐HgUDP‐thiopropyl‐Sepharose affinity chromatography, butylagarose chromatography and hydroxyapatite chromatography. The glucosyltransferase was shown to be separated from other dolichyl‐phosphate‐dependent glycosyltransferases catalyzing the formation of dolichyl diphospho‐N‐acetylglucosamine and dolichyl phosphomannose. Sodium dodecyl sulfate/polyacryl‐amide gradient gel electrophoresis of the purified enzyme under reducing conditions revealed a protein band of Mr 36 000. Protection of the solubilized enzyme against rapid inactivation was achieved by its competitive inhibitor uridine. The purified glucosyltransferase activity exhibited a specific requirement for the presence of phospholipids. Phosphatidylethanolamine was the most effective activator of enzyme activity.
An NMR study of anion binding to yeast phosphoglycerate kinase
Authors: FAIRBROTHER Wayne J. , GRAHAM Heidi C. , WILLIAMS Robert J. P.
Published: March 2005

Anion binding to yeast phosphoglycerate kinase has been investigated using 1H‐NMR spectroscopy. The use of anionic paramagnetic probes, [Cr(CN)6]3− and [Fe(CN)6]3−, has enabled the location of the primary anion binding site in the ‘basic‐patch’ region of the amino‐terminal domain. The anions interact most closely with Arg‐65 and Arg‐168. The binding of these and a variety of other anions to this site is directly competitive with the binding of the substrate, 3‐phosphoglycerate. Binding of 3‐phosphoglycerate and 1,3‐bisphosphoglycerate is, however, stronger than expected on the basis of anionic charge and causes conformational changes in the protein not seen with any of the other simple spherical anions investigated. This must be part, at least, of the substrate specificity.

Evidence for a secondary anion binding site involving the side chains of surface lysine residues is also presented. It is suggested that the primary anion site is responsible for the observed activation by anions at low concentrations while the secondary site leads to inhibition at higher anion concentrations. The kinetics fit these deductions and a scheme for kinase activity is presented.
Purification and characterization of lipopolysaccharide‐binding protein from hemolymph of American cockroach Periplaneta americana
Authors: JOMORI Takahito , KUBO Takeo , NATORI Shunji
Published: March 2005

A protein having affinity to lipopolysaccharide of Escherichia coli K12 was purified to homogeneity from the hemolymph of Periplaneta americana. This protein, with an average molecular mass of 450 kDa, was a homooligomer of a 28‐kDa subunit protein. Comparative studies using lipopolysaccharide molecules of E. coli and Salmonella minnesota suggested that this protein recognizes and binds to a specific carbohydrate structure of E. coli lipopolysaccharide. Ca2+ was required for this protein to bind to lipopolysaccharide, but other divalent cations could not replace Ca2+.
Proton NMR studies of apo‐neocarzinostatin from Streptomyces carzinostaticus
Authors: ADJADJ Élisabeth , MISPELTER Joél , QUINIOU Éric , DIMICOLI Jean‐Luc , FAVAUDON Vincent , LHOSTE Jean‐Marc
Published: March 2005

The sequence‐specific resonance assignment of apo‐neocarzinostatin from Streptomyces carzinostaticus was carried out from two‐dimensional proton‐NMR spectra. The assignments were obtained for the backbone protons of 111 of the 113 residues of the protein, missing the two CαH of one glycine but including 3 of the 4 prolines. The majority of side chain protons were also assigned. The secondary structure derived from the analysis of sequential connections corresponds to ten β‐strands separated by clearly identified loops and turns. Inter‐strand connectivities and slowly exchanging amide protons confirm the presence of the two disulfide bridges from Cys37 to Cys47 and from Cys88 to Cys93 and indicate a global folding similar to that of the similar proteins, actinoxanthin and macromomycin, for which crystallographic data are available.
Purification and characterization of serine–glyoxylate aminotransferase from a serine‐producing methylotroph, Hyphomicrobium methylovorum GM2
Authors: IZUMI Yoshikazu , YOSHIDA Toyokazu , YAMADA Hideaki
Published: March 2005

Serine–glyoxylate aminotransferase was purified to complete homogeneity from a serine‐producing methylotrophic bacterium, Hyphomicrobium methylovorum GM2, which possesses the serine pathway. This is the first microbial serine–glyoxylate aminotransferase to be purified. The enzyme has a molecular mass of about 140 kDa and consists of four subunits of identical mass, i.e. 40 kDa. The holoenzyme exhibited absorption maxima at 282 nm and 408 nm, and a shoulder at about 315–345 nm in potassium phosphate pH 7.0; it contained 4 mol pyridoxal 5′‐phosphate/mol enzyme. Isoelectric focusing showed that the enzyme had a pI value of 6.9. The Km values for glyoxylate and L‐serine were 0.23 mM and 4.98 mM, respectively, and the enzyme showed high specificity for these substrates. The transamination between glyoxylate and L‐serine seemed to be nearly irreversible. These data indicated that this serine–glyoxylate aminotransferase plays an essential role in methanol assimilation through the serine pathway in H. methylovorum GM2.
Protein‐decorated micelle structure of sodium‐dodecyl‐sulfate–protein complexes as determined by neutron scattering
Authors: IBEL Konrad , MAY Roland P. , KIRSCHNER Kasper , SZADKOWSKI Halina , MASCHER Erik , LUNDAHL Per
Published: March 2005

The structure of the complex between sodium dodecyl sulfate (SDS) and a deuterated bifunctional enzyme, N‐5′‐phosphoribosylanthranilate isomerase/indole‐3‐glycerol‐phosphate synthase (Mr 49484), has been studied in dilute solution by small‐angle neutron scattering. The complex nearly acquired its final size, as shown by molecularsieve chromatography, at the chosen SDS concentration of 1.6 mM, which is slightly below the critical micelle concentration of 1.8 mM (at the ionic strength of 0.1 M). The 452 amino‐acid residues of the bifunctional enzyme were combined with 216 detergent molecules. The complex was found to be composed of three protein‐decorated SDS micelles of unequal size, connected by short flexible polypeptide segments. The largest of the three micelles was the middle one. The SDS‐protein complex contained the dodecyl hydrocarbon moieties in three globular cores. Each core was surrounded by a hydrophilic shell, formed by the hydrophilic and amphiphilic stretches of the polypeptide chain, and by the sulfate head groups of the detergent. The average thickness of these shells was 0.7–0.8 nm.

The three‐micelle complex was cleaved with trypsin at a single site, possibly in a micelle‐connecting segment, into a single‐micelle fragment at the carboxyl‐terminal which comprised 73 SDS molecules and 163 amino‐acid residues, and a dual‐micelle fragment. One of the micelles within this larger fragment contained 42 SDS molecules and about 90 amino‐acid residues; the other micelle contained 101 SDS molecules and about 190 amino‐acid residues. The individual micelle sizes seemed to be determined by the amino‐acid sequence.
Inhibition of L‐lactate: cytochrome‐c reductase (flavocytochrome b2) by product binding to the semiquinone transient
Authors: TEGONI Mariella , JANOT Jean‐Marc , LABEYRIE Françoise
Published: March 2005

Pyruvate has previously been shown to slow down the rate of intramolecular electron transfer from the flavosemiquinone (Fs) to the cytochrome b2 moiety of flavocytochrome b2 [Tegoni, M., Silvestrini, M. C., Labeyrie, F. & Brunori, M. (1984) Eur. J. Biochem. 140, 39–45] and to stabilize markedly the Fs state of the prosthetic flavin, relative to the oxidized (Fo) and the reduced (Fh) states [Tegoni, M., Janot, J.‐M. & Labeyrie, F. (1986) Eur. J. Biochem. 155, 491–503].

In the present study, we have determined the dissociation constants of pyruvate for the three redox forms of the prosthetic flavin and demonstrated that the Fs‐pyruvate complex is actually much more stable than the Fo‐pyruvate and Fh‐pyruvate complexes.

The inhibition produced by pyruvate has been characterized under steady‐state conditions using both ferri‐cytochrome c and ferricyanide as external acceptor. A detailed analysis and simulations of the suitable reaction scheme, taking into consideration all data from rapid kinetic studies of partial reactions previously published, show that the experimental noncompetitive inhibition results from the sum of a competitive effect due to binding of pyruvate to Fo and an uncompetitive effect due to binding to the Fs intermediate in a dead‐end complex. Pyruvate binding to the semiquinone transient results in a marked loss of the reactivity of this donor in electron transfers to its specific partner, the cytochrome b2 present in the same active site, as to ferricyanide, an external acceptor. A critical evaluation of the parameters involved in the control of such reactivities is presented.
Identification and characterisation of a Leishmania donovani antigen belonging to the 70‐kDa heat‐shock protein family
Authors: MacFARLANE Juan , BLAXTER Mark L. , BISHOP Richard P. , MILES Michael A. , KELLY John M.
Published: March 2005

A Leishmania donovani promastigote cDNA library was screened with serum obtained from a patient infected with visceral leishmaniasis. Sequence analysis of a clone obtained from this library revealed that the 600‐bp insert corresponded to the carboxy‐terminal region of an antigen related to the 70‐kDa heat‐shock protein family. The full‐length sequence of the corresponding gene (1959 nucleotides) was determined after isolation of genomic clones. Genes encoding the antigen are present on a single chromosome as a series of approximately twelve 3.7‐kb direct tandem repeats. The antigen can be identified as a 70‐kDa heat‐shock cognate protein by virtue of its molecular mass, sequence and constitutive expression during heat shock. It is expressed at all stages of the parasite life‐cycle. Antibodies against the λgt11 fusion protein were detected in more than 50% of serum samples obtained from patients with visceral leishmaniasis, but were not detected in sera from patients with cutaneous leishmaniasis or Chagas' disease.
Cloning and sequencing of a Porifera partial cDNA coding for a short‐chain collagen
Authors: EXPOSITO Jean‐Yves , OUAZANA Roland , GARRONE Robert
Published: March 2005

Collagen is present in Porifera, the lowest multicellular animals, but there is no information available on the primary structure of the collagen chains in this phylum. Developing fresh‐water sponges have been used to extract total RNA in order to study in vitro translation products and to construct a cDNA library. Four translated proteins were collagenase‐sensitive (200 kDa, 160 kDa, 81 kDa and 48 kDa). The cDNA library was screened with a human collagen probe and a clone, EmC4, covering 1.2 kb was isolated. Nucleotide sequencing of EmC4 revealed a conceptual open reading frame coding for 366 amino acids terminated by a stop codon TGA with 103 nucleotides downstream. The presumed translation product encoded contained several domains: a non‐collagenous C‐terminal domain of 156 amino acids with 9 cysteines, an uninterrupted collagenous domain of 171 amino acids, a non‐collagenous domain of 16 amino acids with 3 cysteines and a probably incomplete N‐terminal collagenous domain of 23 amino acids. Comparison with other sequences suggested that this collagen chain might belong to a non‐fibrillar collagen family which evolved into several sub‐families giving rise to nematode cuticular collagens, and type IV collagens.
The roles of ATP4− and Mg2+ in control steps of phosphoglycerate kinase
Authors: FAIRBROTHER Wayne J. , GRAHAM Heidi C. , WILLIAMS Robert J. P.
Published: March 2005

1H‐NMR measurements were made of solutions of yeast phosphoglycerate kinase containing the nucleotide substrate, ATP, and Mg2+ in varying concentrations in order to investigate the affect that the metal ion has on the mode of ATP binding to the enzyme. From the change in the chemical shifts of the ‘basic‐patch’ histidine resonances (His62, His167 and His170) and the nucleotide C8H, C2H and C1′H resonances it is apparent that there are at least two ATP‐binding sites on the enzyme. Downfield shifts observed for the above histidine resonances at low nucleotide/enzyme molar ratios indicates that the primary binding site involves electrostatic interactions between the nucleotide triphosphate chain and the basic‐patch region of the N‐terminal domain. The secondary binding site is shown to involve predominantly hydrophobic interactions between the adenosine moiety and the protein. Evidence from previous two‐dimensional NMR experiments [Fairbrother et al. (1990) Eur. J. Biochem. 190, 161–169] suggests that the secondary site is equivalent to the crystallographically observed catalytic site. The affinity of the catalytic site is increased relative to the primary electrostatic site with increasing Mg2+ concentration. The possible importance of these observations in the regulation of this enzyme in vivo are discussed.
Analysis of the promotors of the single‐copy genes for plastocyanin and subunit δ of the chloroplast ATP synthase from spinach
Authors: BICHLER Josef , HERRMANN Reinhold G.
Published: March 2005

The promotors of the single‐copy genes for subunit δ of the chloroplast ATP synthase (atpD) and plastocyanin (PC) from spinach have been sequenced, dissected and analysed in transgenic F0 and F1 tobacco plants using the bacterial GUS gene as a reporter for promotor activity. The transcription of these genes is photo‐controlled. The results have been compared with those obtained for the spinach rbcS‐1 gene, one of the light‐regulated genes encoding the small subunit of ribulose‐1,5‐bisphosphate carboxylase/oxygenase, and for the cauliflower mosaic virus (CaMV) 35S RNA promotor. We find that the 5′ upstream regions of about 1200 nucleotides contain all the sequences required for light regulation, organ‐, tissue‐ and development‐specific expression, and that they are structurally diverse. Their cis‐acting elements are functionally defined. The proximal regions of the spinach promotors contain potential TATA, CAAT and T‐cyt boxes at appropriate positions, but only sequence elements with low similarity to published light‐responsive elements. Positive light‐stimulated regions, regions with constitutive, light‐independent enhancing effects and with ‘silencer’‐like activity in complete darkness are found in proximal and far upstream promotor segments. Highest activity of these promotors is correlated with the presence of chloroplasts but is not confined to photosynthetic tissue. Surprisingly, expression is observed in the phloem regions of transgenic leaves, leaf and floral stems, in the vascular area of anthers and in pollen. No histochemical staining has been detected in roots. The distal region of atpD located between −1137 and −590 contains elements for expression in the outer phloem, the region from −590 to −185 for activity in the inner phloem of floral stems. Similar tissue‐specific patterns are observed with a fusion between the caufliflower mosaic virus 35S RNA promotor and the GUS gene.
The role of copper in the stability of ascorbate oxidase towards denaturing agents
Authors: SAVINI Isabella , D'ALESSIO Silvana , GIARTOSIO Anna , MORPURGO Laura , AVIGLIANO Luciana
Published: March 2005

The susceptibility of native, type‐2 Cu‐depleted and fully Cu‐depleted ascorbate oxidase to thermal and chemical denaturation has been probed by differential scanning calorimetry, fluorimetry and circular dichroism. The data indicate that copper affects the stability, but not the protein conformation.

The unfolding of ascorbate oxidase is characterized by a single endotherm. Calorimetric domains revealed by deconvolution are consistent with the domains identified by X‐ray crystallography.
The two coiled coils in the isolated rod domain of the intermediate filament protein desmin are staggered
Authors: POTSCHKA Martin , NAVE Rüdiger , WEBER Klaus , GEISLER Norbert
Published: March 2005

Desmin protofilaments and the proteolytically derived α‐helical rod domain have been characterized by highresolution gel permeation chromatography (GPC) using columns calibrated for the determination of viscosity radii. Additional characterization by chemical cross‐linking and the determination of sedimentation values allowed the calculation of the molecular dimensions of the molecular species isolated. In dilute buffers GPC separated desmin rod preparations into two complexes: a dimer species (single coiled coil) with a length of 50 ± 5 nm and a tetramer species (two coiled coils) with a length of 65 ± 5 nm. Thus the two coiled coils in the tetramer are staggered by approximately 15 nm. The hydrodynamically derived lengths of the rod dimer and tetramer are supported by electron microscopy after metal shadowing. The hydrodynamic properties of desmin protofilaments follow that of the rod tetramer. The data on the hydrodynamic analysis of the rod tetramer of desmin in solution are in full agreement with the structural information recently deduced from paracrystals of the rod of glial fibrillary acidic protein [Stewart, M., Quinlan, R. A. & Moir, R. D. (1989) J. Cell Biol. 109, 225–234]. Our results explain the inhomogeneity of molecules encountered in previous electron microscopical analyses.
A 31P‐nuclear‐magnetic‐resonance study of NADPH–cytochrome‐P‐450 reductase and of the Azotobacter flavodoxin/ferredoxin‐NADP+ reductase complex
Authors: BONANTS Peter J. M. , MÜLLER Franz , VERVOORT Jacques , EDMONDSON Dale E.
Published: March 2005

31P‐nuclear‐magnetic‐resonance spectroscopy has been employed to probe the structure of the detergentsolubilized form of liver microsomal NADPH–cytochrome‐P‐450 reductase. In addition to the resonances due to the FMN and FAD coenzymes, additional phosphorus resonances are observed and are assigned to the tightly bound adenosine 2′‐phosphate (2′‐AMP) and to phospholipids. The phospholipid content was found to vary with the preparation; however, the 2′‐AMP resonance was observed in all preparations tested. In agreement with published results [Otvos et al. (1986) Biochemistry 25, 7220–7228] for the protease‐solubilized enzyme, the addition of Mn(II) to the oxidized enzyme did not result in any observable line‐broadening of the FMN and FAD phosphorus resonances. The phospholipid resonances, however, were extensively broadened and the line width of the phosphorus resonance assigned to the bound 2′‐AMP was broadened by ∼ 70 Hz. The data show that only the phosphorus moieties of the phospholipids and the 2′‐AMP, but not the flavin coenzymes are exposed to the bulk solvent. Removal of the FMN moiety from the enzyme substantially alters the 31P‐NMR spectrum as compared with the native enzyme. The 2′‐AMP is removed from the enzyme during the FMN‐depletion procedure and the pyrophosphate resonances of the bound FAD are significantly altered. Reconstitution of the FMN‐depleted protein with FMN results in the restoration of the coenzyme spectral properties. Reduction of FMN to its air‐stable paramagnetic semiquinone form results in broadening of the FMN and 2′‐AMP resonances in the detergent‐solubilized enzyme. In agreement with previous results, FMN semiquinone formation had little or no effect on the line width of the FMN phosphorus resonance for the proteolytically solubilized enzyme.

31P‐NMR experiments with Azotobacter flavodoxin semiquinone, both in its free form and in a complex with spinach ferredoxin‐NADP+ reductase, mimic the differential paramagnetic effects of the flavin semiquinone on the line width of the FMN phosphorus resonance, observed by comparison of the detergent‐solubilized and protease‐solubilized forms of the reductase. The data demonstrate that assignment of the site of flavin semiquinone formation to a particular flavin coenzyme may not always be possible by 31P‐NMR experiments in multi‐flavin containing enzymes.
Regulation of glucokinase by a fructose‐1‐phosphate‐sensitive protein in pancreatic islets
Authors: MALAISSE Willy J. , MALAISSE‐LAGAE Francine , DAVIES Dewi R. , VANDERCAMMEN Annick , VAN SCHAFTINGEN Emile
Published: March 2005

In the post‐microsomal supernatant of pancreatic islets, prepared from fasted or fed rats, D‐fructose 1‐phosphate increased the activity of glucokinase by 20–30% as measured in the presence of D‐glucose 6‐phosphate and D‐fructose 6‐phosphate. Such an activation was less marked than that found in liver extracts. The islet cytosol was also found to inhibit purified liver glucokinase, and this effect was antagonized by D‐fructose 1‐phosphate. In the presence of hexose 6‐phosphates, partially purified islet glucokinase was inhibited by the hepatic glucokinase regulatory protein in a D‐fructose‐1‐phosphate‐sensitive manner. In intact islets, D‐glyceraldehyde stimulated the generation of 14C‐labelled D‐fructose 1‐phosphate from D‐[U‐14C]glucose and increased the production of 3H2O from D‐[5‐3H]glucose. These findings suggest that the activity of glucokinase in islet cells may be regulated by a protein mediating the antagonistic effects of D‐fructose 6‐phosphate and D‐fructose 1‐phosphate in a manner qualitatively similar to that operating in hepatocytes, but with lower efficiency.
Modulation of gelsolin content in rat aortic smooth muscle cells during development, experimental intimal thickening and culture
Authors: CHAPONNIER Christine , KOCHER Olivier , GABBIANI Giulio
Published: March 2005

Gelsolin is a Ca2+ and polyphosphoinositol‐phospholipid regulated modulator of actin polymerization present in most mammalian cells and in plasma. Cytoplasmic gelsolin was first described in highly motile cells such as leukocytes, where actin polymerization is dynamic; however arterial smooth muscle cells (SMC), despite their stabilized actin bundles, express high levels of gelsolin. We have investigated gelsolin modulation in rat aortic SMC by immunohistochemistry, Western blotting and Northern hybridization using three models which are known to show modulation of actin isoform expression: development, aortic intimal thickening after experimental endothelial injury, and growth in culture. When related to the protein and mRNA content of adult aortic SMC, gelsolin is expressed about 50% in aortic SMC of five‐day‐old rats, 20–30% in SMC of intimal thickening 15 days after endothelial injury (when SMC are actively replicating) and in SMC growing in culture; in intimal thickening 60 days after injury (when SMC have returned to quiescence), the gelsolin content becomes similar to that of control SMC. The high level of gelsolin in smooth muscle (SM) tissues and the down regulation with proliferation and migration raises the question as to whether gelsolin in these cells has functions other than the dynamic control of actin filament length. The similar modulation patterns of gelsolin and α‐SM actin suggest a preferential interaction between these two proteins.
Computer‐aided assignment of the 1H‐NMR spectrum of the viral‐protein‐genome‐linked polypeptide from cowpea mosaic virus
Authors: van de VEN Frank J. M. , LYCKSELL Per‐Olof , van KAMMEN Ab , HILBERS Cornelis W.
Published: March 2005

The 1H‐NMR spectrum of the viral‐protein‐genome‐linked (VPg) polypeptide from cowpea mosaic virus, has been interpreted via application of 2‐dimensional (2D) NMR techniques. The interpretation of the data was performed by a computer program called ‘PROSPECT’ (PROtein SPECTra), which detects the cross‐peak patterns of the amino acid residues in the spectra, assigns these patterns to amino acid types, and finally performs the sequential assignments using the well‐known ‘sequential walks’ obtained from the NOE spectrum.

Due to the severe overlap of resonances in the NMR spectrum of the VPg polypeptide, several ways of performing these walks existed. The program detected six alternatives for the sequential assignments of backbone NαH‐CβH‐CβH moieties.
Isolation and characterization of a phosphatidylinositol‐glycan‐anchor‐specific phospholipase D from bovine brain
Authors: HOENER Marius C. , STIEGER Susi , BRODBECK Urs
Published: March 2005

In recent years an increasing number of proteins has been shown to be membrane‐anchored by a covalently attached PtdIns‐glycan residue. In mammalian cells little is known about PtdIns‐glycan‐specific phospholipases which might play a role in the metabolism of PtdIns‐glycan‐anchored proteins. In order to identify PtdIns‐glycan‐specific phospholipases, a rapid and sensitive assay for such enzymes was developed using the PtdIns‐glycan‐anchored amphiphilic membrane form of acetylcholinesterase as substrate. The rate of product formation was monitored by the increase in soluble hydrophilic acetylcholinesterase in the aqueous phase after separation in Triton X‐114. With this assay we established the presence of a PtdIns‐glycan‐specific phospholipase in bovine brain. This enzyme was soluble and could be partially purified by a heat step followed by chromatography on DEAE‐cellulose and by gel filtration on Sepharose CL‐6B. PtdIns‐glycan‐specific phospholipase had a high affinity for the PtdIns‐glycan anchor of the substrate (Km= 52 nM) and did not degrade either PtdCho or PtdIns. Hydrophobic labeling of the anchor of the substrate with 3‐trifluoromethyl‐3‐(m‐[125I]iodophenyl)diazirine ([125I]TID) caused a marked decrease in the cleavage rate and methylation of the amino group of the glucosamine residue of the anchor decreased the cleavage rate to zero. Using [125I]TID‐labeled substrate, diradylglycerol phosphate was identified as the second product showing that the cleavage specificity of PtdIns‐glycan‐specific phospholipase was that of a phospholipase D. PtdIns‐glycan‐specific phospholipase D was inhibited by mercurials, o‐phenanthroline and EGTA. It was stimulated by Ca2+ in micromolar concentrations indicating that PtdIns‐glycan‐phospholipase D is a Ca2+‐regulated enzyme.
Properties and partial characterization of the heat‐shock factor from Tetrahymena pyriformis
Authors: do CARMO AVIDES Maria , SUNKEL Claudio E. , MORADAS‐FERREIRA Pedro , RODRIGUES‐POUSADA Claudina
Published: March 2005

A heat‐shock‐factor‐binding activity was identified in Tetrahymena pyriformis whole‐cell extracts and was further purified by sequential heparin‐agarose and sequence‐specific oligonucleotide affinity chromatography.

Tetrahymena heat‐shock factor (HSF) was able to bind to the heat‐shock elements (HSE) both before and after thermal stress, although heat shock altered both the HSE‐binding affinity and the protein · DNA‐complex mobility on polyacrylamide gels. The mobility difference was significantly reduced by treatment of the proteins with phosphatase.

The HSE‐binding proteins, isolated by oligonucleotide‐affinity chromatography, migrated on SDS/polyacrylamide gels as a closely spaced doublet to about 70 kDa. Polypeptides with similar molecular mass were recovered from preparative band‐shift gels indicating that both are components of the protein · DNA complex.
Primary structure and functional properties of cobra (Naja naja naja) venom Kunitz‐type trypsin inhibitor
Authors: SHAFQAT Jawed , BEG Obaid U. , YIN Shih‐Jiun , ZAIDI Zafar H. , JÖRNVALL Hans
Published: March 2005

A trypsin inhibitor from the venom of the cobra Naja naja naja has been isolated by a single step of reverse‐phase high‐performance liquid chromatography. The protein strongly inhibits trypsin (Ki= 3.5 pM). The primary structure was determined by peptide analysis of the [14C]carboxymethylated inhibitor. The 57‐residue polypeptide chain belongs to the family of Kunitz‐type inhibitors, and exhibits 42% residue identity with bovine pancreatic trypsin inhibitor. The structure shows only 70% identity with the corresponding peptide from the Capa cobra (Naja nevia), establishing that the inhibitor molecule exhibits extensive variations. Functionally, a basic residue at position P3′ correlates with strong inhibition.
Hemoglobin Saint Mandé [β102 (G4) Asn→Tyr]
Authors: POYART Claude , SCHAAD Olivier , KISTER Jean , GALACTEROS Frédéric , EDELSTEIN Stuart J. , BLOUQUIT Yves , AROUS Nicole
Published: March 2005

Oxygen equilibrium studies of purified hemoglobin Saint Mandé (Hb SM) [β102 (G4) Asn→Tyr] reveal a decreased oxygen affinity and cooperativity but to a lesser extent than found for Hb Kansas (β102 Thr). The low affinity of Hb SM depends on environmental conditions: eliminating chloride or raising the pH greatly elevated the ratio of p50 of Hb SM to that of Hb A. The alkaline Bohr effect is reduced by about 40%. The effects of anions (chloride, organophosphates) binding to deoxy Hb SM are also reduced. These data indicate that the functional properties of Hb SM are intermediary between Hb A and Hb Kansas. In addition, molecular graphics modeling of Hb SM in the oxy and deoxy structures indicate the possibility of a new hydrogen bond in the T state between β1102 Tyr and α242 Tyr. Stabilisation of the T state in this manner is a plausible explanation for several of the effects observed.
The synthesis of 3‐phosphonoalanine, phosphonopyruvic acid and phosphonolactic acid
Authors: SPARKES Michael J. , ROGERS Karen L. , DIXON Henry B. F.
Published: March 2005

3‐Phosphonoalanine has been made by the Strecker synthesis from phosphonoacetaldehyde, which is easily prepared from vinyl acetate. It gives phosphonopyruvate by transamination when treated with glyoxylate. Phosphonolactate, an analogue of phosphoglycerate, is prepared by reducing phosphonopyruvate. Diazotization of phosphonoalanine was investigated as a route for making phosphonolactate: addition of NaNO2 to the isoelectric form of phosphonoalanine gave much scission of the C‐P bond with release of phosphate; addition of HBr prevented this release and gave largely the bromo acid. The supplement reports the synthesis of arsonolactate, a similar analogue, by treating chlorolactate with alkaline arsenite.
Content, pattern and metabolic processing of rat‐liver gangliosides during liver regeneration
Authors: RIBONI Laura , GHIDONI Riccardo , BENEVENTO Angelo , TETTAMANTI Guido
Published: March 2005

During rat liver regeneration, the ganglioside content and distribution undergo significant changes after partial hepatectomy; total liver gangliosides increase remarkably till the 4th day after surgery, thereafter progressively decreasing to reach the values of sham‐operated controls at the 12th day. The qualitative pattern is characterized by the 95% relative increase of GD1a at the 4th day and the 40% relative decrease of GD1b. In order to investigate the processes of ganglioside penetration into cells, degradation and biosynthesis, radiolabelled GM1 ([Sph‐3H] GM1) was administered. One day after hepatectomy the liver uptake and metabolism of exogenous ganglioside were significantly reduced. Three days post‐surgery these parameters were restored to control values; however an increased radioactivity incorporation was found in GD1a, thus suggesting an enhancement of its biosynthesis around the 4th day. The data reported here suggest that in the first two days after partial hepatectomy, the ganglioside degradation is reduced with a consequent increase of ganglioside content; later on the catabolic routes normalize and some biosynthetic processes leading to GD1a are enhanced. GD1a seems to be a marker of a peculiar transition phase of liver regeneration.
Dialdehyde ATP derivative as an affinity modifier of the Na+,K+‐ATPase active site
Authors: BERNIKOV Lev R. , DZHANDZHUGAZYAN Karine N. , LUTSENKO Svetlana V. , MODYANOV Nikolai N.
Published: March 2005

Interaction of Na+,K+‐ATPase from pig kidney in various conformational states with the dialdehyde analogue of ATP, α,α‐(9‐adenyl)‐α′‐D‐(hydroxymethyl)diglycolaldehyde triphosphate ester (oATP), has been studied. This interaction leads to an enzyme modification which was shown to be of the affinity type according to the following criteria.

oATP can be hydrolyzed by Na+,K+‐ATPase and prevent inhibition of ATPase activity by γ‐[4‐(N‐2‐chloroethyl‐N‐methylamino)]benzylamide ATP, indicating that it interacts with Na+,K+‐ATPase in the enzyme active site.

oATP irreversibly inhibits ATP‐hydrolyzing activity of Na+,K+‐ATPase; the extent of inactivation is decreased in the presence of 20 mM ATP and depends on the ion composition of the modification medium. The inhibition and ATP protection are maximal in Na+,Mg2‐‐containing buffer.

The value of [14C]oATP incorporation into the α subunit is proportional to the degree of enzyme inactivation at low (< 0.1 mM) concentration of oATP and, on extrapolation to complete inhibition, corresponds to incorporation of 1.05 mol reagent/mol α subunit.

Tryptic hydrolysis of the isolated oATP‐modified α subunit and subsequent separation of the peptides revealed only one labelled fragment with a molecular mass of about 10 kDa. Localization of the modified fragment in the α‐subunit polypeptide chain is discussed.

A morpholine‐like structure was shown to be formed as a result of the modification.